Reviewer #1 (Public Review):

In this manuscript, authors found Halo tag become resistant to lysosome degradation upon ligand binding, using this unique property, they developed a highly sensitive assay to monitor the autophagy flux. Measuring autophagy flux is one of the most important assays for studying autophagy, there are a few widely used assays to monitor the autophagy flux, such as p62 degradation, and LC3 processing, however, each of them has its own limitation, which is well known in the field. In this regard, this assay provides a simple, straight forward and sensitive assay for measuring autophagy flux, which I personally think is very likely it will be widely used by the autophagy community. This is a well-controlled, rigorous study and the manuscript is clearly written.

Reviewer #2 (Public Review):

Yim et al have utilised the HaloTag system to generate tools and assays to measure autophagy flux. The assays are highly accessible and straight forward to conduct. The study does not have any major weaknesses, with all key conclusions strongly supported by clear data. A major strength of the study is the robustness of the assay and its ease of use across SDS-PAGE and imaging techniques that I expect will help with its uptake by the research community. The assay utilises the HaLo tag and its inherent stability within lysosomes once pulsed with a HaLo ligand. This enables analysis of autophagy flux over a set period of time. The approach is highly complementary to the recently published study by Rudinskiy et al (2022) MBoC, but also includes additional tools to measure different types of selective autophagy and bulk autophagy. The inclusion of limitations of their approach within the discussion will be very useful for researchers planning to use the assay in their work. Overall, this is an excellent study that has generated very valuable tools for the study of autophagy.

Reviewer #3 (Public Review):

Monitoring autophagy induction and flux in mammalian cells is challenging and depends largely on the mammalian ATG8 proteins (LC3 and GABARAP), typically tagged at the N-terminus with a small tag (HA, flag, myc) or a range of fluorescent tags. When autophagy is induced these ATG8 proteins get captured into autophagosomes and delivered to lysosomes for degradation. Monitoring flux by western blots relies on a molecular weight shift caused by lipidation, and quantification of loss of signal from degradation (analysis of initiation), or accumulation by the addition of inhibition of lysosomal inhibitors (analyses of flux). Fluorescent tags provide similar results but the measurements rely on counting degradation sensitive or resistant fluorescent signals. Image-based analysis is more challenging than western blot but both require significant optimization. In this manuscript these existing assays are modified by the use of a probe (Halo tag) again appended to the N-terminus of ATG8s which becomes resistant to lysosomal degradation after binding a ligand (TMR). The ligand can be pulsed-in to allow detection of acute induction of autophagy eliminating the background from basal accumulation. Generation of the Halo-TMR is then monitored by western blot or using an in gel-fluorescent assay. The authors present data which shows the adaptability of the system for imaging analysis, and for both quantitative analysis using western blot and imaging of selective autophagy or bulk, non-selective autophagy. The authors have developed a robust, useful alternative to existing assay and present the results in a careful, well described brief manuscript. These modifications are important for the field and for those who require quantitative results. The drawbacks are similar to existing assays and will usually require the generation of stable cell lines because over-expressed ATG8s can aggregate and confound the measurements.

SciScore for 10.1101/2022.05.13.491770: (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">4 colonies from each transformed plate were randomly picked and the insert was checked by performing colony PCR using nested PCR primers.</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 incubated with an anti-SARS-CoV spike primary antibody directly conjugated with alexaflour-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">Ten million PBMCs of select COVID-19 recovered donors were stained with RBD-Alexa Fluor 488 for 1 hour at 4°C, followed by washing with PBS containing 2% FBS (FACS buffer) and incubation with efluor780 Fixable Viability (Live Dead) dye (Life Technologies, #65-0865-14) and anti-human CD3, CD19, CD20, CD27, CD38 and IgD antibodies (BD Biosciences) for 30 minutes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human CD3</div><div>suggested: (RayBiotech Cat# CS-11-0105, RRID:AB_1227994)</div></div><div style="margin-bottom:8px"><div>CD19</div><div>suggested: (Agilent Cat# TC67401, RRID:AB_579635)</div></div><div style="margin-bottom:8px"><div>CD20</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD27 , CD38</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">mAb antibody binding was then detected with 50 μl/well of MSD SULFO-TAG anti-human IgG antibody (diluted 1:200) incubated for one hour at room temperature with shaking at 700rpm.</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, 100 pfu of SARS-CoV-2 (2019-nCoV/USA_WA1/2020), Alpha, Beta, Gamma, Delta and Omicron (BA.1 and BA.2) were used on Vero TMPRSS2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero TMPRSS2</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">IC50 titers were calculated by non-linear regression analysis using the 4PL sigmoidal dose curve equation on Prism 9 (Graphpad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad</div><div>suggested: (GraphPad, RRID:SCR_000306)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed using FlowJo software 10</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">CryoEM data analysis and model building: CryoEM movies were motion-corrected either in Motioncorr2 in Relion3.0 (30) or using Patch motion correction implemented in Cryosparc v3.3.1 (31).</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 combined Focused Map tool in Phenix was used to integrate high resolution locally refined maps into an overall map.</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">Glycans with visible density were modelled in Coot (36).</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">Model validation was performed using Molprobity (37).</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">Figures were prepared in ChimeraX(34) and PyMOL (39).</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>

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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.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

---

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2022.05.10.491349: (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">Protein expression of Nsp15 variants: WT Nsp15 was previously synthesized by Genscript (Piscataway, NJ), and contains an N-terminal His tag with thrombin and TEV cleavage sites in pET14b [28].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET14b</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 sequences of Nsp15 isolates were then aligned to that of the original Wuhan isolate (GenBank NC_045512.2) using the nucmer command from MUMmer 4.0 package [53] with default parameters.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MUMmer</div><div>suggested: (MUMmer, RRID:SCR_018171)</div></div></td></tr></table>

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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>

SciScore for 10.1101/2022.05.10.491295: (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: The study was carried out following a protocol 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: The study was carried out following a protocol 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.</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">Golden Syrian hamster infections and assessment of αREPs antiviral activity: Hamster infections: Thirty-two specific-pathogen-free (SPF) 8 weeks-old Golden Syrian hamsters (Mesocricetus auratus, males, provided by Janvier-Labs, Le Genest-Saint-Isle, France) housed under BSL-III conditions were kept according to the standards of French law for animal experimentation.</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 sections were then incubated overnight in PBS with 0.2% BSA and 0.05% Tween-20 with primary antibodies directed against SARS-CoV-2 Nucleocapsid Protein (1:500; mouse monoclonal, # ZMS1075, Merck);</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 Nucleocapsid Protein ( 1:500</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Fluorescence staining was performed using goat anti-rabbit Alexa-Fluor-488 (1:800; Molecular Probes – A32731; Invitrogen, Cergy Pontoise, France) and donkey anti-mouse Alexa-Fluor 555 (1:800; Molecular Probes – A32773; Invitrogen, Cergy Pontoise, France) secondary antibodies.</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# A32731, RRID:AB_2633280)</div></div><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: (Thermo Fisher Scientific Cat# A32773, RRID:AB_2762848)</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-293TT cells (106 cells per P6 well) were transfected with plasmids encoding GAG-POL, F-LUC and SARS-CoV-2 spikes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293TT</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral stocks were prepared by propagation in Vero E6 cells in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% (v/v</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">This mixture was added to Vero-E6 cells (CRL-1586, ATCC) seeded in a 96-well plate one day before.</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">To prepare the virus working stocks, a 25cm2 culture flask of confluent VeroE6 TMPRSS2 cells growing with MEM medium with 2.5% FCS was inoculated at a multiplicity of infection (MOI) of 0.001.</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">EC50 and EC90 determination: One day prior to infection, 5×104 VeroE6/TMPRSS2 cells per well were seeded in 100 µL assay medium (containing 2.5% FCS) in 96 well culture plates.</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">SARS CoV-2 Beta (SA lineage B 1.351) was isolated in France in 2021, The strain is available through EVA GLOBAL: UVE/SARS-CoV-2/2021/FR/1299-ex SA (lineage B 1.351) at https://www.european-virus-archive.com/virus/sars-cov-2-uvesars-cov-22021fr1299-ex-sa-lineage-b-1351. Sars-Cov-2 Gamma (SARS-CoV-2/2021/JP/TY7-503 lineage P.1, ex Brazil) was isolated in Japan in January 2021.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Sars-Cov-2 Gamma</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 the receptor binding domain (RBD) of the SARS-CoV-2 spike: The RBD (223 amino acids starting at position 319 of the spike sequence) coding sequence was cloned in frame behind a sequence encoding a signal peptide and in front of a His-tag coding sequence in the eukaryotic pYD11 expression plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pYD11</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">αReps expression and purification: The αRep genes encoding the RBD binders were subcloned in the bacterial expression vector pQE81 and resulting plasmids used for transforming Rosetta cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pQE81</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 sections were then incubated overnight in PBS with 0.2% BSA and 0.05% Tween-20 with primary antibodies directed against SARS-CoV-2 Nucleocapsid Protein (1:500; mouse monoclonal, # ZMS1075, Merck);</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Merck)</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Images were quantified using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2012) to threshold specific staining of SARS-CoV-2 in the dorso-medial area of the nasal cavity.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All data obtained were analyzed using GraphPad Prism 8 software (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, RRID:SCR_000306)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

