4,064 Matching Annotations
- Apr 2022
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Im Mittelpunkt der emanzipatorischen Kritiksteht die Praxis. Als Gegenwartskritik stellt sie Diagnosen von Un-recht und greift in das Geschehen durch Stellungnahmen ein, denenüber eine Wissenschaftsgemeinschaft hinaus Gehör verschafft wird.
hashtag metoo, FFF, Feministischer Kampftag, Tag der Arbeiter*innen, weitere Beispiele?
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www.biorxiv.org www.biorxiv.org
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Author Response:
Reviewer #1 (Public Review):
The integrated stress response (ISR) controls cellular protein synthesis in response to diverse stimuli. A set of related protein kinases, with distinct regulatory domains that respond to different stress conditions, share a common kinase domain that specifically phosphorylates the translation factor eIF2 on its alpha subunit. Phosphorylation of eIF2 inhibits translation by inactivating eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2. The decameric eIF2B, a dimer of heteropentamers, is the key control hub of the ISR. Previously, a small molecule inhibitor of the ISR called ISRIB was found to bind to eIF2B and was proposed to reverse the impacts of eIF2 phosphorylation by increasing stabilizing the association of eIF2B heteropentamers into the functional decameric complex. However, more recently, an alternative model ISRIB action has been proposed. eIF2B is proposed to toggle between inactivate and active states. Binding of phosphorylated eIF2 to a regulatory site is proposed to trigger the inactive state by allosterically weakening binding of eIF2 at the active site. In the new model, ISRIB has been proposed to favor the active state conformation of eIF2B and thereby overcome the effects of eIF2 phosphorylation.
In this paper, the authors further study a previously described H160D mutation in the eIF2Bbeta subunit. This mutation at one of the dimer interfaces in eIF2B was previously proposed to inhibit eIF2B by weakening dimerization. Consistent with this hypothesis, the H160D mutation impaired dimerization of eIF2B(beta, gamma, delta, epsilon) tetramers. However, in this study, the authors show that the H160D mutation does not impair dimerization when eIF2Balpha is included; thus, the mutation impairs eIF2B activity without impairing dimerization. Using biochemical assays, the authors show that the H160D mutation impairs nucleotide exchange by eIF2B decamers and weakens the binding eIF2 to eIF2B. However, the binding of phosphorylated eIF2 to eIF2B is not weakened.
Cryo-EM structural analysis of the mutant eIF2B complex reveals a partial rocking of the decameric structure that resembles the structure of the eIF2B complex when bound to its inhibitor phosphorylated eIF2. In this partially rocked structure, both the ISRIB binding site at the dimer interface and the functional eIF2alpha binding sites are widened, providing a structural solution to why the mutation weakens eIF2 binding. Interestingly, the inhibitory binding site for phosphorylated eIF2 is not affected the H160D mutation. The authors propose that the H160D mutation in eIF2Bbeta induces an allosteric conformational change that mimics the effects of phosphorylated eIF2 binding to eIF2B.
Finally, the authors generated cell lines that exclusively express the mutant eIF2Bbeta subunit. The mutation impairs total protein synthesis and cell growth rate and leads to elevated expression of the ISR marker ATF4.
This is a high-quality study, the results are convincing and the authors conclusions are supported by the data. As the ISR has been implicated in a variety of diseases, further elucidation of the mechanism of action of eIF2B and ISRIB will be critical in the development of therapeutic interventions.
A weakness of the paper (that hopefully can be easily remedied) would be to show the quality control data to verify the mutant cell lines used in Figure 6. It would be good to see that the mutant allele is present in the cells and that no WT alleles remain. In addition, examination of eIF2alpha Ser51 phosphorylation in Figure 6A would strengthen the conclusion that the eIF2Bbeta mutation is activating ATF4 expression independent of changes in eIF2 phosphorylation. Also, use of ATF4 reporters in Figure 6A, in addition to the presented Western data, would provide a nice quantitative read-out for the impact of the H160D mutation on ATF4 mRNA translation. Finally, as the biochemical and structural data indicate that the H160D mutation impairs ISRIB activity, it would be worthwhile testing whether ISRIB will rescue the slow-growth of the H160D cell lines in Figure 6D (the anticipation is that this slow-growth phenotype will not be rescued by ISRIB).
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The genotype of our cell lines at the EIF2B2 target locus was screened for by PCR + restriction enzyme digest, and later sequence verified by deep sequencing. We used the CRISPResso2 pipeline to calculate allele frequencies and HDR editing efficiencies from the sequencing data, and now also include those results in a supplementary figure (Figure 6 – supplementary figure 1).
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The levels of baseline eIF2 phosphorylation are indeed the same in WT and both H160D clones, both when assessed using a phospho-specific antibody (for eIF2alpha Ser51-P) or through band shift using phospho-retention gels (Phos-tag). We now include a new supplementary figure with this data (Figure 6 – figure supplement 3A-B).
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It is well-established in the field that ATF4 is regulated at the translational level during acute ISR activation, and indeed, reporters with the ATF4 5’ UTR have been instrumental in studying and quantifying this, allowing scientists to forego time-intensive western blots and perform high throughput analyses. Stable integration, however, can notoriously affect genomic integrity and otherwise introduce clonal variation, even when the construct is targeted to a specific locus (for example when using the FlpIn system). We have observed heterogeneity in baseline ATF4 reporter signal even when comparing polyclonal cell lines generated by lentiviral integration. As it is best practice to avoid comparing between reporter cell lines generated in different backgrounds (WT vs H160D), particularly when investigating basal conditions, we consider it more appropriate to directly measure the levels of proteins of interest by western blot, as is also commonly done in the field. By showing that ATF4 protein levels increase (Figure 6A) but its transcript levels do not (Figure 6B), while those of its target genes do (Figure 6B), we equally confirm that ATF4 is translationally upregulated in the eIF2B H160D mutant. Moreover, our Western blot conditions provide enough sensitivity to differentiate no stress (lane 1) from mild stress (lanes 5 and 9) and high stress (lanes 7 and 11). We have added notation of these specific relevant lanes to the text to make the point more accessible to the reader. We therefore consider the generation of reporter cell lines in different genetic backgrounds to be a redundant abstraction of a phenotype that we already directly show.
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Indeed, as predicted from both our in vitro and cellular work, ISRIB did not alter growth half-life of H160D cells. We included these new data in Figure 6 – supplementary figure 3C.
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www.medrxiv.org www.medrxiv.org
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SciScore for 10.1101/2022.04.07.22273565: (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: Ethics: This study was approved (EK Nr: 1064/2021) by the Institutional Review Board (IRB) of the Office of Research Oversight/Regulatory Affairs, Medical University of Innsbruck, Austria, which is responsible for all human research studies conducted in the State of Tyrol (Austria).<br>IRB: Ethics: This study was approved (EK Nr: 1064/2021) by the Institutional Review Board (IRB) of the Office of Research Oversight/Regulatory Affairs, Medical University of Innsbruck, Austria, which is responsible for all human research studies conducted in the State of Tyrol (Austria).<br>Consent: Study population, study design and recruitment: A total of 74 patients infected with Omicron were recruited for the study under 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></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 assay: End-point binding IgG antibody titers to various SARS-CoV-2–derived antigens were measured using the Meso Scale Discovery (MSD) platform.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>End-point binding IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were coated with the specific antigen on spots in the 96 well plate and the bound antibodies in the samples (1:50000 dilution) were then detected by anti-human IgG antibodies conjugated with the MSD SULPHO-TAG which is then read on the MSD instrument which measures the light emitted from the tag.</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">ACE2 binding inhibition (Neutralization) ELISA: The V-PLEX COVID-19 ACE2 Neutralization kit (Meso Scale Discovery, Panel 23 (ACE2) Kit, K15570U) was used to quantitatively measure antibodies that block the binding of ACE2 to its cognate ligands (SARS-CoV-2 and variant spike subdomains).</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">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: For comparison of samples, data were presented as standard deviation in each group and were evaluated with 2-way ANOVA followed by Tukey’s multiple comparisons test on GraphPad Prism software (version 9.0.0).</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: 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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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.04.08.487674: (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 protocol and consent document were reviewed and approved by ethical review boards for all sites, and all subjects provided written informed consent.<br>Consent: The protocol and consent document were reviewed and approved by ethical review boards for all sites, and all subjects 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">Human Subjects: Peripheral blood mononuclear cells (PBMC) were obtained from subjects in study 2019nCoV-101, a phase I/II clinical trial of NVX-CoV2373 carried out in male and female adult subjects in Australia and the United States.</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">Donors of peripheral blood mononuclear cell fractions for the studies reported here were selected randomly from among subjects who had adequate specimens at all three specified dates (baseline, 7 days after dose 1 and 7 days after dose 2) and were treated twice with 5µg SARS-CoV-2 rS antigen plus 50µg Matrix-M™ adjuvant at a 21-day interval, as this was the dose and regimen selected to go forward for further clinical development.</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">PBMC from 5 recipients of placebo were included among the study samples in a blinded fashion.</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">Anti-S IgG ELISAs: Recombinant SARS-CoV-2 S protein was immobilised onto the surface of the 96-well microtiter plates by direct adsorption at 2°C to 8°C, followed by washing and blocking, Diluted reference standard (2-fold dilution series of 12 dilutions starting 1:1000) and human serum samples (3-fold dilution series of 12 dilutions) in assay buffer were then added in duplicate (100 µL per well) to the S protein-coated wells and specific antibodies are allowed to complex with the coated antigen for 2 hours ± 10 minutes at 24°C ± 2°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-S IgG</div><div>suggested: (LSBio (LifeSpan Cat# LS-C132241-1000, RRID:AB_10835882)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing, IgG bound to the rSARS-CoV-2 S protein was detected using a horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (Southern Biotech) incubated for 1 hour ± 10 minutes at 24°C ± 2°C.</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">Anti-rSARS-CoV-2 S protein IgG antibody level in clinical serum samples was quantitated in ELISA unit, EU/mL, by comparison to a reference standard curve.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-rSARS-CoV-2 S protein IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">hACE2 Binding Inhibition Assay: SARS-CoV-2 (rSARS-CoV-2) S protein was immobilised onto the surface of the 96-well microtiter plates by direct adsorption at 2°C to 8°C, followed by washing and blocking, Serial dilutions of human serum samples, including assay quality controls (QCs), were then added to the spike-coated wells and any molecules that could bind to the S protein, presumptively primarily spike-specific antibodies, were allowed to complex with the immobilized S protein (for 1 hour at 24±2°C) After a plate wash step, a fixed concentration of human ACE2 receptor (hACE2) with a polyhistidine-Tag (His-Tag) (SinoBiological) was added to the plate for incubation (1 hour at 24±2°C) during which the hACE2 bound to the S protein residues with binding sites not obstructed by bound antibody.</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">After incubation of the mixtures at 37°C and 5% CO2 for 1 hour, the mixtures were transferred to 96-well plates with confluent VeroE6 cells.</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">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">Samples were diluted inn duplicate to a base dilution of 1:5 or 1:10, followed by 11 × 1:2 serial dilutions in Dulbecco’s minimal essential medium (DMEM, Quality Biologicals) supplemented with 10% fetal bovine serum (heat inactivated, Sigma), 1% penicillin/streptomycin)(Gemini Bio-products) and 2mM L-glutamine (Gibco) resulting in 100µL per well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Quality Biologicals</div><div>suggested: (Aldevron, RRID:SCR_011017)</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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www.zylstra.org www.zylstra.org
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3. Who are you annotating with? Learning usually needs a certain degree of protection, a safe space. Groups can provide that, but public space often less so. In Hypothes.is who are you annotating with? Everybody? Specific groups of learners? Just yourself and one or two others? All of that, depending on the text you’re annotating? How granular is your control over the sharing with groups, so that you can choose your level of learning safety?
This is a great question and I ask it frequently with many different answers.
I've not seen specific numbers, but I suspect that the majority of Hypothes.is users are annotating in small private groups/classes using their learning management system (LMS) integrations through their university. As a result, using it and hoping for a big social experience is going to be discouraging for most.
Of course this doesn't mean that no one is out there. After all, here you are following my RSS feed of annotations and asking these questions!
I'd say that 95+% or more of my annotations are ultimately for my own learning and ends. If others stumble upon them and find them interesting, then great! But I'm not really here for them.
As more people have begun using Hypothes.is over the past few years I have slowly but surely run into people hiding in the margins of texts and quietly interacted with them and begun to know some of them. Often they're also on Twitter or have their own websites too which only adds to the social glue. It has been one of the slowest social media experiences I've ever had (even in comparison to old school blogging where discovery is much higher in general use). There has been a small uptick (anecdotally) in Hypothes.is use by some in the note taking application space (Obsidian, Roam Research, Logseq, etc.), so I've seen some of them from time to time.
I can only think of one time in the last five or so years in which I happened to be "in a text" and a total stranger was coincidentally reading and annotating at the same time. There have been a few times I've specifically been in a shared text with a small group annotating simultaneously. Other than this it's all been asynchronous experiences.
There are a few people working at some of the social side of Hypothes.is if you're searching for it, though even their Hypothes.is presences may seem as sparse as your own at present @tonz.
Some examples:
@peterhagen Has built an alternate interface for the main Hypothes.is feed that adds some additional discovery dimensions you might find interesting. It highlights some frequent annotators and provide a more visual feed of what's happening on the public Hypothes.is timeline as well as data from HackerNews.
@flancian maintains anagora.org, which is like a planet of wikis and related applications, where he keeps a list of annotations on Hypothes.is by members of the collective at https://anagora.org/latest
@tomcritchlow has experimented with using Hypothes.is as a "traditional" comments section on his personal website.
@remikalir has a nice little tool https://crowdlaaers.org/ for looking at documents with lots of annotations.
Right now, I'm also in an Obsidian-based book club run by Dan Allosso in which some of us are actively annotating the two books using Hypothes.is and dovetailing some of this with activity in a shared Obsidian vault. see: https://boffosocko.com/2022/03/24/55803196/. While there is a small private group for our annotations a few of us are still annotating the books in public. Perhaps if I had a group of people who were heavily interested in keeping a group going on a regular basis, I might find the value in it, but until then public is better and I'm more likely to come across and see more of what's happening out there.
I've got a collection of odd Hypothes.is related quirks, off label use cases, and experiments: https://boffosocko.com/tag/hypothes.is/ including a list of those I frequently follow: https://boffosocko.com/about/following/#Hypothesis%20Feeds
Like good annotations and notes, you've got to put some work into finding the social portion what's happening in this fun little space. My best recommendation to find your "tribe" is to do some targeted tag searches in their search box to see who's annotating things in which you're interested.
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niklasblog.com niklasblog.com
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tvrctkmytfln.gitee.io tvrctkmytfln.gitee.io
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.04.06.487306: (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 binding, samples were washed twice in sort buffer and incubated with a 1:100 dilution of α-Myc-FITC (Abcam, #Ab1263) and α-His-PE (Abcam, #Ab72467) antibody at room temperature for 10 minutes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>α-His-PE ( Abcam , #Ab72467 )</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 1 hour at 4 °C, cells were washed twice in sort buffer and stained with α-His-PE antibody (1:100).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>α-His-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">Briefly, 293T cells expressing scFv on the cell surface were detached with trypsin-EDTA (Thermo Fisher Scientific) and washed twice in prechilled sort buffer (1% FCS, 25 mM HEPES-KOH pH 7.9, 1 mM EDTA in PBS).</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">An expression vector for the secretion and purification of His-tagged proteins from mammalian cells was generated by insertion of DNA encoding an IGK leader and 8xHis tag into pCS2-MT+.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCS2-MT+</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A DNA fragment encoding the SARS-CoV-2 RBD (aa 319-591) was amplified from pCAGGS-SARS-CoV-2-Spike vector (a kind gift from Keith Grehan) and cloned in frame with the N-terminal IGK leader sequence and C-terminal 8xHis tag using NheI and XhoI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS-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">The round 3 enriched ultralong scFv library was transferred from pBovShow into this lentiviral vector and lentiviruses were generated by transient transfection of 293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pBovShow</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, 4 μg Lenti-BovShow-IRES-PuroR, 4 μg of pCMVR8.74 packaging vector (Addgene plasmid #22036) and 2 μg of pMD2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMVR8.74</div><div>suggested: RRID:Addgene_22036)</div></div><div style="margin-bottom:8px"><div>pMD2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">G coat protein vector (Addgene plasmid #12259; both gifts from Didier Trono) were mixed with PEI at a 1:3 molar ratio and added to the 10 cm2 dish.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>#12259</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotype Neutralisation assays: Pseudotyped lentiviral particles were generated by transfecting 3 × 106 293T cells in a 10 cm2 dish with a lentivirus backbone plasmid encoding a luciferase reporter gene (BEI: NR-52516 pHAGE-CMV-Luc2-IRES-ZsGreen-W), a plasmid encoding either the SARS-CoV Spike (pCAGGS-SARS-CoV-Spike_Urbani), SARS-CoV-2 Spike (BEI: NR-52514) or VSV glycoprotein (VSV-G; pMD2.G) and the packaging vectors HDM-Hgpm2 (BEI: NR-52517), HDM-tat1b (BEI: NR-52518) and pRC-CMV-Rev1b (BEI: NR-52519).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHAGE-CMV-Luc2-IRES-ZsGreen-W</div><div>suggested: RRID:Addgene_164432)</div></div><div style="margin-bottom:8px"><div>pCAGGS-SARS-CoV-Spike_Urbani</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pMD2 . G</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pRC-CMV-Rev1b</div><div>suggested: RRID:Addgene_164443)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293T cells were transfected with either B9-WT or B9-Mut plasmid DNA and cell-surface expressed scFvs were tested for binding to purified SARS-CoV RBD (200 nM) using the standard staining protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>B9-Mut</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 raw HDX-MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset: PXD032965.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PRIDE</div><div>suggested: (Pride-asap, RRID:SCR_012052)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Disruption of the B9-scFv knob domain: The sequence encoding B9-scFv was mutated in the BovShow cell surface expression vector; residues 123YNCRPAVWY131 of the B9-scFv knob domain (B9-WT) were replaced with the irrelevant amino acid sequence 123ETCYYGSGL131 by site-directed mutagenesis (B9-Mut) with Q5 polymerase (New England Bioloabs)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>New England Bioloabs</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The KD for interactions between cell surface scFvs and recombinant RBD proteins was estimated by non-linear analyses of the log(molarity)-response plots on 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: 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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www.biorxiv.org www.biorxiv.org
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This work has been peer reviewed in GigaScience (https://doi.org/10.1093/gigascience/giac007), which carries out open, named peer-review.
These reviews are published under a CC-BY 4.0 license and were as follows:
Reviewer 3: Mudra Hegde
Summary:
In this manuscript, Poudel et al. present a software, GuideMaker, to rapidly design sgRNAs targeting non-model genomes. Various input parameters such as PAM motif, guide length, length of seed region for off-target searching and so on can be toggled to design a panel of sgRNAs for pooled screening projects. The tool also helps pick control sgRNAs to include in the sgRNA pool. To benchmark the computational performance of their tool, the authors used GuideMaker to design sgRNAs targeting E.coli, P.aeruginosa, Aspergillus fumigatus and Arabidopdis thaliana. They also compared GuideMaker to the existing design tool, CHOPCHOP and reported that the targets identified by GuideMaker were mostly similar to those identified by CHOPCHOP. This tool can be used as a stand-alone web application, command-line software or in the CyVerse Discovery Environment.
Overall, the tool is very well documented and easy to use. In the current version of the manuscript, GuideMaker does not show a clear improvement over the state-of-the-art design tool, CHOPCHOP. The authors do not implement any existing on-target scoring methods to determine the targeting efficacy of the picked sgRNAs. This can lead to picking guides that are highly specific but not effective enough.
Major points:
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Implementing on-target scoring methods, at least for the Cas enzymes that have on-target efficacy information, can help improve the process of picking sgRNAs. This tool will probably be used more often with standard Cas enzymes and it will be useful to have on-target efficacy scores attached to the guide RNAs.
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The authors do a thorough analysis of the computational performance of GuideMaker with various genomes and Cas enzymes but including a comparison of the computational performance of GuideMaker vs. CHOPCHOP will strengthen the manuscript.
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The authors define the PAM sequence of SaCas9 to be NGRRT whereas the canonical PAM sequence of SaCas9 is NNGRRT. This should be modified throughout the manuscript and analyses involving SaCas9 should be redone.
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A good addition to the tool would be to output a file with all the sequences that were designed targeting the region of interest with the specific PAM sequence. This gives the user a sense of the universe from which the final guides were picked.
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Another useful input parameter would be to specify a target region that the user wants to focus on such as letting the user input genomic coordinates or a gene name or locus tag. For example, CRISPy by Blin et al., 2016 takes a GenBank file as input and allows the user to input features specific to the uploaded genome.
Minor points:
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"CyVerse" is misspelled as "CyCVerse" in multiple places in the manuscript.
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Reference Figure 2 in Line 92.
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Line 154: "Ratios between tools were calculated by dividing the number of gRNA identified.."
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In Supplementary Figure 3 "wit haVX2" should be "with aVX2".
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GitHub link in Line 336 does not work.
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Line 225-226: "GuideMaker also creates off-target gRNAs for use as negative controls in highthroughput experiments." "Off-target gRNAs" is misleading in this context.
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.04.05.487060: (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">Aminoadamantane and aminoadamantane nitrate drugs: Aminoadamantane nitrate compounds (blindly coded NMT2, NMT3, NMT5-NMT9, and NMT5-Met (metabolite, sans nitro group) were synthesized by and obtained from EuMentis Therapeutics, Inc. (Newton, MA), and have been described previously6–9, 33.</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 with secondary antibodies (IR-dye 680LT-conjugated goat anti-mouse [1:20,000; Li-Cor, 926-68020] or IR-dye 800CW-conjugated goat anti-rabbit [1:15,000; Li-Cor, 926-32211]), membranes were scanned with an Odyssey infrared imaging system (Li-Cor)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: (LI-COR Biosciences Cat# 926-68020, RRID:AB_10706161)</div></div><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: (Santa Cruz Biotechnology Cat# sc-53804, RRID:AB_783976)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunoprecipitants were eluted and subjected to immunoblotting with anti-ACE2 antibody (1:1000, Cell Signaling, #15983) and anti-SARS-CoV-2 Spike protein antibody (1:2000, Abcam, ab275759)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-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">Primary antibodies and dilutions were as follows: Mouse anti-TNFα (5 µg/ml, Abcam, #ab1793) and rabbit anti-macrophage inflammatory protein 1α (MIP-1α)/CCL3+CCL3L1 (1:250</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-TNFα</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-macrophage inflammatory protein 1α ( MIP-1α)/CCL3+CCL3L1</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, all cells were collected and subjected to biotin switch-assay and immunoblotting with anti-ACE2 antibody to assess the levels of SNO-ACE2 and total input ACE2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-ACE2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>total input 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">Cell lines: HEK293T (System Biosciences, LV900A-1) and HEK293-Spike cells (SARS-CoV-2 Spike (D614)-expressing 293 cells [293-SARS2-S cells, InvivoGen]) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX™ (DMEM, high glucose, GlutaMAX™ Supplement, Life Technologies, 10566016) supplemented with 10% fetal bovine serum (FBS; Sigma, F7524), 100 IU/ml, and 100 µg/ml penicillin-streptomycin (Thermo Fisher Scientific, 10378016) at 37 °C in a 5% CO2 incubator.</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>HEK293-Spike</div><div>suggested: None</div></div><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">SARS-CoV-2 virus generation: Monkey Vero E6 cells were plated in a T225 flask with complete DMEM containing 10% FBS, 1×PenStrep, 2 mM L-glutamine and incubated for overnight at 37 °C in a humified atmosphere of 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></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HeLa-ACE2 cells were seeded in the assay-ready plates at 1.6×103 cells/well in assay medium, and plates were incubated for 24 h at 37 ℃ with 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa-ACE2</div><div>suggested: JCRB Cat# JCRB1845, RRID:CVCL_B3LW)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Homogenized lungs were titrated 1:10 over 6 steps and layered over 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><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">. Monkey Vero E6 cells (ATCC CRL-1586) were maintained in complete DMEM (Corning, 15-013-CV) containing 10% FBS, 1×PenStrep (Corning 20-002-CL), 2 mM L-glutamine (Corning, 25-005-CL) at 37 °C in a 5% CO2 incubator. Plasmids: hACE2 was a gift from Hyeryun Choe (Addgene plasmid #1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786)46.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_1786)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The C262A, C498A, C261/498A mutant ACE2 constructs were generated using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies, 210514) according to the manufacturer’s protocol. pGBW-m4252984 (SARS-CoV-2 E [envelope]) was a gift from Ginkgo Bioworks (Addgene plasmid #153898; http://n2t.net/addgene:153898; RRID:Addgene_153898).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_153898)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MLV-gag/pol, MLV-CMV-Luciferase, SARS-CoV-2, and VSV-G plasmids were a gift from David Nemazee</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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of human ACE2 protein: The N-terminal peptidase domain of human ACE2 (residues 19 to 615, GenBank: BAB40370.1) was cloned into phCMV3 vector and fused with C-terminal His-tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>phCMV3</div><div>suggested: RRID:Addgene_173431)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, HEK293T cells were transiently co-transfected with MLV-gag/pol, MLV-CMV-Luciferase plasmid, and SARS- CoV-2 Spike (D614) or VSV-G plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MLV-CMV-Luciferase</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For transient expression in HEK293T cells, we used a transfection reagent (Fugene® HD, Promega) to co-transfect plasmids containing cDNAs for SARS-CoV-2 E protein (pGBW-m4133502, Addgene) and green fluorescent protein (GFP) at a ratio of 1:0.1 (0.5:0.05 µg/well, respectively).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGBW-m4133502</div><div>suggested: RRID:Addgene_153565)</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">Maximum intensity projection of images was generated, and fluorescence intensity was analyzed with ImageJ software (https://imagej.nih.gov/ij/download.html) as previously described50.</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 (MD) simulations were performed on the Frontera supercomputer at the Texas Advanced Supercomputing Center (TACC) using NAMD 2.1461 and CHARMM36m all-atom additive force fields62–64.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NAMD</div><div>suggested: (NAMD, RRID:SCR_014894)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Molecular Devices) with a 10× objective, and total live cells per well quantified in the acquired images using the Live Dead Application Module (MetaXpress).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MetaXpress</div><div>suggested: (MetaXpress, RRID:SCR_016654)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analyses were performed using GraphPad Prism 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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www.ddbj.nig.ac.jp www.ddbj.nig.ac.jpDDBJ1
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Ya están anotadas todas las bases de datos de este hub
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SciScore for 10.1101/2022.04.03.486854: (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: Data collection, structure determination, and refinement: The X-ray diffraction data were collected at the beamlines BL18U (RBD-Fab2303) and BL02U (Fab2212) of the Shanghai Synchrotron Research Facility.</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: Immunofluorescence imaging and analysis: For viral nucleocapsid detection in Vero-TMPRSS2 cells by immunofluorescence, cells were washed twice with PBS x1 and fixed in 4% formaldehyde for 30 min at RT.</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 plates were then washed 3 times with washing buffer before adding secondary anti-IgG (Jackson ImmmunoResearch) antibody conjugated to horseradish peroxidase (HRP) diluted 1:5000 in blocking buffer, and incubation for 1 h at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-IgG</div><div>suggested: (Vector Laboratories Cat# FI-5000, RRID:AB_2336128)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Monolayers were observed for at least 4 days for appearance of CPE and then fixed as above and stained with antibody against SARS-CoV-2 Nucleocapsid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody against SARS-CoV-2 Nucleocapsid.</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Fixed cells were washed with PBS x1 and permeabilized for immunofluorescence using BD Cytofix/Cytoperm according to the manufacturer’s protocol for fixed cells, and then stained for SARS-CoV-2 with a primary nucleocapsid antibody (GeneTex GTX135357) and a secondary anti-rabbit AF647 antibody (ThermoFisher A20185), The nuclei were counterstained with Sytox Green.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit AF647</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 soluble RBD, spike and antibodies for crystallization: SARS-CoV-2 RBD (residues Arg319 to Lys529) was expressed by using the Bac-to-Bac Baculovirus System (Invitrogen)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Lys529</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Supernatant containing S2P was collected four days after transfection, S2P was affinity purified by anti-Flag antibody beads and was eluted by 0.1 mg/mL 3×Flag peptide in 20 mM HEPES at pH 7.6 and 150 mM NaCl.</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><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 stably expressing hACE2 were seeded into 0.1% gelatin-coated 96-well plates (Greiner) at an initial density of 0.75 × 105 cells per well.</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">Virus titers were validated using a combination of fluorescent focus assay and tissue culture infectious dose (TCID)50 assays on Vero-TMPRSS2 and Calu-3 (ATCC) monolayers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu-3</div><div>suggested: KCLB Cat# 30055, RRID:CVCL_0609)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For TCID50 assays, Vero-TMPRSS2 or Calu3 cells were infected as above and 100µL DMEM or MEM with 2% FBS was subsequently added per well.</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">Immunofluorescence imaging and analysis: For viral nucleocapsid detection in Vero-TMPRSS2 cells by immunofluorescence, cells were washed twice with PBS x1 and fixed in 4% formaldehyde for 30 min at RT.</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 S2P or S6P pCMV plasmids were used to transiently transfect HEK293F cells with polyethylenimine (PEI) (Polysciences, #24765).</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">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">Expression and purification of soluble RBD, spike and antibodies for crystallization: SARS-CoV-2 RBD (residues Arg319 to Lys529) was expressed by using the Bac-to-Bac Baculovirus System (Invitrogen)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data collection, structure determination, and refinement: The X-ray diffraction data were collected at the beamlines BL18U (RBD-Fab2303) and BL02U (Fab2212) of the Shanghai Synchrotron Research Facility.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RBD-Fab2303</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">Antibody inhibition of hACE2 binding to cell-expressed spike: Expi293F cells were transfected with pcDNA 3.1 containing SΔC19 of wild type, Alpha, Beta, or Delta variants, using the ExpiFectamine 293 Transfection Kit (Thermo Fisher).</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">Pseudo-particle preparation and neutralization assays: SARS-CoV-2-spike pseudo-particles were obtained by co-transfecting Expi293F cells with pCMV delta R8.2, pLenti-GFP (Genecopoeia), and pcDNA 3.1 SΔC19 (Thermo Fisher) at a ratio of 1:2:1, respectively, according to the manufacturer’s instructions.</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><div style="margin-bottom:8px"><div>pLenti-GFP</div><div>suggested: RRID:Addgene_172394)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD containing the gp67 signal peptide and a C-terminal 6×His tag was inserted into pFastBac1 to form the plasmid pFastBac1-RBD.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFastBac1</div><div>suggested: RRID:Addgene_1956)</div></div><div style="margin-bottom:8px"><div>pFastBac1-RBD</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">Fixed cells were washed with PBS x1 and permeabilized for immunofluorescence using BD Cytofix/Cytoperm according to the manufacturer’s protocol for fixed cells, and then stained for SARS-CoV-2 with a primary nucleocapsid antibody (GeneTex GTX135357) and a secondary anti-rabbit AF647 antibody (ThermoFisher A20185), The nuclei were counterstained with Sytox Green.</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 logged from the Incucyte analysis modules and graphed with GraphPad Prism 8.</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">SerialEM was used for the data collection.</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">The CTF parameters of the micrographs were determined by using the program Gctf with local defocus variations taken into consideration59.</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">90569 particles were picked by Gautomatch and then were subjected to 2D classifications with RELION 3.0.</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: 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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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.04.01.486719: (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">B cells were incubated for 30 min at 4°C with biotinylated tri-S and DyLight 650-coupled RBD, washed once with 1% FBS-PBS (FACS buffer), and incubated for 30 min at 4°C with a cocktail of mouse anti-human antibodies: CD19 Alexa 700 (HIB19, BD Biosciences, San Jose,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human antibodies: CD19</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cTfh antibody panel included: CD3 BV605 (SK7), CD4 PE-CF594 (RPA-T4), CD185/CXCR5 AF-488 (RF8B2), CD183/CXCR3 PE-Cy™5 (1C6/CXCR3), CD196/CCR6 PE-Cy™7 (11A9), CD197/CCR7 AF647 (3D12) (BD Biosciences), CD279/PD1 BV421 (EH12.2H7, BioLegend), and CD278/ICOS PE (ISA-3, Thermo Fisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD3</div><div>suggested: (SouthernBiotech Cat# 9515-31, RRID:AB_2796843)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The purified parental IgG1 antibody versions of benchmarked mAbs [REGN10933, REGN10987 (Hansen et al., 2020), CB6 (Shi et al., 2020), LY-CoV555 (Jones et al., 2021), CT-P59 (Kim et al., 2021), COV2-2196, COV2-2130 (Zost et al., 2020b), ADG-2 (Garrett Rappazzo et al., 2021) and S309 (Pinto et al., 2020)] were prepared as described above after cloning of synthetic DNA fragments (GeneArt, Thermo Fisher Scientific) coding for the immunoglobulin variable domains.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CB6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>LY-CoV555</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>S309</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Comparative ELISA binding of Cv2.1169 IgG1 and IgA1 antibodies was performed at a concentration of 70 nM, and 7 consecutive dilutions in PBS.</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">To quantify blood-circulating human Cv2.1169 IgA1 and IgG1 in treated K18-hACE2 mice and golden hamsters, high-binding 96-well ELISA plates (Costar, Corning) were coated overnight with 250 ng/well of purified goat anti-human IgA or IgG antibody (Jackson ImmunoResearch, 0.8 µg/ml final).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>purified goat anti-human IgA</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG antibody</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washings, the plates were revealed by incubation for 1 h with goat HRP-conjugated anti-mice IgG, anti-golden hamster IgG, anti-human IgG or anti-human IgA antibodies (Jackson ImmunoReseach, 0.8 µg/ml final) and by adding 100 µl of HRP chromogenic substrate (ABTS solution, Euromedex) after washing steps.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mice IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-golden hamster IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human 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">HEp-2 IFA assay: Recombinant SARS-CoV-2 S-specific and control IgG antibodies (mGO53 and ED38) at 100 µg/ml were analyzed by indirect immuno-fluorescence assay (IFA) on HEp-2 cells sections (ANA HEp-2 AeskuSlides®, Aesku.Diagnostics, Wendelsheim, Germany) using the kit’s controls and FITC-conjugated anti-human IgG antibodies as the tracer according to the manufacturer’ instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>control IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>ED38</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Membranes were inserted into a Miniblot apparatus (Immunetics) and then incubated with human mAbs (at a concentration of 1 µg/ml) and mouse anti-Hisx6 antibody (1 µg/ml, BD Biosciences) in PBS-T 5% dry milk in each channel for 2 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Hisx6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Microarrays were blocked for 1 h in blocking solution (Thermo Fisher), washed and incubated for 1h30 with IgG antibodies at 2.5 µg/ml as previously described (Grzelak et al., 2020).</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">Antibodies (Cv2.1169, Cv2.1353, Cv2.3194, Cv2.3235 and Cv2.5213) and ACE2 ectodomain were covalently coupled to CM5 sensor chips (Biacore) using amino-coupling kit (Biacore) according to the manufacturer’s procedure.</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">Antibody-dependent cellular phagocytosis (ADCP) assay: PBMC were isolated from healthy donors’ blood (Etablissement Français du Sang) using Ficoll Plaque Plus (GE Healthcare)</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">Phagocytic scores were calculated by dividing the fluorescence signals (% FITC-positive cells x geometric MFI FITC-positive cells) given by anti-SARS-CoV-2 spike antibodies by the one of the negative control antibody mGO53.</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">Antibody-dependent cellular cytotoxicity (ADCC) assay: The ADCC activity of anti-SARS-CoV2 S IgG antibodies was determined using the ADCC Reporter Bioassay (Promega) as previously described (Dufloo et al., 2021).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV2 S IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 5×104 Raji-Spike cells were co-cultured with 5×104 Jurkat-CD16-NFAT-rLuc cells in presence or absence of SARS-CoV2 S-specific or control mGO53 IgG antibody at 10 µg/ml or 50 µg/ml and 10 consecutive 1:2 dilutions in PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>control mGO53 IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Crystallization and structure determinations: The Fab of anti-SARS-CoV-2 S antibody CR3022 (Ter Meulen et al., 2006), served as a crystallization chaperone molecule, and was produced and purified as described above (section with heading Single B-cell FACS sorting and expression-cloning of antibodies) (Koide, 2009).</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">Six or 22 h post-inoculation, mice received an intraperitoneal (i.p.) injection of 5, 10, 20 or 40 mg/kg of Cv2.1169 IgG or IgA antibody, and of mGO53 control IgG or IgA antibody.</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">Four or 24 h post-intranasal inoculation, hamsters received an intraperitoneal (i.p.) injection of 10 or 5 mg/kg of Cv2.1169 IgG or IgA antibody, as well as the mGO53 control antibody or PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>mGO53</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 were amplified by one or two passages in Vero E6 cell cultures and titrated.</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">Drosophila S2 cells were stably co-transfected with pT350 and pCoPuro (for puromycin selection) plasmids.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>S2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEp-2 IFA assay: Recombinant SARS-CoV-2 S-specific and control IgG antibodies (mGO53 and ED38) at 100 µg/ml were analyzed by indirect immuno-fluorescence assay (IFA) on HEp-2 cells sections (ANA HEp-2 AeskuSlides®, Aesku.Diagnostics, Wendelsheim, Germany) using the kit’s controls and FITC-conjugated anti-human IgG antibodies as the tracer according to the manufacturer’ instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEp-2</div><div>suggested: CLS Cat# 300397/p694_Hep-2, RRID:CVCL_1906)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 5×104 Raji-Spike cells were co-cultured with 5×104 Jurkat-CD16-NFAT-rLuc cells in presence or absence of SARS-CoV2 S-specific or control mGO53 IgG antibody at 10 µg/ml or 50 µg/ml and 10 consecutive 1:2 dilutions in PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat-CD16-NFAT-rLuc</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 5×104 Raji-Spike cells were cultivated in the presence of 50% normal or heat-inactivated human serum, and with or without IgG antibodies (at 10 µg/ml or 50 µg/ml and 10 consecutive 1:2 dilutions in PBS).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Raji-Spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The supernatants were titrated on Vero-E6 cells by classical plaque assays using semisolid overlays (Avicel, RC581-NFDR080I, DuPont) and expressed and PFU/100 mg of tissue (Baer and Kehn-Hall, 2014).</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">SARS-CoV-2 infection and treatment in K18-hACE2 mice: B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (stock #034860) were imported from The Jackson Laboratory (Bar Harbor, ME, USA) and bred at the Institut Pasteur under strict SPF conditions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><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">Expression and purification of viral proteins: Codon-optimized nucleotide fragments encoding stabilized versions of SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43-CoV, HKU1-CoV, 229E-CoV, NL63-CoV (2P) and BA.1 spike (HexaPro) (S) ectodomains, and SARS-CoV-2 S2 domain, followed by a foldon trimerization motif and C-terminal tags (Hisx8-tag, Strep-tag, and AviTag) were synthesized and cloned into pcDNA3.1/Zeo(+) expression vector (Thermo Fisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1/Zeo(+</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For crystallographic experiments, a codon-optimized nucleotide fragment encoding the SARS-CoV-2 RBD protein (residues 331-528), followed by an enterokinase cleavage site and a C-terminal double strep-tag was cloned into a modified pMT/BiP expression vector (pT350, Invitrogen)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pMT/BiP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Drosophila S2 cells were stably co-transfected with pT350 and pCoPuro (for puromycin selection) plasmids.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pT350</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCoPuro</div><div>suggested: RRID:Addgene_17533)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For Cryo-EM experiments, a codon-optimized nucleotide fragment encoding the SARS-CoV-2 spike (S) protein (residues 1-1208) was cloned with its endogenous signal peptide in pcDNA3.1(+) vector, and expressed as a stabilized trimeric prefusion construct with six proline substitutions (F817P, A892P, A899P, A942P, K986P, V987P), along with a GSAS substitution at the furin cleavage site (residues 682–685), followed by a Foldon trimerization motif (Hsieh et al., 2020), and C-terminal tags (Hisx8-tag, Strep-tag and AviTag).</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 dimeric form of Cv2.1169 IgA1 was produced by co-transfection of Freestyle™ 293-F cells with a human J chain pcDNA™3.1/Zeo(+) vector as previously described (Lorin and Mouquet, 2015)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA™3.1/Zeo(+</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To evaluate spike cross-reactivity, Freestyle™ 293-F were transfected with pUNO1-Spike-dfur expression vectors (Spike and SpikeV1 to V11 plasmids, Invivogen) (1.2 µg plasmid DNA per 106 cells) using PEI-precipitation method.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pUNO1-Spike-dfur</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 S-Fuse neutralization assay: S-Fuse cells (U2OS-ACE2 GFP1-10 or GFP 11 cells) were mixed (ratio 1:1) and plated at a density of 8 × 103 per well in a μClear 96-well plate (Greiner Bio-One) as previously described (Buchrieser et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GFP1-10</div><div>suggested: RRID:Addgene_68715)</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">Cv2.1169 were also cloned into human Igγ1NA, Igγ1LALA [N297A and L234A/L235A mutations introduced by Site-Directed Mutagenesis (QuickChange, Agilent Technologies)], Igα1 and Fab-Igα1-expressing vectors (Lorin and Mouquet,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Agilent Technologies</div><div>suggested: (Agilent Technologies, RRID:SCR_013575)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry binding assays: SARS-CoV-2 specificity validation of cloned human IgG antibodies was performed using the S-Flow assay as previously described (Grzelak et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: (Active Motif Cat# 91351, RRID:AB_2847848)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were acquired using a CytoFLEX flow cytometer (Beckman Coulter), and analyzed using FlowJo software (v10.7.1; FlowJo LLC).</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">HEp-2 sections were examined using the fluorescence microscope Axio Imager 2 (Zeiss, Jena, Germany), and pictures were taken at magnification x 40 with 5000 ms-acquisition using ZEN imaging software (Zen 2.0 blue version, Zeiss) at the Imagopole platform (Institut Pasteur)</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 each antibody, Z-scores were calculated using ProtoArray® Prospector software (v5.2.3, Thermo Fisher Scientific), and deviation (σ) to the diagonal, and polyreactivity index (PI) values were calculated as previously described (Planchais et al., 2019).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ProtoArray® Prospector</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The evaluation kinetic parameters of the studied interactions were performed by using BIAevaluation version 4.1.1 Software (Biacore).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BIAevaluation</div><div>suggested: (BIAevaluation Software, RRID:SCR_015936)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IC50 values were calculated using Prism software (v.9.3.1,</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">The final models were built by combining real space model building in Coot (Emsley et al., 2010) with reciprocal space refinement with phenix.refine.</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 models were validated with Molprobity (Williams et al., 2018).</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">Superpositions and figures were rendered using Pymol and UCSF Chimera (Pettersen et al., 2004)</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">CryoSPARC blob picker was used for automated particle picking and the resulting particles used to obtain initial 2D references, which were then used to auto-pick the micrographs.</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">Volcano plot comparing gene features (n=206 parameters) of tri-S+ B cells and normal memory B-cells (mB) was also performed using GraphPad Prism software (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">Circos plot linking antibody sequences with at least 75% identity within their CDRH3 was performed using online software at http://mkweb.bcgsc.ca/circos.</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">GraphPad Prism 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">Principal component analysis (PCA) was performed using the prcomp() function in R Studio Server (v1.4.1103)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>R Studio Server</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">NCT04325646</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">Sero-epidemiological Study of the SARS-CoV-2 Virus Responsib…</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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SciScore for 10.1101/2022.03.30.486313: (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">Membranes were first incubated with the primary antibody (Anti-Glutathione antibody [D8], ab19534,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Glutathione</div><div>suggested: (Abcam Cat# ab19534, RRID:AB_880243)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">; Mouse anti DDDDK-Tag (FLAG-tag) mAb, AE005, ABclonal) in 5% BSA TBST for 16 h at 4 °C, washed with TBST three times, then incubated with the secondary antibody (HRP-labeled Goat Anti-Mouse IgG(H+L), A0216, Beyotime) in 5% BSA TBST (1 h, 25 °C), and washed with TBST three times.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti DDDDK-Tag (FLAG-tag</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-Mouse IgG(H+L)</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 the present study, HEK293T cells were seeded in 12-well plates overnight.</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">For bacterial expression, the cDNA encoded the SARS-CoV-2 PLpro with E. coli codon optimization was ordered from GenScript and cloned into the pET15b expression vector with an N-terminal 6 × His-SUMO2 fusion tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET15b</div><div>suggested: RRID:Addgene_129689)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For transfection of mammalian cell, the cDNA encoded the SARS-CoV-2 PLpro with mammalian codon optimization was also ordered from GenScript and cloned into the pcDNA 3.1 with an C-terminal FLAG tag.</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">The sequence of pcDNA3-PL-flipGFP-T2A-mCherry was designed based on plasmid pcDNA3-TEV-flipGFP-T2A-mCherry (Addgene catalog NO.124429) where TEV cleave site was replaced by SARS-CoV-2 PLpro cleavage site (LRGGAPTK), and ordered from GenScript.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3-PL-flipGFP-T2A-mCherry</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA3-TEV-flipGFP-T2A-mCherry</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 resulting kobs values were then plotted versus compound concentrations ([C]), then kinact and Ki or Ka values were calculated according to the equation: kobs = kinact × ([C]/([C] + Ki or Ka)) 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: 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 for 10.1101/2022.03.24.22272837: (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: Ethics: This study was approved (EK Nr: 1064/2021) by the Institutional Review Board (IRB) of the Office of Research Oversight/Regulatory Affairs, Medical University of Innsbruck, Austria, which is responsible for all human research studies conducted in the State of Tyrol (Austria).<br>IRB: Ethics: This study was approved (EK Nr: 1064/2021) by the Institutional Review Board (IRB) of the Office of Research Oversight/Regulatory Affairs, Medical University of Innsbruck, Austria, which is responsible for all human research studies conducted in the State of Tyrol (Austria).<br>Consent: Study population, study design and recruitment: A total of 57 patients infected with Omicron, 34 with no history of prior vaccination and 23 patients who had received 2-3 doses of the BNT162b2 vaccine (Table S1), were recruited for the study under 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></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 assay: End-point binding IgG antibody titers to various SARS-CoV-2–derived antigens were measured using the Meso Scale Discovery (MSD) platform.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>End-point binding IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were coated with the specific antigen on spots in the 96 well plate and the bound antibodies in the samples (1:50000 dilution) were then detected by anti-human IgG antibodies conjugated with the MSD SULPHO-TAG which is then read on the MSD instrument which measures the light emitted from the tag.</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">ACE2 binding inhibition (Neutralization) ELISA: The V-PLEX COVID-19 ACE2 Neutralization kit (Meso Scale Discovery, Panel 23 (ACE2) Kit, K15570U) was used to quantitatively measure antibodies that block the binding of ACE2 to its cognate ligands (SARS-CoV-2 and variant spike subdomains).</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">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 raw data were subjected to QC analyses using the FastQC tool (version 0.11.9) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FastQC</div><div>suggested: (FastQC, RRID:SCR_014583)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">mRNA-seq read quality control was done using Trimmomatic11 (version 0.36) and STAR RNA-seq12 (version STAR 2.5.4a) using 150 bp paired-end mode was used to align the reads (hg19).</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">HTSeq13 (version 0.9.1) was to retrieve the raw counts and subsequently, Bioconductor package DESeq214 in R (https://www.R-project.org/) was used to normalize the counts across samples and perform differential expression gene analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Bioconductor</div><div>suggested: (Bioconductor, RRID:SCR_006442)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quantification and statistical analysis: Differential expression gene (DEG) identification used Bioconductor package DESeq2 in R.</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">P-values of antibody between two groups were calculated using one-tailed Wilcoxon rank t-test on GraphPad Prism software (version 9.0.0).</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: 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 for 10.1101/2022.03.29.486190: (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 statement: This research has been determined to be exempt by the Institutional Review Board of the Boston University Medical Center since it does not meet the definition of human subjects research, since all human samples were collected in an anonymous fashion and no identifiable private information was 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">Contamination: All cell lines are routinely tested for mycoplasma contamination and confirmed 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">For CD169 blocking experiments, primary MDMs from 3 different donors were pre-incubated with 20 μg/ml anti-CD169 antibody (HSn 7D2, Novus Biologicals) or IgG1k (P3.6.2.8.1, eBioscience) for 30 min at 4°C prior to infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-CD169</div><div>suggested: (Thermo Fisher Scientific Cat# MA1-16891, RRID:AB_568734)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This is followed by secondary staining for 30 min at 4°C with APC-conjugated mouse anti-His antibody (BioLegend, #362605, 1:50) or isotype control.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His</div><div>suggested: (BioLegend Cat# 362605, RRID:AB_2715818)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cells were incubated overnight at 4°C with a rabbit antibody directed against the SARS-CoV nucleocapsid protein (Rockland; 1:1000 dilution in 5% goat serum), which cross-reacts with the SARS-CoV-2 nucleocapsid protein, as previously described (99).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV nucleocapsid protein</div><div>suggested: (Creative Diagnostics Cat# DMAB8869, RRID:AB_2392503)</div></div><div style="margin-bottom:8px"><div>SARS-CoV-2 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 cells were washed four times in PBS and incubated with goat anti-rabbit antibody conjugated with AlexaFluor594 for 1 hour at room temperature (Invitrogen; 1:200 dilution in blocking reagent). 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was used at 200 ng/ml for nuclei staining.</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></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For dsRNA staining (61), anti-dsRNA (Pan-Enterovirus Reagent, clone 9D5, Light Diagnostics, Millipore) antibody was used 1:2 overnight and anti-mouse-AF488 (Invitrogen) 1:200 dilution as secondary antibody with DAPI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-dsRNA ( Pan-Enterovirus Reagent , clone 9D5 , Light Diagnostics , Millipore )</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-dsRNA ( Pan-Enterovirus Reagent ,</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse-AF488</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry: To examine cell surface expression of CD169 or ACE2 in transduced THP1 or primary MDMs, approximately 0.5x106 cells were harvested with CellStripper (Corning), stained with Zombie-NIR (BioLegend, #423105, 1:250) followed by staining for 30 min at 4°C with the following antibodies; Alexa647-conjugated mouse anti-CD169 antibody (BioLegend, #346006, 1:50), Alexa647-conjugated</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: (Enzo Life Sciences Cat# BML-SA445, RRID:AB_2273641)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">mouse anti-ACE2 antibody (R&D systems, 1:200), or unconjugated goat anti-ACE2 polyclonal antibody (R&D systems, #AF933, 1:200) followed by Alexa488-conjugated chicken anti-goat antibody (Invitrogen, #A-21467, 1:100).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-goat</div><div>suggested: (Molecular Probes Cat# A-21467, RRID:AB_141893)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunoblot Analysis: To assess expression of endogenous or transduced proteins, cell lysates containing 30- 40 µg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and the membranes were probed with the following antibodies: mouse anti- TMPRSS2 (Santa Cruz, #515727, 1:1000), mouse anti-Cathepsin-L (Santa Cruz, #32320, 1:1000), goat anti-ACE-2 (R&D systems, #AF933, 1;1000), rabbit anti-STING (Cell Signaling, #13647, 1:1000), rabbit anti-MAVS (Thermo Fisher, #PA5-17256, 1:1000), mouse anti-RIG-I (AdipoGen, #20B-0009, 1:1000)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti- TMPRSS2 ( Santa Cruz , #515727</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Cathepsin-L ( Santa Cruz , #32320</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-ACE-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-STING ( Cell Signaling , #13647</div><div>suggested: (Cell Signaling Technology Cat# 13647, RRID:AB_2732796)</div></div><div style="margin-bottom:8px"><div>anti-MAVS</div><div>suggested: (Thermo Fisher Scientific Cat# PA5-17256, RRID:AB_10979584)</div></div><div style="margin-bottom:8px"><div>anti-RIG-I ( AdipoGen , #20B-0009</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Specific staining was visualized with secondary antibodies, goat anti-mouse-IgG-DyLight 680 (Thermo Scientific, #35518, 1:20000), goat anti-rabbit-IgG-DyLight 800 (Thermo Scientific, #SA5-35571, 1:20000), or a donkey anti-goat-IgG-IR-Dye 800 (Licor, #926-32214, 1:20000).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse-IgG-DyLight 680</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit-IgG-DyLight</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>#SA5-35571</div><div>suggested: (Thermo Fisher Scientific Cat# SA5-35571, RRID:AB_2556775)</div></div><div style="margin-bottom:8px"><div>anti-goat-IgG-IR-Dye</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">THP1 cells (ATCC) were maintained in RPMI/1640 (Gibco) containing 10% FBS and 1% pen/strep (50)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>THP1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate HEK293T/ACE2+, THP1/ACE2+ and THP1/CD169+/ACE2+ cells, HEK293T, THP1 or THP1/CD169 cells were transduced with pLenti-ACE2-IRES-puro lentivector and cultured in puromycin-containing media (2 μg/ml).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>THP1/CD169</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 titer was determined in Vero E6 cells by tissue culture infectious dose 50 (TCID50) assay using the Spearman Kärber algorithm.</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">Infection: For RNA analysis, 1x106 cells (THPI/PMA, MDMs, HEK293T) were seeded in 12-well plates.</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">S binding: To evaluate SARS-CoV-2 S binding to various THP1 monocytes expressing different surface receptors, approximately 0.25x106 cells from parental THP1 or those expressing wt CD169, mutant CD169 (R116A), ACE2, or both wt CD169 and ACE2 were incubated for 30 min at 4 °C with 2 μg of spike glycoprotein (stabilized) from Wuhan-Hu-1 SARS- CoV-2 containing a C-terminal Histidine Tag, recombinant from HEK293F cells (BEI resources, #NR-52397).</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">For ACE2 cloning, the NotI-XhoI fragment from pLenti-ACE2- IRES-puro was inserted into the LV-3’LTR backbone.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLenti-ACE2- IRES-puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For cloning CD169 into LV-3’LTR vector, a BglII-AgeI fragment from LNC-CD169 was inserted into LV-3’LTR vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>LV-3’LTR</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HIV-1 packaging plasmid psPAX2 and VSV-G expression constructs have been previously described (39).</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</div><div>suggested: RRID:Addgene_138479)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All lentiviral vectors (pLKO.1) expressing shRNAs used for knockdown of host proteins were purchased from Sigma</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLKO.1</div><div>suggested: RRID:Addgene_13425)</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 analysis was performed using FlowJo software (FlowJo).</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">63x oil immersion objective; numerical aperture 1.4) controlled by Metamorph image acquisition software (Molecular Devices, San Jose, CA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Metamorph</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistics: All the statistical analysis was performed 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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github.com github.com
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This tool watches Hypothesis URLs, groups, tags, or users, and alerts on new annotation activity to Slack, email, or RSS. It runs as a standalone Python program, ideally on a server, but alternatively on an always-connected desktop computer. It periodically queries the Hypothesis API along one or more axes -- url (or wildcard_uri), user, group, tag -- and sends notifications by way of Slack, email, or RSS.
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- Mar 2022
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If I use tags for topics I would tag everything that is relevant for the topic of diet with #diet. A note about carbohydrate intake and insulin sensitivity would definitely fulfill this criterion. If I use tags for objects, I would only tag notes with #diet when these notes are specifically on the concept of dieting. I would not tag the note on insulin sensitivity with #diet.
Tags linked in the body of my notes are for topics, this note is somehow related to that. Tags linked in the "Topics:": field are for objects, this note is primarily about this topic.
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.03.27.485958: (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 studies were approved in accordance with the Weizmann Institute’s Animal Care and Use Committee (IACUC) guidelines and regulations (approval number 00580120-3).<br>Euthanasia Agents: The mice were measured under general isoflurane inhalation anesthesia (5% induction, 1% maintenance) up to four hours after EVs injection using a gradient echo Fast Low Angle Shot (FLASH) with the following parameters: TR = 300 ms; TE = 2 ms; resolution of 0.23×0.23×0.7 mm3.</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 overnight blocking in 5% milk in TBST, specific antibodies were applied to the membranes for 1 h to detect markers: CD81 (B-II, Santa Cruz, USA); c-myc (9E10, Santa Cruz, USA) (all 1:500); and beta-actin (C4, Santa Cruz, USA) (1:1000).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD81 (B-II, Santa Cruz, USA)</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>c-myc</div><div>suggested: (Covance Cat# MMS-150P-1000, RRID:AB_291322)</div></div><div style="margin-bottom:8px"><div>9E10</div><div>suggested: (Covance Cat# MMS-150R-500, RRID:AB_291327)</div></div><div style="margin-bottom:8px"><div>beta-actin</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>C4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HRP-conjugated anti-mouse secondary HRP goat anti-mouse IgG antibody (#4053, Biolegend, USA) (1:5000 in TBST) was applied for 1 h before imaging using enhanced chemiluminescence substrate EZ-ECL Kit (Biological industries, catalog no. 20-500-120).</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-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The expression of ACE2 protein in cells was also confirmed by Western blot analysis, as described above, using a rabbit monoclonal ACE2 antibody (ab239924; 1:1000; Abcam, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: (Abcam Cat# ab239924, RRID:AB_2861381)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then the cells were detached from wells by PBS and incubated with ACE2 primary antibody (ab239924; 1:1000; Abcam, USA) for 1 hour on ice, washed with PBS and then incubated with anti-rabbit fluorescently-conjugated secondary antibody (Alexa Fluor 647 nm) for 1 hour on ice.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit fluorescently-conjugated secondary</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 stably expressing ACE2 HEK293T cell line was kindly obtained from the lab of Dr. Ron Diskin (Weizmann Institute of Science) and kept under puromycin antibiotics (0.5 μg/ml, Invitrogen, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: RRID:CVCL_HA71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK-pAGDisplay-RBD stable cell line generation: The pAGDisplay-based plasmids expressing RBD (1 ug of DNA) were transfected in a 60 mm culture dish with 80% confluent HEK293 cells by a JetPrime transfection reagent (Polyplus, France) according to the manufacturer’s protocol.</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">Binding assays and affinity curve determination using cell-display: The HEK-pAGDisplay-RBD stable cells grown to 80% confluency were gently detached by Accutase (1/2 solution in PBS, 3 min, Sigma Aldrich cat.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-pAGDisplay-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">Colony PCR and sequencing were used for analysis and verification. pAGDisplay vector construction: pDisplay Mammalian Expression vector was purchased from Invitrogen (V66020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pAGDisplay</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pAGDisplay vector backbone was assembled by combining a pET28b fragment bearing KanR and origin of replication, a pLVX vector fragment bearing WPRE, PuroR, and IRES sequences, and a pDisplay CMV promoter with a PDGFRβ expression cassette by a restriction-free three-component assembly 58.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX</div><div>suggested: RRID:Addgene_174088)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein production, purifications, and labeling procedures: The designed ALFA-tag binding nanobody (DnbALFA) and its mNeonGreen fusion were expressed by using expression plasmid pET28bdSUMO 62 and E.coli BL21(DE3) cells as described previously 60.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET28bdSUMO</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Soluble his-tagged peptidase domain of ACE2 protein (Q18 – S740), inserted in pHLsec plasmid, was expressed in an Expi293F cell system with an ExpiFectamine 293 Transfection Kit (ThermoFisher, USA) according to the manufacturer’s protocol and purified as described previously 45.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHLsec</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK-pAGDisplay-RBD stable cell line generation: The pAGDisplay-based plasmids expressing RBD (1 ug of DNA) were transfected in a 60 mm culture dish with 80% confluent HEK293 cells by a JetPrime transfection reagent (Polyplus, France) according to the manufacturer’s protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pAGDisplay-based</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">Mean FL-4 fluorescence signal values of RBD+ cells, subtracted by RFnano and the nonspecific signal of the RBD-population, were used to determine the KD of binding constants using a non-cooperative Hill equation and a nonlinear least-squares regression using Python 3.7 45.</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 fluorescence signal (APC filter) of cells was measured by a FACS machine (LRS-II) and analyzed by the 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">MR images were analyzed by the ImageJ software or by a custom-made script written in MATLAB (MathWorks, USA).</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">After tuning and matching to the1H frequency, shimming of the magnetic field, and B0 correction, the tumor area was measured with the following parameters: both axial and coronal images were acquired; T2 and T2* maps were reconstructed in the Paravision software; and were followed by analysis in the ImageJ software.</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">Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software Inc., 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: 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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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.03.25.485832: (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: Animals: Animal research was conducted at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID).<br>IACUC: Ethics statement: These experiments and procedures were reviewed and approved by the United States Army Medical Research Institute for Infectious Diseases Institutional Animal Care and Use Committee (IACUC).</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">Genders were mixed male and female, and all animals were SARS-CoV-2 naïve at the outset of the study.</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: R4719 was determined to have no detectable mycoplasma, endotoxin or adventitious agents based on the assays and techniques used.</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">Serum binding and ACE-2 inhibitory antibody assessment: SARS-CoV-2-specific binding IgG antibody responses were measured using MULTI-SPOT® 96-well plates, V-PLEX SARS-CoV-2 Panel 7 Kit (Meso Scale Discovery (MSD), Rockville, MD).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2-specific binding IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MSD SULFO-TAG™ conjugated anti-IgG antibody was added to each well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><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">Stimulations consisted of two pools of peptides spanning the S protein of SARS-CoV-2 or SARS-CoV-1 (1 µg/mL, JPT, PM-WCPV-S and PM-CVHSA-S respectively) in the presence of Brefeldin A (0.65 µL/mL, GolgiPlug™, BD Cytofix/Cytoperm Kit, Cat. 555028), co-stimulatory antibodies anti-CD28 (BD Biosciences Cat.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-1 (1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following stimulation, cells were stained serially with LIVE/DEAD Fixable Blue Dead Cell Stain (ThermoFisher #L23105) and a cocktail of fluorescent-labeled antibodies (BD Biosciences unless otherwise indicated) to cell surface markers CD4-PE-Cy5.5 (S3.5, ThermoFisher #MHCD0418, Lot 2118390 and 2247858), CD8-BV570 (RPA-T8, BioLegend #301038, Lot B281322), CD45RA BUV395 (5H9, #552888, Lot 154382 and 259854), CD28 BUV737 (CD28.2, #612815, Lot 0113886), CCR7-BV650 (GO43H7, # 353234, Lot B297645 and B316676) and HLA-DR-BV480 (G46-6, # 566113, Lot 0055314).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD8-BV570</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>RPA-T8</div><div>suggested: (BD Biosciences Cat# 563795, RRID:AB_2722501)</div></div><div style="margin-bottom:8px"><div>CD45RA</div><div>suggested: (BD Biosciences Cat# 552888, RRID:AB_394517)</div></div><div style="margin-bottom:8px"><div>CD28</div><div>suggested: (BD Biosciences Cat# 612815, RRID:AB_2870140)</div></div><div style="margin-bottom:8px"><div>CCR7-BV650</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>B316676</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>G46-6</div><div>suggested: (BD Biosciences Cat# 566113, RRID:AB_2739515)</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">Infectivity and neutralization titers were determined using ACE2-expressing HEK293 target cells (Integral Molecular, Philadelphia, PA) in a semi-automated assay format using robotic liquid handling (Biomek NXp Beckman Coulter, Brea, CA).</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, biotinylated SARS-CoV-2 Spike trimer (Hexapro) was incubated with red streptavidin-fluorescent beads (Molecular Probes, Eugene, OR) for 2h at 37°C. 10 μl of a 100-fold dilution of beads–protein was incubated 2h at 37°C with 100μl 900-fold diluted plasma samples before addition of THP-1 cells (25,000 cells per well; Millipore Sigma, Burlington, MA).</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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293F-Spike-S2-WT cells were incubated with 10-fold diluted heat-inactivated (56°C for 30 min) plasma samples for 30 min at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293F-Spike-S2-WT</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">SARS-CoV-2 pseudovirions (PSV) were produced by co-transfection of HEK293T/17 cells with a SARS-CoV-2 S plasmid (pcDNA3.4), derived from the Wuhan-Hu-1 genome sequence (GenBank accession number: MN908947.3) and an HIV-1 (pNL4-3.Luc.R-E-, NIH HIV Reagent Program, Catalog number 3418).</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><div style="margin-bottom:8px"><div>pNL4-3.Luc.R-E-</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, SARS-CoV-2 Spike-expressing 293 FreeStyle (293F) cells were generated by transfection with linearized plasmid (pcDNA3.1) encoding codon-optimized full-length SARS-CoV-2 Spike protein matching the amino acid sequence of the IL-CDC-IL1/2020 isolate (GenBank ACC# MN988713).</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">Reduced data in the NSS files was extracted into Microsoft Excel workbooks using Notocord-derived formula add-ins, and the 30-minute (min) averages were calculated for each parameter for each subject.</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">Assay equivalency was established by participation in the SARS-CoV-2 Neutralizing Assay Concordance Survey (SNACS) run by the Virology Quality Assurance Program and External Quality Assurance Program Oversite Laboratory (EQAPOL) at the Duke Human Vaccine Institute, sponsored through programs supported by the National Institute of Allergy and Infectious Diseases, Division of AIDS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Quality Assurance Program</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Quality Assurance Program Oversite Laboratory</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sample staining was measured on a FACSymphony(tm) A5 SORP (Becton Dickenson) and data was 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">Display of multicomponent distributions were performed with SPICE v6.0 (NIH, Bethesda, MD).</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></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">NCT04784767</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">SARS-COV-2-Spike-Ferritin-Nanoparticle (SpFN) Vaccine With A…</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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Its core theme - segregation. It's done in such an ingenious and innocent way - colour.
new tag: not so much sneaky, but clever way of communicating an idea/message/theme
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Local file Local file
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To investigatethis possibility, we performed ChIP-seq experiments on WT andmutant T-47D and MCF7 clones, grown for 5 days in hormone-depleted media followed by 1-hour E2 or DMSO treatments, using anantibody that recognizes the FLAG epitope tag.
1 hour after E2 treatment --> To see where it's binding, no second effects from downstream.
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To characterizeendogenous ER mutations, we created multiple isogenic clonal linesthat heterozygously express FLAG-tagged mutant or WT (control) ERfrom the endogenous locus (Supplementary Fig. S1A; ref. 22). Wedeveloped two clones each for WT and the two most common ER LBDmutations (Y537S and D538G) in both T-47D and MCF7 breast cancercell lines (Supplementary Fig. S1B). Engineered lines included a FLAGepitope tag at the C-terminus to allow for downstream analyses. Theheterozygous expression of FLAG-tagged mutant or WT ER and theavailability of multiple clones per genotype provided a robust system toinvestigate mutant ER’s molecular effects.
ER is an ER regulated gene, gives the extent of estrogen receptor effects. Uses multiple clones because all of the cells are genetically identical. Want to rule out that clones are doing something that is a clone effect independent of your study. This also helps to reduce likelihood that the CRISPR mutation didn't have off target effects that influenced the results.
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SciScore for 10.1101/2022.03.23.485576: (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">Expression of different coronavirus E proteins, the SARS-CoV-2 S, and SARS-CoV S protein were confirmed by transfection with the Turbofect transfection reagent (ThermoFisher) followed by radiolabeling and immunoprecipitation analysis using a mouse monoclonal antibody directed against the HA-tag (Thermo-Fisher, #26183) or an antibody against SARS-CoV Spike protein (Sino Biologicals, #40590-T62).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody against SARS-CoV 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 cultures were then incubated at 4C overnight with an and a rabbit monoclonal antibody against either Golgin-97 (Abcam, #ab84340) or Calnexin (Cell Signaling Technology, #2679).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calnexin (Cell Signaling Technology, #2679</div><div>suggested: (Cell Signaling Technology Cat# 2679, RRID:AB_2228381)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cells were washed in PBS, incubated with a secondary goat anti-rabbit antibody conjugated to AlexaFluor™-488 (Invitrogen, A11008) for 1 h at room temperature, washed, and reacted with rabbit anti-mouse conjugated to AlexaFluor™- 594 (Invitrogen, A11062) for 1 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: (Thermo Fisher Scientific Cat# A-11008, RRID:AB_143165)</div></div><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: (Innovative Research Cat# A11062, RRID:AB_1500656)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression of E was detected using an HA-tag antibody (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HA-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Aliquots of cells from the same co-transfections were also analyzed for protein expression by immunoblots using antibodies directed against the HA-tag (E proteins, SARS-CoV-2 S protein, and Vpu), the SARS-Cov-1 S protein, or against GFP to monitor SARS-CoV-2 N-GFP expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 S protein,</div><div>suggested: None</div></div><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">The lysates were transferred to new tubes and BST-2 (using an anti-BST-2 antibody), the E proteins (all having a C-terminal HA-tag), and SARS-CoV and SARS-CoV-2 S proteins, and N-GFP proteins, or GAPDH (to equalize loading) immunoprecipitated using appropriate antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-BST-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>GAPDH</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">Immunofluorescence studies: To examine the intracellular localization of the SARS-CoV-2 E protein, COS-7 cells grown on 13 mm cover slips were transfected with either the empty pcDNA3.1(+) vector or one expressing the SARS-CoV-2 E protein using Turbofect transfection reagent (ThermoFisher).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COS-7</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">At 48 h post-transfection (pt), the culture medium was collected, clarified by low-speed centrifugation, and the supernatants analyzed for infectious virus by titration on TZM-bl cells (29, 30, 91–95).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>TZM-bl</div><div>suggested: RRID:CVCL_B478)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Levels of infectious virus were determined by preparing a series of 10-fold dilutions of the culture supernatant followed by inoculation of 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">At 24 h, equal levels of HIV-1ΔEnv-/VSV-G pseudotyped virus (M.O.I. of 0.1) were used to infect HEK293 cells for 48h.</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">Plasmids with the entire HIV-1 NL4-3 genome (pNL4-3) and pNL4-3Δvpu were obtained from the NIH AIDS Reagent Branch.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pNL4-3Δvpu</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293 cells were co-transfected with empty pcDNA3.1(+) or the vector expressing E proteins and pNL4-3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pNL4-3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293 cells were transfected with either the empty pcDNA3.1(+) vector or one expressing SARS-CoV-2 E protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1(+)</div><div>suggested: RRID:Addgene_129020)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis of the phosphorylation of eIF-2α: To determine if the expression of the E protein of SARS-CoV-2 resulted in phosphorylation of eIF2α, HEK293 cells seeded in 6-well plates were either left untransfected or transfected with 1 μg of pUC19 or of a SARS-CoV-2 E-expressing plasmid.</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><div style="margin-bottom:8px"><div>SARS-CoV-2 E-expressing</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293 cells were co-transfected with either empty pcDNA3.1(+) vector and pcDNA3.1(+) expressing the human BST-2 protein, or pcDNA3.1(+) expressing each of the proteins described above and BST-2.</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">Plasmids expressing the SARS-CoV and SARS-CoV-2 S proteins were purchased from Sino Biologicals (catalog # VG40150-G-N; VG40589-CY).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Sino Biologicals</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:
- No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
- 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 for 10.1101/2022.03.21.485247: (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 experiments: All the animal experiments were performed in accordance with protocols approved by the Icahn School of Medicine at Mount Sinai (ISMMS) Institutional Animal Care and Use Committee (IACUC).</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">Mouse immunization and challenge studies: Female BALB/c mice were used in 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">All samples were analyzed in a blinded manner.</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 HN protein was detected by a mouse monoclonal antibody 8H2 (MCA2822, Bio-Rad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MCA2822</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ELISA plates were afterwards washed 3 times with PBST and 50 μL of anti-mouse IgG-horseradish peroxidase (HRP) conjugated antibody (Cytiva, GE Healthcare) was added at a dilution of 1:3,000 in blocking solution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG-horseradish</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">During this time the primary antibody was biotinylated according to manufacturer protocol (Thermo Scientific EZ-Link NHS-PEG4-Biotin)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NHS-PEG4-Biotin</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blocking solution was removed and 100 μl/well of 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</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">The plaques were immuno-stained with an anti-SARS-CoV-2 NP primary mouse monoclonal antibody 1C7C7 kindly provided by Dr. Thomas Moran at ISMMS.</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">An HRP-conjugated goat anti-mouse secondary antibody was used at 1:2000 and the plaques were visualized using TrueBlue™ Peroxidase Substrate (SeraCare Life Sciences Inc.)</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">VERO-TMPRSS2 cells (BPS Biosciences, #78081) were maintained in DMEM (Gibco) containing 10% (vol/vol)</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">Specifically, transfected BSRT7 and DF-1 cells were washed with warm PBS and trypsinized.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DF-1</div><div>suggested: RRID:CVCL_XF08)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BSRT7 cells were mixed with DF-1 cells (~1: 2.5) in a 10-cm dish.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BSRT7</div><div>suggested: CCLV Cat# CCLV-RIE 0583, RRID:CVCL_RW96)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero-E6 cells or Vero-TMPRSS2 were seeded onto 12-well plates in growth media at 1:5 and cultured for two days.</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">Mouse immunization and challenge studies: Female BALB/c mice were used in 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">Briefly, the variant HXP-S were inserted into the pNDV_LS/L289A rescue plasmid (between P and M genes) by in-Fusion cloning (Clontech, CA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pNDV_LS/L289A</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The recombinant product was transformed into MAX Efficiency™ Stbl2™ Competent Cells (Thermo Fisher Scientific, MA, USA) to generate the pNDV-HXP-S rescue plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pNDV-HXP-S</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, cells were transfected with 2 μg of pNDV-HXP-S, 1 μg of pTM1-NP, 0.5 μg of pTM1-P, 0.5 μg of pTM1-L and 1 μg of pCI-T7opt and were re-suspended in 250 μl of a modified Eagle’s Minimum Essential Medium (Opti-MEM; Gibco).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTM1-NP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pTM1-P</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pTM1-L</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCI-T7opt</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the mammalian-cell codon-optimized nucleotide sequence of a soluble spike protein (amino acids 1-1,213) lacking the polybasic cleavage site, carrying two stabilizing mutations (K986P and V987P), a signal peptide, and at the C-terminus a thrombin cleavage site, a T4 fold-on trimerization domain, and a hexahistidine tag was cloned into the mammalian expression vector pCAGGS. https://www.beiresources.org/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS.</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">Briefly, the mammalian-cell codon-optimized nucleotide sequence of a soluble spike protein (amino acids 1-1,213) lacking the polybasic cleavage site, carrying two stabilizing mutations (K986P and V987P), a signal peptide, and at the C-terminus a thrombin cleavage site, a T4 fold-on trimerization domain, and a hexahistidine tag was cloned into the mammalian expression vector pCAGGS. https://www.beiresources.org/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>https://www.beiresources.org/</div><div>suggested: (BEI Resource Repository, RRID:SCR_013698)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed using GraphPad Prism 7 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><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analyses were performed using Prism 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><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">NCT04871737</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">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.