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

Results from rtransparent:

---

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

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

---

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About SciScore

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SciScore for 10.1101/2022.05.09.491254: (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 MODELS AND SUBJECT DETAILS: MATERIALS AND METHODS: Human subjects: This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (Ref No. UW 21-452).<br>Consent: Written informed consent and questionnaire of vaccination and infection were obtained from these 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">Antigen-specific B cells: To characterize the SARS-CoV-2 Spike-specific B cells, PBMCs from each vaccinee were first stained with an antibody cocktail contained dead cell dye (Zombie aquae), CD3-Pacific Blue, CD14-Pacific Blue, CD56-Pacific Blue, CD19-BV785, IgD-BV605, IgG-PE, CD27-BV711, CD21-PE/Cy7, CD38-Percp/Cy5.5, CD11C-APC/Fire750 and His-tag Spike protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgD-BV605, IgG-PE</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD38-Percp/Cy5.5</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD11C-APC/Fire750</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>His-tag Spike protein.</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 with FACS buffer (PBS with 2% FBS) and further stained with the secondary antibodies including APC anti-His and DyLight 488 anti-his antibodies.</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">In brief, different SARS-CoV-2 pseudotyped viruses were generated through co-transfection of 293T cells with 2 plasmids, pSARS-CoV-2 S and pNL4-3Luc_Env_Vpr, carrying the optimized SARS-CoV-2 S gene and a human immunodeficiency virus type 1 backbone, respectively.</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 plasma-virus mixtures were then added into pre-seeded HEK293T-hACE2 cells.</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><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, different SARS-CoV-2 pseudotyped viruses were generated through co-transfection of 293T cells with 2 plasmids, pSARS-CoV-2 S and pNL4-3Luc_Env_Vpr, carrying the optimized SARS-CoV-2 S gene and a human immunodeficiency virus type 1 backbone, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><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">For intracellular staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) prior to staining with the mAbs against IFN-γ-PE, TNF-α-AF488 and IL-2-PE-Cy7.</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">Stained cells were acquired by FACSAriaIII Flow Cytometer (BD Biosciences) and analyzed with FlowJo software (v10.6) (BD Bioscience).</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">Correlation plots and heatmap visualizations: Correlograms plotting the Spearman rank correlation coefficient (r), between all parameter pairs were generated with the corrplot package (v0.84) (Wei and Sikmo, 2017) running under R (v3.6.1) in Rstudio (1.1.456).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Rstudio</div><div>suggested: (RStudio, RRID:SCR_000432)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Spearman rank two-tailed P values were calculated using corr.test (psych v1.8.12) and graphed (ggplot2 v3.1.1) based on *p<0.05, **p<0.01, ***p<0.001.</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><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Statistical analysis was performed using PRISM 8.0.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PRISM</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

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Limitations of the study: There are some limitations in our study. First, we were unable to obtain blood samples from our subjects after they became infected and quarantined. We, therefore, could not determine the B and T cell activation post BA.2 infection. Nevertheless, vaccine breakthrough infections often recall rapid NAbs and T responses against various VOCs, including Omicron (Collier et al., 2022; Suntronwong et al., 2022; Zhou et al., 2022). Second, most of our infected vaccinees were confirmed infection by self-RAT, thus the effect of different vaccine regimens on controlling viral loads was not determined. It remains necessary to compare the dynamics and magnitudes of recalled immune responses among vaccinees with BA.2 breakthrough infection patients in the future. In summary, we report that 3×BNT and 3×CorV provided better protection against SARS-COV-2 BA.2 than 2×BNT and 2×CorV. High frequencies of S-specific activated memory B cells and cross-reactive T cell responses induced by the third vaccination are critical for protection and illness reduction during the Omicron BA.2 breakthrough infection.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2022.05.10.491301: (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 experiments with mice, hamsters, and macaques were carried out in accordance with the Regulations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China.<br>IACUC: All animal procedures were approved by the Institutional Animal Care and Use Committee of the Institute of Medical Biology, Chinese Academy of Medical Science.</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 6- to 8-year-old male or female rhesus macaque experiments were performed in the animal biosafety level 4 (ABSL-4) facility at Wuhan Institute of Virology (WIV), Hubei, China.</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 following primary antibodies were used in this study: anti-SARS-CoV-2 (2019-nCoV) Spike Antibody (40589-T62, Sino Biological), and anti-GAPDH Antibody (60004, Proteintech).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-GAPDH</div><div>suggested: (Proteintech Cat# 60004-1-Ig, RRID:AB_2107436)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The secondary antibodies used were Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (111-035-003, Jackson ImmunoResearch), Peroxidase</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Rabbit IgG</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 111-035-003, RRID:AB_2313567)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For determination of S-specific antibody response, plates were incubated with goat anti-mouse IgG HRP (for mouse sera, Proteintech Cat: SA00001-1) or goat anti-Syrian hamster IgG HRP (for hamster sera, abcam Cat: ab6892) or goat anti-monkey IgG HRP (for NHP sera, Invitrogen Cat: PA1-84631) at 37°C for 1 hour and then substrate tetramethylbenzidine (TMB) solution (Invitrogen) was used to develop.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: (Proteintech Cat# SA00001-1, RRID:AB_2722565)</div></div><div style="margin-bottom:8px"><div>anti-Syrian hamster IgG</div><div>suggested: (Abcam Cat# ab6892, RRID:AB_955427)</div></div><div style="margin-bottom:8px"><div>anti-monkey IgG</div><div>suggested: (Thermo Fisher Scientific Cat# PA1-84631, RRID:AB_933605)</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 and antibodies: HEK 293F cells were grown in FreeStyle Media (Gibco-Thermo Fisher Scientific) and transiently transfected using polyethylenimine (PEI) (Polysciences, Inc.) in an 8% CO2 environment at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293F</div><div>suggested: RRID:CVCL_6642)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK 293A and Vero E6 cells were maintained in high glucose DMEM(GIBCO) supplemented with 10% FBS(GIBCO) and 1% penicillin/streptomycin (P/S) (GIBCO) in a 5% CO2 environment at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293A</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Virus titration: Virus titrations were performed by endpoint titration in Vero E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 1 hour of incubation, 100 μL mixtures were inoculated onto monolayer Vero cells in a 24- well plate for 1 hour with shaking every 15 minutes.</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">Animal vaccination and serum collection: Mice: For mouse vaccination, groups of 6- to 8-week-old female BALB/c mice or female K18-hACE2 Transgenic Mice were intramuscularly immunized with LNP vaccine candidates or a placebo in 50 μL, and 3 weeks later, a second dose was administered to boost the immune responses.</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">Cloning, expression, and preparation of the RQ3013 encoded Spike proteins: The gene encoding the RQ3013 was fused with a C-terminal twin Strep-tag (LEVLFQGPSGS WSHPQFEK GGGSGGGSGGSA WSHPQFEK) and cloned into a mammalian cell expression vector pcDNA3.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting plasmid, pcDNA3.1-RQ3013-Twinstrep, was transformed into HEK 293F cells using polyethylenimine (PEI) in FreeStyle Media (Gibco-Thermo Fisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-RQ3013-Twinstrep</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">Other procedures of cryo-EM data processing were performed within RELION v3.1 or CryoSPARC v3 using the dose-weighted micrographs23, 24.</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><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">Briefly, the initial templates were fit into the map using Chimera and Coot28, followed by a ten-cycle rigid body refinement using 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">Then, a combined manual refinement and real-space refinement were carried out for both prefusion state and postfusion state S structures in Coot and Phenix29.</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 dose-response curves were plotted from viral RNA copies versus the drug concentrations using GraphPad Prism 8.0 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 Analysis: All statistics data were performed and graphed using GraphPad Prism8.0.</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>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

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

---

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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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

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Reply to the reviewers

Manuscript number: RC-2021-01219

Corresponding author(s): Rajan, Akhila

1) General Statements [optional]

This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

The goal of this study is to:

• Define how prolonged exposure to a high-sugar diet (HSD) regime alters both the lipid landscape and feeding behavior.
• Determine how changes in lipid classes within the adipose tissue regulates feeding behavior. Key findings:

In this study, by taking an unbiased systems level and genetic approach, we reveal that phospholipid status of the fat tissue controls global satiety sensing.