<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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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.03.21.485243: (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">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 recombinant antibodies: A pair of plasmids separately expressing the heavy- and the light-chain of antibodies were transiently co-transfected into HEK293F cells.</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">Serial 1/3 dilutions of antibodies were incubated with pseudoviruses at 37°C for 1 hour, and then the mixtures were added in ACE2 expressed Huh-7 cells (104 per well in 96-well plates).</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><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 D614G and variants spike ectodomain: The gene encoding SARS-CoV-2 S ectodomain (residues 1-1208, Gene Bank: MN908947) was synthesized (GeneScript) and cloned into mammalian expression constructs pcDNA-3.1, proline substitutions at residues 986 and 987, “GSAS” substitution at furin cleavage site (residues 682-685), a T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrepTag, and an 8XHisTag at C-terminal were introduced simultaneously by MultiS one step cloning kit (Vazyme).</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">Using the SARS-CoV-2 WT plasmid as the template, mutations such as D614G, B.1.17 (Del 69H70V, N501Y, P681H, S982A, Del 145Y, A570D, T716I, D1118H), B.1.351 (K417N, E484K, N501Y) and P1 (K417T, E484K, N501Y) was introduced by Multi Site-Directed Mutagenesis Kit (Yeasen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 WT</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The expression plasmid of Omicron S with HexaPro mutations(26), “GSAS” substitution at furin cleavage site (residues 682-285), a T4 fibritin trimerization motif, a TwinStrep Tag, and a C-terminal 8 × His Tag was constructed into pcDNA3.1 vector by MultiS one step cloning kit (Vazyme).</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">Briefly, whole spike glycoprotein sequences of SARS-CoV, wild type or variants of SARS-CoV-2 were inserted into the vector of pcDNA3.1+ and severally co-transfected into 293 T cells (ATCC, Manassas, VA, USA) with a defective HIV-1 genome that encodes luciferase reporter.</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">A nonlinear regression analysis was performed on the resulting curves using Prism (GraphPad) to calculate half-maximal inhibitory concentration (IC50) values.</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">Automated data acquisition was carried out with SerialEM software(27).</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">Cryo-EM image processing: All the data processing was carried out using either modules on, or through, RELION v3.0 and cryoSPARC(28).</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">For S-15 complex (Fig.S10), a total of 6,943 movie stacks was binned 2 × 2, dose weighted, and motion corrected using MotionCor2(29).</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">After blob-picking in cryoSPARC and 2D classification, trimer and monomer particles were observed.</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">For model building of S-15 and S-60, the apo-S trimer model and the antibody (15 and 60) model generated by swiss-model were fitted into the map using UCSF Chimera(31) and followed by manually adjustment in COOT(33), as well as real space refinement 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></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Model validation was performed using 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></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.
</footer>
-
-
www.biorxiv.org www.biorxiv.org
-
SciScore for 10.1101/2022.03.22.485248: (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: Studies using primary human cells were approved by the University of Queensland Human Medical Research Ethics 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">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">THP-1 cells (TIB-202; ATCC) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), 2 mM GlutaMAX (Life Technologies) and 50 U/ml penicillin–streptomycin (Life Technologies).</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">Calu-3 cells purchased from ATCC (HTB-55) were maintained in Minimal Essential Media (Invitrogen), containing 10% heat-inactivated foetal bovine serum (Cytiva), 50 U/ml penicillin and streptomycin (Life Technologies Australia), and were seeded at 300 000 cells per well in a 12-well plate 48 h prior to experiments.</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">HEK-293T cells were transfected with the expression vectors according to the manufacturer’s protocol with PEI 2500 (BioScientific) and transduced target THP-1 cells were selected with puromycin (1 μg/mL) after 24 h and used for assays after 72 h.</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">Virus was grown on Vero E6 TMPRSS2 cells for 48 h in DMEM with 2% FBS, and cell debris was cleared by centrifugation at 500G for 5 minutes at room temperature.</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">For Calu3 cells, virus was added to cells to give a total volume of 500 μL of RPMI 1640 with 2% FBS (HMDM and THP-1) or MEM with 2% FBS (Calu3) per well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For studies involving SARS-CoV-2 infection of BAL macrophages, the SARS-CoV-2 isolate hCoV-19/Australia/VIC01/2020 (kindly provided by the Victorian Infectious Diseases Reference Laboratory) was grown in Vero cells for 72 h in serum-free MEM with 1 μg/ml TPCK trypsin.</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">Lentiviral transduction: A lentiviral construct containing human ACE2 (Addgene 155295), or mScarlet (Addgene 85044) was cloned into pLV-CMV-MCS-IRES-Puro-Sin (48) and packaged into lentivirus in HEK-293T cells by means of third generation lentiviral packaging plasmids (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLV-CMV-MCS-IRES-Puro-Sin</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Mpro expression vector was generated by cloning the Mpro PCR product into a modified pEF6 plasmid, with an HA-tag N-terminal of the multiple cloning site, by standard restriction digest cloning techniques.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pEF6</div><div>suggested: RRID:Addgene_70765)</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: Statistics were calculated using GraphPad Prism using tests indicated in 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: 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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Local file Local file
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I’ve established the habit of asking myself: Is this information or oppor-tunity to communicate worth my attention at all, given my goal for today? Is it something I want to look at later, but not right now while I am focus-ing on a task toward a larger goal (in which case, maybe I’ll open a tab on my browser)? Is it information that I don’t want to distract myself with at the moment and don’t want to burden my near-term reading agenda, but might want to refer to later, because it is about a subject that interests me (in which case I’ll tag and bookmark)? Like basic mindfulness, paying attention to microdecisions—and learning to make them more and more quickly—is easy enough to start, and yields increasing power to diligent, regular practitioners. It’s an exercise in strategic goals (What am I setting out to do?), attention (What am I about to click on?), and intention (I’m going to ignore this, or open a tab or bookmark, because I intend to return to focus) that deepens the more you do it.
Beispiel für eine Gewohnheit und für konkrete Fragen, die man sich während seiner online/onlife Zeit stellen kann
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hree steps to new habits: Make it tiny, find a spot, train the cycle.”102 What could be tinier than writing a one-sentence goal for the day before you get online, and reviewing it once or twice?
Empfehlung, bevor wir online gehen: Einen Satz, ein Ziel für den Tag notieren!
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diamond.mirror.xyz diamond.mirror.xyz
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dive into trends and patterns that emerge from applying network analysis and data science on the digital traces collected from Snapshot and DAOHaus. Finally, in Part 3, I take the MetaCartel DAO as a case study of governance and look at some of the properties that drive participation. In each section, you will find a collectible NFT. The proceeds from each NFT will go directly towards supporting current and future Diamond Dao community resea
This is super interesting because blah blah blah
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paul.kinlan.me paul.kinlan.me
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outraged that we wanted to add a new tag (especially in the head)
add new tag in the head
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.03.21.485084: (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 mouse anti-SARS-CoV-2 S2 domain (1:1000, GTX632604, GeneTex, Irvine, CA, USA), rabbit anti-Flag-tag (1:1000, PM020, MBL, Woburn, MA, USA), mouse anti-tubulin (1:1000, CP06, Millipore, Billerica, MA, USA), and mouse anti-VSVM (1:1000, 23H12, absolute antibody, Oxford, UK).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 S2 domain</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Flag-tag</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-tubulin</div><div>suggested: None</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 secondary antibodies used were horseradish peroxidase (HRP)-linked donkey anti-rabbit IgG (NA934; GE Healthcare) and HRP-linked donkey anti-mouse IgG (NA931V; GE Healthcare).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HRP)-linked donkey anti-rabbit IgG</div><div>suggested: None</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">After 24 h infection, fixed cells were incubated with anti-SARS-CoV-2 nucleocapsid (1:1000, GTX135357) primary antibody for 16 h at 4 °C and detected with anti-rabbit-Alexa488 (1:200, A11008, Invitrogen, Carlsbad, CA, USA) secondary antibodies for 40 min at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 nucleocapsid (1:1000</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">Cells and viruses: VeroE6 (CRL-1586), 293T (CRL-3216), A704 (HTB-45) and Calu-3 (HTB-55) cells were obtained from American Type Culture Collection (Rockville, MD, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div><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">Caco-2 cells (RCB0988) were obtained from RIKEN BioResource Research Center (Tsukuba, Japan).</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></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunofluorescence staining: Immunofluorescence analysis of HEC50B cells was performed as described previously [21].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEC50B</div><div>suggested: JCRB Cat# NIHS0391, RRID:CVCL_2929)</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">OVISE (JCRB1043), HEC50B (JCRB1145), and VeroE6-TMPRSS2 (JCRB 1819) cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS2 (JCRB 1819</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VeroE6-TMPRSS2 (JCRB 1819) 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: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</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">Caco-2 cells (RCB0988) were obtained from 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></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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wiki.elte-dh.hu wiki.elte-dh.hu
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tagállam
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groups.google.com groups.google.com
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I've been using the Hypothesis Obsidian annotator to annotate PDFs in an Obsidian vault—so I have a bunch of annotations as markdown files. I am now attempting to publish the Obsidian vault as a website at movement-ontology.brandazzle.net, but the plugin apparently isn't supported on website, as the annotations do not render. Is there any way I could host the Hypothesis tools on the website and connect my annotations so that viewers can see my annotations?
Brandon,
Obsidian Annotator (https://github.com/elias-sundqvist/obsidian-annotator), which I'm presuming you're using, looks like it's working from within Obsidian instead of a web page and is very clever looking, but without some significant work, I don't think it's going to provide you with the results you're looking for. It sounds like you want an all-public chain of work and Obsidian Annotator currently defaults to an all-private chain.
From my brief perusal of what's going on, the plugin appears to be tied to a single Hypothes.is account (likely the developer's) which defaults all annotations to private (only you) and as a result, even if you had the permalink to the annotations you'd not be able to see them presented on the web as they're all private and you wouldn't have access to the account. You could try filing some issues on the related Github repository to see if the developer might add the ability to make public annotations using your own personal account, which I'm sure would require your personal API key for Hypothes.is to be put into the settings page for the plugin in Obsidian. Another issue I see is that it's taking Hypothesis tags and turning them into Obsidian tags, which is generally fine, but the developer isn't accounting for multi-word tags which is creating unintended tag errors along the way that will need to be manually fixed.
If you're open to an alternate method of annotating and doing so in public, I can recommend a workflow that will allow you to do what it sounds like you're attempting. It starts with annotating .pdf files (either on the web, or as local files in your browser) in public using your own Hypothes.is account. (Most of this also works with private annotations, but if you want them to appear on public versions of web-hosted .pdf files with the same fingerprints, you'll want them to be public so others can see/interact with them.) Next set up the Hypothesidian script described here: https://forum.obsidian.md/t/retrieve-annotations-for-hypothes-is-via-templater-plugin-hypothes-idian/17225. There are some useful hints further down the thread on that page, so read the whole thing. The Github repository for it is here: https://github.com/SilentVoid13/Templater/discussions/191 if you need it. I've documented a few modifications I've made to the built-in template to suit my particular needs and which might serve as a template if you find it useful: https://boffosocko.com/2021/07/08/hypothes-is-obsidian-hypothesidian-for-easier-note-taking-and-formatting/.
You can then use the functionality of Hypothesidian to pull in the annotations you want (by day, by document, only your annotations, all the annotations on a document, etc.) For .pdf files, you may require Jon Udell's facet tool https://jonudell.info/h/facet/ to search your personal account for the name of the file or one of the tags you used. When you find it, you can click on it and it will open a new browser window that contains the appropriate urn file "key" you'll need to put into Hypothesidian to grab the annotations from a particular .pdf file. It will be in the general form: urn:x-pdf:1234abcd5678efgh9101112ijkl13. I haven't found an easier means of pulling out the URN/fingerprint of pdf files, though others may have ideas.
When you pull in your annotations you can also get/find permalinks to the annotations on the web if you like. I usually hide mine in the footnotes of pages with the labels "annotation in situ" and "syndication links", a habit I've picked up from the IndieWeb community (https://indieweb.org/posts-elsewhere). You can see a sample of how this might be done at https://notes.boffosocko.com/where-are-the-empty-spaces-on-the-internet where I've been doing some small scale Hypothes.is/Obsidian/Web experiments. (I'm currently using Blot.im to get [[wikilinks]] to resolve.)
Another strong option you're probably looking for is to use "via" links (https://web.hypothes.is/blog/meetvia/) on the URLs for your pdf files so that people can automatically see the annotation layer. (This may require whitelisting on Hypothes.is' end depending on where the files are hosted; alternately https://docdrop.org/ may be useful here.) Then if you've annotated those publicly, they'll also be able to see them that way too.
Another side benefit of this method is that it doesn't require the Data View plugin for Obsidian to render your annotations within Obsidian which also means you'll have cleaner looking pages of annotations in your web published versions. (ie. none of the %% code blocks which don't render properly on the web)
As I notice you're using some scanned .pdf files which often don't have proper OCR and can make creating annotations with appropriate Hypothes.is anchors, you might also appreciate the functionality of docdrop for this as well.
Given your reliance on documents and the fact that you've annotated some in what looks like Adobe Acrobat or a similar .pdf program, you might additionally enjoy using Zotero with Obsidian, Zotfile, and mdnotes as outlined here: https://forum.obsidian.md/t/zotero-zotfile-mdnotes-obsidian-dataview-workflow/15536. It's relatively slick, but requires additional set up, reliance on more moving pieces, and isn't as nice an overall user interface in comparison to Hypothes.is. It also misses all of the potential useful social annotation you might get with Hypothes.is.
Hopefully this is all reasonably clear and helpful. I'd be interested in hearing about options from others who are using Hypothes.is in conjunction with Obsidian or other related note taking tools and publishing them to the web after-the-fact.
Best, Chris Aldrich
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ludocode.com ludocode.com
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I believe this is partly due to a militant position on free software. Some advocates believe so strongly that users should be able to recompile their software that they force them to do so. They break libraries seemingly on purpose just to say, “Recompile! Oh you can’t? That’ll teach you to use binary software!” Of course users don’t want to recompile their software, but what users actually want is usually lost on GNOME developers.
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hypothes.is hypothes.is
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ich werde nicht gefunden also bin ich nicht ich werde nicht 00:31:16 gesucht also bin ich nicht es hat schon ein bisschen was davon wenn man eben nach etwas sucht und es gibt es nicht
Was bedeutet es für das Internet wenn es einen Tag lang von niemandem, von keinem einzigen Menschen genutzt würde? Gedankenexperiment - lonely net
Tags
Annotators
URL
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not as good/useful as some other gem options/approaches, such as the one that adds a
datamethod per migration, or that lets you tag with:post_deploy, etc.
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If you need to ensure migrations run in a certain order with regular db:migrate, set up Outrigger.ordered. It can be a hash or a proc that takes a tag; either way it needs to return a sortable value: Outrigger.ordered = { predeploy: -1, postdeploy: 1 } This will run predeploys, untagged migrations (implicitly 0), and then postdeploy migrations.
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class PreDeployMigration < ActiveRecord::Migration tag :predeploy end
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docs.google.com docs.google.com
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dlsoftex.com_0 : 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blogs.baruch.cuny.edu blogs.baruch.cuny.eduUntitled1
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my ingenious home-made typewriter, in perfect order except for two faulty characters, was knocked down for four and tuppence. I
Dehumanizing a memory by putting a price tag on it, turning it into an object.
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github.com github.com
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In any significant project I worked in the last 15 years, logging text messages resulted in a large amount of strings which was hard to make sense of, thus mostly ignored.
hard to make sense of, thus mostly ignored
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.03.14.484208: (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: Handling and working with SARS-CoV-2 were conducted in a BSL3 facility in accordance with the biosafety guidelines of the Israel Institute for Biological Research (IIBR), or in a BSL3 facility at The University of Texas Medical Branch (UTMB).<br>Euthanasia Agents: Golden Syrian hamsters (females; 4-5 weeks old) were intranasally infected with 1000 pfu of virus in 50 μl of inoculum while under isoflurane anesthesia.</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 DIG-labeled random-primed probe, corresponding to nucleotides 28,999 to 29,573 of the SARS-CoV-2 genome (Kamitani et al., 2006; Xie et al., 2020), was used to detect SARS-CoV-2 mRNAs and visualized by DIG luminescent detection kit (Roche, Indianapolis, IN), according to the manufacturer’s protocol.</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: Calu3 and 293T cells were authenticated by ATCC using STR profiling.<br>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">Briefly, paraffin-embedded sections were firstly subjected to immunostaining with a commercially available and diluted (1:5,000) rabbit-anti-SARS-CoV-2 spike (S) protein (ab272504, Abcam Plc., Cambridge, UK), followed by staining with a peroxidase-conjugated secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>rabbit-anti-SARS-CoV-2 spike ( S ) protein ( ab272504</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the detection of N protein, an affinity-purified rabbit anti-SARS-CoV N polyclonal antibody (Harcourt et al., 2020) was used as the primary antibody, followed by Alexa Fluor 488-conjugated secondary antibody (Invitrogen) and DAPI counterstaining, using SlowFade Gold antifade reagent (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the detection of nsp1 protein, the anti-SARS-CoV-1 nsp1 antibody was used as the primary antibody, followed by Alexa Fluor 594-conjugated secondary antibody (Invitrogen) and DAPI counterstaining.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-1 nsp1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were washed and labeled with anti-rabbit FITC conjugated antibody at a 1:200 dilution and with DAPI (4′,6-diamidino-2-phenylindole) at a 1:200 dilution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit FITC</div><div>suggested: (Bioss Cat# bs-0708R-FITC, RRID:AB_11075028)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Secondary antibodies used were Goat anti-rabbit or Goat anti-mouse (IRDye 800CW or IRDye 680RD, Li-Cor).</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">An affinity-purified rabbit anti-SARS-CoV-1 nsp1 polyclonal antibody (Narayanan et al., 2015) was used as the primary antibody, and an HRP-linked anti-rabbit IgG (Cell Signaling Technology) was used as the secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG ( Cell Signaling Technology )</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">African green monkey kidney clone E6 cells (Vero E6, ATCC® CRL-1586™) were grown in growth medium DMEM containing 10% FBS, MEM NEAA, 2 mM L-glutamine, 100 Units/ml penicillin, 0.1 mg/ml streptomycin, 12.5 Units/ml nystatin (P/S/N), all from Biological Industries, Israel]</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">Calu3 and 293T cells were authenticated by ATCC using STR profiling.</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">Viral titers: For determination of growth rate of SARS-CoV-2 WT or SARS-CoV-2 mut viruses, Vero E6 and Calu3 cells were seeded in 12-well plates (5E5 and 1E6 cells/well respectively).</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">Virus titers in the tissues were determined on confluent Vero E6/TMPRSS2 cells obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (1819).</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">SARS-CoV-2 nsp1 ORF, carrying a C-terminal myc-tag, was cloned into pCAGGS-MCS, resulting in pCAGGS-nsp1-WT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS-MCS</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCAGGS-nsp1-WT</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Similarly, SARS-CoV-2 nsp1 carrying a deletion of the amino acids 155 to 165, and SARS-CoV-2 nsp1 carrying R124A and K125A mutations, were cloned into pCAGGS to generate pCAGGS-nsp1-ΔRB and pCAGGS-nsp1-CD, respectively.</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><div style="margin-bottom:8px"><div>pCAGGS-nsp1-ΔRB</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCAGGS-nsp1-CD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Luciferase assay: Vero E6 cells in 24-well plates were co-transfected, in triplicate, with pRL-EMCV-FL reporter plasmid and the indicated pCAGGS-based nsp1 expression plasmids using TransIT-LT1 reagent (Mirus).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRL-EMCV-FL</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmids and cloning: pLVX-EF1alpha-SARS-CoV-2-nsp1-2XStrep-IRES-Puro (nsp1-WT) was kindly provided by N. Krogan.</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">The mutations to generate nsp1-ΔRB and nsp1-CD were introduced by amplifying the nsp1-WT plasmid so that it became a linear fragment containing the desired mutations (primers 1-4 listed in Supplementary Table 1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>nsp1-WT</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 for SARS-CoV-2 N protein were collected using an Olympus BX53 microscope with a 40X objective lens at UTMB and processed with ImageJ (NIH) software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>UTMB</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Alignment was performed using Bowtie v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Bowtie</div><div>suggested: (Bowtie, RRID:SCR_005476)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Differential expression analysis was done using DESeq2 (Love et al., 2014)</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">Output.bam files from STAR were used as input for the GRAND-SLAM analysis(Jürges et al., 2018) with default parameters and with trimming of 5 nucleotides in the 5′ and 3′ends of each read.</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">Reactive bands were detected by Odyssey CLx infrared imaging system (Li-Cor) and quantified using Fiji (ImageJ) as detailed in https://imagej.nih.gov/nih-image/manual/tech.html.</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><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">Graphics: Figure 1a and extended figure 2c were created using BioRender.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BioRender</div><div>suggested: (Biorender, RRID:SCR_018361)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Fluorescence-activated cell sorting figures were created with FlowJo and the rest of the figures were drawn with ggplot2 in R.</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>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:
However, a major limitation of these measurements is that viral mRNA replication and transcription is massive, and occurs within a unique endomembrane system (Wolff et al., 2020) that would thus render much of the viral mRNA inaccessible to ribosomes. This inaccessibility of potentially large portions of viral RNA will skew our measurements and it is likely the true translation efficiency of viral transcripts is significantly higher. Comparing the translation efficiencies of viral and cellular transcripts in cells infected with CoV2-wt or with CoV2-mut allowed us to overcome this limitation and to demonstrate that in infected cells nsp1 indeed specifically promotes the translation of viral transcripts compared to cellular transcripts, as we observed that in cells infected with CoV2-mut, viral genes are translated even less efficiently compared to their cellular counterparts. However, at the same time our measurements of the overall translation capacity in infected cells, using incorporation of OPP into nascent chains, show that nsp1-WT leads to a major reduction in the absolute levels of translation in infected cells. Since the vast majority of mRNA in infected cells is viral mRNA this means that viral mRNAs translation is likely also inhibited in the presence of nsp1. Together these results support a model in which nsp1 acts as a strong inhibitor of translation that tightly binds to ribosomes and thus inhibits the translation of both viral and cellular mRNAs. Viral transcrip...
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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Author Response
Reviewer #2 (Public Review):
Fukuda et al. use whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) data obtained from sperm and human primordial germ cells (hPGCs), as well as KRAB-ZFP protein ChIP-seq data obtained from HEK293T cells, to study the relationship between DNA methylation, KRAB-ZFP binding and genome-wide transcription of LINE-1 (L1), SVA and LTR12 retrotransposons.
This work aims overall to elucidate pathways silencing retrotransposons in the male germline, in particular making new (and known) links between ZFPs and DNA methylation. The focus here ends up being on immobile retrotransposons, as L1s (bound by ZNF93 and ZNF649) and SVAs (bound by ZNF28 and ZNF257) capable of mobilization either do not have binding sites for the identified ZFPs, or have far fewer than their older relatives. The relationships between L1, ZNF93 and ZNF649 has been reported previously (Jacobs et al., 2014, Nature; Fernandes et al., 2018, bioRxiv). That older retrotransposons have more binding to these ZFPs, and are more methylated in hPGCs, is based mainly on correlation. Overall, I thought the subject matter was interesting but I have substantial reservations around the analyses, particularly the more novel results related to SVA. As the work stands it is not clear whether ZNF257 or ZNF28 are reinforcing DNA methylation on SVA. The claims around this repression being transcriptionally-directed or varying significantly amongst individuals, for biological as opposed to technical reasons, appear preliminary at this stage.
Specific comments:
1) The use of WGBS to analyse very young retrotransposons, like L1HS and SVA_F, has potential caveats. One of the most important of these is that the CpG islands most likely to be differentially methylated for these elements, in somatic cells at least, are internal to their sequences (e.g. PMID: 33186547). This includes the SVA VNTR sequence, which is where the vast majority of proposed ZNF28 and ZNF257 binding motifs reside (Fig. 2F). Does WGBS, using only uniquely mapped reads as done here, resolve these regions sufficiently to identify differential methylation?
First of all, thank you for your valuable comments on our manuscript. We confirmed that VNTR in SVA_A derived sequences can be uniquely mapped and DNA methylation in VNTR in SVA_A could be analyzed (please see New Supplementary Fig. S1I and J).
2) Why does Fig. 1D have only 36 full-length L1HS copies? The definition of a full-length L1 here (>90% consensus length) should yield (from memory) >300 reference L1HS copies.
According to our threshold for full-length, we obtained 319 full-length L1HS copies. However, the most of them could not measure DNA methylation levels due to low mappability (please see New Supplementary Fig. S1B).
3) It would be useful to explain the inclusion of LTR12 as a representive ERV, as opposed to, say HERVK, which has been studied in hPGC like cells recently (PMID: 35075135).
Thank you for your comment. We added the following sentence. “A subset of LTR transposons, including LTR12, function as enhancers (Deniz et al., 2020). It was recently reported that LTR5s, which are Hominidae-specific LTR-type transposons and hypomethylated in hPGCs (DNA methylation levels < 10%), can function as enhancers to promote hPGC differentiation (Xiang et al., 2022). Therefore, in the case of LTR12C, maintaining DNA methylation might be beneficial for hPGC development because it suppresses inappropriate activation of transposon-embedded enhancer function.” p18, 311-316
4) The exo ChIP-seq for a variety of ZFPs was obtained from published data generated using HEK293T cells, whereas the WGBS is from sperm and hPGCs. What evidence can the authors point to be reasonably sure that the ZFP binding patterns from HEK293Ts carry over to the male germline in vivo?
It is not known whether ZNF binding pattern in HEK293Ts is same, similar or totally different in male germline in vivo, and we do recognize this is the strong shortage of this paper. Good antibody for KRAB-ZNFs, availability of human PGCs and establishment of low-cell number input ChIP-seq (or Cut and TAG) for KRAB-ZNFs are required to address this issue. From our analysis, all we can say is KRAB-ZNFs we identified are candidate factors for retroelement silencing during human male germ cell development.
5) In Fig. 3C it appears quite a few L1PA3s have ZNF649 peaks and yet the motif for ZNF649 has two mismatches to the L1PA3 consensus (Fig. 3F). Yes, L1HS has one more mismatch than L1PA3. It would be useful to explain further why two mismatches are acceptable whereas 3 completely abolishes binding.
We added following sentences. “Although highly methylated L1 copies had two mismatches within the ZNF649 binding motif, one at the third position (T→G) and one at the sixth position (A→T) (Figure 3G), a minor fraction of the ZNF649 binding motif had the same base composition at these sites (Figure 3D). Thus, these two mismatches may not abrogate ZNF649 binding.” p11, 181-185
6) Line 244: "More than 90% of full-length SVA_B-F copies could be analyzed by SVA amplicon-seq". What is the basis for this calculation? Presumably the amplicon-seq doesn't give information as to where on the genome the SVA resides.
The criterion of analyzed copy is more than 10 CpGs within each copy are covered by at least five reads. I mapped computationally generated 100-bp reads from full-length SVA copies to investigate how much reads from each copy are uniquely aligned to genome. Even in the youngest SVA type, SVA_F, more than 10% of reads can be uniquely mapped in the most of copies (please see New Supplementary Fig .S1A).
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SciScore for 10.1101/2022.03.12.484088: (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: In all studies, written informed consent was obtained from each participant.<br>IRB: Blood samples were obtained after written informed consent from adults with PCR-confirmed SARS-CoV-2 infection who were enrolled in a prospective cohort study at the Africa Health Research Institute approved by the Biomedical Research Ethics Committee at the University of KwaZulu–Natal (reference BREC/00001275/2020).</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">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">These measurements were used in downstream steps to computationally filter library variants that were highly deleterious for RBD expression or ACE2 binding and would likely represent spurious antibody-escape mutations (see below for details).</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">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">S4B), and are the counts of variant v in the RBD library after and before enriching for antibody-escape variants with a pseudocount of 0.5 added to all counts, and and are the total counts of all variants after and before the antibody-escape enrichment.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-escape enrichment .</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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The multidimensional scaling in Fig 4 and 5 that projects the antibody-escape maps into two dimensions was performed using the Python scikit-learn package, version 1.0.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-escape</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 generate spike-pseudotyped lentiviral particles (58), 6 × 105 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">100 µL of serially diluted spike-pseudotyped lentiviral particles were added to 1.25 × 104 293T-ACE2 cells (BEI Resources NR-52511), grown overnight in 50 µL of D10 growth media in a 96-well black-walled poly-L-lysine coated plate (Greiner Bio-One, 655936)</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">Note that these assays were performed in 293T cells over-expressing human ACE2, which emphasize contributions of ACE2-competitive antibodies to viral neutralization (3,34,60).</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">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 (58), 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 (59) and the D614G mutation that is now predominant in human SARS-CoV-2 (25).</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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Depletion of RBD-binding antibodies from polyclonal sera: Magnetic beads conjugated to the SARS-CoV-2 Wuhan-Hu-1 RBD (ACROBiosystems, MBS-K002) or Delta RBD (ACROBiosystems, MBS-K037) were prepared according to the manufacturer’s protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 Wuhan-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">AWY101 yeast containing an empty vector pETcon plasmid were grown overnight, shaking, at 30°C in SG-CAA 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">PacBio HiFi circular consensus sequences (CCSs) were generated on-instrument (Sequel IIe control software version 10.1.0.119549) and demultiplexed with lima using SMRTLink version 10.1.0.119588.</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">The multidimensional scaling in Fig 4 and 5 that projects the antibody-escape maps into two dimensions was performed using the Python scikit-learn package, version 1.0.1.</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><div style="margin-bottom:8px"><div>scikit-learn</div><div>suggested: (scikit-learn, RRID:SCR_002577)</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. First, each cohort in this study consists of a relatively small sample size. Due to patterns of SARS-CoV-2 viral circulation and competition among variants (e.g., early 2020, Beta, and Delta viruses circulated during different time periods and in different geographic regions), as well as the introduction of SARS-CoV-2 vaccination in 2021, the cohorts differ with regards to geographic location, age, and time of sample collection. Second, the antibody-escape mapping experiments were performed in a yeast-displayed deep mutational scanning system which only measures antibody binding to the RBD. There is not always a direct relationship between antibody binding and neutralization, as some epitopes are more immunodominant with regards to neutralization than others, and the relative contributions to neutralization of antibodies targeting different spike epitopes may depend on cell type and ACE2 expression level used in the experimental assays (3,34,35). Despite these caveats, our results have important implications for future viral evolution and vaccine design. Due to the rapid antigenic evolution of SARS-CoV-2, public health experts must decide if and when to update SARS-CoV-2 vaccines (51,52). But because each variant elicits an antibody response with its own susceptibilities to erosion by mutation, the impacts of mutations in future variants may depend on prior exposure history. This knowledge will help to understand which vulnerabilities may ex...
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 for 10.1101/2022.03.13.484037: (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">Pseudovirions were generated by co-transfection of HEK293T cells with MLV-gag/pol (Addgene #14887) and MLV-Luciferase (Addgene #170575) plasmids and SARS-CoV-2 spike WT or variants with an 18-AA truncation at the C-terminus.</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 incubation, 10,000 Hela-hACE2 cells were added to the mixture with 20 µg/ml Dextran (Sigma, 93556-1G) to enhance infectivity.</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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mixture was incubated for 1 h at 37 °C, 5% CO2 before adding to a 24-well plate coated in a subconfluent Vero E6 cell monolayer.</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">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 IgGs and Fabs: The 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: RRID:Addgene_173431)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the RBD (residues 333-529) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1) was cloned into a customized pFastBac vector (38), and fused with an N-terminal gp67 signal peptide and C-terminal His6 tag (9).</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">Iterative model building and refinement were carried out in COOT (42) and PHENIX (43), 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><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">Fifty percent maximal inhibitory concentrations (IC50), the concentrations required to inhibit infection by 50% compared to the controls, were calculated using the dose-response-inhibition model with 5-parameter Hill slope equation in GraphPad Prism 7 (GraphPad 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><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 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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Local file Local file
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If you have advanced email marketing features that allow you to segment and tag your subscribers, depending on the free resource, you could tag people who click on this link as being interested in whatever that free resource is about.
I could have the free recourse be a entire list of some of my favorite books, podcasts, and articles ranked and ordered under themes that I think they should read.
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code.visualstudio.com code.visualstudio.com
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Note that this is a breaking API change in the libraries (more information in the README.md). It does not affect the backwards compatibility of the protocol itself.
annotation meta: may need new tag: backwards compatibility of the protocol backwards compatibility for [libraries that use [it?]]
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www.biorxiv.org www.biorxiv.org
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Author Response:
Evaluation Summary:
This work provides new insights into how surface-exposed lipoproteins of Gram-negative bacteria reach their destination in the outer membrane. Authors find that the outer membrane protein complex Slam serves as a translocon for the lipoproteins and the periplasmic chaperone Skp mediates their targeting to Slam. This work may contribute to the elucidation of host invasion mechanisms by pathogenic bacteria, in which surface lipoproteins play an important role.
Reviewer #1 (Public Review):
Previously, using rigorous genetic, bioinformatic and cell-based biochemical analyses, the same group discovered SLAM1, an outer membrane protein in Neisseria spp., which mediates the membrane translocation of surface lipoproteins (SLPs) (Hooda et al. 2016 Nature Microbiology 1, 16009). Here, authors reconstituted this system in proteoliposomes using minimal purified components including the translocon Slam1 and the client lipoprotein TbpB. Authors further coupled the system to TbpB-expressing E. coli spheroblasts and LolA, the Slam1-specific periplasmic shuttle system. Using the digestion pattern of TbpB by Proteinase K as a readout, authors confirmed that Slam1 indeed served as a translocon for SLPs. As a step forward, authors found that Skp, a periplasmic chaperone (holdase), was critical to the membrane-assembly and translocation of TbpB. Strengths: Overall, this is a solid biochemical study that demonstrates the role of Slam1 as a translocon for SLPs. The experimental design is neat and straightforward. The specific role of Skp in SLP translocation is interesting. This reconstituted system will serve as a novel platform for further elucidation of the Slam1-mediated SLP translocation mechanisms. The manuscript is overall well written. Weakness: There are several major concerns, however. 1) It is not fully convincing whether these findings are novel and significantly advance the field. Identification of minimal components in a biological process and their reconstitution are always challenging and thus, this study is an achievement. Nonetheless, I am not sure whether we have learned novel molecular insights besides the confirmation of the group's previous discovery. The specific role of Skp in translocation is interesting but not surprising, considering that periplasmic holdases are already known to be extensively involved in the biogenesis of periplasmic and outer membrane proteins.
We thank the reviewer for their time and thorough review of the manuscript. In the previous paper (Hooda et al. 2016 Nature Microbiology 1, 16009), we discovered that the outer membrane protein Slam is “important/responsible” for the surface display for SLPs (TbpB, LbpB, fHbp). In this mechanism focused manuscript, we were able to demonstrate Slam’s role as an outer membrane translocon. One of the achievements in this paper is to demonstrate that Slam as an autonomous translocon – importantly this is unlike the two-partner secretion systems, as it does not require the Bam complex for the translocation of TbpB.
2) Although authors developed nice assays (Figs. 1 and 2), it was not verified whether TbpB protected from Proteinase K digestion had "correct" conformation and membrane-topology. Authors performed a functional assay on TbpB (Fig. 5a), but this result was obtained from a cell-based assay, not from the reconstituted system.
We have performed pulldown assay for the TbpB that has been translocated into Slam-proteoliposomes using human transferrin conjugated beads to show that this TbpB protein is correctly folded and functional. Blots and explanations are attached in the revised manuscript (see new Figure 2 – figure supplement 2 and line 197-207). (As addressed in major scientific concerns point 2-i).
Although the data in Figs. 1 and 2 clearly show that the membrane association of TbpB depends on Slam1, it does not mean that the "translocation" has actually occurred in the proteoliposomes. Probably, more rigorous analysis on the Proteinase K-protected portion of TbpB (for example, mass spec) seems necessary (that is, whether the proteolytic product is expected based on the predicted topology).
The TbpB is flag-tag at its C-terminus and the protected band on our blots (detected by α-flag antibody) corresponds to the expected Mw (~75kDa) for Mcat TbpB flag tagged protein. Therefore, we believed the band at 75kDa is our full length processed TbpB. Moreover, we have confirmed that TbpB can be detected at the top of the sucrose gradient with our Slam-proteoliposomes in this assay. This would only occur if TbpB was actually translocated inside the intact liposomes, otherwise we should not see any TbpB in the top layer of the sucrose gradient (Figure 4d). Furthermore, we have performed a pulldown assay for TbpB in proteoliposomes to check for their functional binding to human transferrin beads after translocation. These results are explained in the updated new Figure 2 – figure supplement 2 and line 197-207.
3) The manuscript has a couple of missing supporting data. 3a) Lines 87-89: "From our analysis, we found that the Slam1 from Moraxella catarrhalis (or Mcat Slam1) expressed well and the purified protein was more stable than other Slam homologs." I cannot find the expression and stability data of various homologs supporting this sentence.
In general, what we meant was that we chose Mcat Slam as the target of this study because it is more stable during the purification and resulted in a higher yield of protein. We needed higher yields of Slam to be able to reconstitute the protein into the liposomes for the translocation assay. We have purification data for Mcat Slam1, Nme Slam1 and Ngo Slam2 but we think including them in the supplementary is not necessary. We have changed and rewritten this section dedicated to Mcat Slam1 purification (Figure 1 – figure supplement 1 and 2).
3b) "Lines 216-219: Furthermore, the processing of TbpB by signal peptidase II and subsequence release from the inner membrane was unaffected suggesting the defect in surface display by Skp occurs after the release of TbpB from the inner membrane (Fig. 4a)." The result supporting this sentence seems missing or this sentence points to a wrong figure.
Yes, this sentence is misleading. What we meant was that the processed TbpB (TbpB has 2 bands, unprocessed TbpB – upper band and signal peptidase processed, lipidated TbpB - lower band) is similar for all samples indicating that the knockout of Skp did not affect the expression or processing of the signal peptide of TbpB up until it is ready (processed and lipidated in the periplasm) for translocation by Slam to the surface. We have added an explanation in the figure legend of Figure 4a –line 267-269.
4) Some statistical analysis results are not clear, making some conclusions not convincing. 4a) Figure 4a top "Exposure of TbpB on the surface of K12 E. coli" Apparently, all three data points for (Delta_DegP+Slam1+TbpB) are very closely distributed. Accordingly, (WT+Slam1+TbpB) vs (Delta_DegP+Slam1+TbpB) data look significantly different (difference is ~0.2). But the two data were assigned as "Not Significant". Similarly, in the comparable in vitro data (Figure 4b), the intensity for Slam1 (WT+Proteinase K - Triton) looks larger than that for Slam1 (Delta_DegP + Proteinase K - Triton). So, the DegP contribution should not be ignored.
For figure 4a, the ONE WAY ANOVA test was performed using Prism with 4 biological replicates (we can include the analysis report in the revised submission if this is requested we have updated the figure to include data points. In general, both our in vitro liposomes translocation assay and in vivo surface exposure assay for TbpB showed that delta-DegP only slightly reduces the translocation of TbpB to the surface but could not detect statistically significant differences.
4b) Figure 5a top "Exposure of TbpB on the surface of N. meningitidis" What is the p-value for WT vs Delta_Skp data? Are the two data significantly different? The p-value range for (*) is not shown.
We have included the p-value range for (*) in the revised manuscript, figure 5a.
Reviewer #2 (Public Review):
The article addresses the function of SLAM, a protein which the authors have shown previously to be involved in the traffic of lipoproteins to the bacterial surface. The authors have performed a series of experiments to assess the impact of SLAM on the delivery into proteoliposomes of the model lipoprotein TbpB either added exogenously or presented by E coli spheroplasts. They identify a periplasmic chaperone, Skp, which enhances transport of TbpB and other lipoproteins to proteoliposomes, and show the contribution of endogenous Skp to lipoprotein transport in Neisseria meningitidis. The authors set up an in vitro translocation assays using purified components from different bacteria. This is reasonable as the assays can be challenging to establish and require proteins that can be expressed and are stable. It would be helpful however if the sources of the proteins and how they are tagged (for their detection) is clearly documented in the article and the figures. In keeping with this, the figures describing the assays could be improved (ie 1A, 2A, 3A and C). Despite this, the results presented in Fig 1 and 2 clearly demonstrate the role of SLAM as a translocase, and the authors have included appropriate controls for their assays; the translocation of a OmpA to demonstrate that the Bam complex is functional in their hands in an important control and should be included in the main figures. Experiments outlined in Figure 3 and Table 1 demonstrate the interaction specific of TbpB and another lipoprotein HpuA with Skp, a previously characterised periplasmic chaperone. This is performed by pull-downs and MS as well as immunobloting. A critical result is shown in Figure 4 in which SLAM and TbpB are introduced into E coli, and the role of endogenous Skp is assessed. Importantly, the absence of Skp reduces but does not eliminate TbpB surface expression. The authors could speculate on the nature of Skp-indendent surface expression of TbpB, as this result mirrors what they find in a meningococcal strain lacking Skp (Figure 5A). It appears that Skp might be required for the correct insertion/folding of lipoproteins given their result in Figure 5B (currently, this could be changed into 5C) which tests the binding of transferrin to the bacterial surface. Clearly this could be influenced by an effect of Skp on TbpA, which acts as a co-receptor with TbpB. In summary, the authors have used appropriate assays to reach their conclusions about the role of SLAM as a translocase and the contribution of Skp to the localisation of lipoproteins to the surface of bacteria. The findings presented are robust and shed new insights into the sorting of proteins in bacteria, an incompletely understood process which is central to microbial physiology, viurlence and vaccines.
Reviewer #3 (Public Review):
Slam was identified as an outer membrane protein involved in the translocation of certain lipoproteins to the cell surface in Neisseria meningitidis. Slam homologs were also identified in other proteobacteria. However, direct evidence that Slam is an outer membrane translocation device is still missing. In this paper, the authors set up an in vitro translocation assay to probe the role of Slam proteins in the translocation of the lipoprotein TbpB. Although they provide strong data supporting the role of Slam in lipoprotein translocation, further molecular dissection is required to unambiguously establish Slam as a lipoprotein translocator. The work is interesting and the paper clearly written. The authors also discovered a functional link between the periplasmic chaperone Skp and Slam-dependent lipoproteins, which is a novel and interesting finding.
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SciScore for 10.1101/2022.03.01.22271507: (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 trial protocol was reviewed and approved by the Independent Ethics Committees of the involved sites and the Ethics Council of the Ministry of Health of the Russian Federation.<br>Consent: To be included, participants needed to be able to understand the content of the informed consent documents and be willing to sign the informed consent form.</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">Men and women aged 18–85 years with a body mass index (BMI) between 18.5 and 30.0 kg/m2 were selected to participate if they had no indication of a current or previous SARS-CoV-2 infection (e.g., respiratory infection in last 14 days, axillary temperature ≥37.0 °C) or close contact with a suspected or confirmed case of SARS-CoV-2 infection.</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: Prometheus is a multicentre, randomised, double-blind, placebo-controlled, clinical trial being conducted in six centres in the Russian Federation.</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">Initially, as part of the interim analysis and to ensure 90% power for the between-group comparison of the primary variable, 180 subjects were to be included.</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: Eligible participants were randomly allocated to the Ad5-nCoV group or the Placebo group, in a 3:1 ratio, by an independent statistician using a validated system including a pseudorandom number generator with a seed value; allocation used block randomisation and stratification by study site.</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">Screening included the detection of SARS-CoV-2 RNA using real-time polymerase chain reaction (PCR) via a swab, and SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) antibody testing to ensure negative results, as well as testing for human immunodeficiency virus (HIV), syphilis, hepatitis B and hepatitis C viruses via blood serum.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 immunoglobulin M (IgM</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>immunoglobulin G (IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Determination of serum antibodies against the S protein and RBD of SARS-CoV-2 and the presence of neutralising antibodies against SARS-CoV-2 were conducted on Day 0, Day 14, Day 28 and after Month 6; neutralising antibodies against Ad5 were assessed on Day 0, Day 28 and after Month 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>Ad5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Horseradish-peroxidase conjugated mouse monoclonal anti-human IgG antibody was used to detect antibodies, visualised with tetramethylbenzidine substrate solution.</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">Anti-coronavirus neutralising antibodies were determined with a microneutralisation assay in which Vero cell (#ССL-81, American Type Culture Collection) monolayers were incubated in 96-well plates with 2-fold serial dilutions (1:10 to 1:1280) of participant serum.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-coronavirus neutralising antibodies</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-coronavirus neutralising</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-adenovirus neutralising antibodies were also determined using microneutralisation assay; A549 cell monolayers were incubated in 96-well plates with serial dilutions (1:10 to 1:1280) of participant serum and working dilutions of Ad5 (Adenovir; Smorodintsev Research Institute of Influenza).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-adenovirus neutralising</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 twice with PBS, washed twice with PBS + 0.05% Tween-20, and then incubated at room temperature with biotinylated anti-human IFN-γ detection antibody for 2 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IFN-γ</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The primary and secondary endpoints used the quantitative definition: the proportion of participants with at least a 4-fold increase in antibody titres against SARS-CoV-2 S protein and/or its RBD, specifically.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 S 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">Anti-coronavirus neutralising antibodies were determined with a microneutralisation assay in which Vero cell (#ССL-81, American Type Culture Collection) monolayers were incubated in 96-well plates with 2-fold serial dilutions (1:10 to 1:1280) of participant serum.</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">Anti-adenovirus neutralising antibodies were also determined using microneutralisation assay; A549 cell monolayers were incubated in 96-well plates with serial dilutions (1:10 to 1:1280) of participant serum and working dilutions of Ad5 (Adenovir; Smorodintsev Research Institute of Influenza).</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">Sequence encoding ΔFurin variant of SARS-CoV-2 S protein (amino acids 1–1213) containing a C-terminal Gly-Gly-6xHis tag was subcloned into the pMCAG-2T vector using the GeneArt Type IIs Assembly Kit, BbsI (Thermo Fisher Scientific), according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pMCAG-2T</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">Statistical Methods: Statistical analysis was performed using SPSS Statistics, Version 26.0 (IBM Corp.).</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></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:
This study has several limitations. First, the participants included in this study were all white, although conversely, this also provided the first data in a non-Chinese, European population. Second, this trial did not include children or pregnant women, and there were only a small number of older adults (35 were ≥60 years in the Ad5-nCoV group). An ideal candidate vaccine for COVID-19 should cover vulnerable populations of all ages. Anti-S protein-specific antibodies have been reported to decline rapidly in individuals who have recovered from COVID-19, especially those who were asymptomatic or had mild symptoms [23]. Third, the sample size was relatively small and some of the calculated 95% CIs were wide. Finally, virus mutation, an emerging problem, may reduce the effectiveness of current vaccines [24]. It is not known whether participants of this study were exposed to any COVID-19 variants. Further study is underway to determine neutralising antibodies to the widely circulating variants following vaccination with Ad5-nCoV, which include but are not limited to the Alpha (B.1.1.7), Beta (B.1.351) and Gamma (P.1 ) variants. Conclusions: Analysis of data from this phase 3 trial demonstrated the immunogenicity and safety of this Ad5-vector based COVID-19 vaccine. More data are required to determine whether this vaccine reduces infections and transmission. Overall, this stable, single-dose vaccine could contribute to the global fight against the evolving SARS-CoV-2 virus.
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">NCT04540419</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">Clinical Trial of Recombinant Novel Coronavirus Vaccine (Ade…</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.
<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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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.03.10.483772: (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">tween-20 (TBS-T) before probing overnight at 4°C with antibodies raised to the following targets: mouse anti-puromycin (Millipore-Sigma, MABE343), mouse anti-OC43 N (CoV antibody, OC43 strain, clone 541-8F, Millipore-Sigma, MAB9012), rabbit anti-BiP (Cell signaling Technologies (CST), mouse anti-XBP1s (CST, #12782), mouse anti-CHOP (CST, #2895), rabbit anti-α-Tubulin (CST, #2125), rabbit anti-SARS-CoV-2 S1 RBD (Elabscience, E-AB-V1006), anti-SARS-CoV-2 E (abbexa, abx226552), anti-SARS-CoV-2 M (Novus Biologicals, NBP3-05698), rabbit anti-SARS-CoV-2 N (Novus Biologicals, NBP3-05730)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-puromycin ( Millipore-Sigma , MABE343</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-OC43 N ( CoV</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-BiP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-XBP1s</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-CHOP</div><div>suggested: (Cell Signaling Technology Cat# 2895, RRID:AB_2089254)</div></div><div style="margin-bottom:8px"><div>anti-α-Tubulin ( CST</div><div>suggested: None</div></div><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 E</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 M</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 N</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>NBP3-05730</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">HCT-8 cells were cultured as above with additional 1X MEM Non-Essential Amino Acids (NEAA, Gibco</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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Baby hamster kidney (BHK-21) were maintained as above yet supplemented with 5% FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BHK-21</div><div>suggested: ATCC Cat# CRL-6281, RRID:CVCL_1914)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">African green monkey kidney Vero’76 and human lung adenocarcinoma Calu-3 cells were maintained in DMEM (Sigma-Aldrich, D5796; St. Louis, MO, USA) supplemented with 10% fetal bovine serum (ThermoFisher Scientific, 16000-044) and 1X Penicillin-Streptomycin (Gibco, 15140148).</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></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">1A) or Vero E6 cells (SARS-CoV-2/SB3-TYAGNC, used in Figs.</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">Stocks were titered by plaque assay on Vero E6 cells (68) or TCID50 on Vero’76 cells calculated using the Spearman-Kärber method.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero’76</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Stocks of human coronavirus OC43 (hCoV-OC43; ATCC, VR-1558) were propagated in 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">After 1 h, the inoculum was removed, replaced with DMEM/Pen/Strep/Gln containing 1% FBS and NEAA (HCT-8), 2% FBS (Calu-3), 2.5% FBS (Huh-7.5, 293A or 293T), or 5% FBS (BJ) supplemented with DMSO or drug at the indicated concentration, and maintained for an additional 23 h at the indicated temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Huh-7.5</div><div>suggested: RRID:CVCL_7927)</div></div><div style="margin-bottom:8px"><div>293A</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pLJM1-ACE2-BSD was cloned by from cDNA of generated from Caco-2 cells.</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">SARS-CoV-2 Spike-pseudotyped lentiviruses: To generate Spike-pseudotyped lentivirus particles, 293T cells were seeded in a 10 cm dish and co-transfected with 3 µg pLJM1-Luc2, 2 µg pSPAX2 (a kind gift from Didier Trono; Addgene #12260), and 2 µg of pcDNA3-SΔ19 with PEI, then treated with 6-TG or DMSO as described above.</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">To generate 293A-ACE2 cells, 293A cells were stably transduced with a lentivirus vector encoding ACE2 (pLJM1-ACE2-BSD) or an empty vector control (pLJM1-B*) (67), then selected and maintained in 10 µg/mL Blasticidin S HCl (ThermoFisher, A11139030)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLJM1-ACE2-BSD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pcDNA3-SARS-CoV-2-Spike plasmid contains a codon-optimized ORF for Spike from GenBank NC_045512 that was synthesized by GenScript (a kind gift from David Kelvin) then cloned between the KpnI and BamHI sites of pcDNA3.1(+).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3-SARS-CoV-2-Spike</div><div>suggested: None</div></div><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">pcDNA3-Membrane (M) and pcDNA3-Envelope (E) were cloned from pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro and pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro (kind gifts from Neven Krogan, available from Addgene #141386 and #141385) using PCR to remove the 2xStrep epitope tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3-Membrane</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA3-Envelope</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro</div><div>suggested: RRID:Addgene_141386)</div></div><div style="margin-bottom:8px"><div>pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro</div><div>suggested: RRID:Addgene_141385)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pCMV3-MERS-FLAG was purchased from SinoBiologicals (VG40069-CF).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3-MERS-FLAG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pLJM1-Luc2 used in generating Spike-pseudotyped lentiviruses was generated by cloning Luc2 from pGL4.26 (Promega) into pLJM1-B* (67).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGL4.26</div><div>suggested: RRID:Addgene_68742)</div></div><div style="margin-bottom:8px"><div>pLJM1-B*</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of SARS-CoV-2 virus-like particles: To generate SARS-CoV-2 virus-like particles, 293T cells were seeded in a 10 cm dish and co-transfected with 2 µg of each pcDNA-Spike, pcDNA-M, pcDNA-E, and pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro (a kind gift from Neven Krogan, available from Addgene #141391) with PEI, then treated with 6-TG or DMSO as described above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA-Spike</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA-M</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA-E</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro</div><div>suggested: RRID:Addgene_141391)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 Spike-pseudotyped lentiviruses: To generate Spike-pseudotyped lentivirus particles, 293T cells were seeded in a 10 cm dish and co-transfected with 3 µg pLJM1-Luc2, 2 µg pSPAX2 (a kind gift from Didier Trono; Addgene #12260), and 2 µg of pcDNA3-SΔ19 with PEI, then treated with 6-TG or DMSO as described above.</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-SΔ19</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quantitation of SARS-CoV-2 Spike surface expression: 293T cells were co-transfected with pEGFP-N1 and plasmids expressing SARS-CoV-2 Spike (FL) or SARS-CoV-2 Spike-Δ19 for 24 h using FugeneHD (Promega).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pEGFP-N1</div><div>suggested: RRID:Addgene_172284)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Gaussia luciferase secretion assays: For short-term treatments, 293T were transfected with pCMV-Gaussia Luc Vector (ThermoFisher Scientific, 16147) as described above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-Gaussia</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For long-term treatments, 293T cells were co-transfected with pCMV-Gaussia Luc and pLJM1-Luc2 and treated 6 h post transfection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLJM1-Luc2</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">Values were imported into GraphPad PRISM (v.9) and CC50 values were calculated by fitting a non-linear curve to the data.</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">Z-stacks were imaged on a Zeiss LSM880 and processed into maximum intensity projections using Zen Black (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">tween-20 (TBS-T) before probing overnight at 4°C with antibodies raised to the following targets: mouse anti-puromycin (Millipore-Sigma, MABE343), mouse anti-OC43 N (CoV antibody, OC43 strain, clone 541-8F, Millipore-Sigma, MAB9012), rabbit anti-BiP (Cell signaling Technologies (CST), mouse anti-XBP1s (CST, #12782), mouse anti-CHOP (CST, #2895), rabbit anti-α-Tubulin (CST, #2125), rabbit anti-SARS-CoV-2 S1 RBD (Elabscience, E-AB-V1006), anti-SARS-CoV-2 E (abbexa, abx226552), anti-SARS-CoV-2 M (Novus Biologicals, NBP3-05698), rabbit anti-SARS-CoV-2 N (Novus Biologicals, NBP3-05730)</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">Graphics: Molecular models were created with NCBI PubChem (http://pubchem.ncbi.nlm.nih.gov/) The model presented in Fig.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PubChem</div><div>suggested: (PubChem, RRID:SCR_004284)</div></div><div style="margin-bottom:8px"><div>http://pubchem.ncbi.nlm.nih.gov/</div><div>suggested: (PubChem BioAssay, RRID:SCR_010734)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">7 was adapted from “Coronavirus Replication Cycle”, by BioRender.com (2022).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BioRender</div><div>suggested: (Biorender, RRID:SCR_018361)</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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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
Manuscript number: RC-2021-01015
Corresponding author(s): Jordan, Raff
1. General Statements [optional]
We thank the reviewers for their thoughtful and constructive comments and have now revised our manuscript accordingly. We apologise that it has taken so long to send in these revisions, but this is in part because both first authors have now left the lab.
2. Point-by-point description of the revisions
Reviewer #1
This reviewer was generally supportive. They note that it is unfortunate that our data suggests the CP110/Cep97 complex does not play a major part in controlling daughter centriole growth—although we believe that this is an important negative result—but feel that other aspects of our data are interesting. They requested no further experiments, but did comment that it would be interesting to determine when g-tubulin is incorporated into growing centrioles. Unfortunately, we cannot test this as the centrioles in these embryos recruit large amounts of g-tubulin to their PCM, so we cannot specifically assay the small amount of protein in the centriolar fraction.
Reviewer #2
Major Points:
__Figure 1: The reviewer notes that Sas-4 and CP110 have antagonistic roles in promoting/repressing centriole growth and asks if Sas-4 is involved in promoting centriole elongation and whether it also oscillates. __It is unclear if Sas-4 directly promotes centriole elongation in flies. We have previously shown that centriolar Sas-4 levels do oscillate during S-phase, but with a timing that is distinct from CP110/Cep97 (Novak et al., Curr. Biol., 2014). These observations do not shed much light on the potential antagonistic relationship between CP110/Cep97 and Sas-4, so we do not comment on this here.
Figure S1B: The reviewer requests that we image the centrioles with greater laser intensity to test whether some residual CP110 or Cep97 protein can be recruited in the absence of the other protein. The quantification of this data suggests that some residual CP110 or Cep97 can still be recruited to centrioles in the absence of the other (Graphs, Figure S1B,C), so we do not think it necessary to repeat this experiment at higher laser intensity to further test this point. We now state that the centriolar recruitment of one protein may not be completely dependent of the other (p6, para.2).
Figure 3: The reviewer questions whether the reduction in CP110/Cep97 levels at the mother centriole that we observe during S-phase could be due to photobleaching. This is an interesting point that we now analyse in more detail (p8, para.2). We do not think the decrease in mother centriolar CP110/Cep97 levels is due to photobleaching as our new analysis (which includes more data points during mitosis) strongly suggests that centriolar levels on the mother rise again at the start of the next cycle (New Figure 3C,D).
The reviewer asks whether the CP110/Cep97 oscillations occur at the tip of the growing centriole, and whether we can use super-resolution imaging to address this. A large body of evidence indicates that CP110/Cep97 are highly concentrated at centriole distal tips, and all our experiments suggest that it is this fraction that is oscillating. In Figure 3, for example, we use Airy-scan super-resolution imaging to follow the oscillation on Mother and Daughter centrioles in living embryos. Although the resolution in these experiments is not as high as we can achieve using 3D-SIM in fixed cells, it seems reasonable to assume that the dots of fluorescence we observe oscillating on these centrioles (Fig. 3) are the same fluorescent dots we observe localised at the distal tips of the mother and daughter using 3D-SIM in fixed cells (Fig. 1A).
The reviewer requests additional quantification of the western blots shown in Figure S1 that we use to judge relative expression levels. As we now describe in more detail in the M&M, these ECL blots are very sensitive, but highly non-linear, so we usually estimate relative expression levels by comparing serial dilutions of the different fractions (see, for example, Figure 1B, Franz et al., JCB, 2013). As we now clarify, the key point is not precisely by how much these proteins are over- or under-expressed, but that we observe a similar oscillatory behaviour when they are either over- or under-expressed.
__The reviewer points out that our statement that the CP110/Cep97 oscillation is entrained by the Cdk/Cyclin oscillator (CCO) is too strong as it is based only on a correlation. __We agree and apologise for this overstatement. To address this, we have now perturbed the CCO by halving the dose of Cyclin B (New Figure 5E—H). This extends S-phase length and we now show that the period of the CP110/Cep97 oscillation is also extended. This suggests that the CCO directly influences the period of the CP110/Cep97 oscillation.
The reviewer notes that our conclusion that the centriole cartwheels are longer or shorter when CP110 or Cep97 are absent or overexpressed, respectively, is based only on Sas-6-GFP fluorescence intensity. They ask if this fluorescence intensity perfectly reflects cartwheel length, and if we can confirm these conclusions using EM. Sas-6 is the main structural component of the cartwheel, so the amount of Sas-6 at the centriole should be proportional to cartwheel length, and we have published two papers that support this conclusion and that use the incorporation of Sas-6 as a proxy to measure cartwheel length (Aydogan et al., JCB, 2018; Aydogan et al., Cell, 2020). Importantly, our previous EM studies support our conclusions about the relationship between cartwheel length and CP110/Cep97 levels: the centrioles in wing-disc cells are slightly longer in the absence of CP110 and slightly shorter when CP110 is overexpressed (Franz et al., JCB, 2013). The new findings reported here provide a potential explanation for this EM data, which was puzzling at the time.
Minor Points:
Figure 1C: The reviewer noted that our schematic illustrations in this Figure could be misleading____. We agree and have now redrawn them.
Reviewer #3
Major points:
The reviewer requested that we clarify our use of the term oscillation, pointing out that oscillations are repetitive variations in levels/activity over time, whereas the “oscillations” we describe here occur during each round of centriole assembly. This is a fair point, and one that is often debated in the oscillation field, with many believing that too many biological processes are termed “oscillations”, when they are not truly driven by the passage of time. To avoid any ambiguity, we now no longer describe the behaviour of CP110/Cep97 as an oscillation (although, for ease of discussion, we still use the term in this letter).
The reviewer thought that the data we show in Figure 1 was not relevant as we largely analyse centrioles in living embryos whereas the data in Figure 1 is derived from fixed wing-disc cells—and similar fixed-cell data has been shown in previous studies. The reviewer suggests we use super-resolution methods to analyse Cp110/Cep97 dynamics in the syncytial embryo, and show this relative to Sas-6 and Plk4. They ask if Plk4 and CP110/Cep97 colocalise at any time. While CP110/Cep97 localisation has been analysed by super-resolution microscopy previously (e.g. Yang et al., Nat. Comm., 2018; LeGuennec et al., Sci. Adv., 2020), CP110/Cep97 was a minor part of these studies and our data is the first to show that this complex sits as a ring on top of the centriole MTs in fly centrioles (that lack the complex distal and sub-distal appendages present in the previously analysed systems). As this localisation is important in thinking about how CP110/Cep97 might influence centriole MT growth, we would like to include it. We cannot show this detail in living embryos as the movement of the centrioles reduces resolution and we cannot observe the ring structure.
Although we do use Airy-scan super-resolution microscopy to study CP110/Cep97 dynamics in living embryos (Figure 3), we cannot do this in two colours (to compare these dynamics to Sas-6 or Plk4 dynamics) as red-fluorescent proteins bleach too quickly. We now show the relative dynamics of CP110/Cep97 and Plk4 recruitment using standard resolution microscopy (New Figure S2). While it is well established that Plk4 and CP110/Cep97 are concentrated at opposite ends of centrioles, they are all recruited to the nascent site of daughter centriole assembly, effectively “colocalising” at this timepoint. This could provide an opportunity for the crosstalk we observe here, and we now mention this possibility (p17, para.1).
The Reviewer questioned whether the loading of Sas-6-GFP onto centrioles can be used as a proxy for cartwheel length, pointing out that Sas-6 could load into centrioles in a way that does not change the cartwheel structure, and that EM is required to test this. As described in our response to Reviewer #2, Sas-6 is the main structural component of the cartwheel, and we have published two papers that use the incorporation of Sas-6 into the cartwheel as a proxy to measure cartwheel length (Aydogan et al., JCB, 2018; Aydogan et al., Cell, 2020). While we cannot exclude that Sas-6 might also associate with the cartwheel in a way that does not involve its incorporation into the cartwheel, it is not clear how EM might address this question. Moreover, even if such a fraction existed, it should not affect our conclusions—as long as Sas-6 is binding to the cartwheel in some way, then the amount bound should remain proportional to the length of the cartwheel. Perhaps the reviewer is suggesting that we perform an EM time course of cartwheel growth to back up our conclusions from the Sas-6 incorporation assay? If so, we think this impractical. The changes in cartwheel length shown in Figure 6 are revealed from analysing several thousand images of centrioles compared at precise relative time points. Such an analysis cannot be done in fixed embryos by EM.
Similar to the point above, the reviewer notes that we use the length of the cartwheel to infer centriole MT length, but we never directly measure MT length. They suggest we perform either an EM analysis or use MT markers to directly measure the kinetics of centriole MT growth. In flies (and many other organisms), the centriole MTs grow to the same length as the centriole cartwheel (Gonzalez, JCS, 1998), so we can be confident that the final length of the cartwheel reflects the final length of the centriole MTs. Moreover, we previously measured the distance between the mother centriole and the GFP-Cep97 cap that sits at the distal tip of the centriole MTs as a proxy for centriole MT length, and found that the inferred kinetics of MT growth were similar to the kinetics of cartwheel growth (inferred from Sas-6 incorporation) (Aydogan et al., 2018). This manual analysis was very time consuming, and we have tried to implement computational analysis methods, but so far without success. For similar reasons to those described in the point above, it is not feasible to accurately measure centriole MT growth kinetics by EM (nobody has been able to do this). Moreover, the centrosomes in these embryos are associated with too much tubulin and the centriole MTs are not yet modified (e.g. by acetylation) as the cycles are so fast—so we cannot directly stain the centriole MTs in fixed embryos. We have now toned down our conclusions about MT length throughout the paper, and we make it clear that we cannot directly measure this.
All of the experiments shown here are performed in the presence of endogenous untagged proteins, and the reviewer wonders if recruitment dynamics might be influenced by competition for binding from the endogenous protein. We have compared the behaviour of many centriole and centrosome proteins in the presence and absence of the untagged WT protein. In all cases, less tagged-protein binds to centrioles/centrosomes in the presence of untagged protein, presumably due to competition. Apart from this, however, we usually observe no real difference in overall dynamics and in Reviewer Figure 1 (see below) we show that CP110-GFP and GFP-Cep97 both oscillate even in the absence of any endogenous protein. As we feel this result is not very surprising, we do not show it in the manuscript.
The reviewer correctly noted that our data was not strong enough to conclude that the CP110/Cep97 oscillation is influenced by the CCO. This was also raised by Reviewer #2 and, as described above (p2, para.3 above), we have now performed additional experiments to more directly demonstrate this point (new Figure 5G—H).
The reviewer requests more discussion of why our conclusion that CP110/Cep97 levels oscillate on the growing daughter centrioles during S-phase is different to that reached by Dobbelaere et al, (Curr. Biol., 2020), who conclude that Cep97-GFP only starts to incorporate into the new daughter centrioles late in S-phase when the daughters are fully grown. We have discussed this discrepancy with these authors and they kindly shared their reagents with us (so our endogenous Cep97-GFP oscillation data comes from the same line they used in their experiments), but we have not come to a clear conclusion on this point. We have shown robust oscillations for CP110 and Cep97 by quantifying many hundreds of centrioles using multiple transgenes (both over- and under-expressed) in multiple backgrounds. Cep97 dynamics were a very minor part of the Dobbelaere et al., study, and they analysed a much smaller number of centrioles. We now briefly mention this discrepancy (p9, para.1), but do not discuss it in detail as we have no definitive explanation for it.
The reviewer requests more experiments or more discussion to address the mechanism(s) of crosstalk between CP110/Cep97 and Plk4, and they suggest several avenues for further investigations. These are excellent ideas, and we are working hard on these approaches. These are all long-term experiments, however, and we feel it is important that the field be made aware of these surprising findings as soon as possible, as others may be better-placed to provide mechanistic insight into how this system ultimately works. We now briefly mention some of the future directions the reviewer highlights in the Discussion.
The reviewer thought we should highlight the previous publications showing that Plk4-induced centriole amplification requires CP110 and that Plk4 can phosphorylate CP110. These studies (Kleylein-Sohn et al, Dev. Cell, 2007; Lee et al., Cell Cycle, 2017) were mentioned, but we now discuss them more prominently (p17, para.2).
Minor Points:
The reviewer raised a number of minor concerns that we have now addressed: (1) We discuss the model the reviewer suggests; (2) we no longer state that the crosstalk between CP110/Cep97 and Plk4 is unexpected; (3) We have clarified our description of the shift in timing of the peak levels of CP110/Cep97, which we no longer refer to as an oscillation; (4) We define mNG as monomeric Neon Green; (5) We have changed our schematics in Figure 1 as suggested by the reviewer; (6) We have corrected the mistake in the legend to Figure 8.
Reviewer #4
Major points:
- The reviewer noted that the amplitude of the CP110/Cep97 oscillations depended on protein expression levels, so the oscillations might not reflect the behaviour of the endogenous proteins. They requested that we either repeat our experiments with CRISPR knock-in alleles, or conduct experiments with the lines driven by the endogenous promotors but in their respective mutant backgrounds. We have not generated CRISPR knock-ins for CP110/Cep97, but have done so for many other centriole/centrosome proteins (>8) and found that most such lines are expressed at higher or lower levels than the endogenous allele (and sometimes very significantly so). This is also true for our standard transgenic lines, where genes are expressed from their endogenous promoters, but are randomly integrated into the genome. The blots in Figure 4 show that CP110-GFP and GFP-Cep97 expressed from a ubiquitin (u) promoter or from their endogenous promoters (e) are expressed at ~2-5X higher or ~2-5X lower levels than the endogenous proteins, respectively. As we observe CP110/Cep97 oscillations in all cases, it seems unnecessary to generate new CRISPR knock-ins (that are also likely to be somewhat over- or under-expressed) to show this again. As the reviewer asks, we show that Cep97-GFP and CP110-GFP still oscillate in in the absence of the endogenous proteins (Reviewer Figure 1). As this does not seem a surprising result, we do not show this in the main manuscript. In the same point the reviewer requests that we use antibody staining in fixed embryos to show that the untagged proteins also oscillate. Analysing protein dynamics is much harder in fixed embryos, as the levels of fluorescent staining are more variable and we can only approximately infer relative timing, rather than precisely measuring it (as we can in living embryos). Moreover, as both proteins in the CP110/Cep97 complex exhibit a very similar oscillatory behaviour when tagged with either GFP or RFP (e.g. Figure 2C), and this behaviour is distinct to that observed with several other GFP- or RFP-tagged centriole proteins (e.g. Novak et al., Curr. Biol., 2014; Conduit et al., eLife, 2015; Aydogan et al., JCB, 2018; Aydogan et al., Cell, 2020) it seems very unlikely that this behaviour is induced by the GFP (or RFP) tag.
The reviewer also suggests that we show the data with the endogenous promoter before we show the data with the ubiquitin promoter. As we now explain better (and show in Figure 4), this seems unnecessary as the proteins expressed from the ubiquitin promotor are probably actually expressed at levels that are more similar to the endogenous protein.
The reviewer questions whether the oscillations we observe might be due to the centrioles simply moving up and down in the embryo during the cell cycle, and they suggest we monitor Asl behaviour to rule this out. We have previously shown that Asl-GFP levels do not oscillate; they remain constant throughout the cell cycle on old-mother centrioles, and grow approximately linearly throughout S-phase on new-mother centrioles (see Figure 1D in Novak et al., Curr. Biol., 2014).
We were not sure we understood this point properly, so we copy the reviewers comment in full here: ____The authors mention (for instance on p. 3) that the inner cartwheel and the surrounding microtubules assemble at opposite ends of the daughter centriole. However, my understanding is that the short centrioles present in the fly embryo have an inner cartwheel that extends throughout the organelle, such that it might be moot to make a distinction between the two ends in this case. Moreover, it is also my understanding that this inner cartwheel is itself surrounded by microtubules, so that microtubule assembly might not be expected to occur strictly at the distal end no matter what. The reviewer is correct that Drosophila centrioles are short (~150nm) and that the cartwheel extends throughout the centriole. We think the reviewer is suggesting that it may not be relevant therefore whether the cartwheel and centriole MTs grow from opposite ends—as the activities that govern their growth may not be spatially separated? However, because cartwheels grow preferentially from the proximal-end (Aydogan et al., JCB 2018) while centriole MTs are assumed to grow preferentially from the distal (plus) end, there is an intrinsic problem in ensuring they grow to the same size—no matter how short or long the centrioles are. The reviewer is correct that one possible solution to this problem is that the centriole MTs actually grow from their minus ends, but this is not widely accepted (or even proposed). We have tried to explain this issue more clearly throughout the revised manuscript.
The reviewer points out that the schematic illustrations in Figure 1A and 1C are inaccurate and unhelpful. We agree and have now redrawn these.
The reviewer asks that we provide information about the eccentricities of the centrioles in the different datasets used to calculate the protein distributions shown in Figure 1, particularly as the data for Sas-4-GFP and Sas-6-GFP were obtained previously using a different microscope modality, making comparisons complicated. The point that comparing distance measurements across different datasets is difficult is an important one, and we now state that such comparisons should be treated with caution. However, we have not provided information on the distribution of centriole eccentricities in the different experiments as it wasn’t clear to us how this information could be used to make such comparisons more accurate (presumably the reviewer is suggesting we could apply a correction factor to each dataset?). The very tight overlap in the positioning of CP110/Cep97 fusions (Figure 1C) strongly suggests that any difference in the average centriole eccentricities of the different populations of centrioles analysed, which are already tightly selected for their en-face orientation (i.e. eccentricity
The reviewer requested that we show the “noisy data” we obtained during mitosis that we excluded from our analysis in Figure 3. As we now explain in more detail (p8, para.2), there are two reasons why the data for mitosis in this experiment is “noisy”: (1) The protein levels on the centrioles are low in mitosis and the centrioles are more mobile, so they are hard to track; (2) The Asl-mCherry marker used to identify the mother centriole starts to incorporate into the daughter (now new mother) centriole during mitosis, making it difficult to unambiguously distinguish mothers and daughters. As a result, we cannot track and assign mother/daughter identity to very many centrioles during mitosis—although we now include some extra data-points during mitosis for the centrioles where we could do this (revised Figure 3C,D). Importantly, it is clear that this “noisy” data hides no surprises: one can see (Figure 3C,D) that the signal on the centrioles is simply low during mitosis and then starts to rise again as the embryos enter the next cycle. This is confirmed in the normal resolution data (Figure 2B,C; Movies S1 and S2) where we can track many more centrioles due to the wider field of view and because we do not have to discard centrioles in mitosis that we cannot unambiguously assign as mothers or daughters.