Impact of Key findings:

By uncovering a critical role for adipose tissue phospholipid balance as a key regulator of organismal feeding, our work raises the possibility that the rate-limiting enzymes in phospholipid synthesis, including Pect, are potential targets for therapeutic interventions for obesity and feeding disorders.

Peer review comments:

This study has immensely benefited from the thoughtful peer-review of three reviewers. As per their recommendations, we have performed a major revision by performing additional experiments (see summary table below in next section) and strived to address the major concerns raised. Based on our reading, there were two major concerns that overlapped between all three reviewers raised. They are as follows:

• Does the genetic disruption of Pect in fly fat body alter phospholipid levels? Two reviewers (#2 and #3) recommended that we perform lipidomic analyses on adult flies with adipose tissue specific knockdown of For the revised version, we have completed this lipidomic experiment, and present results as a new main Figure 6, Supplemental S7 and S9.
• Is the dampened HSD induced hunger-driven feeding (HDF) behavior because of increased baseline feeding (#1 and #3)? In addition, reviewer #1, asked us whether HSD flies experience an energy-deficit? In other words, we were asked to uncouple whether what we observed was HSD-driven allostasis or indeed, as we had interpreted, that HSD dampened hunger-driven feeding response.

Hence, they recommended that we:

1. Re-analyze our hunger-driven feeding datasets and present non-normalized data (also requested by Reviewer #3) and show baseline feeding behavior on HSD. To address this, we have completed this analysis and present our results in Figure 1B-D and S1.
2. Determine whether the HSD fed flies display an energy deficit on starvation. To this end, we performed an assayed starvation-induced fat mobilization on HSD, results for this are now presented on Figure 1E-G and S2. Conclusions after the revision:

First, it is important to note here that the additional experiments have not caused a significant revision of the major conclusions of the original version of our study. In fact, we hope that the revised version provides clarity and further substantiation to our original arguments.

• The lipidomics experiments on Pect fat-specific knock-down flies show that reducing Pect in fat-body causes a significant reduction in certain PE lipid species (PE 36.2 specifically- Figure 6B). This is consistent with a prior report on lipidomics of the Pect null allele by Tom Clandinin’s group (PMID: 30737130). Furthermore, we note that when Pect is knocked down in the fat body, there is a significant increase in two other classes of phospholipids LPC and LPE (Figure 6A). Together, this suggests that an imbalance in phospholipid composition in the absence of Pect activity in fat.
• The starvation-induced fat mobilization experiments show that despite being fed a prolonged HSD, adult flies sense starvation and effectively mobilize fat stores, at a level comparable to Normal food (NF) fed adult flies, suggesting that even despite HSD exposure, adult flies experience an energy deficit on starvation.
• In our non-normalized data, we find that the baseline feeding events are not significantly altered between HSD and NF-fed flies (Figure 1D). This suggests that the effects we observe are not due to an increase in the “denominator”, but a dampening of hunger-driven feeding on HSD. With regard to our original version, all three peer-reviewers found that the study was interesting, significant, important, and novel – Reviewer #1: “The work is potentially novel and interesting”; #2 : “I find the study to be potentially very important - the authors combine a longitudinal study that would be difficult in any other model with the powerful genetic tools available in the fly. The conclusions are mostly convincing”; #3: “This manuscript demonstrates how fat body Pect levels affect HSD induced changes in hunger-driven feeding response. I agree with all the reviewers points; potentially very interesting”. But had requested that we provide further substantiation and clarification.

We sincerely hope that the peer-reviewers find that our revised version with additional new experimental datasets, improved data visualization, and the presentation of non-normalized raw data points, makes this study clear, compelling, and well-substantiated.

• Point-by-point description of the revisions This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

Below we summarize in Part A, the key experiments that were performed to address the major concerns. In Part B, we provide a point-point response to each reviewer with embedded datasets.

Part a:

We performed several new experiments, including:

• To address the primary concern of Reviewer #1 regarding whether the HSD flies have a similar energy deficit to Normal food (NF) fed flies, we performed analysis of stored neutral fat Triacylglycerol (TAG) reserves and how HSD fed flies mobilized fat stores on starvation. We present these results in Figure 1E-G, S2. These results show that HSD-flies despite accumulating more TAG (S2), breakdown a similar amount of fat reserves as NF-fed flies on starvation at any time-point (Figure 1E-G). This suggests that HSD-fed flies do sense and respond to energy deficit.
• To address concerns of reviewer #2 and #3 on whether Pect genetic manipulation affects specific phospholipid classes, we performed lipidomic analyses. The table below summarizes the new 3 new figures and 4 supplemental figures (blue text are all new figure numbers and figure panels) and three new Supplementary files as per reviewer’s request.

Figure #

Main point

New datasets in revision

Companion Supplement

1

HSD alters feeding behavior, but flies still breakdown TAG on starvation.

TAG storage and breakdown over longitudinal HSD shows that HSD and NF fed flies show similar levels of TAG breakdown on starvation, despite consistently elevated TAG on HSD. This supports the idea that flies do sense starvation even on HSD, but there is a uncoupling of the feeding behavior after Day 14. Revised the data representation of Figure 1 to show non-normalized data over time. S1 and S2 companions are new in the revision. Panels 1D to 1E are new for the revision.

S1- Raw data of feeding events plotted.

S2 Elevated TAG at all time points.

2

HSD causes insulin resistance

S3A added to show that insulin transcript levels remain the same in response to reviewer #3’s concerns.

S3

3

Phospholipid concentration raw data from lipidomic on Day 7 and Day 14 HSD suggest that PC, PE levels are increased on Day 14 HSD.

Figure 3 revamped to show new data visualization and non-normalized raw data to address Reviewer #2’s major concerns. S4A and S4B added. In addition Supplementary File 1 and 2 provided with raw lipidomics data as per reviewer #2’s request.

S4.

S4A- non normalized raw data of all other lipid classes on HSD.

S4B- fatty acid species data on Day 14 added as per request of rev.#2.

4

HSD regulate Apo-I levels in the IPCs and phenocopies Pect KD.

Added Figure 4A to show that HSD phenocopies Pect-KD in terms of delivery to brain

S5 showing the validation of the Apo-I antibody.

S6 validation of Pect KD and over-expression and Pect mRNA levels dysregulation on HSD.

5

Pect RNAi is insulin resistant

N/A

N/A

6

Pect knockdown shows significant increase in LPC and LPE, and a non-significant reduction in PC, PE levels. Specifically, the PE lipid class PE36.2 is downregulated.

Fig 6, S7, S9 are completely new based on reviewer #2 and #3 requests. In addition Supplementary File 3 provided with raw lipidomics data as per reviewer #2’s request

S7, S8, S9#.

S7- new Pect KD other classes

S8- new PE classes for day 14 and Pect associated classes.

S9- Pect OE lipidomics

7

Pisd and Pect activity in adipocytes are required for hunger-driven feeding behavior in normal diets

Pisd RNAi data was moved from supplement to main figure.

N/A

Note on revised text: We have revised text not only in the results section, but also as per reviewer #2’s recommendation, we have revamped our introduction and discussion as well. Since the manuscript has been significantly revised to include a main figure 6, fully altered Figure 1 and 3, multiple new supplemental figures, the changes in text are extensive. Hence, they are unmarked in the main text. Nonetheless, we hope that the reviewers will be able to evaluate these changes, as we have provided the specific locations in text and embed key figures in the point-point response below.