The reviewer requests that we conduct a super-resolution Airy-scan analysis of CP110/Cep97 driven from their endogenous promoters (eCP110 or eCep97) to ensure that the oscillations we see with these lines (shown in Figure 4C,D) are also occurring at the daughter centriole—as we already show for the oscillations observed with the uCP110 and uCep97 lines (shown in Figure 4C,D, and analysed at super-resolution on the Airy-scan in Figure 3). This is technically very challenging as super-resolution techniques require a lot of light and the centriole signal in the eCP110/Cep97 embryos is very dim compared to uCP110/Cep97 embryos (Figure 4C,D). We have managed to do this for eCep97-GFP and confirmed that—even in these embryos that express Cep97-GFP at much lower levels than the endogenous protein (Figure 4A)—the “oscillation” is primarily on the daughter (Reviewer Figure 2). As this data is very noisy, and as the ubiquitin uCP110/Cep97 lines express these fusions at levels that are closer to endogenous levels (Figure 4A,B), we do not show this data in the main text.
The reviewer also asks for clarification as to why we use the Airy-scan for some experiments and 3D-SIM for others. As we now explain (p8, para.1), 3D-SIM has better resolution than the Airy-scan, but it takes more time and requires more light—so we cannot use it to follow these proteins in living embryos. Thus, for tracking CP110/Cep97 throughout S-phase in living embryos we had to use the Airy-scan.
The reviewer questions why in some experiments we analyse the behaviour of 100s of centrioles, whereas in others the numbers are much smaller (1-14 in Figure 3—note, the reviewer quoted this number as coming from Figure 4, but it actually comes from Figure 3, so we have assumed they mean Figure 3). We apologise for not explaining this properly. The super-resolution experiments in Figure 3 are performed on a Zeiss Airy-scan system, which has a much smaller field of view than the conventional systems we use in other experiments. Thus, we inherently analyse a much smaller number of centrioles in these experiments. In addition, as explained in point 6 above, in these experiments we need to analyse mother and daughter centrioles independently, and in many cases we cannot unambiguously make this assignment, so these centrioles have to be excluded from our analysis.
The reviewer questions why we selected the 10 brightest centrioles for the analysis shown in Figure S1B,C (note, the reviewer states Figure S2 here, but it is the data shown in Figure S1B,C that is selected from the 10 brightest centrioles, so we assume this is the relevant Figure). We apologise for not explaining this properly. In these mutant embryos very little CP110-GFP localises to centrioles in the absence of Cep97, and vice versa, so we cannot track centrioles using our usual pipeline and instead have to select centrioles using the Asl-mCherry signal. As the difference between the WT and mutant embryos is so striking, we simply selected the brightest 10 centrioles (based on Asl-mCherry levels) in both the WT and mutant embryos for quantification. We could select more centrioles, or select centrioles based on different criteria, but our main conclusion—that the centriolar localisation of one protein is largely dependent on the other—would not change.
The reviewer also questioned why we performed the analysis shown in Figure S2 (new Figure S3) during S-phase of nuclear cycle 14, when the rest of the manuscript focuses on nuclear cycles 11-13. We apologise for not explaining this properly. In cycles 11-13 centriolar CP110/Cep97 levels rise and fall during S-phase, whereas both proteins reach a sustained plateau during the extended S-phase (~1hr) of nuclear cycle 14—making it easier to analyse CP110/Cep97 levels in embryos when their centriole levels are maximal. We now explain this.
The reviewer requests that we quantify the western blots shown in Figure 4 in the same way we do in figure 8. To do this we would need to perform multiple repeats of these blots and we did not perform these because the blots shown in Figure 4 largely recapitulate already published data (Franz et al., JCB, 2013; Dobbelaere et al., Curr. Biol., 2020). Moreover, as described in our response to Reviewer #2, these ECL blots are very sensitive, but highly non-linear, so we always compare multiple serial dilutions of the different extracts to try to estimate relative levels of protein expression. We now explain this in the M&M.
The reviewer suggests the data shown in Figure 8 is a “straw man”: we really want to test whether modulating CP110/Cep97 levels modulates centriolar Plk4 levels, but instead we test how they modulate cytoplasmic Plk4 levels. The language here is harsh, as it suggests that our intention was to mislead readers into thinking that we have addressed a relevant question by addressing a different, irrelevant, one. We apologise if we have missed something, but we believe we do perform exactly the experiment that the reviewer thinks we should be doing—quantifying how centriolar Plk4 levels change when we modulate the levels of CP110 or Cep97 (Figure 7). It is clear from this data that modulating the levels of CP110/Cep97 does indeed modulate the centriolar levels of Plk4. In Figure 8 we seek to address whether this change in centriolar Plk4 levels occurs because global Plk4 levels in the embryo are affected—a very reasonable hypothesis, which this experiment addresses quite convincingly (although negatively).
Minor Points:
The reviewer highlights a small number of mistakes and omissions, all of which have been corrected.
Finally, we would like to thank the reviewers again for their detailed comments and suggestions. We hope that you and they will agree that the changes we have made in response to these comments have substantially improved that manuscript and that it is suitable for publication in The Journal of Cell Science.
Sincerely,
Jordan Raff
__Reviewer Figure 1. CP110/Cep97 dynamics remain cyclical even when Cep97-GFP and CP110-GFP are expressed from their endogenous promotors in the absence of any endogenous protein. __Graphs show how the levels (Mean±SEM) of centriolar CP110/Cep97-GFP change during nuclear cycle 12 in (A) Cep97-/- embryos expressing eCep97-GFP or (B) CP110-/- embryos expressing eCP110-GFP. CS=Centrosome Separation, NEB=Nuclear Envelope Breakdown. N≥11 embryos per group, average of n≥15 centrioles per embryo.
__Reviewer Figure 2. ____The cyclical recruitment of Cep97-GFP expressed from its endogenous promoter occurs largely at the growing daughter centriole. __The graph quantifies the fluorescence intensity (Mean±SD) acquired using Airy-scan microscopy of eCep97-GFP on mother (dark green) and daughter (light green) centrioles in individual embryos over Cycle 12. CS = Centrosome Separation, NEB = Nuclear Envelope Breakdown. Data was averaged from 3 embryos as the number of centriole pairs that could be measured was relatively low (total of 2-8 daughter and mother centrioles per time point; in part due to the much dimmer signal of eCep97-GFP in comparison to uGFP-Cep97).
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Referee #4
Evidence, reproducibility and clarity
The authors report that CP110 and Cep97 localize near the distal end of centrioles in Drosophila embryos. CP110 and Cep97 tagged with GFP exhibit an oscillatory distribution, with levels on the daughter centriole being maximal in mid S-phase. These oscillations correlate with cell cycle progression. The authors also show that modulating CP110 or Cep97 levels changes the rate at which Sas6-GFP incorporates in the daughter centriole, as well as aspects of the previously reported oscillatory behavior of Plk4.
These results could be of potential interest if the stated conclusions were backed up by more convincing data than that which is provided at present. The issues delineated hereafter must be addressed in full before further consideration of the manuscript.
Major points
1) The oscillatory amplitude of CP110/Cep97 tagged with GFP is much smaller when expression is driven by the endogenous promoters than upon overexpression (see Figure 4), raising the possibility that oscillation might not reflect, or only reflect in part, the behavior of the endogenous proteins. To address this issue, the authors could GFP tag the endogenous loci using CRISPR/Cas9. If this is too demanding, they should at the minimum conduct experiments with the extant lines driven by the endogenous promoters, but in the background of the available CP110 or Cep97 null mutants. Moreover, the authors should stain staged wild-type embryos with antibodies against CP110 and Cep97 to ensure that the mild oscillations reported in Figure 4 do not merely reflect the behavior of the tagged proteins, for example due to the presence of GFP. Related to this point, the authors should considering showing first the data with CP110-GFP GFP-Cep97 driven from the endogenous promoters (current Figure 4), perhaps relegating the results upon overexpression (current Figure 2) to a Supplementary Figure.
2) In repeating the above experiments, the authors must ensure that potential mild oscillations do not simply reflect the fact that centrioles are located at a slightly different distance from the coverslip as a function of cell cycle stage. This could be addressed for example by simultaneously imaging a mother centriole marker such as Asl-mCherry.
Other important points
3) The authors mention (for instance on p. 3) that the inner cartwheel and the surrounding microtubules assemble at opposite ends of the daughter centriole. However, my understanding is that the short centrioles present in the fly embryo have an inner cartwheel that extends throughout the organelle, such that it might be moot to make a distinction between the two ends in this case. Moreover, it is also my understanding that this inner cartwheel is itself surrounded by microtubules, so that microtubule assembly might not be expected to occur strictly at the distal end no matter what.
4) Partially related to the point above, the schematic representations in Figure 1 are somewhat confusing. The schematic in Figure 1A represents CP110/Cep97 strictly at the distal end of the centriole, yet the actual immunofluorescence data on the left suggests that CP110/Cep97 are in fact present very close to Asl-mCherry. This apparent difference must be resolved. Moreover, Figure 1C seems to indicate that all the depicted proteins are present throughout the centriole, which I guess is not what the authors wanted to convey.
5) For the quantification of the data reported in Figure 1, the authors considered only centrioles for which CP110/Cep97 ring eccentricity was less than 1.2, to ensure that only near top views are considered (see p. 23). This is entirely reasonable, but the authors should report the distribution of eccentricities in the data set for the two proteins, and compare them to those of the Sas6-GFP and Sas4-GFP data set, all the more since the latter two were obtained previously with a different microscope modality, potentially complicating thorough comparisons. A slight difference in the fraction of centrioles with a slight tilt could easily skew the data when dealing with such small dimensions.
6) In Figure 3, the authors chose not to report the "Noisy data" observed during mitosis. While it is understandable that the data is noisier at this stage, it must nevertheless be reported, as this may have bearing on assessing oscillations between cycles 12 and 13.
7) The authors should conduct Airy-scan analyses of CP110/Cep97 oscillations driven from the endogenous promoters, to ensure that the variations across the cell cycle reported in Figure 4 reflect changes in the daughter centriole. Moreover, it was not clear why the authors used the Airy-scan for some super-resolution experiments and 3D-SIM for others.
8) Why are solely 1-14 centrioles per embryo considered in the experiments reported in Figure 4 as compared to over 100 per embryo in Figure 2? And how were these centrioles chosen? This needs to be explained, justified and, potentially, rectified.
9) Likewise, why are only the 10 brightest centriole pairs in each embryo retained for the analysis reported in Figure S2? And would the conclusion differ if more centrioles than that were included? Moreover, S phase of cycle 14 is analyzed in Figure S2 for Sas6-GFP, whereas the remainder of the manuscript analyzes CP110/Cep97 during cycles 11 through 13 (with an emphasis on cycle 12). This must be resolved.
10) The Western blots in Figure 4A, 4B, as well as in Figure S1A, should be quantified in the same manner as those in Figure 8C, to achieve a better assessment of the differences in protein levels between conditions.
11) The set up for the experiment reported in Figure 8 comes across as a straw man. What one would really like to find out is whether levels of Plk4 at centrioles are modulated by CP110/Cep97 levels, as the authors themselves acknowledge. Since this does not appear to be feasible, the authors set out to test whether cytoplasmic levels of Plk4 differ, finding that this is not the case. Since this experiment does not address what should be tested, it could be reported as a Supplementary Figure, not as the last main figure of the manuscript.
Minor points
- The authors forgot to mention the Tang et al. paper (doi: 10.1038/ncb1889) when referring to Sas-4/CPAP (for instance on p. 4).
- On p. 9, the authors conclude that the "recruitment of CP110/Cep97 to centrioles is regulated by the CCO". Figure 5 shows that the two correlate, not that the latter regulates the former. A related comment holds for the discussion (bottom of p. 13).
- It is not clear why the authors sometimes report SDs (Figure 7) and sometimes SEMs (Figure 3), or fail to report what is being shown (Figure 2). This needs to be clarified.
- The legend of Figure 8A mentions Pie charts and other things that are not featured in the current rendition of the figure.
Significance
These results could be of potential interest if the stated conclusions were backed up by more convincing data than that which is provided at present.
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Reply to the reviewers
This manuscript was evaluated at Review Commons by four individual reviewers. There was a consensus amongst reviewers that the localization behavior of altORF peptide to the Golgi is a compelling observation and that, with some additional characterization, would provide an effective cell biological tool for use in labeling and studying the Golgi. Our primary goal for this paper was to explore this surprising alternative protein hidden within the sequence of a centromere component and to establish this peptide as a cell biological tool that can be used to study the Golgi. However, the reviewers also highlighted some interesting open questions regarding the nature of this peptide. Below we summarize these core comments our current changes and plans
- Where within the Golgi does the peptide localize? In the work currently included in the paper, we demonstrate that the altORF peptide robustly colocalizes with markers for the Golgi (GM130/TGN46), but not with markers for the Endoplasmic Reticulum (KDEL). However, the resolution at which we imaged the localization of the peptide was not sufficient to determine in which compartment of the Golgi the peptide resides. To address reviewer comments on the specificity of the peptide’s localization within the Golgi, we will attempt to use higher resolution imaging such as confocal or spinning disk microscopy to attempt to better resolve this.
- How does the peptide target to the Golgi? In this manuscript, we show that the localization of the altORF peptide relies on a Cysteine residue present within in a minimal 10 amino acid sequence. Through treatment with 2-Bromopalmitate (2-BP; a palymityltransferase inhibitor) to disrupt its localization, our work suggests that the peptide is palmitoylated. In addition to this observation, the reviewers asked for an additional demonstration that this peptide is palmitoylated in cells. To test this, we have attempted to identify this modification using mass spectrometry of the isolated (IP) GFP tagged peptide from cells. However, we were unable to identify peptides that coincide with the modified peptide cysteine residue. Secondly, we have attempted to identify the modification using Click-chemistry labeling strategy, but this has proved to be technically challenging and infeasible. As an alternative approach for the revised version, we will attempt to perform hydroxylamine treatment followed by SDS-PAGE analysis to determine whether this results in a shift in migration of the GFP tagged altORF, as suggested by a reviewer, to provide additional evidence that the peptide is modified.
- Can this peptide be used to ectopically target proteins to the Golgi? The reviewers asked whether the altORF peptide can be used to ectopically target proteins to the Golgi. In this manuscript, we demonstrate that the peptide sequence is sufficient to target both GFP and the Halo tag (two very different proteins) to the Golgi, and can be tagged at either terminus of the peptide, suggesting that it can be used as a powerful strategy to recruit other proteins to the outer surface of the Golgi. We have emphasized this point in the updated version that is included in this revision.
- Does this peptide alter Golgi structure? For this peptide to provide a useful cell biological marker, it would be preferential for it not to alter cellular physiology. Our work demonstrates that expression of the altORF peptide does not affect the growth of cultured cells. For this updated version, we have performed additional analysis to test whether induced expression of the altORF peptide alters the structure of the Golgi or the localization of other Golgi-associated proteins. Based on a qualitative analysis of these cells, we do not detect any obvious changes in Golgi organization or morphology. This is now included as Supplemental Figure 2D.
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- Is this peptide expressed in human cells? *We have analyzed published ribosome profiling data that suggests that this altORF can be translated, although it is produced to a much lower degree than the full-length CENP-R protein. The short length of the peptide as well as the nature of the amino acid sequence makes this peptide highly challenging to identify via mass spec. It is also possible that this peptide would be expressed in different cell types in the human body, but not robustly expressed in cultured cells. We believe that these are beyond the scope of this paper. However, we now comment on these important points in the updated version.
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- Is this peptide “functional”? *Based on our deliberate analysis of the evolutionary conservation of this altORF within the CENPR transcript, it is clear that this peptide acquired the ability to localize to the Golgi only recently during evolution (only old world primates have this capacity). We believe that this peptide represents a great example of evolution in action, with minor sequence changes resulting in the acquisition of a new capacity and trait. However, as this peptide is not broadly conserved across mammals, it is unlikely to facilitate a core biological function that can be analyzed in cell culture. It is certainly possible that this peptide would contribute to a feature of human biology on the organismal level, but it is not feasible to test this experimentally. The functional nature of this peptide, and particularly the recent evolutionary acquisition of this novel trait are interesting points that we have now commented on in the updated manuscript (text changes in blue).
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SciScore for 10.1101/2022.03.08.483451: (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 washing with 5% milk in TBS-Tween, the membranes were incubated with HRP conjugated anti-mouse antibodies for N and S protein, and with HRP conjugated anti-rabbit antibody for M protein and alpha-actinins for 2h at room temperature, then washed in TBS-Tween buffer, incubated with ECL reagent (Amersham cat#RPN2236) and imaged using a Chemidoc Imager (Biorad)</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-rabbit</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Incubation with primary antibodies anti-SARS-CoV2 rabbit membrane (M) protein (1:100) was performed for 2 hours at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV2</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 infection: Human pulmonary Alveolar A549-hACE2 cells were obtained from original A549 (ECACC) transduced with a lentiviral vector expressing human ACE2 receptor (manufactured by FlashTherapeutics company, Toulouse, France) and sorted by cytometry for having more than 80% hACE2 on their surface.</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">The sorted A549-hACE2 cells were maintained in RPMI supplemented with 10% heat inactivated fetal bovine serum (FBS), 1% sodium Pyruvate, 0.5% HEPES and antibiotics (penicillin/Streptavidin) and cultivated at 37°C with 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549-hACE2</div><div>suggested: RRID:CVCL_A5KB)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For virus production, VeroE6 cells were obtained from (ECACC) and maintained in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) at 37°C with 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6</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">Raw reads were visualized by FastQC to determine the quality of the sequencing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FastQC</div><div>suggested: (FastQC, RRID:SCR_014583)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">High quality reads were mapped using with HISAT2 v2.1.0 with reads corresponding to the transcript with default parameters.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HISAT2</div><div>suggested: (HISAT2, RRID:SCR_015530)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After mapping, Tag libraries were obtained with MakeTaglibrary from HOMER (default setting).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HOMER</div><div>suggested: (HOMER, RRID:SCR_010881)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Read trimming was performed using trimmomatic (v 0.39) with the following parameters “ILLUMINACLIP:all_adapters_v0.38.fa:2:30:10 AVGQUAL:30 LEADING:0 TRAILING:0 SLIDINGWINDOW:6:30 MINLEN:38”.</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">Trimmed reads where then aligned to the SARS-CoV-2 reference genome NC_045512.2.fasta (downloaded May 2021 from https://www.ncbi.nlm.nih.gov/nuccore/NC_045512) using the STAR (v 2.7.9a) aligner; STAR parameters where the following “--outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --outSJfilterOverhangMin 12 12 12 12 --outSJfilterCountUniqueMin 1 1 1 1 --outSJfilterCountTotalMin 1 1 1 1 -- outSJfilterDistToOtherSJmin 0 0 0 0 --outFilterMismatchNmax 999 -- outFilterMismatchNoverReadLmax 0.04 --scoreGapNoncan -4 --scoreGapATAC -4 -- chimOutType WithinBAM HardClip --chimScoreJunctionNonGTAG 0 –alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 -- alignSJstitchMismatchNmax -1 -1 -1</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">Samtools (v 1.12) was used to handle the alignments, and bedtools coverage was used to count reads in each viral feature (gene) using the genomic coordinates from GCF_009858895.2_ASM985889v3_genomic.gff (downloaded May 2021 from https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/009/858/895/GCF_009858895.2_ASM985889v3/GCF_009858895.2_ASM985889v3_genomic.gff.gz) only for protein coding features.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Samtools</div><div>suggested: (SAMTOOLS, RRID:SCR_002105)</div></div><div style="margin-bottom:8px"><div>bedtools</div><div>suggested: (BEDTools, RRID:SCR_006646)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All the images processed with ImageJ/Fiji.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ/Fiji</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Z stack was processed using ImageJ/Fiji, Imaris viewer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Imaris</div><div>suggested: (Imaris, RRID:SCR_007370)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Statistical tests were performed using Origin 2021 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Origin</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">Cell area and cell volume and height were calculated using 3D viewer plugin from Fiji image J.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Fiji image J</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div><div style="margin-bottom:8px"><div>image</div><div>suggested: (NIH Image, RRID:SCR_003073)</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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github-actions released this 14 Dec 2021 v0.1.6 3cdc3c3 This commit was created on GitHub.com and signed with GitHub’s verified signature. GPG key ID: 4AEE18F83AFDEB23 Learn about vigilant mode. Compare Choose a tag to compare View all tags
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Die sind dafür konzipiert, Tag und Nacht, sommers wie winters durchzulaufen, möglichst mit hundert Prozent ihrer Leistung. In einem vorrangig auf Sonne und Wind gegründeten Stromsystem kommen die Back-up-Kraftwerke jedoch nur noch stundenweise zum Zuge, wenn Wind und Sonne ausfallen. Das macht Atomkraftwerke, erstens, unwirtschaftlich. Zweitens und wichtiger: Atomkraftwerke, alte wie künftige, sind für den so genannten Lastfolgebetrieb, der im System mit den Schwankungen von Wind- und Sonnenstrom wie nie gefragt ist, nicht konzipiert. Gerade die sicherheitstechnisch entscheidenden Komponenten im Reaktorkern halten den ständigen Last- und Temperaturwechseln nicht dauerhaft stand.
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tomcritchlow.com tomcritchlow.com
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How might we create easier ways for small groups to publish / maintain knowledge in public?
It is at this junction that I become sad I can't tag in Flancian as such.
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SciScore for 10.1101/2022.03.07.481785: (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">Twenty micrograms of cell lysate and an equal volume of purified virus were separated by SDS-PAGE, transferred to nitrocellulose membrane and membranes probed using the following primary antibodies: anti-SARS-CoV Spike (100% overlap with the SARS-CoV-2 epitope) (Invitrogen, PA1-41165, 1:1000)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>SARS-CoV-2 epitope </div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>PA1-41165</div><div>suggested: (Thermo Fisher Scientific Cat# PA1-41165, RRID:AB_1087210)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, anti SARS-CoV nucleoprotein (N) monoclonal antibody (clone CR3009, gift from Laura McCoy, 0.5 μg/ml), anti-HA-tag (Biolegend, 16B12, 1:2000)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti SARS-CoV nucleoprotein ( N</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-HA-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibodies were incubated overnight at 4°C with agitation and detected with fluorescent secondary antibodies: anti-Rabbit IgG (IRDye 800CW, Abcam, ab216773, 1:10,000), anti-Mouse IgG (IRDye 680RD, Abcam, ab216776, 1:10,000) and anti-Human IgG (IRDye 800CW, Licor, 925-68078, 1:10,000), and imaged with an Odyssey Cxl Infrared Imager (Licor).</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><div style="margin-bottom:8px"><div>ab216776</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Human IgG</div><div>suggested: (LI-COR Biosciences Cat# 925-68078, RRID:AB_2814915)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were then washed and permeabilised with Perm buffer (Biolegend) for 5 min at RT and stained with primary antibodies (20 min at RT): anti-HA-tag-APC (Biolegend, 16B12, 1:150), anti-Gag-PE (Beckam Coulter, KC57, 1:250).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-HA-tag-APC</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Gag-PE</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>KC57</div><div>suggested: (Beckman Coulter Cat# 6604667, RRID:AB_1575989)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 infection was detected by intracellular staining for nucleoprotein (N) using anti-SARS-CoV N monoclonal antibody (human, clone CR3009, gift from Laura McCoy, 1 μg/ml) and secondary anti-Human IgG-AF488 (Jackson labs, 1:400).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV N</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Human IgG-AF488</div><div>suggested: (SouthernBiotech Cat# 2040-30, RRID:AB_2795650)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For analysis of cell surface spike infection, 293T cells were harvested with 5 mM EDTA (Sigma), washed, stained with Zombie NIR and anti-SARS-CoV-2 Spike antibody from human convalescent sera (NIBSC, 20/130 1:200) on ice for 30 min.</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">Primary antibody incubation was carried out for 1h at RT with rabbit anti-HA antibody (H6908, Sigma-Aldrich) to label IFITM1, IFITM2 and IFITM3 and mouse anti-CD63 (IB5, a gift from M. Marsh, UCL) to label endogenous CD63.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-HA</div><div>suggested: (Sigma-Aldrich Cat# H6908, RRID:AB_260070)</div></div><div style="margin-bottom:8px"><div>IFITM1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IFITM2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IFITM3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IB5</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD63</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibodies were detected with secondary anti-rabbit AlexaFluor-488 and anti-mouse AlexaFluor-568 conjugates (Jackson Immuno Research) for 1h.</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><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: HEK293T/17 cells (abbreviated herein as 293T cells) were obtained from American Type Culture Collection (ATCC,</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">Vero.E6 cells were obtained from the National Institute for Biological Standards and Control (NIBSC).</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">Vero.E6-TMPRSS2 cells (stable cell line expressing TMPRSS2 under G418 selection) were a gift from Mala Maini (UCL).</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">HeLa-TZM-bl cells (expressing luciferase and beta-galactosidase under the control of HIV-1 LTR) were obtained from the Centre for AIDS Reagents (CFAR).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa-TZM-bl</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus infection: Target cells were seeded into white 96-well plates 24h before infection (Caco2, Vero.E6 and Vero.E6-TMPRSS2 cells seeded at 1.5×104 cells/well, 293T-ACE2, HeLa-ACE2 and HeLa-TZMbl cells seeded at 1×104 cells/well).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa-ACE2</div><div>suggested: JCRB Cat# JCRB1845, RRID:CVCL_B3LW)</div></div><div style="margin-bottom:8px"><div>HeLa-TZMbl</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IFITM inhibition assay: Caco2 cells, WT or stably expressing IFITM1/2/3 (described above), were infected with indicated spike PVs and luciferase expression was measured 48 h post-infection as described above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Caco2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infection of Caco2-IFITM1/2/3 cells with live SARS-CoV-2 virus was done as described above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Caco2-IFITM1/2/3</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: SARS-CoV-2 Spike expression vectors were originally synthesised by Genewiz and subcloned into 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: RRID:Addgene_117272)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentiviral backbone packaging plasmid (expressing HIV Gag, Pol, Tat and Rev) p8.91 and the reporter plasmid encoding luciferase gene pCSLW were a gift from Greg Towers (UCL).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p8.91</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus production: Spike pseudoviruses (PVs) were made by co-transfection of spike, p8.91 and pCSLW plasmids as described previously (Dicken et al., 2021).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCSLW</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For HIV-1 Env PV, 293T cells were seeded as described above and transfected with 240 ng p8.91, 240 ng pCSLW,120 ng pSVIII-JRFL Env and indicated doses of GBP or empty vector control.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCSLW,120</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pSVIII-JRFL</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">Plasmids: SARS-CoV-2 Spike expression vectors were originally synthesised by Genewiz and subcloned into pCDNA3.1+ vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Genewiz</div><div>suggested: (GENEWIZ, RRID:SCR_003177)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Scale bars and RGB composite images were created in in FIJI ImageJ software package (Schindelin et al., 2012).</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">Statistical Analysis: Statistical significance was calculated using Prism 9 (GraphPad Prism) using indicated statistical tests and significance was assumed when P < 0.05.</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.
<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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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.03.01.482536: (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">The blot was blocked with 5% skim milk in 1xPBS and probed with the anti-mono-ADP-ribose binding reagent/antibody MABE1076 (α-MAR), and anti-GST tag monoclonal antibody MA4-004 (ThermoFisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mono-ADP-ribose</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>α-MAR</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-GST</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The primary antibodies were detected with secondary anti-rabbit and anti-mouse antibodies (LI-COR Biosciences).</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><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 evaluation and Tm determination involved use of the Roche LightCycler® 480 Protein Melting Analysis software, and data fitting calculations involved the use of single site binding curve analysis on 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: 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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For each subject that you might have ongoing thoughts about, start a separate “Thoughts On” journal. Whenever you have some thoughts on this subject, open up that file, write today’s date, then start writing. To give you an example, here are my “Thoughts On” journals as of today: Accounting Addiction Airports Alcohol Ambition Animation AppDevelopment Artist
Whoa. Taking the journaling to another level. Writing under specific topic areas. He has 97 topics!! I wonder how he remembers which topic ... like would it be 'airports' or 'planes'?... 'BeingSocial' or 'Social'.
Part of me wonders if you could just tag your journal entries with these. But I rather like the intentionality of writing on one topic. It's not just a journal entry that happens to take place in an airport. It's a journal entry ABOUT AIRPORTS.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The authors investigated spindle growth dynamics during anaphase B in S. pombe, a unicellular eukaryote which undergoes closed mitosis. In order to accurately quantify spindle growth speeds, the authors tagged Alp7, a protein that localizes to plus ends of microtubules, with 3xGFP, and tracked its position in the dividing cell during mitosis, resulting in precise measurements of both the duration and velocity of microtubule growth events for the first time. By performing these experiments across a set of different genotypes (e.g. in strains with knock-outs of specific microtubule-associated proteins (MAPs)) the authors conclude that (1) microtubule rescues preferentially occur at the midzone edges, (2) microtubule growth speed decreases throughout anaphase B, independent of several known anaphase MAPs and (3) wrapping of the nuclear membrane around the spindle is responsible for this reduction in MT growth speed.
The data in this study is of very high quality and the conclusions are largely well supported by the data, but some changes could be made in the interest of simplicity and clarity, and some additional experiments should ideally be performed to strengthen the third claim.
Many changes occur in the nuclear bridge in late anaphase, including the disassembly of nuclear pore complexes and the fenestration of the nuclear envelope (Lucena et al. 2015, Exposito-Serrano et al. 2020, Dey et al. 2020). This opens up the possibility for a different interpretation of some of the authors' data - for example, that local alterations in the permeability barrier directly alter microtubule polymerisation dynamics, rather than the wrapping of the nuclear envelope in the bridge per se. This could help explain the ark1-as3 data, for example, in which (non-physiological) membrane tubes wrap around the spindle but local NEB is prevented (Dey et al. 2020).
The authors state that 'transition from fast to slow microtubule growth occurs in the absence of known anaphase MAPs' (heading paragraph 3 (239-240) and Figure 2), yet two paragraphs later, they show that the MAP Ase1 does in fact impact this transition. This distinction might prove confusing for readers, especially those unfamiliar with the S. pombe spindle.<br /> The authors state that Ase1 is required for the decrease in growth speed during anaphase. While the example kymograph in Figure 5B looks convincing, there seem to be too few points at the right side of Figure 5F to properly see the two distributions. Without any statistics to compare wt to ase1∆, it is difficult to tell if the apparent absence of decrease is real. In addition, as the authors also show in Figure 1C-E, the spindle collapses, causing the final spindle length to be shorter than wt. It could be that the spindle collapses before the characteristic drop in growth rate could be observed.<br /> One of Ase1's direct interactors, Cls1, was shown to not have an impact on the transition of fast to slow microtubule growth (Figure 2). In Ase1's case, a full deletion of the gene was necessary to reveal loss of the transition. The ase1off strain with reduced Ase1 expression showed a similar transition to wt, similar to the cls1off strain. cls1 is an essential gene and cannot be deleted, but similar to its interactor, its phenotype might be hidden even at low expression values.
Some observations, such as Figure 2G-L, lack associated statistics. In addition, since Alp7 tagged with 3xGFP is used throughout the paper, it seems important to test whether this tag influences microtubule dynamics.
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www.biorxiv.org www.biorxiv.org
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Reviewer #2 (Public Review):
In this manuscript, Pimm and colleagues report their development of a functional probe that enables in vivo visualization of profilin, a cytoskeletal protein that interacts with both actin and microtubules. Because of its dual impacts on actin and microtubule dynamics, profilin is a critical regulatory hub for cytoskeletal assembly and cellular morphology. Through systematic characterization, the authors find that attachment of a fluorescent protein (or Halo tag) and 10 amino acid linker at the N-terminus of human profilin-1 does not impact profilin's lipid-, actin-, or poly-L-proline-binding functions, or its ability to influence actin and microtubule assembly. Further, expression of Halo-tagged profilin-1 rescues neuroblastoma cells from profilin-1 knockout-induced morphological and cytoskeletal defects. Finally, the localization of Halo-tagged profilin-1 to microtubule networks in these cells requires an intact tubulin-binding interface but is unaffected by disruption of its actin- or poly-L-proline functions.
Strengths
This study undertakes an extensive characterization of profilin's molecular interactions and complex effects on cytoskeletal filament assembly. Notably, the authors use a clever approach to measure profilin's affinity for actin monomers. Using both competition and direct titration assays, the authors demonstrate that binding of both untagged and fluorescently labeled profilin can be robustly detected via fluorescence anisotropy. The use of complementary biochemical and cell biological approaches demonstrates the broad utility of this tagged profilin, and the introduction of point-mutations to probe profilin's localization patterns in live cells provides insight into the dynamic nature of its interactions.
Weaknesses
Overall, the experiments are well designed, and the data are robust. My only recommendations seek to elicit additional information to further highlight the potential utility of this probe.
It would be helpful for the authors to expand on the design of their tagged profilin to provide context for readers who might like to use this probe in their own studies. For example, were various linkers sampled? If so, did the authors find that the length and/or sequence impacted profilin's functions?
It would also be helpful to have additional information about the morphology measurements carried out by the authors. For example, does the morphology index for actin and microtubules report fraction of the cell area that is occupied by polymerized actin or microtubules? Is this value normalized relative to the cell size or fluorescence intensity (i.e., density of the cytoskeletal network)? Also, do profilin knockout cells expressing Halo-G118V have altered microtubule morphologies relative to cells in which Halo-profilin-1 is expressed?
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lad.saras.uniroma1.it lad.saras.uniroma1.it
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storiche
Anche la bibliotheque nationale de france ha una ricchissima collezione di mappe storiche, ordinate per paese e accompagnate da metadati dettagliati. Molte mappe sono in pubblico dominio e possono essere riutilizzate liberamente. I documenti possono essere scaricati a una risoluzione media o condivisi in formato IIIF. Oltre ai metadati tecnici e storici, le mappe sono anche corredate di tag semantici e link ad altre mappe della collezione che incoraggiano la scoperta. Sito: https://gallica.bnf.fr/html/und/cartes/cartes?mode=desktop
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Then we’re free. We’ve stalked the prey, secured it for later nourishment. We can safely forget. We’ve insured against faulty memories. Now on to the next quest, finding something new to stash.
I do have to admit I use Evernote and Obsidian has a sort of digital hoarding. Keeping it all these articles, just in case I want to refer to them later.
And indeed, there are times where I want to pull up an article I read, because it comes up in conversation with a friend. In those times Evernote and Obsidian come in handy.
I also keep the hope alive that I'll go back to my 1,400+ blog post drafts and publish some of them. Some day. Maybe if I only tag them better, I'll uncover these previous ideas more often. If only. If only.
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So my idea was to create a machine-tag format based on Wikipedia topics, allowing any content creator to tag content with any topic in Wikipedia. By using Wikipedia as an index, this format provides very specific identification of content across a vast knowledge domain. Call it the Dewey Decimal System for the web: “The Wiki Decimal System.” In general, the problem with machine tags is how to make them easy to add for regular folks. Although the format itself is simple, the tags are typically lengthy and require you to know the data ID for what you want to tag. Enter my hack: A web page that takes your text and builds the list of Wikipedia machine tags automatically.
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.02.24.481778: (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: , location AMC, in the Netherlands and approved by the local ethical committee of the AMC (NL 73281.018.20).<br>Consent: All individuals included in this study gave written informed consent before participating.</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 the protein was subsequently produced in human embryonic kidney (HEK) 293F cells (Thermo Fisher Scientific) and purified as previously described (4, 8).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK</div><div>suggested: RRID:CVCL_6642)</div></div><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">Pseudo-viruses 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 described (13).</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 Bieniasz (13), 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">The genes were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned Pst I/Not I in a pPPI4 expression vector containing a hexahistidine (his) tag with Gibson Assembly (Thermo Fisher Scientific).</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">Pseudo-virus construction: The WT, Alpha, Beta and Gamma pseudovirus S constructs were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned using SacI and Apal in the pCR3 SARS-CoV-2-SΔ19 expression plasmid (13) using Gibson Assembly (ThermoFisher).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCR3 SARS-CoV-2-SΔ19</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudo-viruses 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 described (13).</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 LC was directly coupled to an Orbitrap Exploris 480 mass spectrometer with BioPharma option (Thermo Fisher Scientific, Bremen, Germany).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BioPharma</div><div>suggested: (TransCelerate BioPharma, RRID:SCR_003728)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data analysis: The retention times and masses of each of the Fab molecules were retrieved from the generated RAW files using BioPharmaFinder 3.2 (Thermo Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BioPharmaFinder</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Further data analysis was performed using in-house scripts using Python 3.8.3 (with libraries: Pandas 1.0.5, Numpy 1.18.5, Scipy 1.5.0, matplotlib 3.2.2 and seaborn 0.11.0).</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><div style="margin-bottom:8px"><div>Numpy</div><div>suggested: (NumPy, RRID:SCR_008633)</div></div><div style="margin-bottom:8px"><div>Scipy</div><div>suggested: (SciPy, RRID:SCR_008058)</div></div><div style="margin-bottom:8px"><div>matplotlib</div><div>suggested: (MatPlotLib, RRID:SCR_008624)</div></div></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 plasmadilution 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: 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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blog.readwise.io blog.readwise.io
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consistent use of the concatenate action tag will elevate your reading practice to the next level. Analyzing a text to decide what is and isn't worth saving is a form of actively engaging with what you're reading.