__Part B: __Point-Point responses to reviewer comments.

Reviewer #1 comments in Blue, author response in black.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

In this manuscript, Kelly et al. show that the difference between the feeding behavior of fed and starved flies (hunger-driven feeding; HDF) is absent in animals fed a high-sugar diet (HSD) for two weeks or more. The disappearance of HDF with HSD coincides with changes in phospholipid profiles caused by HSD. Furthermore, RNAi-mediated downregulation of Pect in the fat body-a key enzyme in the PE biosynthesis pathway-phenocopies physiological effects of HSD. Moreover, downregulation or overexpression in the fat body abolishes or induces HDF, respectively, abolishes or induces HDF, respectively, independent of HSD treatment.

Overall, the manuscript is well-written and the phenotypes are clear. However, I have major concerns regarding the authors' interpretation of the data and their conclusion. Most importantly, while it is clear that the authors' high-sugar dietary treatment affects feeding behavior and physiology, I am not convinced that the changes can be considered "hunger-driven"-which is central to the main point of the manuscript. Therefore, it is my recommendation that the authors substantially revise the manuscript by either showing additional/re-analyzed data that rule out alternative hypotheses, or rewriting the manuscript keeping alternative interpretations in mind.

We are thankful to this reviewer for their thoughtful critique, and constructive and specific suggestions on how we can redress these concerns. We have taken on board the concerns of this reviewer regarding our interpretation of whether the changes in feeding behavior can be considered hunger-driven or not. Based on their advice, we have made significant changes by addressing: i) does HSD increased baseline feeding- we now show non-normalized raw data and data supports conclusion that baseline feeding is not higher; ii) whether HSD- fed flies can sense an energy deficit at levels similar to NF fed flies- we show that HSD flies sense energy deficit. We have provided detailed response below, and we hope the reviewer finds the additional datasets and re-analyzed data are consistent with the interpretation that prolonged HSD dampens starvation induced feeding. In addition to this key concern this reviewer has made a many other salient points that we have addressed with additional data or by clarifying the text.

Major comments: 1) The data do not sufficiently show that the long-term HSD regime disrupts "hunger-sensing." The manuscript should address alternative hypotheses by showing raw instead of normalized data, rewriting the manuscript with a new central conclusion, or running additional experiments that actually show a defect in hunger-driven response. a. The main results that the authors rely on for the argument is that the ratio of feeding events that the starved and non-starved flies eat is different between the groups fed normal or HSD. However, because the authors only show normalized data (normalized to non-starved flies; Fig. 1), it is difficult to tell whether the change is due to a chronically increased feeding in non-starved HSD flies-maybe in perpetual hunger-like allostasis-or dampened starvation response. Indeed, the data shown in Fig S1 show that flies fed HSD for as short as 5 days show more frequent feeding events compared to age-matched controls fed normal food. It is possible that because the HSD-fed flies eat more than NF-fed flies, even without being starved, the ratio of starved/non-starved feeding is lower in the HSD-fed group-due to changes in the denominator, rather than the numerator.

We have taken onboard this concern regarding presenting only normalized data, and that clouded the interpretation and left open other possibilities. In the completely revised figure 1 and S1. We now show non-normalized data, as a function of time. First we note that HSD-fed flies, do not show higher baseline feeding that NF fed flies, except on Day 10 of HSD, when there is a modest but significant elevation (Figure 1D).

Nonetheless, on Day 10 HSD, flies still display increased hunger-driven feeding HDF (Figure 1C), it is only after Day 14 HSD that HSD dampens the starvation induced feeding.

1. It is also possible that the HSD-fed flies are simply not in as big an energy deficit physiologically, due to the increased fat deposits they've accumulated (as the authors show later in the manuscript). It may take longer for the fat HSD flies to reach substantial energy deficiency than the NF flies, but they still may eventually be able to appropriately respond to hunger, just like NF flies. In such case, it would be a misnomer to call this behavioral change a 'defect in hunger-driven feeding behavior.' Maybe an experiment with a dose-response curve of "hunger driven feeding response" as a function of duration of starvation would help? Prompted by this reviewers question, we asked whether HSD fed flies, that have a higher baseline neutral fat store (Triacylglycerol-TAG) level, and if HSD-fed flies can sense energy deficit. For this, we revisited the longitudinal assays for neutral fat triacylglycerol (TAG) storage that our lab had generated, along with the HSD-HDF studies. We now present this evidence as Figure 1E-1G and Figure S2. Overall, our experiments point to the idea that adult flies fed HSD, are able to sense and mobilize TAG stores effectively throughout the 28-day time point that we analysed.

First as shown in Figure S2, flies fed HSD display an increase in TAG levels. But it is to be noted that while TAG stores increase, the increase is not linear with time. This suggests that adult flies exposed to HSD store excess energy as TAG, but the increased TAG stores stay within a certain range despite the length of HSD exposure. This suggests that adult flies on HSD still display TAG homeostasis.

Next, to directly address the reviewers point about HSD fed flies not sensing an energy deficit, we subject HSD-fed flies to an overnight starvation, same regime as used in the overnight feeding experiments, and asked whether they mobilize TAG. We noted that flies exposed to HSD breakdown TAG throughout the 28-day exposure at statistically significant levels for Day 3- Day 28, except on 14 and 21 days (Figure 1F). While there is TAG mobilization on Day 14 and 21, the difference is not statistically significant. Nonetheless, we note the same levels TAG breakdown for normal lab food (NF) fed flies on Day 14 and 21 (Figure 1E). Overall, HSD fed flies sense and display energy deficit, as measured by TAG store mobilization, throughout the 28 days of HSD exposure, at levels comparable to NF-fed flies (Figure 1G).

Taken together, these results suggest that while HSD-fed flies experience an energy deficit on starvation, at levels comparable to NF-fed flies, throughout the 28-day time point assayed. But, their starvation driven feeding-response is dampened by Day 14 and by Day 28, the HSD-fed flies display more feeding events than HSD starved flies. These results are consistent with the interpretation that in HSD-fed flies the starvation-induced feeding behavior becomes desynchronized from the starvation induced TAG-mobilization, suggesting that there is an absence of hunger-driven feeding.

2) How can you be sure that lower Dilp5 immunofluorescence is indicative of increased Dilp5 secretion? Wouldn't decreased production of dilp5 also have the same results?

It has been shown previously in HSD fed larvae are hyperinsulinemic, i.e., they have 55% increase in circulating Dilp2 ( PMID: 22567167). Additionally, we have shown that ectopic activation of the insulin-producing neurons by expressing TRPA1, an ion channel that activates neurons, reduces Dilp5 accumulation without a change in Dilp5 mRNA levels (PMID: 32976758), suggesting that reduced Dilp5 accumulation, without alterations to mRNA levels is a proxy for increased secretion. Now, in response to this concern, in the revised manuscript, we have added qPCR data of Dilp2 and 5 (Figure S3A), which show no difference in expression levels after 14 days on HSD. Therefore, there is no dip in Dilp5 mRNA production. Given that Dilp2 and Dilp5 mRNA levels remain the same, but we see reduced Dilp5 accumulation, we interpret this to mean that Dilp5 secretion is increased.

1. Also, the authors should state in the main text that it is Dilp5, not just any Dilp. Thanks for this suggestion and we have fixed this and referred to Dilp5 specifically throughout the text in the results section.

3) Data presentation: a. Sometimes the data are normalized to NF (Fig 4B-C), sometimes not (ex. Fig 4A, S4C). Unless there is a specific rationale for the data transformation, it would be more appropriate to show untransformed data (ex. Fig 4A, S4C), especially as the authors use two-way ANOVA to determine significance. Only showing the differences implies comparison against a hypothetical mean (i.e. μ0=0), not between two group means.

We thank the reviewers for bringing this issue to our attention. We updated all the figures to show untransformed data in the revised manuscript.

1. Some figures show both individual data points and summary statistics (mean, SD, ... ex. Fig 2A)-which I believe is ideal-but some show only one or the other (ex. Fig 2B, no summary statistics; Fig. 3, no data points. The manuscript would read more convincing if data visualization is consistent across figures. We thank the reviewers for their feedback. We have made changes to all the figures in the revised manuscript to improve visual consistency.

Minor comments: 1) High sugar diet: what is the actual sugar concentration in the NF v. HSD diets? The authors write that the HSD diet contains "30% more sugar" than the NF, but providing the final sugar concentrations-sucrose or others-would be informative for other scientists studying the effect of high sugar diets.

We thank the reviewer for their suggestion and now we have updated the methods to include this sentence. “After 7 days, flies were either maintained on normal diet or moved to a high sugar diet (HSD), composed of the same composition as normal diet but with an additional 300g of sucrose per liter”.

1. Additionally, the definition of HSD is inconsistent. Main text (Page 5, line 17) states that their HSD is "60% more sugar than normal media," whereas the figure legend (Fig 1) and the Methods state that the HSD contains "30% more sugar." We apologize for this egregious typo in the figure legend! We have now fixed this to say 30% HSD. Only 30% HSD was used throughout this study.

2) Starvation medium: please provide justification for why the authors used 1% sucrose/agar for starvation medium, instead of plain agar/water that most labs use. At least clarify and provide a reference for the claim that the 1% sucrose/agar "is a minimal food media to elicit a starvation response."

We are very grateful for this reviewer identifying this this methods description error and bring it to our attention. We used 0% sucrose agar for overnight starvation in this study as most labs do. The error occurred because we were using another manuscript from the lab to help draft the methods section (PMID: 29017032). In that study, where we assayed the effect of chronic starvation our lab used: “1% sucrose agar for 5 days at 25C”. However, in this current study, because we are testing acute effects of overnight starvation, we are using 0% sucrose agar.

3) Pect mRNA level is higher with HSD. This is surprising because not only, as authors mention, is increased PC32.2 with HSD suggests lower Pect activity, but also because Pect RNAi phenocopies long-term HSD in HDF behavior, lipid morphology, FOXO accumulation in fat body. The authors speculate that the data "likely shown an upregulation in an attempt to mediate the Pect dysregulation occurring at the protein level." If that were true, a western blot may be informative. Zhao and Wang (2020, PLoS Genetics) generated a Pect antibody that seems compatible with western blot applications. That being said, I don't think such data is critical for the manuscript. I mention this simply as a suggestion for the authors. a. page 8, line 22-23, did you mean to write "Given how PC32.2 is elevated after 14 days of exposure to HSD, we assumed that Pect levels would be low for flies under HSD," not "high?" Otherwise the subsequent 2 sentences don't make sense.

We agree that the most confusing aspect of the study was that Pect mRNA levels being very high on Day 14 HSD, but nonetheless the effects of Pect-KD phenocopied HSD. To resolve this, we have now performed lipidomic analyses on whole adult flies, when Pect is knocked-down (KD) by RNAi in the fat tissue. We now present a new dataset in Figure 6. Two striking changes occur. They are:

1. Pect-KD shows increase in the phospholipid classes LPC and LPE (Figure 6A). In contrast, LPE is significantly downregulated on HSD Day 14 (Figure 3).
2. Pect-KD shows a significant reduction in specific class of PE 36.2 (Figure 6B). Our data regarding increase in PE 36.2 agree with a previous lipidomic analyses of Pect mutant retina (PMID: 30737130). In contrast, PE 36.2 trends upwards on 14 day HSD (Figure S7C) though not significantly. On 14-day HSD consistent with extreme upregulation of Pect mRNA fed flies (Figure S6A; Pect mRNA 200-250 fold), PE trends upwards on 14-day HSD (Figure 3) and PE 36.2 trends higher (Figure S7C). We note that on the surface of it PE and LPE per se are contrasting between 14-day HSD lipidome and fat-specifc Pect-KD. But there is a significant commonality that under both states there is an imbalance of phospholipids classes PE and LPE. Hence, we propose that maintaining the compositional balance of phospholipid classes PE and LPE is critical to hunger-driven feeding and insulin sensitivity. Hence, either increase or decrease, of these key phospholipid species, may lead to abnormal hunger-driven feeding.

We agree that a western blot would be informative as well, but we were unable to obtain the reagent from Dr. Wang’s group, precluding us from performing this request. See email snapshot.

To ensure that we appropriately discuss and clarify this issue, we have now included a section in the discussion - Page 14 Lines 26-34- under the subtitle “The implications of relationship between Pect levels and HSD”. We have pasted an excerpt from that subsection below for this reviewers assessment.

“Also, we note that over-expression of Pect cDNA in the fat-body does not alter phospholipid balance (Figure S9) and indeed improves HDF on HSD (Figure 7B). While this may appear inconsistent, it is critical to note that over-expression of Pect cDNA using UAS/Gal4 only increases Pect mRNA expression by 7-fold (Figure S6A), whereas HSD causes its upregulation by 250-fold (Figure S6B). Hence, we speculate that an increased ‘basal’ level of Pect such as by that provided by a cDNA over-expression in fat, may be protective to the negative effects of HSD (Figure 7B) without affecting overall phospholipid levels (Figure S9) , but extreme upregulation Pect on HSD affects the PE and LPE balance (Figure 3).”

Reviewer #1 (Significance (Required)):

The work is potentially novel and interesting, but at this stage it's difficult to interpret what the phenotype signifies. Although the manuscript could be revised simply by modifying the text, experimentally addressing the concerns would significantly improve the work.

In sum, we hope we have addressed the key concern for Reviewer #1 as to whether the behavior we report here is indeed a dampening of starvation-induced feeding, or an effect of increase in baseline feeding. We hope that by reviewing our non-normalized data, they can appreciate that it is the former. Also, we hope that Reviewer #1 appreciates that we have strived to address the concerns by additional experiments, to clarify our findings and improve the impact of the work.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

This intriguing manuscript by Kelly and colleagues uses the fruit fly Drosophila melanogaster as a model to understand how diet-induced obesity alters the feeding response over time. In particular, the authors findings indicate that chronic exposure to a high-sugar diet significantly alters the starvation-induced feeding response. These behavioral studies are complemented by a lipidomics approach that reveals how a chronic high sugar affects many lipid species, including phospholipids. The authors then pursue mechanistic studies that indicate phospholipid metabolism within the fat body appears to remotely affect insulin secretion from the insulin producing cells. Moreover, the changes in phospholipid abundance are associated with changes in insulin-signaling, including increased insulin secretion from the IPCs and elevated levels of FOXO within the nucleus.

I find the study to be potentially very important - the authors combine a longitudinal study that would be difficult in any other model with the powerful genetic tools available in the fly. The conclusions are mostly convincing, but a few follow-up experiments are required:

We are grateful for the reviewers constructive, detail-oriented, and balanced feedback, and their recognition of the value of this study. Now, we have performed additional experiments to address the key concerns raised by all reviewers. We hope that on reading the revised version of our study, that the reviewer continues to feel positive about the message of this study and its potential impact.

1. The key conclusions from the manuscript assume that manipulation of Pect expression levels alters phosphatidylethanolamine (PE) levels. However, the authors make no attempt to verify that the genetic experiments described herein actually affect PE levels. At a minimum, changes in PE levels should be verified for the Pect knockdown and overexpression lines. Similarly, there is no evidence that manipulation of either EAS or Pcyt2 induces the expected metabolic effects. I'm not asking that the longitudinal feeding experiments be repeated, simply that the authors measure the relevant lipid species, preferably with a targeted LC-MS approach.

Prompted by this reviewer, we performed targeted LC-MS on whole adult flies, on normal diet, to assess lipid levels for fat-specific Pect-KD and overexpression. We decided to focus on Pect, as its knock-down even on normal diet causes a dampened hunger-driven feeding behavior (Figure 7A) and phenocopied a 14-day HSD feeding phenotype.

We now present a new dataset in Figure 6. Two striking changes occur:

They are:

Pect-KD shows a significant reduction in specific class of PE 36.2 (Figure 6B). Our data regarding decrease in PE 36.2 agree with a previous lipidomic analyses of Pect mutant retina (PMID: 30737130). It is to be noted that though overall levels of all PE species trend downwards, like the Clandinin lab study on Pect (PMID: 30737130), we did not find a significant change in the overall PC and PE levels.

• Pect-KD shows increase in the phospholipid classes LPC and LPE (Figure 6A). In contrast, LPE is significantly downregulated on HSD Day 14 (Figure 3). On 14-day HSD consistent with extreme upregulation of Pect mRNA fed flies (Figure S6A; Pect mRNA 200-250 fold), PE trends upwards on 14-day HSD (Figure 3) and PE 36.2 trends higher (Figure S7C). We note that on the surface of it PE and LPE per se are contrasting between 14-day HSD lipidome and fat-specifc Pect-KD. But there is a significant commonality that under both states there is an imbalance of phospholipids classes PE and LPE. Hence, we propose that maintaining the compositional balance of phospholipid classes PE and LPE is critical to hunger-driven feeding and insulin sensitivity. Hence, either increase or decrease, of these key phospholipid species, may lead to abnormal hunger-driven feeding.