Identifying what parts of a highlight are important and which are superfluous is another aspect of active reading.
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Rather than capture that extraneous content, use the concatenate action tag to highlight and note the first sentence .c1 and the last sentence .c2.
To concatenate multiple highlights simply add
.c<number in sequence>, for the first highlight you would use.c1, for the second,.c2.
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blog.readwise.io blog.readwise.io
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Not only does inline tagging make it easy to add keywords and categories to your highlights, consistent use will also elevate your reading practice to the next level. Distilling a highlight down to a single keyword or forging an association between a passage and something you're working on are both forms of actively engaging with what you're reading. And actively (rather than passively) reading is essential to getting more of what you want out of books
Adding keywords isn't just to make finding things easier later on, the act of associating a highlight with a keyword or linking it to a project through a tag elevates your reading to be active rather than simply passive.
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In practice, you might not want to type out the full word .probability because typing without a keyboard can be frustrating. To help you type less, we created a shorthand feature. In the example above, you could note the passage .prob instead of .probability. The highlight would initially be tagged prob, but once you rename the shorthand a single time, Readwise will thereafter be trained to automatically convert to all .prob tags to .probability.
Readwise does tag expansion, use a shorthand tag name such as
.proband rename that in Readwise to.probabilityand from then on Readwise will expand the tag name from there. If slashes are okay in a inline tag name this would make it easy to expand.questionto.annotation/question.
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e-vsf.zivit.iv.bfinv.de e-vsf.zivit.iv.bfinv.de
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des Artikels 254
Artikel 254 Endverwendung
(1) In der Endverwendung können Waren aufgrund ihres besonderen Zwecks abgabenfrei oder zu einem ermäßigten Abgabensatz zum zollrechtlich freien Verkehr überlassen werden.
(2) Befinden sich Waren auf einer Herstellungsstufe, in der in wirtschaftlicher Hinsicht lediglich die vorgeschriebene Endverwendung erreicht werden kann, so können die Zollbehörden in der Bewilligung die Bedingungen festlegen, unter denen die Waren als zu den Zwecken verwendet gelten, die für die Anwendung der Abgabenfreiheit oder des ermäßigten Einfuhrabgabensatzes festgelegt wurden.
(3) Sind die Waren zur mehrfachen Verwendung geeignet und halten die Zollbehörden es zwecks Vermeidung von Missbrauch für angebracht, so wird die zollamtliche Überwachung fortgesetzt für einen Zeitraum von höchstens zwei Jahren ab dem Tag der ersten Verwendung dieser Waren zu den Zwecken, die maßgeblich für die Anwendung der Abgabenfreiheit oder des ermäßigten Einfuhrabgabensatzes waren.
(4) Die zollamtliche Überwachung in der Endverwendung endet in allen folgenden Fällen:
a) wenn die Waren zu den Zwecken verwendet wurden, die maßgeblich für die Anwendung der Abgabenfreiheit oder des ermäßigten Einfuhrabgabensatzes waren,
b) wenn die Waren aus dem Zollgebiet der Union verbracht, zerstört oder zugunsten der Staatskasse aufgegeben werden,
c) wenn die Waren zu anderen Zwecken als denen, die maßgeblich für die Anwendung der Abgabenfreiheit oder des ermäßigten Einfuhrabgabensatzes waren, verwendet und die anwendbaren Einfuhrabgaben entrichtet wurden.
(5) Wird eine bestimmte Ausbeute verlangt, so gilt Artikel 255 für die Endverwendung.
(6) Abfälle oder Reste, die bei der Be- oder Verarbeitung von Waren im Rahmen der vorgeschriebenen Verwendung anfallen, sowie Verluste aufgrund natürlichen Schwundes gelten als der vorgeschriebenen Endverwendung zugeführt.
(7) Abfälle oder Reste, die bei der Zerstörung von Waren in der Endverwendung anfallen, gelten als in das Zolllagerverfahren übergeführt.
Fußnoten
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- Feb 2022
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blog.readwise.io blog.readwise.io
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Simply highlight a passage and add a note beginning with a period (.) followed by a single word or abbreviation (with no spaces).
To add a tag to an annotation simple use a
.followed by a single word to create that tag like.productivityor.InProgress.I need to find out if
/characters will break it. -
Keyword tags can help you quickly recall a passage's content, reference relevant material on a topic of interest, or identify interesting patterns in your thinking. And categorical tags can help you organize your highlights into actionable workflows for later use.
Interesting way to classify different kind of tags, keyword tags are topical while categorical tags that are associated with automated actions.
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.02.22.480950: (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">A positive selection follows, comprised of incubation with anti-Myc-AlexaFluor647 antibody (1:100 volume:volume) and anti-AlexaFluor647 magnetic beads (1:10 volume:volume) and flowed over a Milltenyi column on a magnet at 4 °C, such that yeast with bound peptide are retained on the column.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Myc-AlexaFluor647</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-AlexaFluor647</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">This extended product was assembled in yeast with linearized pYal vector at a 5:1 insert:vector via electroporation with electrocompetent RJY100 yeast.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pYal</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Yeast were then washed into 4 °C acid saline (150mM NaCl, 20mM citric acid, pH5) with 1 μM HLA-DM and incubated at 4 °C overnight.</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">Ectodomain sequences of each chain were formatted with a C-terminal poly-histidine purification tag and cloned into pAcGP67a vectors.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pAcGP67a</div><div>suggested: RRID:Addgene_41812)</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">To examine conservation between viruses, viral proteins are aligned using ClustalOmega (Madeira et al., 2019).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ClustalOmega</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Amplicons were sequenced on an Illumina MiSeq using paired-end MiSeq v2 300bp kits at the MIT BioMicroCenter.</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">Paired-end reads were assembled using PandaSeq (Masella et al., 2012).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PandaSeq</div><div>suggested: (PANDAseq, RRID:SCR_002705)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Each vector was individually transfected into SF9 insect cells (Thermo Fisher) with BestBac 2.0 linearized baculovirus DNA (Expression Systems; Davis, CA) and Cellfectin II Reagent (Thermo Fisher), and propagated to high titer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BestBac</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Relative binding curves were then generated and fit in Prism 9.3 to the equation y = 1/(1+[pep]/IC50), where [pep] is the concentration of un-labelled competitor peptide, in order to determine the concentration of half-maximal inhibition, the IC50 value.</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: 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.
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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Author Response:
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.
We thank the reviewer for their enthusiasm and support of our work!
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.
Thank you for the suggestion. We have added a new figure supplement (Figure 6-Figure Supplement 3) with additional example traces for all odorants from Figure 6 for which we found a statistically significant difference between the two genotypes (Ir25a versus wildtype).
- 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.
For these experiments, we followed a standard practice in Drosophila optogenetics to test the same experimental genotype in the presence or absence of the essential cofactor all-trans-retinal. This controls for potential effects from the genetic background. It is possible our description of these experiments was unclear (as also suggested by comments from Reviewer 2). As such, we have clarified our experimental design for the optogenetic experiments in the revised manuscript:
Modified text: “No light-induced responses were found in control flies, which had the same genotype as experimental flies but were not fed all-trans retinal (-ATR), a necessary co-factor for channelrhodopsin function (see Methods).” and “Bottom trace is control animal, which has the same genotype as the experimental animal but was not fed the required all-trans retinal cofactor (-ATR).”
Figure 4-Figure Supplement 1 legend: “In all optogenetic experiments, control animals have the same genotypes as the corresponding experimental animals but have not been fed all-trans retinal.”
Methods: “For all optogenetic experiments, the control flies were of the same genotype as experimental flies but had not been fed all-trans retinal.”
- 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?
We share this reviewer’s excitement regarding the numerous questions our work now raises. While testing additional functional ramifications of chemosensory co-receptor expression is beyond the scope of this work (but will undoubtedly be the focus of future studies), we did expand on what this might mean in the revised Discussion section of the revised manuscript. Previously, we had raised the hypothesis that chemoreceptor co-expression could be an evolutionary relic of Ir25a expression in all chemoreceptor neurons , or a biological mechanism to broaden the response profile of an olfactory neuron without sacrificing its ability to respond to specific odors. We now extend our discussion to raise additional possible ramifications. For example, we suggest that modulating Ir25a coexpression could alter the electrical properties of a neuron, making it more (or possibly less) sensitive to Orco-dependent responses. We also suggest that Ir25a coexpression might be an evolutionary mechanism to allow olfactory neurons to adjust their response activities. That is, that most Orco-positive olfactory neurons are already primed to be able to express a functional Ir receptor if one were to be expressed. Such co-expression in some olfactory neurons might present an evolutionary advantage by ensuring olfactory responses to a complex but crucial biologically relevant odor, like human odors to some mosquitoes.
Reviewer #2 (Public Review):
In the present study, the authors: 1) generated knock-in lines for Orco, Ir8a, Ir25a, and IR7ba, and examined their expression, with a main focus on the adult olfactory organs. 2) confirmed the expression of these receptors using antibody staining. 3) examined the innervation patterns of these knock-in lines in the nervous system. 4) identified a glomerulus, VM6, that is divided into three subdivisions. 5) examined olfactory responses of neurons co-expressing Orco and Ir25a
The results of the first four sets of experiments are well presented and support the conclusions, but the results of the last set of experiments (the electrophysiology part) need some details. Please find my detailed comments below.
We thank the reviewer for their support of our work and appreciating the importance of our findings. In the revised manuscript, we now provide the additional experimental details for the electrophysiology work as requested.
Major points
Line 167-171: I wonder if the authors also compared the Orco-T2A-QF2 knock-in with antibody staining of the antenna.
We did perform whole-mount anti-Orco antibody staining on Orco-T2A-QF2 > GFP antennae (example image below). We saw broad overlap between Orco+ and GFP+ cells, similar to the palps. However, we did not include these results since quantification of these tissues is challenging for the following reasons:
- There are ~1,200 olfactory neurons in each antenna, many of which are Orco+.
- The thickness of the tissue makes determinations of co-localization difficult in wholemount staining.
- Co-localization is further complicated by the sub-cellular localization of the signals: Orco antibodies preferentially label dendrites and weakly label cell bodies, while our GFP reporter is cytoplasmic and preferentially labels cell bodies. For these reasons, we focused on the numerically simpler palps for quantification. For the Ir8aT2A-QF2 and Ir76b-T2A-QF2 lines, palp quantification was not an option as neither knock-in drove expression in the palps (and the available antibodies did not work with the whole-mount staining protocol). This is why we performed antennal cryosections to validate these lines. Below is an example image of the antennal whole-mount staining in the Orco-T2A-QF2 knock-in line, illustrating the quantification challenges enumerated above.
*Co-staining of anti-Orco and GFP in Orco-T2A-QF2 > 10xQUAS-6xGFP antenna *
Lines 316-319 (Figure 4D): It would be better if the authors compare the responses of Ir25a>CsChrimson to those of Orco>CsChrimson.
The goal of the optogenetic experiments was to provide experimental support for Ir25a expression in Orco+ neurons in an approach independent to previous methods. Our main question was whether we could activate what was previously considered Orco-only olfactory neurons using the Ir25a knock-in. These experiments were not designed to determine if this optogenetic activation recapitulated the normal activity of these neurons. For these reasons, we did not attempt the optogenetic experiments with Orco>CsChrimson flies.
Line 324-326: Why the authors tested control flies not fed all-trans retinal? They should test Ir25a-T2A-QF2>QUAS-CsChrimson not fed all-trans retinal as a control.
We apologize for the confusion. The “control” flies we used were indeed Ir25a-T2AQF2>QUAS-CsChrimson flies not fed all-trans retinal as suggested by the reviewer. This detail was in the methods, yet likely was not clear. We have amended the main text in multiple locations to state the full genotype of the control fly more clearly:
Modified text: “No light-induced responses were found in control flies, which had the same genotype as experimental flies but were not fed all-trans retinal (-ATR), a necessary co-factor for channelrhodopsin function (see Methods).” and “Bottom trace is control animal, which has the same genotype as the experimental animal but was not fed the required all-trans retinal cofactor (-ATR).”
Figure 4-Figure Supplement 1 legend: “In all optogenetic experiments, control animals have the same genotypes as the corresponding experimental animals but have not been fed all-trans retinal.”
Methods: “For all optogenetic experiments, the control flies were of the same genotype as experimental flies but had not been fed all-trans retinal.”
Line 478-500: I wonder if the observed differences between the wildtype and Ir25a2 mutant lines are due to differences in the genetic background between both lines. Did the authors backcross Ir25a2 mutant line with the used wildtype for at least five generations?
Yes, the mutants are outcrossed into the same genetic background as the wildtypes for at least five generations. Please see Methods, revised manuscript: “Ir25a2 and Orco2 mutant fly lines were outcrossed into the w1118 wildtype genetic background for at least 5 generations.”
Line 1602-1603: Does the identification of ab3 sensilla using fluorescent-guided SSR apply for ab3 sensilla in Orco mutant flies. How does this ab3 fluorescent-guided SSR work?
In fluorescence guided SSR (fgSSR; Lin and Potter, PloS One, 2015), the ab3 sensilla is GFPlabelled (genotype: Or22a-Gal4>UAS-mCD8:GFP), which allows this sensilla to be specifically identified under a microscope and targeted for SSR recordings. We generated fly stocks for fgSSR identification of ab3 in all three genetic backgrounds (wildtype, Orco mutant, Ir25a mutant).
These three genotypes are described in the methods:
“Full genotypes for ab3 fgSSR were:
Pin/CyO; Or22a-Gal4,15XUAS-IVS-mcd8GFP/TM6B (wildtype),
Ir25a2; Or22a-Gal4,15XUAS-IVS-mcd8GFP/TM6B (Ir25a2 mutant),
Or22a-Gal4/10XUAS-IVS-mcd8GFP (attp40); Orco2 (Orco2 mutant).”
Line 1602-1604: There is no mention of how the authors identified ab9 sensilla.
Information on the identification of ab9 sensilla is under the optogenetics section of the methods: “Identification of ab9 sensilla was assisted by fluorescence-guided Single Sensillum Recording (fgSSR) (Lin and Potter, 2015) using Or67b-Gal4 (BDSC #9995) recombined with 15XUAS-IVS-mCD8::GFP (BDSC #32193).”
Line 1648: what are the set of odorants that were used to identify the different coeloconic sensilla?
We have added the specific odorants used for sensillar identification for coeloconic SSR in the Methods. The protocol and odorants used were:
*2,3-butanedione (BUT), 1,4-diaminobutane (DIA), Ammonia (AM), hexanol (HEX), phenethylamine (PHEN), and propanal (PROP) to distinguish coeloconic sensilla:
o Wildtype flies: Strong DIA and BUT responses identify ac2 and rule out ac4. Absence of strong AM response rules out ac1, absence of HEX response rules out ac3, absence of PHEN response further rules out ac4.
o Ir25a mutant flies (amine responses lost, so cannot use PHEN and DIA as diagnostics): Strong BUT response and moderate PROP response identify ac2 and rule out ac4. Absence of strong AM response rules out ac1, absence of HEX response rules out ac3. Ac4 is further ruled out anatomically based on sensillar location compared to ac2.
Revised text: “Different classes of coeloconic sensilla were identified by their known location on the antenna and confirmed with their responses to a small panel of diagnostic odorants: in wildtype flies, ac2 sensilla were identified by their strong responses to 1,4-diaminobutane and 2,3-butanedione. The absence of a strong response to ammonia was used to rule out ac1 sensilla, the absence of a hexanol response was used to rule out ac3 sensilla, and the absence of a phenethylamine response was used to rule out ac4 sensilla. In Ir25a mutant flies in which amine responses were largely abolished, ac2 and ac4 sensilla were distinguished based on anatomical location, as well as the strong response of ac2 to 2,3-butanedione and the moderate response to propanal (both absent in ac4). Ac1 and ac3 sensilla were excluded similarly in the mutant and wildtype flies. No more than 4 sensilla per fly were recorded. Each sensillum was tested with multiple odorants, with a rest time of at least 10s between applications.
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SciScore for 10.1101/2022.02.21.481262: (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">Serological test systems: All samples collected in the 2021/22 hunting season were tested for the presence of anti-SBV antibodies by a glycoprotein Gc-based ELISA performed as described previously [42].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SBV</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The latter allows for the detection of anti-BVDV antibodies as well as for antibodies against the ovine BDV.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-BVDV</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the detection of antibodies directed against SARS-CoV-1, an ELISA system was established in line with the SARS-CoV-2 test.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-1</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">Expi293 cells were grown in suspension in Expi293 expression medium (Thermo Fisher Scientific) at 37 °C, 8 % CO2 and 125 rpm.</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><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 expression of the recombinant protein, the RBD-SD1 domain (aa 306 – 577) of the SARS coronavirus strain Tor2 (NC_004718.3) was ordered as a synthetic DNA string fragment (GeneArt synthesis; Thermo Fisher Scientific, Darmstadt, Germany) and cloned into the pEXPR103 expression vector (IBA Lifesciences, Göttingen, Germany) in-frame with a C-terminal Strep-tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pEXPR103</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.
<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 for 10.1101/2022.02.21.481223: (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 protocol was approved by the Institutional Review Board of UCSD’s Human Research Protections Program (181180).</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 foreground was a bed file of significant IDR peaks; the background was randomly defined peaks within the same annotated region as the foreground peaks.</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: Cell culture and cell line generation: BEAS-2B, HEK293T and Vero E6 cells were purchased from the American Type Culture Collection and were not further authenticated.<br>Contamination: Cells were routinely tested for mycoplasma contamination with a MycoAlert mycoplasma test kit (Lonza) and were found 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">The remaining lysates were immunoprecipitated using 15 µl anti-Strep or 10 µl anti-FLAG antibody (depending on the epitope tag of the construct; Supplementary Table 4) on Sheep Anti-Mouse IgG Dynabeads M-280 (ThermoFisher) overnight at 4°C.</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>Anti-Mouse IgG</div><div>suggested: (Thermo Fisher Scientific Cat# 11201D, RRID:AB_2783640)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Negative control samples are wild type (WT) BEAS-2B cells, and performed using both anti-Strep and anti-FLAG antibodies (separately).</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></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were fixed with 4% paraformaldehyde for 30 min, which inactivates the virus, before transferring from BSL3 to BSL2, and proceeding with immunofluorescence staining using anti-Nucleocapsid antibody (40143-R019, Sino Biological).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BSL2</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">Next, cells were incubated with primary antibodies (Supplementary Table 4) at 1:250-2000 dilutions in blocking buffer for 16 h at 4 °C, washed with PBS+0.01% Triton X-100 three times for 5 min each at room temperature, and then incubated with secondary antibody (goat anti-rabbit secondary IgG (H+L)</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></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 cell line generation: BEAS-2B, HEK293T and Vero E6 cells were purchased from the American Type Culture Collection and were not further authenticated.</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 ACE2-overexpressing A549 cell line (A549-ACE2) was clonally generated and a gift from Benjamin tenOever52.</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">BEAS-2B cells were cultured on Matrigel (Corning) coated plates and maintained in the PneumaCult-Ex Plus Medium (Stem Cell Technologies), supplemented with 33 µg/ml hydrocortisone (Stem Cell Technologies).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BEAS-2B</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T, Vero E6 and A549-ACE2 cells were cultured in DMEM (ThermoFisher) supplemented with 10% FBS (ThermoFisher) and passaged every three days.</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>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 virus infection: SARS-CoV-2 isolates USA-WA1/2020 (BEI Resources, #NR-52281), hCoV-19/USA/CA_UCSD_5574/2020 (lineage B.1.1.7) and hCoV-19/South Africa/KRISP-K005325/2020 (lineage B.1.351 BEI Resources NR-54009) were propagated and infectious units quantified by plaque assay using Vero E6 cells for the WA1 variant, and Vero-TMPRSS2 cells for variants B.1.1.7 and B.1.351.</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">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">Cell culture and cell line generation: BEAS-2B, HEK293T and Vero E6 cells were purchased from the American Type Culture Collection and were not further authenticated.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BEAS-2B</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 construction: 2xStrep-tagged plasmids in a pLVX vector expressing SARS-CoV-2 proteins were a gift from Nevan Krogan1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX</div><div>suggested: RRID:Addgene_174088)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cloning into the pcDNA3.4 was performed using FastDigest restriction enzymes EcoRI and BshT1 (Invitrogen) and Gibson assembly (NEB).</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">SARS-CoV-2 ORFs were amplified by PCR (KAPA HiFi HotStart ReadyMix, Roche) from the 2xStrep-tagged or 3xFLAG-tagged plasmids with oligonucleotide primers containing attB recombination sites and recombined into pDONR221 using BP clonase II (ThermoFisher) (Supplementary Table 6).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDONR221</div><div>suggested: RRID:Addgene_63743)</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">This study protocol was approved by the Institutional Review Board of UCSD’s Human Research Protections Program (181180).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Human Research Protections Program</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For reads mapped to the SARS-CoV-2 genome, bedgraph densities were generated using SAM tools v1.9 to obtain read densities at each nucleotide position.</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">For reads mapped to the African Green Monkey genome, peaks were called on the usable reads by CLIPper54 and assigned to gene annotations in Ensembl ChlSab1.1 release 102 and Sars_cov_2 ASM985889 v3.101 were used to annotate peaks mapped to the African Green Monkey and SARS-CoV-2 genome.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Ensembl</div><div>suggested: (Ensembl, RRID:SCR_002344)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Gene Ontology analysis of eCLIP target genes was performed using ENRICHR (https://maayanlab.cloud/Enrichr/https://maayanlab.cloud/Enrichr/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ENRICHR</div><div>suggested: (Enrichr, RRID:SCR_001575)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cluster maps were visualized using Cytoscape version 3.8.1</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Cytoscape</div><div>suggested: (Cytoscape, RRID:SCR_003032)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">GENCODE v19 gene annotations and featureCounts (v.1.5.30) were used to create read count matrices.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>featureCounts</div><div>suggested: (featureCounts, RRID:SCR_012919)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequencing reads are first processed as RNA-seq libraries, where RNA-seq reads were trimmed of adaptor sequences using cutadapt (v1.4.0) and mapped to repetitive elements (RepBase v18.04) using STAR (v2.4.0i).</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">Multiple sequence alignment was performed using MAFFT v7.453 and default parameters, and sequence alignment was visualized using Jalview (version 1.0).</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><div style="margin-bottom:8px"><div>Jalview</div><div>suggested: (Jalview, RRID:SCR_006459)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">De novo motif analysis: HOMER was used to identify de novo motifs using reads from IDR peaks.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HOMER</div><div>suggested: (HOMER, RRID:SCR_010881)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The length of each region within the metagene was then scaled to 8%, 62% and 30%, corresponding to the average length of regions from the most highly expressed transcripts in ENCODE HepG2 RNA-seq control datasets.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ENCODE</div><div>suggested: (Encode, RRID:SCR_015482)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Interval features such as introns and exons were extracted from the Gencode v19 coordinates.</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">Images were collected via Zeiss ZEN software and converted to tiff for downstream analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Zeiss ZEN</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Images were analyzed using a custom-developed pipeline in CellProfiler (v.3.1.09).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CellProfiler</div><div>suggested: None</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 64. 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 for 10.1101/2022.02.20.480711: (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">Female BALB/c mice (age 6-8 weeks, Envigo), housed in specific-pathogen free environments, were immunized intramuscularly by injection of 50 μL of vaccine formulated in endotoxin-free PBS (Gibco) into both hind limbs of each animal (100 μL total).</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">Mouse monoclonal anti-hexon antibody (B025/AD51, Thermo-Fisher) was added at 1:1000 dilution in 1% w/v milk in PBS and incubated for 1 h at 25°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-hexon</div><div>suggested: (DSHB Cat# TC31-27F11.C2, RRID:AB_1553403)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were washed with 1% w/v milk in PBS prior to addition of a secondary goat anti-mouse alkaline phosphatase (ALP) conjugated antibody (STAR117A, BioRad) at 1:1000 dilution in 1% w/v milk in PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse alkaline phosphatase (ALP</div><div>suggested: (CEDARLANE Cat# CLF004ALP, RRID:AB_10060171)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Coupling efficiency was assessed by comparing band intensities of unconjugated hexon-Tag in ligand decorated samples to undecorated (control) samples using Image J: Anti-vector antibody neutralization assay: For assessment of vector neutralization by potent neutralising mouse monoclonal antibody (mAb) 9C12 (24) (Developmental Studies Hybridoma Bank, University of Iowa), Ad5 vectors expressing GFP were incubated with serially diluted mAb 9C12 antibody at a 1:1 ratio in complete media for 1 h at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-vector</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For assessment of vector neutralization by serum containing anti-adenovirus antibodies, serum samples were obtained by immunizing C57BL/6 mice with 1E+8 ifu of an Ad5 vector expressing ovalbumin (vector had an unmodified hexon).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-adenovirus</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">Linearized DNA was transfected (Lipofectamine 2000, Invitrogen) into E1-complementing Human Embryonic Kidney (HEK) 293 cell lines; either 293A cells (Invitrogen) for Ad vectors expressing GFP and DogCatcher-NANP18, or 293TREX cells (Invitrogen) for Ad vectors expressing SARS-CoV-2 spike.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>293</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>293A</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>293TREX</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Titration of recombinant adenoviruses: Infectious titer of vector preparations was assessed by single cell infectivity assay on HEK293A cells or 293TREX cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293A</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Diluted serum was incubated with Ad5(GFP) vectors, the mix incubated on HEK293 cells, and bulk GFP fluorescence read 24 h later as described above.</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">Assessment of coagulation Factor X-mediated vector transduction of SKOV3 cells: SKOV3 cells (human ovary adenocarcinoma) were obtained from Public Health England and cultured in McCoy’s 5a media with 2 mM Glutamine and 15% v/v fetal bovine serum (complete McCoy’s).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SKOV3</div><div>suggested: RRID:CVCL_5J11)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 pseudovirus neutralization (pVNT) assay: Pseudotyped HIV-1 viruses incorporating the SARS-CoV-2 full-length spike (Wuhan strain or B.1.1.7, B.1.617.2 or B.1.351 variants of concern) were generated and SARS-CoV-2 pVNT assays performed as previously described using Hela cells stably expressing ACE2 as target cells (28).</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">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 assessment of vector neutralization by serum containing anti-adenovirus antibodies, serum samples were obtained by immunizing C57BL/6 mice with 1E+8 ifu of an Ad5 vector expressing ovalbumin (vector had an unmodified hexon).</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">Female BALB/c mice (age 6-8 weeks, Envigo), housed in specific-pathogen free environments, were immunized intramuscularly by injection of 50 μL of vaccine formulated in endotoxin-free PBS (Gibco) into both hind limbs of each animal (100 μL total).</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">Bacterial artificial chromosome (BAC) sequences from pBELOBAC11 (NEB) were amplified using forward (5’-TTAATTAAcgtcgaccaattctcatg) and reverse (5’-TTAATTAAgtcgacagcgacacacttg) primers to introduce PacI sites at either end of the BAC cassette.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pBELOBAC11</div><div>suggested: RRID:Addgene_60342)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The entire Ad5(GFP) genome was subsequently cloned into the BAC with PacI, to generate pBAC-Ad5(GFP).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pBAC-Ad5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant vectors expressing DogCatcher-NANP18 and SARS-CoV-2 spike (residues 1-1208 Wuhan strain, codon-optimized for mammalian expression and including stabilizing mutations K986P and V987P and mutation of the furin cleavage site 682-GSAS-685 (60)) were generated through cloning of gene constructs into pENTR4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pENTR4</div><div>suggested: RRID:Addgene_26366)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein production and purification: DNA sequences for expression of DogCatcher, DogCatcher-NANP9, DogCatcher-NANP18 and SpyCatcher were cloned into expression plasmid pET45(+) (EMD Millipore) for protein production in BL21(DE3) E. coli (NEB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET45</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">DNA sequences for expression of DogCatcher fused to SARS-CoV-2 spike receptor binding domain (Wuhan strain, residues 319-532) (DogCatcher-RBD), were cloned into mammalian protein expression plasmid pcDNA3.4.</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">Coupling efficiency was assessed by comparing band intensities of unconjugated hexon-Tag in ligand decorated samples to undecorated (control) samples using Image J: Anti-vector antibody neutralization assay: For assessment of vector neutralization by potent neutralising mouse monoclonal antibody (mAb) 9C12 (24) (Developmental Studies Hybridoma Bank, University of Iowa), Ad5 vectors expressing GFP were incubated with serially diluted mAb 9C12 antibody at a 1:1 ratio in complete media for 1 h at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Image J</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">Automated data-collection was performed with Leginon software (65) at a nominal magnification of 28,000×, corresponding to a pixel size of 5.19 Å.</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">Statistics: Statistical analyses were performed 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: We detected the following sentences addressing limitations in the study:
Further investigation will be required to comprehensively determine limitations for capsid display in terms of size and structure of ligands, but similar sized receptor-binding domains of other viral proteins, including influenza hemagglutinin (∼25 kDa), have been expressed independently as recombinant proteins and displayed on VLPs (56) implying that this could be a generalizable concept. Capsid decoration using our protein superglue technology is simple, requiring only co-incubation of spontaneously and irreversibly reacting components with no chemical modification required. A similar conjugation process has already been scaled under good manufacturing practice (GMP) during development of a VLP-based SARS-CoV-2 vaccine currently in Phase I/II clinical trials (19). The ability of our adenovirus-based platform to induce both robust cellular and humoral immunity and to enhance efficacy of multi-shot regimens could be advantageous for applications beyond prophylactic vaccines, including therapeutic vaccines against chronic viral pathogens and cancer. Methods of rapid and customizable covalent decoration of Ad capsids could also be utilized for development of personalized therapies. In prophylactic settings, adenovirus capsid decoration could be utilized in the design of pan-coronavirus and pan-influenza vaccines; combining broad and conserved T cell immunity from encoded antigens with exchangeable capsid ligands delivering potent neutralizing humoral immunity.
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 27. 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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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.02.19.481107: (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 monoclonal antibodies SARS-CoV-1/SARS-CoV-2 Spike Protein S2 (1A9) and SARS-CoV-1/SARS-CoV-2 Nucleocapsid (6H3) were purchased from ThermoFisher Scientific.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-1/SARS-CoV-2 Nucleocapsid ( 6H3 )</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The rabbit polyclonal Anti-GAPDH antibody were purchased from Abcam.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-GAPDH</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The rabbit polyclonal Anti-HIV-1 p24 antibody was purchased from MilliporeSigma.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-HIV-1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mouse anti-N protein antibody (clone 1C7) was purchased from Bioss Antibodies, and the rabbit anti-SARS-CoV-2 spike protein (clone 007) antibody, was purchased from Sino Biological.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-N protein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">protein primary antibody and an anti-mouse IgG HRP secondary antibody in conjunction with SIGMAFAST™ OPD developing solution</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">In parallel and after blocking, the second plate was incubated for one hour with a primary antibody solution formulated in PBS + 1% non-fat milk containing both mouse anti-N protein (1 μg/mL, clone 1C7) and rabbit anti-SARS-CoV-2 spike protein (1:500 dilution, clone 007) antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-N protein ( 1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-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">donkey anti-rabbit IgG Alexa Fluor Plus 594 (2 μg/mL, Invitrogen) antibodies and DAPI (1:1000, Millipore Sigma) in PBS + 0.5% BSA consisting of.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: (Thermo Fisher Scientific Cat# A48284, RRID:AB_2896348)</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, inhibitors, and antibodies: HEK293T (ATCC), HEK293T-ACE2 (kind gift of Hyeryun Choe, Scripps Research), HT1080 cells (ATCC) and Calu3 (ATCC) were cultured in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma), 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.3 mg/mL L-glutamine.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Fusion assays: For the syncytium formation assay HEK293T and HEK293T-Ace2 cells were seeded in 24-well plates and grown to approximately 80% confluency.</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">Cell-cell fusion assay with soluble ACE2, effector HEK293T cells were transiently transfected with plasmid DNA encoding mCherry, and SARS-CoV2 spike and target HEK293T cells were transiently transfected with plasmid DNA encoding LTR-GFP, TMPRSS2 or pCAGGS.</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">Gelatin zymography: HEK293T, HEK293T-ACE2, Calu3 and HT1080 cells were analyzed for MMP2 and MMP9 activity through zymographic analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HT1080</div><div>suggested: CLS Cat# 300216/p517_HT-1080, RRID:CVCL_0317)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HT1080-ACE2 cells were cultured in DMEM supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), HEPES, L-Glutamine (0.3 mg/mL), 10% FBS (all from Thermo Fisher Scientific) and puromycin (1 μg/mL, InvivoGen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HT1080-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Twenty-four hours before infection, 2.5×104 HT1080 ACE2 cells were seeded per well of duplicate 96 well plates in puromycin-deficient DMEM and cultured overnight (37°C/5% CO2) for cell monolayer to adhere.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HT1080 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">HT1080 cells stably expressing ACE2 were generated by infection with lentiviral particles generated with psPAX2, pMDG and pLENTI_hACE2_PURO (gift from Raffaele De Francesco</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>pMDG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLENTI_hACE2_PURO</div><div>suggested: RRID:Addgene_155295)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The full gene, untagged or with a N-terminal FLAG tag, was reconstituted by Gibson assembly, amplified by PCR, and cloned in pCAGGS.</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">Time-course imaging of the syncytia formation was performed using an Incucyte-Zoom (EssenBioscience), and images were analyzed in imageJ to measure the percentage of green surface area over background.</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">Statistical analyses were performed with 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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docdrop.org docdrop.org
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liebe mitbürgerinnen und mitbürger heute ist ein furchtbarer tag für die ukraine und ein düsterer tag für europa wir alle sorgen und zum den frieden
dear fellow citizens. today is a terrible day for ukraine and a gloomy day for europe. we all care and for peace
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people.cs.rutgers.edu people.cs.rutgers.edu
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The "+tag" portion of the email address can then be used both for filing mail and for screening it.
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tutorial.eyehunts.com tutorial.eyehunts.com
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HTML a name Attribute | Jump The name attribute specifies the name of an anchor tag in html. Syntax: <a name=”value”> The name attribute is used in Anchor Tag to “jump” to a specific point on a web page. It is very useful and specially used in large pages or subdivisions.
a name attribute
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Link to anchor on same page
anchor on same page
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www.baltimoresun.com www.baltimoresun.com
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At that time, The Sun was still choosing to identify Black people by race in its coverage — and only Black people — placing the tag “Negro” after individual names, even though many other newspapers had long since stopped similar practices. When a Westminster minister and seminary professor asked the paper to discard “this discriminatory practice” in 1955, according to an article in The Afro-American, the editor-in-chief flat out refused, self-righteously declaring that “the Sunpapers will not be a party to such suppression” of fact and that “the matter of what it is now fashionable to call ‘pigmentation’ is important from both the white and the Negro point of view.”
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post45.org post45.org
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Yet the Goodreads classics depart from these school-sanctioned lists in two particularly striking ways. First, the Goodreads classics are considerably less diverse in terms of the race and ethnicity of their authors. Race is extremely complex and difficult to reduce to data, especially because racial categories differ across different societies.
This part stuck out to me because its quantifying race is a very tricks subject and trying to "tag" it into categories can be scene as very difficult and touchy.
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sakai.duke.edu sakai.duke.edu
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I consider it opportune at this juncture to recall that montage is just as indispensable a component feature of film production as any other element of film effectiveness
Not a thesis, just an assertion of how important this discussion is
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.02.15.480546: (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">Since there was no antibody-hCoV229E spike protein complex in the training set of ScanNet, we used all the 55 networks trained for antibody binding site prediction (including the 11 used elsewhere that were not trained on SARS-CoV-1/2 data).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-hCoV229E</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plate was washed by the washing buffer to remove the unbound hACE2. 1:5,000 diluted Pierce™ High Sensitivity NeutrAvidin™-HRP (Thermo Fisher cat# 31030) or 1:7,500 diluted T7-tag polyclonal antibody-HRP (Thermo Fisher, cat# PA1-31449) were incubated with the plate for 1 hr at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-HRP</div><div>suggested: (Thermo Fisher Scientific Cat# PA1-31449, RRID:AB_1960906)</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 SARS-CoV-2 neutralization assay: The 293T-hsACE2 stable cell line (Integral Molecular, cat# C-HA101, Lot# TA060720MC) and pseudotyped SARS-CoV-2 (Wuhan-Hu-1 strain D614G 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><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 isotonic regression fit was performed using scikit-learn (sklearn.isotonic.IsotonicRegression, default parameters)32. Mice: 8 weeks old female C57BL/6 mice were ordered from The Jackson Laboratory and housed in pathogen-free conditions at the core animal facility at the University of Pittsburgh Medical Center with the approval from the University of Pittsburgh Institutional Animal Care and Use Committee.</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">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">Construction of the MSA: Homologs of the WT RBD were first searched in the UniprotKB using BLAST.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>UniprotKB</div><div>suggested: (UniProtKB, RRID:SCR_004426)</div></div><div style="margin-bottom:8px"><div>BLAST</div><div>suggested: (BLASTX, RRID:SCR_001653)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The isotonic regression fit was performed using scikit-learn (sklearn.isotonic.IsotonicRegression, default parameters)32. Mice: 8 weeks old female C57BL/6 mice were ordered from The Jackson Laboratory and housed in pathogen-free conditions at the core animal facility at the University of Pittsburgh Medical Center with the approval from the University of Pittsburgh Institutional Animal Care and Use Committee.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>scikit-learn</div><div>suggested: (scikit-learn, RRID:SCR_002577)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The raw data were processed by Prism 9 (GraphPad) to fit into a 4PL curve and to calculate IC50/logIC50.</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: 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.
</footer>
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hypothes.is hypothes.is
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A reference to the phrase "useless use of cat". See e.g. https://porkmail.org/era/unix/award.html
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web3.storage in the browser A demo using the web3.storage client in the browser to pre-calculate the CID for an asset then store it on web3.storage. Content addressing, IPFS, Filecoin, web3.storage... it's all pretty rad! Here is gateway URL for the Content ID of this example, (stored via web3.storage of course!) so you can check it out in your browser! https://dweb.link/ipfs/bafybeic5r5yxjh5xpmeczfp34ysrjcoa66pllnjgffahopzrl5yhex7d7i
in browser
This is a perfect way to distibute this capability just add a script tag for it remove original submit event handler and provide your own
https://dweb.link/ipfs/bafybeic5r5yxjh5xpmeczfp34ysrjcoa66pllnjgffahopzrl5yhex7d7i
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cliutils.gitlab.io cliutils.gitlab.io
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Reading files
Use for add build tag in version
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ifttt.com ifttt.com
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Tags & beacons
不懂场景，是说硬件的tag么？
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betasite.razorpay.com betasite.razorpay.com
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This integration method provides a default Razorpay Pay with Razorpay button that invokes the Checkout form. The Checkout form options are passed as data attributes inside a <script> tag. You can add any additional, hidden or visible fields to the form, which will be submitted along with the form.
- This integration method provides a default Razorpay Pay with Razorpay button that invokes the Checkout form.
- The Checkout form options are passed as data attributes inside a <script> tag.
- You can add any additional, hidden or visible fields to the form, which will be submitted along with the form.
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www.biorxiv.org www.biorxiv.org
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Evaluation Summary:
The authors describe the hei-tag, which, when added to a genome editing enzyme, results in increased editing rates in fish embryos and mammalian cell culture. The hei-tag tool could provide a valuable alternative that can potentially boost genome editing efficiency in different species and systems. The wider applicability of this approach still requires further investigation, since the improvement of editing efficiency is so far supported by experimental data on only a few targets. It would also be important to learn how the authors' design decisions affect activity, especially when benchmarked against current state-of-art genome editing tools.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Improving the efficiency of genome engineering tools is a very important and competitive field. Moreover, the design of improved tools with a focus on models outside the mammalian cell editing dominated space is very welcome, as cross species activity should not be taken for granted. Thomas Thumberger et al focus on improving Cas9 activity in medaka and zebrafish embryos by rethinking about the "bells and whistles" attached to the enzyme in its commonly available variants.
They first develop an assay that links Cas9 activity to a phenotypic readout (retinal pigmentation controlled by oc2) in fish embryos. Using the first Cas9 variant reported to edit zebrafish embryos (Hwang et al 2013) as benchmark, they test an improved Cas9 variant (Zhang et al 2014) and their own heiCas9, and report up to 8-fold "enhanced activity" in medaka and 27-fold in zebrafish, when comparing to the Hwang et al variant. Improvements over the Zhang et al variant, tested only in medaka, were more modest.
Lacking a phenotypic assay in mammalian cells, the authors use genome editing outcomes deconvolution software (ICE and TIDE) on Sanger sequencing data to compare the editing efficiency of the Hwang variant, a commercially available Cas9, and heiCas9. HeiCas9 scores better in both analyses, and also shows a clear improvement at the ICE knockout score.
Finally, the authors move away from DSB inducing methods of genome editing and construct a heiBE4 (C to T base editor) variant. By adopting their phenotypic assay in medaka to introduce a stop codon (CAG>TAG) in oc2, they show stronger pigment loss phenotypes in injected embryos when using heiBE4, compared to the original BE4. They further quantify and confirm the high rate of C>T transitions by sequencing.
Overall, the manuscript is well written and the results are clearly presented. Boosting genome editing without modifying the primary sequence of the enzyme is a very interesting approach, and has been reported before (Liu et al 2021). Such methods could also be compatible with artificially evolved Cas9 variants (e.g. high fidelity, relaxed PAM recognition) or even other Cas enzymes (e.g. Cas12), providing an orthogonal approach to increase their activity.
The authors also provide evidence that the hei-tag is not restricted to the conventional DSB inducing approach, by trying a BaseEditor.
How their height-tag works to improve genome editing is not investigated in detail. Knowing the mechanistic underpinnings can help predict the usefulness or lack thereof across different organisms, developmental stages, or cell states. Moreover, the balance between ON- and OFF-target activity is not considered, an important parameter for cell culture experiments where outcrossing is not possible to segregate non-specific modifications of the genome. As a result, the mammalian cell culture data are interesting, but don't add much to the value of the hei-tag.
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Reviewer #2 (Public Review):
The paper focuses on describing a novel tag (named hei-tag) consisting of two optimized NLS sequences and a Myc epitope separated by a linker peptide. This tag fused with Cas9 ORF increases the efficiency of genome editing in fish embryos following mRNA+sgRNA injection. The authors assess these results with a rigorous quantification of pigmentation reduction in fish embryos following the targeting of the pigmentation locus Oca2 in Medaka and zebrafish embryos. The same improved version of Cas9 is tested in mammalian cells in culture comparing the results with older version of Cas9. Finally the authors fuse the hei-tag to the base editor BE4-Gam showing also in this case an increased activity. The data would be more significant for the community if the injection of the Cas9 optimized construct would be tested as protein as this is the most efficient and commonly used approach in fish experiments. Similarly the comparison with the BE4-Gam should be extended to the more recent family of improved Cytidine Base Editors including the ancBE4Max that was optimized for nuclear shuttling among other properties. In the present manuscript the presence of the Myc epitope in the hei-tag is not tested in any of its possible applications and it remains unclear what is the utility of this part of the hei-tag system.
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Reviewer #3 (Public Review):
In this manuscript, Thumberger et al. developed a novel high-efficiency tag to be used with existing CRISPR/Cas9 tools that boosted the editing efficiency in fish (medaka, zebrafish) and mammalian cell culture. Compared to the baseline gene editing methods chosen by the authors, hei-tag improved bi-allelic editing efficiency in medaka fish by about 30% and resulted in about 10% more indels in editing mammalian cells. The authors have also shown that hei-tag can be added to other Cas9-based techniques such as base editors to boost editing efficiency.
The authors have shown convincing evidence that hei-tag improves editing efficiency compared to the baseline methods they have chosen (JDS246-Cas9, myc-Cas9) in fish, demonstrating a boost in bi-allelic targeting efficiency of the oca2 gene in making eye pigment in medaka and zebrafish. Applications beyond gene knockout in fish have been carried out in mammalian cell culture, and also in base editing tools in medaka, implicating the potential broad application of the tool.
However, it is not clear under what scenarios hei-tag carries out significant and practical improvement compared to the state-of-art gene editing techniques. Especially concerning its original purpose of editing earlier and more efficiently in early embryos, the common strategy is to inject Cas9 protein rather than mRNA, which the authors did not account for. Even in the realm of RNA-based editing tools, it is questionable whether JDS246-Cas9, a construct originally made for mammalian gene editing, is the best baseline to compare to. Part of the ambiguity originates from a lack of systematic comparison of existing editing tools in the field, but the authors would need to ensure they are comparing to the state-of-art, and demonstrate the universality and limitation of hei-tag in practical use.
Overall, I think hei-tag would be a good addition to the exiting gene editing tools and has a potential to boost editing efficiency in many systems, although its practical improvement is yet to be solidly demonstrated. Further investigation of the impact of N-terminal tags and linker structure on Cas9 specificity and efficacy will be useful to guide future improvement of protein engineering.
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.02.08.479664: (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: Stock cells were tested for mycoplasma contamination and authenticated by short-tandem repeat (STR) profiling.<br>Authentication: Stock cells were tested for mycoplasma contamination and authenticated by short-tandem repeat (STR) profiling.</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">Primary antibodies used were: anti-HIV-1 p24 (ARP432, CFAR), anti-GFP (sc-9996, SCBT), anti-HA (C29F4, CST), anti-Strep-tag</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Primary antibodies used were: anti-HIV-1 p24 ( ARP432 , CFAR) , anti-GFP ( sc-9996 , SCBT) , anti-HA ( C29F4 , CST)</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-HIV-1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-GFP</div><div>suggested: (Santa Cruz Biotechnology Cat# sc-9996, RRID:AB_627695)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Secondary antibodies include, anti-mouse (61-6520, Thermo Fisher)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: (Innovative Research Cat# 61-6520, RRID:AB_138451)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were labelled with the following primary antibodies, anti-Nup98 or anti-ORF6 diluted in 4% BSA in PBS for 1 hour at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Nup98</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-ORF6</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 the following secondary antibodies, donkey anti-rabbit-AF568 (ab175692, Abcam) and donkey anti-sheep AF568 (A-21099, Invitrogen) diluted in 4% BSA in PBS for 1 hour at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit-AF568</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>donkey anti-sheep AF568 (A-21099, Invitrogen) diluted in 4% BSA in PBS for 1 hour at RT.</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, HeLa, Mus dunni tail fibroblast (MDTF) and Vero cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher), supplemented with 10% heat-inactivated foetal bovine serum (FBS, Biosera) and 1% Penicillin/Streptomycin (Sigma).</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>Vero</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunoprecipitation: HEK293T cells were grown in 10 cm2 culture plates transfected and incubated for 16 hrs after which media was replaced.</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">Viruses were titrated by plaque assay on 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><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 plasmids used to generate HIV-1 and MLV VLPs for transduction of ORFs, pCMVΔ8.91 and pHIT60 respectively, and pVSV-G have been described before [38, 39].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMVΔ8.91</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pVSV-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">For the initial experiments on Gag expression (Figs 1A-C and S1A and B Figs) and ORF6 co-expression with Rae1 (Fig 2A), a pLVX-IRES-Puro vector encoding an individual ORF was used.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-IRES-Puro</div><div>suggested: RRID:Addgene_140240)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pLVX-StrepII-ORF-IRES-Puro plasmids were then cloned using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB) from the pTriEX-6 plasmids, using the primers listed in Table 1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-StrepII-ORF-IRES-Puro</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pTriEX-6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the remaining assays that required ORF detection or immunoprecipitation, a pLVX-EF1α-SARS-CoV-2-ORF-2xStrep/2xStrep-ORF -IRES-Puro encoding one of the nine ORFs or pLVX-EF1α-eGFP-2xStrep-IRES-Puro plasmid was used, referred to as twin-strep-tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-EF1α-SARS-CoV-2-ORF-2xStrep/2xStrep-ORF -IRES-Puro</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLVX-EF1α-eGFP-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">These plasmids were kindly gifted from Nevan Krogan (Addgene plasmid #141383-4, #141387-90, #141392-95); http://n2t.net/addgene:141383-4, 141387-90, 141392-95; RRID:Addgene_141383-4, 141387-90, 141392-95) [5].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>#141383-4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate the pLVX-EF1α-CoV-1-ORF6-2xStrep-IRES-Puro plasmid, the ORF6(CoV-1)-2XStrep gene was synthesised by GeneArt and cloned in using the EcoRI and BamHI restriction sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-EF1α-CoV-1-ORF6-2xStrep-IRES-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ORF6 mutations were introduced into the pLVX-StrepII-SARS-CoV-2-ORF6-IRES-Puro and pLVX-EF1α-SARS-CoV-2-ORF6-2xStrep-IRES-Puro plasmids using the QuickChange II-XL site-directed mutagenesis kit (Agilent) according to the manufacturer’s instructions using SDM primers listed below.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-StrepII-SARS-CoV-2-ORF6-IRES-Puro</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLVX-EF1α-SARS-CoV-2-ORF6-2xStrep-IRES-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The following plasmids were sourced from Addgene, psfGFP-N1 (Addgene, #54737) and pmApple-N1 (Addgene, #54567).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pmApple-N1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pCMVsport6Rae1 (MGC:117333) was purchased from Horizon Discovery Biosciences Limited.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMVsport6Rae1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Virus-like particles (VLP) production: HIV-1 and MLV virus-like particles were generated by co-transfecting HEK293T cells with plasmids, pCMVΔ8.91 (HIV-1) or pHIT60 (MLV) and pVSV-G using lipofectamine 2000 (11668019, Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIT60</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For generating GFP reporter VLPs, pCSGW (HIV-1) or pczCFG2fEGFPf (MLV) were also added [39, 40].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCSGW</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Subcellular fractionation: HEK293T cells were transfected with psfGFP-N1 and either treated with LMB (Merck) at 5nM; or co-transfected with plasmids expressing pLVX-EF1α-ORF6, ORF6(M58A) or ORF9b [5].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psfGFP-N1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLVX-EF1α-ORF6</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 following primers and probes were used; sfGFP: for 5’-GCGCACCATCAGCTTCAAGG, rev 5’-GTGTCGCCCTCGAACTTCAC and probe 5’-FAM-CGGCACCTACAAGACCCGCGC-TAMRA. mRNAseq: The quality of extracted RNA was checked by Agilent TapeStation 42000 on Agilent RNA ScreenTape (Agilent technologies) before proceeding to library preparation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Agilent TapeStation</div><div>suggested: (Agilent TapeStation Laptop, RRID:SCR_019547)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RNA-seq libraries were generated using the NEBNext Ultra Directional RNA Library Prep kit for Illumina with NEBNext® Poly(A) mRNA Magnetic Isolation Module (both New England BioLabs).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NEBNext®</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Poly(A</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Enriched GO terms (Biological process) were filtered and mapped using the REViGO tool available at http://revigo.irb.hr/ [41].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>REViGO</div><div>suggested: (REViGO, RRID:SCR_005825)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Normalisation and differential expression analyses were conducted within DESeq2 [43]: normalisation factors for gene-level read counts were produced using counts for the D. melanogaster spike-in controls and differential gene expression calculations were subsequently performed for Human genes alone using the pre-calculated size factors.</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">Statistics: Statistical analysis was performed using GraphPad Prism 9 software. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, ns – not significant).</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 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>
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.02.07.479477: (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">P43 crystals: Protein expression and purification: The gene encoding SARS-CoV-2 NSP3 Mac1 (residues 3-169) was cloned into a pET-22b(+) expression plasmid with a TEV-cleavable N-terminal 6-His tag (Genscript).</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">crystals: Protein expression and purification: The gene encoding SARS-CoV-2 2-170 NSP3 Mac1 was cloned into a pET-11a plasmid (Bio Basic) and transformed into E. coli (BL21-DE3).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-11a</div><div>suggested: RRID:Addgene_154201)</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 five macrocycles of refinement, the 2mFO-DFC (unfilled) and the mFO-DFC NSL density maps were inspected in Coot and modifications made to the coordinates.</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">Phases were obtained by molecular replacement with Phaser using the same search model as the lower resolution X-ray experiment.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phaser</div><div>suggested: (Phaser, RRID:SCR_014219)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The number of contacts made by water molecules was defined as the number of protein nitrogen or oxygen atoms within 3.5 Å of a water, with distances calculated using 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></table>Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
One caveat to using the neutron-determined orientations is that only orientations of highly ordered water molecules could be determined with high confidence (Fig. 5D/E). Fortunately, these waters are most frequently involved in bridging interactions. Simulation-based methods might yield an improved model of the orientations of disordered water molecules (65). We found that the conformational ensemble of Mac1 was unusually robust to temperature and pH perturbation in the crystal. Room temperature crystallography can reveal low occupancy, functionally relevant conformational states of proteins (51, 66–68). The 1.1 Å room temperature structure of Mac1 reported here, in combination with the previously reported 0.85 Å structure determined using the same crystal form at 100 K (14), allowed a detailed analysis of temperature dependent changes in structure and function. Overall, the structure and flexibility at 100 and 293 K were remarkably similar, indicating that unlike several recently reported examples (68–70), Mac1 displays minimal temperature dependent structural changes in crystallo (Fig. 4C). Similarly, assuming that the crystals have equilibrated to the target pH in the ∼4 hour soaks conducted here (71), the pH shift experiments revealed that the Mac1 active site residues and water networks are remarkably robust (Fig. 6I/J), and this is matched by the invariance in ADPr binding across the entire pH range (Fig. 6G/H). One caveat to the pH-shift experiments is the pH-dependent...
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 for 10.1101/2022.02.07.479468: (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 procedures were performed under the approvals of the Institutional Animal Care and Use Committee (IACUC) of University of Washington, Seattle, WA.<br>Euthanasia Agents: Mice were injected intramuscularly into the quadriceps muscle of each hind leg using a 27-gauge needle (BD, San Diego, CA) with 50 μL per injection site (100 μL total) of immunogen under isoflurane anesthesia.<br>IRB: This study 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">BALB/c mice for RBD-NP, S ‘2P’, and HexaPro immunizations: Female BALB/c mice (Stock # 000651, BALB/c cByJ mice) four weeks old were obtained from Jackson Laboratory, Bar Harbor, Maine, and maintained at the Comparative Medicine Facility at the University of Washington, Seattle, WA, accredited by the American Association for the Accreditation of Laboratory Animal Care International (AAALAC).</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">One day post-transfection, cells were infected with VSV(G*ΔG-luciferase)29 and after 2 h were washed five times with DMEM before adding medium supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant, 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: (LSBio (LifeSpan Cat# LS-C51761-50, RRID:AB_1277784)</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, HEK293T cells in DMEM supplemented with 10% FBS, 1% PenStrep seeded in 10-cm dishes were transfected with the plasmid encoding for the corresponding S glycoprotein using lipofectamine 2000 (Life Technologies) following manufacturer’s indications.</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">Pseudovirus Neutralization: HEK293-hACE2 cells15 or VeroE6-TMPRSS214 were cultured in DMEM with 10% FBS (Hyclone) and 1% PenStrep and 8ug/mL puromycin for TMPRSS2 maintenance with 5% CO2 in a 37°C incubator (ThermoFisher).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS214</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate VSVΔG-based SARS-CoV-2 pseudovirus, BHK-21/WI-2 cells were transfected with the spike expression plasmid and infected by VSVΔG-firefly-luciferase as previously described30.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BHK-21/WI-2</div><div>suggested: RRID:CVCL_HB78)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentivirus encoding hACE2-P2A-TMPRSS2 was made to generate A549-hACE2-TMPRSS2 cells which were maintained in DMEM supplemented with 10% fetal bovine serum and 1µg/mL puromycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549-hACE2-TMPRSS2</div><div>suggested: RRID:CVCL_A5KB)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Both viruses were propagated on Vero-TMPRSS2 cells and subjected to deep sequencing to confirm the presence of expected substitutions.</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">Immune complexes were added to VeroE6-TMPRSS2 cell monolayers and incubated for 1 h at 37°C prior to the addition of 1% (w/v) methylcellulose in MEM.</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><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">BALB/c mice for mRNA-1273 immunizations: Female BALB/c mice (6 to 8 weeks old) were obtained from Charles River Laboratories.</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">1292S mice for mRNA-1273 immunizations: 129S2 mice (strain: 129S2/SvPasCrl, Cat # 287) were obtained from Charles River Laboratories and housed in a pathogen-free animal facility at Washington University in St. Louis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>129S2</div><div>suggested: RRID:IMSR_CRL:287)</div></div><div style="margin-bottom:8px"><div>129S2/SvPasCrl</div><div>suggested: RRID:IMSR_CRL:287)</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 construction: 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">The SARS-CoV-2 S ‘2P’ ectodomain trimer (GenBank: YP_009724390.1, BEI NR-52420) was synthesized by GenScript into pCMV with an N-terminal mu-phosphatase signal peptide and a C-terminal TEV cleavage site (GSGRENLYPQG)</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 RBD-16GS-I53-50A fusion was cloned into pCMV/R using the Xba1 and AvrII restriction sites and Gibson assembly25.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV/R</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus Neutralization Assay for BALB/c mRNA-1273 samples: Codon-optimized full-length spike genes (Wuhan-Hu-1 with G614, Beta, or Gamma) were cloned into 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><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">Animals were housed and maintained at the New Iberia Research Center (NIRC) of the University of Louisiana at Lafayette, an AAALAC International accredited institution, in accordance with the rules and regulations of the Guide for the Care and Use of Laboratory Animal Resources.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NIRC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Relative luciferase units were plotted and normalized in Prism (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">Antibody-dose response curves were analyzed using non-linear regression analysis (with a variable slope) (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: 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 10. 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 for 10.1101/2022.02.07.22270617: (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: Study population, study design and recruitment: 51 SARS-CoV-2 naïve healthy volunteers (Supplementary Table 1) were recruited for the study under informed consent.<br>IRB: This study was approved (IRB No 2021-0898) by the Institutional Review Board (IRB) of Asan medical center in Korea.<br>Field Sample Permit: All methods were carried out in accordance with relevant guidelines and regulations.</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">COVID-19 serology and ACE2-neutralization assay: A multiplexed solid-phase chemiluminescence assay (Meso Scale Discovery, MD) was evaluated for the detection of IgG binding to various SARS-CoV-2–derived antigens (V-PLEX SARS-CoV-2 Panel 17 (IgG) Kit, K15524U, and Panel 23 (IgG) Kit, K15567U) and the quantification of antibody-induced ACE-2 binding inhibition to various variants’ spike antigens (pseudo-neutralization assay) (V-PLEX SARS-CoV-2 Panel 18 (ACE2) Kit, K15535U, and Panel 23 (ACE2) Kit, K15570U).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>V-PLEX SARS-CoV-2 Panel 17 (IgG) Kit, K15524U,</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Panel 23 (IgG) Kit, K15567U</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were coated with the specific antigen on spots in the 96 well plate and the bound antibodies in the samples were then detected by anti-human IgG antibodies or ACE2 conjugated with the MSD SULPHO-TAG which is then read on the MSD instrument which measures the light emitted from the tag.</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><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 raw data were subjected to QC analyses using the FastQC tool (version 0.11.9) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FastQC</div><div>suggested: (FastQC, RRID:SCR_014583)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">mRNA-seq read quality control was done using Trimmomatic37 (version 0.36) and STAR RNA-seq38 (version STAR 2.5.4a) using 150 bp paired-end mode was used to align the reads (hg19).</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">HTSeq39 (version 0.9.1) was to retrieve the raw counts and subsequently, Bioconductor package DESeq240 in R (https://www.R-project.org/) was used to normalize the counts across samples and perform differential expression gene analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Bioconductor</div><div>suggested: (Bioconductor, RRID:SCR_006442)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Differential expression gene (DEG) identification used Bioconductor package DESeq2 in R.</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">P-values of cytokines were calculated using two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli on GraphPad Prism software (version 9.0.0).</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:
Limitations of the study: There are several limitations to this study. First, our study population was limited to a specific geographic area (South Korea) and a specific genetic population. Second, most study subjects were healthy females of normal weight. Third, the homologous ChAd-ChAd cohort was smaller than the heterologous ChAd-BNT cohort. Fourth, our study did not investigate neutralizing antibodies using live or pseudo-SARS-CoV-2 virus and variants.
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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I did a spike to come up with a PoC for introducing this into the codebase of a product that I'm working on (matteeyah/respondo#225) by monkey-patching ActiveRecord with delegated types. It's amazing how can a small code change in ActiveRecord facilitate a big change in the domain model.
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SciScore for 10.1101/2022.02.06.479332: (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">Briefly, 96 well plates were coated with His-Tag capture antibody and incubated at 4°C overnight then blocked with 1% BSA in PBS for 1 hour at 37°C.</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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Serial dilutions of the selected antibodies were loaded in duplicates and incubated for 1 hour at room temperature, followed by the addition of biotinylated human 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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bead-based multiplex assay method for SARS-CoV-2 spike protein binding: A custom assay was designed using Magplex-Avidin microspheres (Luminex), which were coated in anti-HIS antibody [Biotin] (GenScript A00613, mouse IgG1k clone 6G2A9) at 2.5 μg/mL, washed, and then each microsphere was coated with 2.5 μg/mL of either SARS-CoV-1 S1-HIS, MERS S1-HIS, and SARS-CoV-2 S1-HIS or SARS-CoV-2 RBD-HIS variant proteins (Sino Biological) to create bead stocks.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-HIS</div><div>suggested: (GenScript Cat# A00613, RRID:AB_915572)</div></div><div style="margin-bottom:8px"><div>mouse IgG1k</div><div>suggested: (GenScript Cat# A00186, RRID:AB_914704)</div></div><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>S1-HIS</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies were incubated with microspheres for 30 minutes at RT with shaking at 300 rpm, washed 3x with PBS, then incubated with Goat anti-Human IgG Fc Secondary Antibody PE, eBioscience (minimal cross-reactivity to bovine/horse/mouse serum proteins)(Thermo Fisher Scientific 12-4998-82) diluted 1:200 in PBS/1% BSAfor 30 minutes.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Human IgG</div><div>suggested: (Thermo Fisher Scientific Cat# 12-4998-82, RRID:AB_465926)</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 recommended amount of particles/well was incubated in DMEM + 10% FBS with varying amounts of serially-diluted antibody for 1 hour at 37°C, and then 20,000 ACE2-HEK293T cells (Integral Molecular, see Extended Table 5) were added.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2-HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mutant library was arrayed in 384-well microplates and transiently transfected into HEK-293T cells.</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">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">Results were analyzed in GraphPad PRISM Version 9.3.0.</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><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Structural analysis: The figures were rendered using PyMOL Version 2.4.1 (The PyMOL Molecular GraphicsSystem; http://www.pymol.Org).</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">Multiple alignment of related lineage B betacoronavirus spike protein sequences: Multiple alignment was made using Clustal Omega (Madeira, 2019) and image rendered using Geneious software (Kearse, 2012).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Clustal Omega</div><div>suggested: (Clustal Omega, RRID:SCR_001591)</div></div><div style="margin-bottom:8px"><div>Geneious</div><div>suggested: (Geneious, RRID:SCR_010519)</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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You may want to jump straight to the Examples section if formal stuff annoys you.
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hypothes.is hypothes.is
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user: roberthambly Narrow your search: user: search by username tag: search for annotations with a tag url: search by URLfor domain level search add trailing /* eg. example.com/* group: show annotations associated with a group roberthambly Groups ▾ Groups Create new group ▾ Settings Account details Edit profile Notifications Developer Sign out [{"tag": "getting started", "count": 2}] [] roberthambly More info 3 Matching Annotations Last 7 days hypothes.is hypothes.is Hypothesis 3 roberthambly 03 Feb 2022 in Public Getting started Now you have the extension up and running. It's time to start annotating some documents. Create an account using the sidebar on the right of the screen. Pin the Hypothesis extension in Chrome (1 and 2), then activate the sidebar by clicking the button in the location bar (3). Three steps to get started getting started roberthambly 03 Feb 2022 in Public Annotation Types There are a few types of annotations that can be created with the application: Notes Create a note by selecting some text and clicking the button Highlights Highlights can be created by clicking the button. Try it on this sentence. Replies You can reply to any annotation by using the reply action on every car Annotation Types, getting started roberthambly 03 Feb 2022 in Public Privacy Annotations are either public and visible to everyone or private and visible only to you. Public These annotations are visible to everyone both in the document itself and our public stream. Private Private annotations are visible only to you when logged in. Hypothes.is getting started, privacy Visit annotations in context Tags getting started Annotators roberthambly URL hypothes.is/welcome/9adb709b3ce8f461 Collapse view
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Tagging is very useful but the lack of control and meaning breaks things down fast. “a (controlled term) is worth a thousand tags” as one article put it. Part of my pitch here is to say that i8n organizing your PKB you should favor controlled vocabularies and favor classification systems when you can.
One thing I find interesting here is how intimidating I find it to consider a proper controlled term / hierarchical system. Maybe the improvements here need to be around UX to curate tags toward proper controlled vocabularies -- do many PKM systems have the concept of a tag redirect, such that my "vampire" can be bucketed in with "vampires" (but unbucketed if necessary)?
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SciScore for 10.1101/2022.02.03.479007: (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">AlexaFluor-647-conjugated goat anti-human IgG (H+L) Ab (Invitrogen) and AlexaFluor-conjugated donkey anti-goat IgG (H+L) Ab (Invitrogen) was used as secondary antibodies.</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>AlexaFluor-conjugated donkey 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">An anti-mouse SARS-CoV-2 nucleocapsid protein (Clone 1C7, Bioss Antibodies)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse SARS-CoV-2 nucleocapsid protein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following extensive washing (3×) with PBS, an anti-mouse IgG HRP secondary antibody solution was formulated in PBS + 1% non-fat milk.</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><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 293T-ACE2 cell line was previously reported (Prevost et al., 2020)</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">Briefly, 293T cells were transfected by the calcium phosphate method with the lentiviral vector pNL4.3 R-E-Luc (NIH AIDS Reagent Program) and a plasmid encoding for SARS-CoV-2 Spike at a ratio of 5:4.</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">All virus-compounds supernatant was removed from wells without disrupting the Vero E6 monolayer.</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">Cell viability test: To measure the cytotoxicity of VE607 and its stereoisomers on 293T-ACE2 or Vero-E6 cells, a cell viability assay using CellTiter-Glo® One Solution Assay (Promega) was performed.</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">The codon-optimized RBD sequence (encoding residues 319-541) fused to a C-terminal hexahistidine tag was cloned into the pcDNA3.1(+) expression vector and was reported elsewhere (Beaudoin-Bussieres et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1(+)</div><div>suggested: RRID:Addgene_129020)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmid encoding the Delta (B.1.617.2) and Omicron (B.1.1.529) Spikes were generated by overlapping PCR using a codon-optimized wild-type SARS-CoV-2 Spike gene (GeneArt, ThermoFisher) that was synthesized (Biobasic) and cloned in pCAGGS as a template (Chatterjee et al., 2021; Gong et al., 2021b; Tauzin et al., 2022).</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">The vesicular stomatitis virus G (VSV-G)-encoding plasmid (pSVCMV-IN-VSV-G) was previously described (Emi et al., 1991).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VSV-G)-encoding</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pSVCMV-IN-VSV-G</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 transfected by the calcium phosphate method with the lentiviral vector pNL4.3 R-E-Luc (NIH AIDS Reagent Program) and a plasmid encoding for SARS-CoV-2 Spike at a ratio of 5:4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pNL4.3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell surface staining and flow cytometry analysis: Using the standard calcium phosphate method, 10 μg of Spike expressor and 2.5 μg of a green fluorescent protein (GFP) expressor (pIRES-GFP) were transfected into 2 × 106 293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pIRES-GFP</div><div>suggested: RRID:Addgene_78264)</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">VE607 compound was structurally preprocessed using LigPrep (Schrödinger, 2020) to generate multiple states for stereoisomers, tautomers, ring conformations, and protonation states at a selected pH range.</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">Samples were acquired on a LSRII cytometer (BD Biosciences, Mississauga, ON, Canada) and data analysis was performed using FlowJo vX.</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">Densitometry data were acquired with a Typhoon Trio Variable Mode Imager (Amersham Biosciences) in storage phosphor acquisition mode and analyzed using ImageQuant 5.2 (Molecular Dynamics).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageQuant</div><div>suggested: (ImageQuant, RRID:SCR_014246)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quantification and statistical analysis: Statistics were analyzed using GraphPad Prism version 8.0.2 (GraphPad, 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></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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Author Response:
Reviewer #2 (Public Review):
This work aimed to advance knowledge of the roles of polycystin-1 and polycystin-2 (PC-1, PC-2) in the vascular endothelium. For this, the authors developed tamoxifen-inducible Cre-lox models to delete PC-1, PC-2 or both specifically in endothelial cells of mice. Evidence is presented that flow or sheer stress activates PC-1-dependent current in endothelial cells, which is associated with NOS and KCa channel activation, smooth muscle hyperpolarization, and flow-dependent vasodilation. The Jaggar laboratory has recently reported that deletion of endothelial PC-2, a member of the TRP family, leads to loss of flow-induced Ca2+ influx, NOS and SK/IK activation, reduced vasodilation, and higher blood pressure. Thus, the novelty of the current work is the finding that PC-1 is similarly critical for activation of this pathway by flow, and that it is a physical interaction between membrane-localized PC-1 and PC-2 that underlies complex activation by flow.
Strengths of the current study include the use of powerful inducible knockout models in combination with a wide array of in vivo and ex vivo methods to test hypotheses. Thus, conclusions are based on multiple approaches and are mostly well supported. However, there are some concerns, specifically related to a lack of clarity on the interactions and purported interdependence between PC-1 and PC-2 that warrant further consideration.
1.The prospective impact of the current study is based on the suggestion that interactions between PC-1 and PC-2 via coiled-coil domains are required for activation of inward current by flow. However, the authors did not show evidence, via fluorescence imaging or otherwise (e.g., coIP), that peptides generated to disrupt this interaction actually do so. Does treatment with the coiled-coil domain peptides cause a shift in the PC-1-to-PC-2 distance (using TIRF-SMLM as in Fig 5)?
We have performed new experiments and now show that scrambled peptides of either the PC-1 or PC-2 coiled-coil domains do not alter flow-activated I_Cat in endothelial cells (Figure 6E-G, Figure 6 – figure supplement 2). In contrast, peptides corresponding to the coiled-coil domains present in PC-1 or PC-2 similarly inhibit flow-activated cation currents in endothelial cells.
Multiple different domains in PC-1 and PC-2 physically interact to form the heteromeric complex. Several groups have demonstrated that PC-1 and PC-2 couple via their C-terminal coiled-coils (Qian et al, Nat. Genet. 1997; Zhu et al., PNAS 2011; Yu et al., PNAS 2009; Tsiokas et al., PNAS 1997). Recombinant PC-1 and PC-2 lacking coiled-coils also interact via N-terminal loops (Babich et al, JBC 2004; Feng et al., JBC 2008). The structure of a PC-1/PC-2 heterotetramer that lacked N- and Ctermini was resolved using cryo-EM and indicated that a region between TM6 and TM11 of PC-1 interdigitates with PC-2 (Su et al, Science 2018). As such, it is unlikely that that the coiled-coil domain peptides physically separate PC-1 and PC-2 subunits. Rather, these data suggest that coiled-coil domain coupling in PC-1 and PC-2 is required for flow to activate non-selective cation currents in endothelial cells. In response to your comment, we have expanded discussion of this point in the manuscript.
2.The use of immunoFRET to test for PC-1/PC-2 proximity is not ideal. At minimum, proper negative controls (e.g., use of cells from KO models) should be provided to demonstrate the specificity of this technique for PC-1/PC-2 interactions in endothelial cells.
We agree. As suggested, we have performed new experiments and now provide immunoFRET data for Pkd1 ecKO and Pkd2 ecKO endothelial cells (Figure 4B, C; Figure 4 - figure supplement 1A, B). These data show that N-FRET between PC-1 and PC-2 antibodies is extremely low in Pkd1 ecKO and Pkd2 ecKO endothelial cells.
3.The authors conclude that PC-1/PC-2 clusters in KO cells in SMLM experiments are likely due to non-specific antibody binding. While I agree with this, it raises a question as to the meaning of cluster size data. Considering that the approach relies on fluorophore-tagged antibodies, which cannot be assumed to be in 1:1 stoichiometry with proteins of interest, how relevant is cluster size?
This is an interesting question. All immunofluorescence techniques rely on the use of antibodies to tag proteins. We recognize that the size of clusters reflects the size of both the proteins and antibodies. We now include text in the Discussion stating that the size of the PC-1 and PC-2 clusters reported is the size of both the proteins and the antibodies.
4.Based on data shown in Figure 1, the authors conclude that there is a reduction in inward current with flow. Since the applied technique measures total current, couldn't this result also reflect an increase in outward current (e.g., K+) due to flow that depends on the presence of Ca2+? Also related to these data, the magnitude of initial flow-induced transient current was quite variable (~8 - ~45 pA). Was this due to differences in cell size? The authors should consider expressing data from current recordings in terms of density (pA/pF).
We agree that presenting data as current density is useful and now do so throughout the manuscript, including in figure 1. The results in figure 1 do reflect a flow-activated increase in K^+ current as this response is partially inhibited by apamin/tram-34 (Mackay et al. eLife 2020). We state that there is “a reduction in inward current” as the entire current range in these experiments is negative of 0 pA.
5.Endothelium-specific deletion of PC-1 increased blood pressure, implying that the proposed role for PC-1 is generally applicable to the resistance arterial network; yet here, only small mesenteric vessels were studied. Given the known heterogeneity in the regulation of vascular tone by sheer stress among different arterial beds, is the identified role of PC-1 observed outside of the mesenteric circulation?