Finally, fat-specific Pect-OE did not cause significant changes to lipid species (Figure S9). This could either be due to the fact that in fat-specific Pect-OE flies under normal food and that we were assaying whole body lipid levels and not fat-specific lipid changes. But to counter that, even a 60% reduction in Pect mRNA levels (Figure S6A), was sufficient to produce an effect on whole body phospholipid balance (Figure 6). Hence, we speculate that by maintaining a basally higher (7-fold higher Pect mRNA level Figure S6A), might allow 14-day HSD-fed flies to buffer the negative effects of HSD and we predict that it might take longer to disrupt the phospholipid balance and HDF response.

We have now included a section in the discussion - Page 14 Lines 26-34- under the subtitle “The implications of relationship between Pect levels and HSD”. We have pasted an excerpt from that subsection below for this reviewers assessment.

“Also, we note that over-expression of Pect cDNA in the fat-body does not alter phospholipid balance (Figure S9) and indeed improves HDF on HSD (Figure 7B). While this may appear inconsistent, it is critical to note that over-expression of Pect cDNA using UAS/Gal4 only increases Pect mRNA expression by 7-fold (Figure S6A), whereas HSD causes its upregulation by 250-fold (Figure S6B). Hence, we speculate that an increased ‘basal’ level of Pect such as by that provided by a cDNA over-expression in fat, may be protective to the negative effects of HSD (Figure 7B) without affecting overall phospholipid levels (Figure S9), but extreme upregulation Pect on HSD affects the PE and LPE balance (Figure 3).”

A central hypothesis in the study is that the HSD over a period of 14 days results in insulin resistant and that these changes are leading to changes in hunger dependent feeding. I would encourage the authors to determine if Foxo mutants are resistant to these HSD-induced effects on HFD.

We thank the reviewers for this suggestion. However, given that dFOXO nuclear localization rather than expression levels regulate insulin sensitivity, we feel that disrupting dFOXO levels via mutation or knockdown will produce a plethora of indirect effects including developmental abnormalities (PMID: 24778227, PMID: 16179433, PMID: 29180716, PMID: 12893776). Our data suggest that chronic HSD treatment and Pect affect insulin sensitivity in fat tissue. However, we feel that investigating whether insulin sensitivity/FOXO signaling in fat tissue regulates feeding behavior is outside the scope of our work.

1. In lines 25-30, the authors draw the conclusion that an increase in unsaturated fatty acid species is associated with the HSD and that these changes results in a more fluid lipid environment. While I agree with the model, the manuscript contains no evidence to support such a model. Either test the hypothesis or move the last line of the section to the discussion.

We thank the reviewer for this important and insightful comment. We agree that the data we presented and discussed in the original version is at the moment speculative. Addressing the hypothesis that increase in unsaturated fatty acid species result in a more fluid lipid environment will require us to build tools and expertise. Hence, this hypothesis is better suited for exploration in a future study. Given this, we have moved this out of the results section into the Discussion section titled “HSD and fat-specific PECT-KD causes changes to phospholipid profile” (See excerpt below from page 13, lines 24-35).

“In addition to changes in phospholipid classes, we found that HSD caused an increase in the concentration of PE and PC species with double bonds (Figure S4C and S4D). Double bonds create kinks in the lipid bilayer, leading to increased lipid membrane fluidity which impacts vesicle budding, endocytosis, and molecular transport14,92. Hence it is possible that a mechanism by which HSD induces changes to signaling is by altering the membrane biophysical properties, such as by increased fluidity, which would have a significant impact on numerous biological processes including synaptic firing and inter-organ vesicle transport.”

Also, as per the reviewer’s guidance, given that we are speculating here, we have also shifted this dataset from Main figure 4 to supplement S4C and S4D.

In addition, lines 25-30 state that FFAs are increased after 14 days of a HSD. Figure 3A shows the exact opposite - FFAs are significantly decreased in 14 day fed animals despite being elevated in the 7 day fed animals. This is an interesting result that warrants discussion. Moreover, I would encourage to examine the lipidomic data more carefully to ensure that the text accurately portrays the lipid profiles.

We apologize for misstating that FFAs are decreased on 14-day HSD in the lines 25-30. It was an error and we have corrected this. We agree with the reviewer that the reduction of FFA on Day 14-HSD is an intriguing and unexpected observation that needs to be emphasized and further discussed. To this end, we have added figure S4B, wherein we have provided the difference in FFA concentration (by species) after days 7 and 14.

Furthermore, we have discussed what the potential meaning of reduced FFA at Day 14 implies in page 12, lines 19-27 of the Discussion section titled “HSD and fat-specific PECT-KD causes changes to phospholipid profile”. We have stated the following-

“We speculate that this reduction in FFA maybe due to their involvement in TAG biogenesis (PMID: 13843753). We were interested to see if the decrease in FFA correlated to a particular lipid species, as PE and PC are made from DAGs with specific fatty acid chains. However, further analysis of FFAs at the species level did not reveal any distinct patterns. The majority of FFA chains decreased in HSD, including 12.0, 16.0, 16.1, 18.0, 18.1, and 18.2 (Figure S4B). This data was more suggestive of a global decrease in FFA, likely being converted to TAG and DAG, rather than a specific fatty acid chain being depleted.”

The processed lipidomics data should also be included as supplementary data table so that they can be independently analyzed by the reader.

We thank the reviewer for this suggestion. As per the reviewers request, we have included the raw data as an attachment in our supplementary material (Supplementary Files 1-3.), so that interested readers can use the datasets generated in this study for future work and further analysis.

Beyond these experimental suggestions, the manuscript needs significant editing for clarity. While I won't provide a comprehensive list, the authors need to provide accurate descriptions and annotation of genotypes (including w[1118], which is written as W1118), typos, and formatting. I've listed a few examples below:

1. Page 3, Line 1 and 2: "...have been shown to impact feeding behavior and metabolism that leads to..." This is an awkward and grammatically incorrect sentence.
2. Page 3, Lines 7-32 is one very large paragraph but contains concepts that should be broken down over at least three paragraphs.
3. Page 3, Line 25: A description of the reaction catalyzed by Pect would be helpful for a manuscript focused on Pecte activity.
4. Page 4, Line 10: "previously characterized method of eliciting diet induced feeding behavior." As stated in the text, the method is previously described yet the manuscript characterizing the method isn't cited.
5. Figure legend 3 contains a random assortment of capitalized lipid species. Also, the names of lipid species are inappropriately broken into multiple names. Please use correct nomenclature throughout the manuscript.

The list above is nowhere near comprehensive. The manuscript requires significant editing.

We are grateful to the reviewer for drawing our attention to these errors. We have made significant edits to the revised manuscript to address the above-mentioned concerns, as well as made additional textual changes throughout and copyedited it. We hope that the reviewer will find the manuscript reads better and the clarity and preciseness is significantly improved.

Reviewer #2 (Significance (Required)):

I find the study to be potentially very important - the authors combine a longitudinal study that would be difficult in any other model with the powerful genetic tools available in the fly. The findings will significantly advance our understanding of how lipid metabolism links dietary nutrition with feeding behavior.

Once again, we are grateful for this reviewer’s thoughtful critique and encouraging words regarding our work and its potential impact.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary: This manuscript uses Drosophila to investigate how diet-induced obesity and the changes in the lipid metabolism of the fat boy modulate hunger-driven feeding (HDF) response. The authors first demonstrate that chronic exposure (14 days) of high sugar diet (HSD) suppresses HDF response. Through lipidome analysis, the authors identify a specific class of lipids to be elevated upon chronic HSD feeding. This coincided with the changes in expression of Pect, an enzyme that regulates the biosynthesis of these lipids. Modulating the expression of Pect specifically in the fat body affected HDF response.

We thank this reviewer for their rigorous and thoughtful critique and for identifying a key issue with our original study pertaining to a gap in how Pect mRNA levels on 14-day HSD are elevated but the Pect-KD phenocopies the HDF. Now by performing whole-body adult fly lipidomic on fat-specific Pect-KD we have resolved this issue and provided clarity on role of Pect in maintaining phospholipid homeostasis and thus subsequently impacts hunger-driven feeding. We hope the reviewer finds that the revised manuscript provides further clarity to the functional link between Pect’s role in fat-body and hunger-driven feeding.