We agree that the blood pressure phenotype in Pkd1 ecKO mice suggest that flowactivates PC-1 in endothelial cells of other vascular beds to induce vasodilation. We have now discussed this concept in the manuscript.
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SciScore for 10.1101/2022.02.01.478647: (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 PVDF membranes were incubated with primary antibodies against HA-Tag (C29F4) (3724, CST, USA) or GAPDH (30201ES20, Yeasen, China), then incubated with second antibodies against IgG (Yeasen, China)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HA-Tag</div><div>suggested: (Cell Signaling Technology Cat# 3724, RRID:AB_1549585)</div></div><div style="margin-bottom:8px"><div>GAPDH</div><div>suggested: None</div></div><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">Then, second antibodies FITC-AffiniPure Goat Anti-Rabbit IgG (33107ES60, Yeasen, China) and Cy3-AffiniPure Goat Anti-Mouse IgG (33208ES60, Yeasen, China)were used to incubate the cells at a 1:1,000 dilution.</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><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 treatment: Vero E6 and Raw 264.7 cells were grown in 90% DMEM basal medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C under 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Raw 264.7</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">It was propagated and titrated with Vero E6 cells, and its associated operations were performed in a biosafety level 3 (BSL-3) facility.</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">Determination of lysosome pH: Vero cells were transfected with Lyso-pHluorin (addgene, 70113) and 2-E plasmid.</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">Vector pET28a was used for protein purification; vector pcDNA5 and pcDNA3.1 were used for cell survival assay and cell imaging.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET28a</div><div>suggested: RRID:Addgene_114145)</div></div><div style="margin-bottom:8px"><div>pcDNA5</div><div>suggested: RRID:Addgene_49428)</div></div><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">Determination of lysosome pH: Vero cells were transfected with Lyso-pHluorin (addgene, 70113) and 2-E plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>2-E</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 processed using pClamp 10.2 software (Molecular Devices, US).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pClamp</div><div>suggested: (pClamp, RRID:SCR_011323)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The percentages of differently labeled cells were calculated by FlowJo 7.6.</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">Flow cytometric data were collected and quantified using CytoExpert 2.4 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CytoExpert</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These ten structures were employed to construct ten homology models of the mutant envelope protein using Modeller [A. Sali, T.L. Blundell, Comparative protein modelling by satisfaction of spatial restraints, J Mol Biol 234 (1993) 779-815. 10.1006/jmbi.1993.1626.].</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">Statistics were performed in 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: 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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web.hypothes.is web.hypothes.is
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urn:x-pdf:a08d875ad57045edf70d283087a0e339 (PDF fingerprint)
To find this urn address for your .pdf files and their fingerprints (particularly local/private files not otherwise hosted in the cloud):
- Visit https://jonudell.info/h/facet/
- Search for your Hypothes.is username (and perhaps a tag) to find the document name and annotations you made on it.
- You should be able to click on the document title which will take you to a non-loading web page with an address that looks something like this:
urn:x-pdf:1ab23cd45e678fgh9012i34j56k78l90
If others know of alternate/faster methods of finding URIs for local pdf files I'd be happy to hear about them.
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forum.obsidian.md forum.obsidian.md
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pupka12Dec '21Does anyone know a way to do this with my annotations on locally stored pdfs? That would be lovely. I know I could probably move them to some cloud, but I have no idea which one could work (the most popular ones seem not to)
If you've annotated local (private) files within your browser using Hypothes.is, you'll need to find the uri path (a rough equivalent to http address for web pages) for your .pdf file with its "fingerprint" (a long unique number).
To do this 1. Visit https://jonudell.info/h/facet/ 2. Search for your Hypothes.is username (and perhaps a tag) to find the document name and annotations you made on it. 3. You should be able to click on the document title which will take you to a non-loading web page with an address that looks something like this:
urn:x-pdf:1ab23cd45e678fgh9012i34j56k78l904. Copy and paste that address you get into Hypothesidian as the address for an appropriate option like "Retrieve my annotations for a web article or web pdf". 5. Hit enter/return. 6. Hypothesidian should return the appropriate annotations for that document.If others know of alternate/faster methods of finding URis for local pdf files I'd be happy to hear about them.
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hypothes.is hypothes.is
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Suppose the server were sending rel=canonical in a Link header for non-document resources (e.g. images like this one). Is the Hypothesis bookmarklet/extension thorough enough to deal with this?
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.01.31.478460: (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">Blots were incubated either with anti-His antibody (6x-His Tag Monoclonal Antibody (HIS.H8), Alexa Fluor 488; ThermoFisher Scientific-MA1-21315-A488; 1:5000) or with anti-Strep-tag mouse monoclonal antibody (anti-Strep-tag mouse monoclonal, C23.21; PROGEN-910STR; 1:5000) overnight at 4°C in dilution buffer (TBS-T containing 5% bovine serum albumin (BSA).</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-Strep-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">Briefly, HEK 293T cells were seeded onto 96-well white plates before 24 h of transfection.</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">For this, fragments BstXI-mNG-Mpro-Nter-auto-NLuc-XhoI and BstXI-mNG-Mpro-Nter-auto-L-NLuc-XhoI were synthesized (Integrated DNA Technologies, IDT; Iowa, USA) and inserted into pIDTSmart (Kan) vectors to generate the plasmid constructs pIDT-mNG-Mpro-Nter-auto-NLuc and pIDT-mNG-Mpro-Nter-auto-L-NLuc, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pIDT-mNG-Mpro-Nter-auto-NLuc</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pIDT-mNG-Mpro-Nter-auto-L-NLuc</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Restriction enzymes BstX-I and XhoI were used to excise the two DNA fragments of interests from entry clones pIDT-mNG-Mpro-Nter-auto-NLuc and pIDT-mNG-Mpro-Nter-auto-L-NLuc and ligated into similarly digested destination plasmid pmNeonGreen-DEVD-NLuc [Addgene: 98287]49 and further confirmed by Sanger sequencing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pmNeonGreen-DEVD-NLuc</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For bacterial expression and purification of the Mpro sensor, the mNG-Mpro-Nter-auto-NLuc plasmid construct was digested with HindIII and XhoI and the mNG-Mpro-Nter-auto-NLuc fragment was subcloned into similarly digested pET-28b(+) plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>mNG-Mpro-Nter-auto-NLuc</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pET-28b(+ )</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Live cell, BRET-based Mpro proteolytic cleavage activity assays: Live cell Mpro proteolytic cleavage activity assays were performed by co-transfecting HEK 293T cells with either the pmNG-Mpro-Nter-auto-NLuc or the pmNG-Mpro-Nter-auto-L-NLuc Mpro sensor plasmid constructs along with either pLVX-EF1alpha-SARS-CoV-2-nsp5-2xStrep-IRES-Puro (Mpro WT) (a gift from Nevan Krogan (Addgene plasmid # 141370; http://n2t.net/addgene:141370 ; RRID:Addgene_141370)85or pLVX-EF1alpha-SARS-CoV-2-nsp5-C145A-2xStrep-IRES-Puro (C145A mutant Mpro) plasmid (a gift from Nevan Krogan (Addgene plasmid # 141371 ; http://n2t.net/addgene:141371 ; RRID:Addgene_141371)85 in 96-well white flat bottom plates (Nunc; 136101).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pmNG-Mpro-Nter-auto-L-NLuc</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_141370)</div></div><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_141371)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For dose-response experiments, the filler plasmid (a pcDNA3.1-based plasmid) is also co-transfected.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-based</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Live cell Mpro proteolytic cleavage inhibitor assay: HEK 293T cells were co-transfected with either pmNG-Mpro-Nter-auto-NLuc or pmNG-Mpro-Nter-auto-L-NLuc plasmid along with either pLVX-EF1alpha-SARS-CoV-2-nsp5-2xStrep-IRES-Puro (Mpro WT) (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pmNG-Mpro-Nter-auto-NLuc</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Live cell, FlipGFP-based Mpro proteolytic assay: For live cell FlipGFP-based Mpro proteolytic activity assays, HEK 293T cells were seeded onto 24-well plates and co-transfected with the FlipGFP sensor plasmid (pcDNA3 FlipGFP(Mpro)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3</div><div>suggested: RRID:Addgene_15475)</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">Mpro N-terminal autocleavage sequence analysis: A total of 1984 sequences for the SARS-CoV-2 pp1a polyprotein available at the NCBI Virus database (https://www.ncbi.nlm.nih.gov/genome/viruses/) were downloaded and aligned using MAFFT server (https://mafft.cbrc.jp/alignment/server/)63, 64.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>https://www.ncbi.nlm.nih.gov/genome/viruses/</div><div>suggested: (NCBI Viral Genomes, RRID:SCR_013789)</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">Models were generated using MODELLER (10.1 release, Mar. 18, 2021)66.</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">The NAMD output structure was then used as an input for Gaussian accelerated molecular dynamics (GaMD) simulation utilizing the integrated GaMD module in NAMD and its default parameters73, 74 which included 2 ns of conventional molecular dynamics (cMD) equilibration run in GaMD, to collect potential statistics required for calculating the GaMD acceleration parameters, and another 50 ns equilibration run in GaMD after adding the boost potential74, 75, and finally GaMD production runs for 1000 ns.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NAMD</div><div>suggested: (NAMD, RRID:SCR_014894)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Restriction enzymes BstX-I and XhoI were used to excise the two DNA fragments of interests from entry clones pIDT-mNG-Mpro-Nter-auto-NLuc and pIDT-mNG-Mpro-Nter-auto-L-NLuc and ligated into similarly digested destination plasmid pmNeonGreen-DEVD-NLuc [Addgene: 98287]49 and further confirmed by Sanger sequencing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Addgene</div><div>suggested: (Addgene, RRID:SCR_002037)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The ImageJ macro script used for the analysis is provided in the Supporting Text.</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">Data analysis and figure preparation: GraphPad Prism (version 9 for macOS, GraphPad Software, La Jolla California USA; www.graphpad.com), in combination with Microsoft Excel, was used for data analysis and graph preparation.</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><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">Figures were assembled using Adobe Illustrator.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><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.
</footer>
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.02.01.478657: (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: Mice: BALB/c mice (Taconic Biosciences Inc.) 2-3 months age (female) were purchased and housed in the Emory University Division of Animal Resources (DAR) facility and used according to the University IACUC guidelines.</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">Mice: BALB/c mice (Taconic Biosciences Inc.) 2-3 months age (female) were purchased and housed in the Emory University Division of Animal Resources (DAR) facility and used according to the University IACUC guidelines.</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">Antibodies and proteins: Purified anti-mouse GM-CSF (clone MP1-22E9) and anti-mouse IL-12 (clone C17.8) were from BioXcell and used for affinity chromatography purification of GPI-RBD-GM-CSF fusion protein and GPI-IL-12, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse GM-CSF</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>GPI-IL-12</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-spike RBD antibody (clone MM57) obtained from Sino Biologicals (Cat#40592).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-spike RBD antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-spike RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FITC-conjugated goat secondary antibody against mouse IgG/IgM was purchased from BD Pharmingen (Cat# 555988).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>mouse IgG/IgM</div><div>suggested: (Novus Cat# NBP1-75215, RRID:AB_11005727)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Peroxidase (HRP)-conjugated goat anti-mouse IgG F(ab’)2 specific antibody was from ThermoFisher Scientific/Pierce (Cat#31436).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: (Thermo Fisher Scientific Cat# 31436, RRID:AB_228313)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HRP-conjugated donkey anti-human IgG antibody was obtained from Jackson Immunoresearch (Cat#709-036-098).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HRP-conjugated donkey anti-human IgG antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 709-036-098, RRID:AB_2340497)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression of both the S1 RBD and GM-CSF on the surface of transfected CHO-S cells was confirmed by flow cytometry using fluorophore conjugated antibodies against RBD, Clone MM57 (Sino Biologicals) and GM-CSF, Clone MP1-22E9 (BioLegend).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GM-CSF</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After transfer, the membranes with the proteins were blocked for 1 hr at room temperature with 5 % milk in phosphate buffered saline and 0.2 % Tween 20 (PBS-T) and incubated with a primary antibody (anti-RBD, anti-mouse GM-CSF or anti-mouse IL-12) overnight at 4 °C in PBS-T with on a shaker at low speed.</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">Next day, membranes were washed three times with PBS-T and then incubated with appropriate secondary antibody conjugated with alkaline phosphatase that provides a visual color change upon addition of the chromogenic substrate (mixture of BCIP (5-bromo-4chloro-3-indolyl phosphate-catalog# 34040) and NBT (nitro-blue tetrazolium chloride, catalog# 34035 from Thermo Scientific)).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>5-bromo-4chloro-3-indolyl phosphate-catalog# 34040</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Resulting incorporation was detected by western blot and flow cytometry analysis using anti-RBD mAb, clone MM57 (Sino Biologicals), anti-mouse GM-CSF (clone MP1-22E9, BioLegend), and anti-mouse IL-12 (clone C17.8, Invitrogen) antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IL-12</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Enzyme-linked immunosorbent assay (ELISA): SARS-CoV-2 S protein RBD specific antibodies of different subtypes (IgG, IgG1, IgG2a) were determined in sera by enzyme-linked immunosorbent assay (ELISA) using an approach similar to one previously described using PR8 or WSN as targets (44, 45).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG1, IgG2a</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 and diluted secondary antibody (HRP-conjugated) against mouse total IgG or immunoglobulin isotypes (IgG1, IgG2a) were added and incubated for 30 minutes at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>mouse total 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 influenza antigen-specific antibody levels in immune sera, inactivated A/PR8 H1N1 virus (200 ng/well) was coated onto ELISA plates, followed by addition of diluted immune sera.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antigen-specific</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IgG isotypes were measured using goat anti-mouse immunoglobulin (Ig) G, IgG1 and IgG2a, and horse-radish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotechnology).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse immunoglobulin (Ig) G, IgG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plaque Reduction Neutralization Test (PRNT): The titers of anti-SARS-CoV-2 neutralizing antibodies were measured in the serum of BALB/c mice using PRNT assay as described previously (41).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 neutralizing</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">For mouse sera, CHO-S cells were incubated with diluted serum samples (100-100,000 times diluted in FACS buffer).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO-S</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 mixture was then added to Vero 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</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">Mice: BALB/c mice (Taconic Biosciences Inc.) 2-3 months age (female) were purchased and housed in the Emory University Division of Animal Resources (DAR) facility and used according to the University IACUC guidelines.</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">This construct was cloned into the pCHO 1.0 vector using the AvrII and BstZ17l sites (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCHO 1.0</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">Peroxidase (HRP)-conjugated goat anti-mouse IgG F(ab’)2 specific antibody was from ThermoFisher Scientific/Pierce (Cat#31436).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ThermoFisher Scientific/Pierce</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After incubation with secondary antibody, cells were washed with FACS buffer and resuspended in FACS buffer and acquired in a FACSCalibur (BD Biosciences) flow cytometer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FACSCalibur</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data analyzed using FlowJo software (FlowJo LLC).</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">The FRNT-mNG50 titers were interpolated using a 4-parameter nonlinear regression in GraphPad Prism 8.4.3.</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: 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 limitation of the current study is that a comparative analysis of GPI-RBD-GM-CSF and GPI-RBD was not carried out to demonstrate the contribution of GM-CSF as an adjuvant in VLP vaccine. Attempts to make GPI-RBD in CHO cells were not successful. Further, our study did not investigate whether incorporating VLPs with different amounts of GPI-anchored fusion protein results in stronger immune response to SARS-CoV-2. However, the comparison of antibody response induced by purified GPI-RBD-GM-CSF molecule with RBD-His-Tag suggests that GM-CSF stimulated antibody production against RBD. Further, our study focused only on the RBD domain of SARS-CoV-2 S protein but not the full-length S protein which may limit the breadth of protective immune response. In summary, our results demonstrate that influenza VLP-based delivery of SARS-CoV-2 RBD protein in combination with cytokine adjuvants can be used as a platform to develop multivalent vaccines targeting the variant strains of viruses which are currently observed in ongoing SARS-CoV-2 pandemic. Our fusion protein vaccine design also allows for creation of fusion proteins with new variant sequences and quickly purify using anti-GM-CSF mAb affinity chromatography. Further, use of immobilized cytokines as adjuvants will provide a safer way to induce anti-viral immunity with minimal side-effects.
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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Author Response:
Reviewer #1 (Public Review):
This manuscript is a follow-up of an earlier manuscript using the LRET technology, but extends the study by identifying a new "open" state and using experimental distance constraints to provide molecular models of the different states. All in all, the manuscript is well written, the experiments are described in sufficient details and experiments are done to high quality with the appropriate controls. The data corroborate the partially open state as published early, but extend the study to a second, open state. It is very good to see that the observed states are not only present in the catalytic head but the authors also use the full-length protein and find similar states. However, in the present manuscript, I find the conceptual advance with respect to the mechanism of MR somewhat limited. The authors curiously do not include any DNA in their structural studies, so the observed states are only relevant for the free MR complex, but not the complex "in action" bound to DNA where quite different conformations might occur. As one consequence, the structurally proposed states do not directly correlate with the functional nuclease states that are necessarily bound to DNA. Perhaps as a consequence, in the author's model, Rad50 is merely a gate-keeper for Mre11, but this is not the case as recent structural work shows that Rad50 forms a joint DNA binding surface with Mre11. Likewise, biochemical studies are done with physiologically unclear/less relevant 3' exonuclease activity only, but not with the physiological important 5' endonuclease activity. In my opinion, it is important for a publication in a journal with the scope of eLife and addressed to a broad audience to provide structural analysis in the presence of DNA and validate the structures using the endonuclease activity.
We thank the reviewer for these comments.
Specific recommendations:
1) Instead of using the physiological unclear exo activity, I suggest to use the more relevant endonuclease activity to validate the mutants.
We now include plate- and gel-based endonuclease activity assays, using a variety of DNA substrates, for all of the validation mutants. We have expanded Fig. 3 and included a new Supplemental Fig. S4 to show this data. We have expanded the Results section of the modified manuscript to present and discuss these findings.
2) Since the authors mutated one side of newly identified/proposed salt-bridges, I also suggest to test whether a charge reversal on both sides of the salt bridge rescues the phenoptype. I find this important because MR has quite many conformations, and mutating a single residue might not unambiguously validate the proposed conformation, a rescue by a charge reversed salt bridge is much stronger.
We thank the Reviewer for this suggested experiment, and we tried to do it. Although we were successful in generating each of the charge reversal mutations in full-length Rad50, all of the mutants unfortunately had issues with either expression or purification. For example, the 6x His-tag for several of the new Rad50 mutants was not accessible to the TEV protease for cleavage indicating that the mutated proteins were mis-folded (the His-tag of the WT full-length Rad50 is readily cleaved off by TEV). As such, we did not feel confident using these proteins in subsequent MR activity assays.
3) Since all LRET experiments are done without DNA, the authors do not capture relevant DNA processing states and comparison of structural (w/o DNA) and biochemical data (w/ DNA) is not really justified, in my opinion. Also, they might miss critical conformations. Is there a technical reason for not including DNA in the LRET studies?
We have collected LRET data on ATP-bound MRNBD in the presence of a hairpin DNA or a ssDNA as substrates. We still observe three states in the presence of both DNAs; however, the open conformation appears to be slightly more compact (i.e., closer distance between Rad50NBD protomers) in the presence of ssDNA. As described above, we have added to the Results section of the modified manuscript and included a new figure (Fig. 4) describing these data.
4) If the authors want to claim processive movement coupled to partially open/open state interchanges, they should provide experimental evidence. Where would the energy come from for such a movement, this is not clear from the model?
On the surface, ATP hydrolysis by Rad50 would seem to be the perfect source of energy for the conformational changes that drive the sequential and/or processive nuclease functions of the MR complex. However, the D313K mutant is not as good at ATP hydrolysis as the wild type enzyme (Fig. 3E), and the data in Fig. 3 and Supplemental Fig. S4 clearly demonstrate that D313K is by far the best nuclease. If the free energy for the movement does not come from ATP hydrolysis, where else could it come? Richardson and co-workers measured a release of -5.3 kcal mol-1 (-22.17 kJ mol-1) of free energy for the hydrolysis of a DNA phosphodiester bond (Dickson, K.S. et al. 2000 J. Biol. Chem. 275:15828–15831). Thus, the free energy released from the Mre11 nuclease activity could be the driving force for the conformational changes we propose. We have made this point in the Discussion of the revised manuscript.
5) The SAXS data for the "open" state do not validate the model, in my opinion. Experimental data and model are not inconsistent, but the curve looks to me as if the open state is perhaps much more flexible (i.e. an ensemble) or extended? Please comment.
We agree with the Reviewer on this point. We have updated Fig. 5A (original Fig. 4) to include the two-state fits to the experimental SAXS data. Although the multi-state fit to the apo MR SAXS data is better than any of the single model fits (2 = 1.05 vs. 1.26, respectively), the 2 is still larger than the multi-state fits to the ATP-bound MR SAXS data. Thus, an additional unobserved conformation (perhaps the so-called “extended”) might be present in solution for apo MRNBD. We have added a sentence to the revised manuscript with this point.
To explore the possibility that the previously described “extended” structure might be contributing to the SAXS data, we built a model of the extended conformation of Pf MRNBD based on the Tm MRNBD structure (PDB: 3QG5) and used Rosetta to connect the coiled-coils and add the linker to the Mre11 HLH. When this model was used in the FoXS calculations for the apo SAXS data, the 2 was 4.77 (versus 2 of 1.26 for the “open” model). The MultiFoXS two-state fit gave 90% open + 10% closed (2 of 1.04), whereas the three-state fit gave 65% open + 20% extended + 15% part open (2 of 0.84). Thus, there is some improvement when using the extended model, but since that model is not measurable in our LRET experiments and we are unsure of its validity as we have modeled it for Pf MR, we have chosen to omit it from the analysis.
6) Distance errors for the full complex are much smaller than those for the catalytic module only (Fig. 1d). Does that mean that the full complex is more rigid, please comment?
From looking at the data presented in Fig. 1D, it is logical to suggest that the full-length complex may be more rigid or better defined by the LRET data. However, we note that there are nearly as many distance errors which are similar between MRNBD and MR as there are MR errors less than MRNBD. And although many are not identical, most are of a similar magnitude. Because of this, we do not think the variations in LRET errors are systematic (i.e., related to a more rigid full-length complex).
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SciScore for 10.1101/2022.01.20.22269581: (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 review was not required for this review.</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">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">We searched the COVID-19 living evidence database [20], which uses automated workflow processes to: (1) provide daily updates of searches of four electronic databases (Medline, PubMed,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Medline</div><div>suggested: (MEDLINE, RRID:SCR_002185)</div></div><div style="margin-bottom:8px"><div>PubMed</div><div>suggested: (PubMed, RRID:SCR_004846)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, Ovid Embase, bioRxiv and medRxiv), using medical subject headings and free-text keywords for SARS-CoV-2 infection and COVID-19; (2) de-duplicate the records; (3) tag records that are preprints; and (4) allow searches of titles and abstracts using Boolean operators.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>bioRxiv</div><div>suggested: (bioRxiv, RRID:SCR_003933)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two authors independently assessed the risk of bias, using a customised online tool, which saved responses into the REDCap database.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>REDCap</div><div>suggested: (REDCap, RRID:SCR_003445)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We used the metaprop and metabin functions from the meta package (version 4.11-0) [26] and the ggplot2 package (version 3.3.5) in R (version 3.5.1) to display the study findings in forest plots and synthesise their results, where appropriate.</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 code and data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Strengths and weaknesses of the living systematic review methods: A strength of the methodology of this review is the transparent reporting, with openly available data and changes over different versions reported in the protocol. Our inclusion criteria attempted to reduce risks of bias and we developed a new tool to address potential biases in the studies included in this review. In contact investigations, we subtracted index cases from the total number of people with SARS-CoV-2 to avoid underestimation of the proportion asymptomatic [14]. We examined heterogeneity in detail and, as a result of the wide prediction interval, we chose not to report an overall summary estimate [19, 146]. A limitation of the methods for this living systematic review is that this update only includes published studies up to 2 February 2021. Although we made extensive efforts to comply with the planned 3-monthly updates, with weekly searches and a continuous process of screening, data extraction and risk of bias assessment, the pace of publications about SARS-CoV-2 exceeds the capacity of our crowd of reviewers [8, 20]. In reviews of observational epidemiological studies, search terms are broad so the number of studies that needs to be screened is high, but the yield of included studies is low. We are in the process of updating our findings and preliminary analyses show that the main findings do not change when including studies published up to April 2021. The four databases that we searched are no...
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 for 10.1101/2022.01.26.22269848: (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: Patients and samples: The study was approved by the Institutional Review Board of Washington University School of Medicine (Approval # 202104138).<br>Consent: Inclusion criteria included males and females over 18 years of age, health care provider-documented PAD syndrome including common variable immunodeficiency (CVID), specific antibody deficiency, or hypogammaglobulinemia, and the ability to give 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">Inclusion criteria included males and females over 18 years of age, health care provider-documented PAD syndrome including common variable immunodeficiency (CVID), specific antibody deficiency, or hypogammaglobulinemia, and the ability to give informed consent.</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">Exclusion criteria included participation in an investigational study of SARS-CoV-2 vaccines within the past year, history of HIV infection, an active cancer diagnosis, treatment with immunosuppressive medications, history of hematologic malignancy, treatment with anti-CD20 monoclonal antibody, receipt of live-attenuated vaccine within 30 days or any inactivated vaccine within 14 days of SARS-CoV-2 vaccination, blood or blood product donation within 30 days prior to study vaccination, and planned blood donation at any time during or 30 days after the duration of subject study participation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-CD20</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Of the 30 subjects, 9 were diagnosed with a prior SARS-CoV-2 infection with a positive nasal swab RT-PCR test, and one received treatment with an anti-SARS-CoV-2 monoclonal antibody (bamlanivimab) 90 days prior to study enrollment.</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">Unbound antibodies were washed away, and antigen-bound antibodies were detected by using a PE-coupled detection antibody for each subclass and isotype (IgG1, IgG2, IgG3, IgA1, and IgM; Southern Biotech), and FcγRs were fluorescently labeled with PE before addition to immune complexes (FcγR2a, FcγR2b, FcγR3a, FcγR2b; Duke Protein Production facility).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antigen-bound</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG2, IgG3</div><div>suggested: None</div></div><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">PE median fluorescent intensity (MFI) is reported as a readout for antigen-specific antibody titers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antigen-specific</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 and sequentially incubated with an oligoclonal pool of SARS2-2, SARS2-11, SARS2-16, SARS2-31, SARS2-38, SARS2-57, and SARS2-7148,49 anti-spike antibodies and HRP-conjugated goat anti-mouse IgG (Sigma) in PBS supplemented with 0.1% saponin and 0.1% BSA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS2-57</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>SARS2-7148,49</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-spike</div><div>suggested: (Sigma-Aldrich Cat# ZMS1076, RRID:AB_2893440)</div></div><div style="margin-bottom:8px"><div>anti-mouse 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">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><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">SARS-CoV-2 spike and RBD protein expression: Genes encoding SARS-CoV-2 spike protein (residues 1-1213, GenBank: MN908947.3) and RBD (residues 319-514) were cloned into a pCAGGS mammalian expression vector with a C-terminal hexahistidine tag.</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">Quantification and statistical analysis: Statistical significance was assigned using Prism Version 9 (GraphPad) when P < 0.05.</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:One limitation of our study is the heterogeneity in the PAD patient cohort, which included those with CVID, hypogammaglobulinemia, or specific antibody deficiency. Although this was a limitation of the cohort available for study, we did not observe substantive differences between the patient subgroups. Instead, the most significant differences in antibody response to mRNA vaccines were between patients that had or lacked a prior history of SARS-CoV-2 infection. Many of our PAD patients who historically had poor immune responses to bacterial and other protein antigens (e.g., Streptococcus pneumoniae polysaccharides, tetanus toxoid, and diphtheria toxin) as part of their initial immune workup (Table S3) responded to mRNA vaccines. The basis for this difference remains unclear, although it could be due to the unique adjuvant properties of the lipid nanoparticle or in vitro-synthesized mRNA35-37. In comparison, although numbers in our cohort were clearly small (n = 3), we detected little to no antibody response at 35, 60 or 90 days after immunization of COVID-naive PAD patients with the Ad26.COV2.S adenoviral-vectored vaccine (Fig S3). Because PAD is a heterogeneous clinical entity, with many of the genetic defects unknown7-11, certain classes of adjuvants or antigens may overcome specific deficiencies and promote B cell responses, albeit at lower levels than healthy counterparts. Our data suggest that the mRNA platform may have utility for vaccination of PAD patients. That said,...
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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pressbooks.online.ucf.edu pressbooks.online.ucf.edu
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Sankhya-Yog,
According to Bhagavad Gita Society, Sankhya-yog means "a philosophy established by sage Kapila. Essence of Sankhya yoga is removal of suffering by cultivating discrimination and by releasing the soul (purusha) from its entanglement in matter (Prakriti)".
Sankhya Yoga | Bhagavad Gita Society. bhagavadgitasociety.com/tag/sankhya-yoga/.
Samkhya Yoga." Samkhya or Sankhya means number. Yoga means union. Samkhya yoga means the union of numbers. The numbers are with regard to the number of realities (tattvas) that are present in existence. Samkhya Yoga deals with the union or the combination of a number of hidden realities, which manifest the existential reality. Those who are familiar with the Indian philosophies know that the Samkhya Philosophy is one of the six schools (Darshanas) of Hinduism. It is said to have been founded by Kapila and expounded by Isvara Krishna (6th Century AD) in his work, the Samkhya Karika.
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courses.lumenlearning.com courses.lumenlearning.com
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Within the
practice tag
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www.biorxiv.org www.biorxiv.org
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SciScore for 10.1101/2022.01.26.477937: (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: Crystallization, X-ray diffraction data collection, and processing: Crystallization was carried out by the sitting-drop vapor diffusion method at 20 °C.</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 serum dilution or antibody concentration causing a 50% reduction of RLU compared to control (ED50 or IC50, respectively) were reported as the neutralizing antibody titers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ED50</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IC50</div><div>suggested: (GeneTex Cat# GTX21096, RRID:AB_384608)</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 vector was transfected into Expi293 cells and incubated at 37°C for 4 days.</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">Lentiviral vector, pWPI-ffLuc-P2A-EGFP for luciferase reporter assay and pTRC2puro-ACE2-P2A-TMPRSS2 for the generation of 293T cell line susceptible to SARS-CoV-2 infection was created from pWPI-IRES-Puro-Ak-ACE2-TMPRSS2, a gift from Sonja Best (Addgene, no.154987) by In-Fusion® technology (Takara Bio).</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">Pseudovirus production and neutralization: Pseudoviruses bearing SARS-Cov2 S-glycoprotein and carrying a firefly luciferase (ffLuc) reporter gene were produced in LentiX-293T cells by transfecting with pWPI-ffLuc-P2A-EGFP, psPAX2, and either of S variant from Wuhan, D614G</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>LentiX-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus titers were measured by infecting 293T/TRCAT cells for 72 hours before measuring luciferase activity (ONE-Glo™ Luciferase Assay System, Promega, Madison, WI)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T/TRCAT</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization assay with authentic SARS-CoV-2 viruses: VeroE6/TMPRSS2 cells (African green monkey kidney-derived cells expressing human TMPRSS2, purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, JCRB1819) were maintained in DMEM containing 10% FBS and 1 mg/mL G418 at 37 °C in 5% CO2.</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><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 fragments were assembled into a linearized pcDNA vector using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the 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_66792)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pcDNA3 (Invitrogen) vectors containing an Ig light chain gene and the pcDNA4 (Invitrogen) vectors containing an Ig heavy chain gene were simultaneously transfected into Expi293 cells using Expi293 Expression System Kit (Thermo Fisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3</div><div>suggested: RRID:Addgene_15475)</div></div><div style="margin-bottom:8px"><div>pcDNA4</div><div>suggested: RRID:Addgene_45907)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A soluble version of the S protein (amino acids 1–1213), including the T4 foldon trimerization domain, a histidine tag, and a strep-tag, was cloned into the mammalian expression vector pCMV.</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">psPAX2 (Addgene, no.12260) was a gift from Didier Trono. pCDNA3.3_CoV2_B.1.1.7 (Addgene, no.170451) for Alpha-S and pcDNA3.3-SARS2-B.1.617.2 (Addgene, no.172320) for Delta-S proteins, were gifts from David Nemazee25.</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>pCDNA3.3_CoV2_B.1.1.7</div><div>suggested: RRID:Addgene_170451)</div></div><div style="margin-bottom:8px"><div>pcDNA3.3-SARS2-B.1.617.2</div><div>suggested: RRID:Addgene_172320)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentiviral vector, pWPI-ffLuc-P2A-EGFP for luciferase reporter assay and pTRC2puro-ACE2-P2A-TMPRSS2 for the generation of 293T cell line susceptible to SARS-CoV-2 infection was created from pWPI-IRES-Puro-Ak-ACE2-TMPRSS2, a gift from Sonja Best (Addgene, no.154987) by In-Fusion® technology (Takara Bio).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pWPI-IRES-Puro-Ak-ACE2-TMPRSS2</div><div>suggested: RRID:Addgene_154987)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pcDNA3.4 expression plasmids encoding SARS-CoV-2 S proteins with human codon optimization and 19 a.a deletion of C-terminus (C-del19) from Wuhan, D614G, and Omicron were generated by assembly of PCR products, annealed oligonucleotides, or artificial synthetic gene fragments (Integrated DNA Technologies, IDT) using In-Fusion® technology.</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">lentiviral vector VSV-G-pseudotyped lentivirus carrying ACE2 and TMPRSS2 genes were produced in LentiX-293T cells by transfecting with pTRC2puro-ACE2-P2A-TMPRSS2, psPAX2 (gag-pol), and pMD2G-VSV-G (envelope) using PEI-MAX (Polysciences)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTRC2puro-ACE2-P2A-TMPRSS2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pMD2G-VSV-G</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus production and neutralization: Pseudoviruses bearing SARS-Cov2 S-glycoprotein and carrying a firefly luciferase (ffLuc) reporter gene were produced in LentiX-293T cells by transfecting with pWPI-ffLuc-P2A-EGFP, psPAX2, and either of S variant from Wuhan, D614G</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pWPI-ffLuc-P2A-EGFP</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">ED50 or IC50 were calculated using a nonlinear regression curve fit (GraphPad Prism software Inc., La Jolla, CA)</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">Paired-end, 300 bp sequencing was performed using MiSeq (Illumina) with the MiSeq reagent kit v3 (Illumina, MS-102-3003).</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">Structure determination and analyses: Phase determinations were carried out by the molecular replacement method using the program Phaser in the PHENIX package32 and the program Molrep33 with the combination between RBD structure (PDB ID:7EAM) and Fab structures (PDB ID:7CHB and 7CHP) as search models.</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 figures of structures were generated by the program pymol (The PyMOL Molecular Graphics System, Version 1.2r3pre, 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></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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hypothes.is hypothes.is
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Name chosen as a nod to the use of Template:Should be SVG on Wikipedia
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www.medrxiv.org www.medrxiv.org
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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.
<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 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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www.biorxiv.org www.biorxiv.org
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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 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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www.biorxiv.org www.biorxiv.org
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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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www.biorxiv.org www.biorxiv.org
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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.
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www.biorxiv.org www.biorxiv.org
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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.
<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 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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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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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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hal.archives-ouvertes.fr hal.archives-ouvertes.fr
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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.).
Tags
Annotators
URL
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www.biorxiv.org www.biorxiv.org
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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.
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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.
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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.
<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 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.
<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 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.
<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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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.
Tags
Annotators
URL
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stackoverflow.com stackoverflow.com
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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.
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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?
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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....
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Local file Local file
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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.
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anagora.org anagora.org
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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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www.medrxiv.org www.medrxiv.org
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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.
<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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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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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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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.
<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 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.
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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.
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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
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support.google.com support.google.com
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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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support.mozilla.org support.mozilla.org
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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.
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sspai.com sspai.com
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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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www.biorxiv.org www.biorxiv.org
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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.
<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 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.
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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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
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www.medrxiv.org www.medrxiv.org
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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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www.biorxiv.org www.biorxiv.org
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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.
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www.medrxiv.org www.medrxiv.org
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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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kit.svelte.dev kit.svelte.dev
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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?
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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.
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www.biorxiv.org www.biorxiv.org
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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.
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www.biorxiv.org www.biorxiv.org
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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.
<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>
-
-
www.biorxiv.org www.biorxiv.org
-
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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web.hypothes.is web.hypothes.is
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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.
"Export" annotations
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www.balisage.net www.balisage.net
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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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www.biorxiv.org www.biorxiv.org
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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.
<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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www.biorxiv.org www.biorxiv.org
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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.
<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 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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www.biorxiv.org www.biorxiv.org
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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.
</footer>
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commoncrawl.org commoncrawl.org
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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...
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blog.readwise.io blog.readwise.io
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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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www.biorxiv.org www.biorxiv.org
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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.
<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 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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www.biorxiv.org www.biorxiv.org
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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.
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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.
<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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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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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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www.biorxiv.org www.biorxiv.org
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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.
<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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-
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www.biorxiv.org www.biorxiv.org
-
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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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.
<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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