Major comments: The author claim that the HDF response in HSD is distinct between early (5d, 7d) and chronic (day 14) HSD feeding. However, the data seem to indicate that HDF response is significantly decreased at all time points in HSD. For example, at day 5 HDF response was increased only 3-fold in HSD (Figure 1C) compared to around 50-fold increase in NF (Figure 1B). The scale of the Y-axis in Figure 1B and 1C is an order of magnitude different. Including the starved data (NFstv and HSDstv) in Figure S1, normalized to NF fed group, would better visualize the overall trends. Related to this, having the source data for the actual number of feeding events would be useful (e.g., to see the baseline changes in feeding in different time points in Figure 1 and the effect of genetic manipulations in Figure 7).

As per the reviewers request, we now have modified our graphs to show source data (Figure S1) and show the raw feeding events.

Then in the non-normalized graphs we plot, over a longitudinal time course, baseline and hunger-driven feeding events (Figure 1B-D). We also show that HSD fed flies do not display increased baseline feeding (Figure 1D) suggesting that the effect we see on HDF are no clouded by increased baseline feeding.

Yes, the reviewer makes an important point that HDF response on HSD fed flies is of a lower magnitude than NF fed flies. We think that is a biologically meaningful observation, as it suggests that flies have a remarkably fine-tuned ability to coordinate food-intake with nutrient store levels.

­­Now we have included a paragraph in the Discussion, Page 11 Lines 23-27, that say the following to ensure the readers appreciate this salient point raised by this reviewer.

*It is to be noted that the HDF response of HSD-fed flies (Figure 1C, Days 3-10) is of lower order of magnitude than the NF-fed flies. This suggests that that in addition to sensing an energy deficit and mobilizing fat stores (Figure 1F, 1G, S1), HSD fed flies calibrate their starvation-induced feeding to compensate only for the lost amount of fat. Overall, this suggests that flies have a remarkably fine-tuned ability to coordinate food-intake with nutrient store levels. *

The association between fat body Pect level and phospholipid levels is not clear. Day 14 of HSD feeding shows high expression of Pect in the fat body and elevated levels of PC32.0 and PC32.2. The authors assume the high expression of Pect in the fat body is due to the compensatory response, but there are no data indicating downregulation of Pect levels at the earlier time points of HSD feeding. A previous study demonstrated that Pect mutant flies have lower levels of PC32.0 but higher PC32.2 (PMID: 30737130).

We agree that one puzzling aspect of the original version of this study was that Pect mRNA levels being very high on Day 14 HSD, but nonetheless the effects of Pect-KD phenocopied HSD. To resolve this, prompted by Reviewer #2 and #3 concerns, for this revised version we have now performed lipidomic analyses on whole adult flies, when Pect is knocked down (KD) by RNAi in the fat tissue. We now present a new dataset in Figure 6. Two striking changes occu. They are:

1. Pect-KD shows increase in the phospholipid classes LPC and LPE (Figure 6A). In contrast, LPE is significantly downregulated on HSD Day 14 (Figure 3).
2. Pect-KD shows a significant reduction in specific class of PE 36.2 (Figure 6B). Our data regarding increase in PE 36.2 agree with a previous lipidomic analyses of Pect mutant retina (PMID: 30737130). In contrast, PE 36.2 trends upwards on 14 day HSD (Figure S7C) though not significantly. On 14-day HSD consistent with extreme upregulation of Pect mRNA fed flies (Figure S6A; Pect mRNA 200-250 fold), PE trends upwards on 14-day HSD (Figure 3) and PE 36.2 trends higher (Figure S7C). We note that on the surface of it PE and LPE per se are contrasting between 14-day HSD lipidome and fat-specifc Pect-KD. But there is a significant commonality that under both states there is an imbalance of phospholipids classes PE and LPE. Hence, we propose that maintaining the compositional balance of phospholipid classes PE and LPE is critical to hunger-driven feeding and insulin sensitivity. Hence, either increase or decrease, of these key phospholipid species, may lead to abnormal hunger-driven feeding.

On day 14, HDF response was increased 70-fold in w1118 flies in NF (Figure 1B; w1118), but only 2.5-fold in lpp>LucRNAi control flies in NF (Figure 7A). This suggests that lpp-gal4 driver lines have a significant effect on HDF response. Using a different fat-body specific Gal4 line would be necessary to validate conclusions.

Regards reduced HDF magnitude, in our experience using UAS-Gal4 reduces HDF response magnitude consistently and cannot be compared to w1118 which is more robust. To account for background differences, we use Uas-Gal4 with control RNAi. It clearly shows differences in HDF response on starvation, but Pect and Pisd RNAi does not (Figure 7A). Hence, given that this experiment internally controls for any changes in HDF response for UAS-Gal4>RNAi, we conclude that HDF response in disrupted in Pect and PISD KD (Figure 7).

We only presented the Lpp-driver in our study, as this driver is the only fat-specific driver that has no leaky expression in other tissues, and is specific to fat as apolpp promoter used to generate this Gal4 line is only expressed in fat tissue (Eaton and colleagues, PMID: 22844248). Other widely used fat-specific drivers, including the pumpless-Gal4 (ppl-Gal4) driver has leaky expression in gut or other tissues (See Table 2 of this detailed study by Dr. Drummond- Barbosa https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642949/). If the reviewer is aware of a fat-specific Gal4 line, other than Lpp-Gal4, which has a highly specific expression in the fat tissue without leaky expression in other tissues, then we are happy to take onboard the reviewer’s suggestion and try that fat-specific Gal4 that they suggest.

HSD feeding promotes Pect expression (Figure S3C) and global changes in phospholipid levels (Figure 3, 4). Therefore, shouldn't Pect overexpression (not Pect RNAi) in a normal diet mimic HSD feeding state and promote loss of HDF response? Conversely shouldn't knockdown of Pect in HSD rescue loss of HDF response?

We agree that a puzzling aspect is that Pect mRNA levels are significantly elevated in HSD Day-14, but Pect-KD showed displays the inappropriate HDF response. As we have described in our response to this reviewer on Page 19, we believe that Pect-KD and HSD disrupt PE and LPE balance overall but in different ways. Whereas Pect-OE using cDNA expression in fat body does not cause a significant change to any lipid class (Figure S9), and our results suggest that basally higher level of PECT is likely to be protective on HSD with respect to HDF(Figure 7B).

To ensure that we appropriately discuss and clarify this issue, we have now included a section in the discussion - Page 14 Lines 26-33- under the subtitle “The implications of relationship between Pect levels and HSD”. We have pasted an excerpt from that subsection below for this reviewers assessment.

“Also, we note that over-expression of Pect cDNA in the fat-body does not alter phospholipid balance (Figure S9) and indeed improves HDF on HSD (Figure 7B). While this may appear inconsistent, it is critical to note that over-expression of Pect cDNA using UAS/Gal4 only increases Pect mRNA expression by 7-fold (Figure S6A), whereas HSD causes its upregulation by 250-fold (Figure S6B). Hence, we speculate that an increased ‘basal’ level of Pect such as by that provided by a cDNA over-expression in fat, may be protective to the negative effects of HSD (Figure 7B) without affecting overall phospholipid levels (Figure S9) , but extreme upregulation Pect on HSD affects the PE and LPE balance (Figure 3).”

We would have liked to test Pect protein expression on HSD, but since we were unable to access antibodies for Pect published in a prior study (PMID: 33064773) from Dr. Wang’s lab (see Page 10-11, of response to Reviewer #1). Hence, we were unable to test how the proteins levels of Pect correlate with the 250-fold increase mRNA expression.

In conclusion, we hope the reviewer appreciates that our results regarding Pect function are consistent with the main conclusion that achieving the right phospholipid balance between PE and LPE, is critical for an organism to display an appropriate HDF response.

Minor comments: All graphs should plot individual data points and showed as box and whisker plot as much as possible.

Thanks for this suggestion, we have added individual data points to the vast majority of figures in the paper. We have made exceptions to graphs such as seen in figure 1 and FigureS4B-D where we find individual data points add an unnecessary layer of complexity. We hope these changes provide additional clarity and strength to the claims made in this manuscript.

Data for day 14 missing in Figure S4A and S4B.

We have provided Day 14 for the PC composition and PE composition, due to changes in Figures, they are now S7A and S7B.

Reviewer #3 (Significance (Required)):

The interactions between diet-induced obesity, peripheral tissue homeostasis and feeding behavior is an interesting topic that can be addressed using Drosophila. This manuscript demonstrates how fat body Pect levels affect HSD induced changes in hunger-driven feeding response. However, at this point, the functional association between fat body Pect level, global phospholipid level, and loss of hunger-driven feeding response in chronic HSD feeding is not clear.

We hope the revised data, and discussion of the paper, provides well-substantiated functional association on the importance of maintaining phospholipid balance, driven by Pect enzyme, as a critical regulator of hunger-driven feeding behavior. As stated in the revised discussion, the key take home message of our manuscript is that on prolonged HSD exposure PC, PE and LPE levels are dysregulated, the loss of phospholipid homeostasis coincided with a loss of hunger-driven feeding. Following this lead on phospholipid imbalance, we then uncovered a critical requirement for the activity of the rate-limiting PE enzyme PECT within the fat tissue in controlling hunger-driven feeding.

Reviewer #1 (Public Review):

The work, mostly performed in yeast S. cerevisiae, shows that the knockout of DIP2 leads to accumulation in cells of some DAG subspecies (36:0 and 36:1), and also a deficit of similar TAG subspecies (something which mostly occurs, as they showed, in early to mid log growth phase). Accordingly, over-expression of DIP2 leads to the opposite outcome (lower DAG and higher TAG subspecies levels). ∆DIP2 cells showed increased ER stress and UPR, which can be counterbalanced by incubating cells with oleic acid. Moreover, the authors show that the absence of DIP2 causes vacuole fusion defects, which they ascribe to a localization of the protein in the vacuole and possibly to the fact that enhanced levels of DAG in the vacuole membrane can promote vacuole fusion. Although it is true that neither of these claims are fully supported by the experimental results, the data that the authors show serves as a starting point for future, more robust studies to test those claims. Finally, the authors show that the DBD1 domain is not necessary and that the two FLD domains are key for the observed lipid metabolism induced by DIP2 expression. Altogether this manuscript presents interesting new data on an uncharacterized protein that seems to be regulating the metabolism of relatively low abundant DAG/TAG subspecies in cells, and by doing so possibly control cell homeostasis.

Reviewer #3 (Public Review):

This study examines a family of poorly defined enzymes that contain fatty acyl-AMP ligase like domains (FAALs). The study reveals that these DISCO-interacting protein 2 (DIP2) enzymes are required to maintain a specific pool of diacylglycerol (DAG) lipids containing primarily C36 acyl chain lengths in budding yeast. Using primarily yeast, the study shows that deletion of ScDIP2 significantly increases C36 DAG pools while leaving the more abundant C32 and C34 DAG pools generally unaltered. Triglyceride (TAG) is also reduced in this deletion. Conversely, ScDIP2 over-expression promotes C36 inclusion in TAG. The ScDIP2 KO yeast manifests ER stress that can be relieved by the addition of oleic acid, but not other fatty acids. In the last section of the study, ScDIP2 is proposed to localize to the vacuole and mitochondria, where it maintains a specific DAG pool to enable proper vacuole morphology and fusion, as well as proper osmoregulation of the vacuole.

This is a well executed study that begins to characterize a conserved and generally poorly understood family of enzymes. However, questions still remain about some of the conclusions of the study. There are two general issues with the study. The first is the specificity of the effect of loss of ScDIP2. The study beautifully shows that loss of ScDIP2 (or its over-expression) affects a specific sub-pool of DAG (mainly the C36 species). TAG levels are also somewhat lower. However, how ScDIP2 impacts other lipid precursors to DAG synthesis such as PA and lyso-PA is under-examined, and should be looked at as they can also affect ER stress. Whether the change in DAG/TAG is primarily driven by decreased synthesis versus increased lipolysis also required additional analysis.

The second issue relates to how ScDIP2 relates to the yeast vacuole. It is proposed that some of the ScDIP2 enzyme is vacuole localized, and influences vacuole morphology. The evidence presented here does not strongly support that model. From imaging at least, it appears that ScDIP2 is primarily mitochondria localized. It is therefore possible that it influences vacuole lipid composition and morphology distally from the mitochondria. Resolving ScDIP2's native subcellular localization would strengthen the manuscript.

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SciScore for 10.1101/2022.05.07.491022: (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: Reagents: Cell lines: All cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U ml−1 penicillin and 100 mg ml−1 streptomycin and regularly tested and found to be mycoplasma free.</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-rabbit IgG, HRP-linked Antibody (7074); Cyclin D3 Mouse mAb (DCS22, 2936); from Cell Signaling.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-rabbit IgG</div><div>suggested: (Cell Signaling Technology Cat# 7074, RRID:AB_2099233)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody: Alexa 488 (A-11001), Alexa 594 (A-11032), Alexa 647 (A-21236); Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody: Alexa 488 (A-11034), Alexa 405 (A-48254); Rabbit polyclonal SARS-CoV-2 Spike (PA1-41165); Rabbit monoclonal SARS-CoV-2 Nucleocapsid (MA5-29982) from Thermo Fisher Scientific.</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# A-11001, RRID:AB_2534069)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Rabbit Polyclonal Cyclin A2 antibody (GTX103042); Rabbit Polyclonal Cyclin D1 antibody (N1C3, GTX108824); Rabbit Polyclonal Cyclin E1 antibody (GTX103045); Rabbit Polyclonal Cyclin B1 antibody (GTX100911); monoclonal SARS-CoV-2 Spike (GTX632604) from GeneTex.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Cyclin A2</div><div>suggested: (GeneTex Cat# GTX103042, RRID:AB_1949884)</div></div><div style="margin-bottom:8px"><div>Cyclin D1</div><div>suggested: (GeneTex Cat# GTX108824, RRID:AB_10618686)</div></div><div style="margin-bottom:8px"><div>Cyclin E1</div><div>suggested: (GeneTex Cat# GTX103045, RRID:AB_10731259)</div></div><div style="margin-bottom:8px"><div>Cyclin B1</div><div>suggested: (GeneTex Cat# GTX100911, RRID:AB_1949886)</div></div><div style="margin-bottom:8px"><div>GTX632604</div><div>suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pre-cleared cell lysates were incubated with a-HA magnetic beads, MagStrep beads (IBA-Lifescience, Gottingen, Germany) or anti-cyclin D3 monoclonal antibody (sc-xx) bound Protein G Dynabeads for 1h at 4°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-cyclin D3</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">Following cells were a gift from: A549 ACE2/TMPRSS2 40 Massimo Palmerini, Vero E6 ACE2/TMPRSS2 from Emma Thomson, HeLa-ACE2 from James Voss, 293T (a human embryonic kidney cell line, ATCC CRL-3216).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div><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">293T GFP11 cells and Vero-GFP10 cells for Split GFP assay were a gift from Leo James41.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T GFP11</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Vero-GFP10</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293Tv cells were transfected with pEXN-MNCX-Fucci, CMVi and pMD2.G.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293Tv</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: pBOB-EF1-FastFUCCI-Puro was a gift from Kevin Brindle & Duncan Jodrell (Addgene plasmid # 86849 ; http://n2t.net/addgene:86849 ; RRID:Addgene_86849) 29. pCMV5 cyclin D3 HA was obtained from MRC-PPU Reagents and Services. Rc/CMV cyclin D1 HA was a gift from Philip Hinds (Addgene plasmid # 8948 ; http://n2t.net/addgene:8948 ; RRID:Addgene_8948) 44. pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro (Addgene plasmid # 141385 ; http://n2t.net/addgene:141385 ; RRID:Addgene_141385); pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro (Addgene plasmid # 141386 ; http://n2t.net/addgene:141386 ; RRID:Addgene_141386).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_86849)</div></div><div style="margin-bottom:8px"><div>pCMV5</div><div>suggested: RRID:Addgene_15002)</div></div><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_8948)</div></div><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_141385)</div></div><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_141386)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pLVX-EF1alpha-SARS-CoV-2-nsp9-2xStrep-IRES-Puro (Addgene plasmid # 141375 ; http://n2t.net/addgene:141375 ; RRID:Addgene_141375); pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro (Addgene plasmid # 141391 ; http://n2t.net/addgene:141391 ; RRID:Addgene_141391) were a gift from Nevan Krogan 34. pEXN-MNCX, MLV vector encoding N-terminal double HA tag 45.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_141375)</div></div><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_141391)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pCAGGS_SARS-CoV-2_Spike was obtained from NIBS.</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">Cell cycle analysis using fluorescence ubiquitination cell cycle indicator (Fucci): Fucci cassete was cloned from pBOB-EF1-FastFucci-Puro vector to pEXN-MNCX using BamHI/NotI restriction sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pEXN-MNCX</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293Tv cells were transfected with pEXN-MNCX-Fucci, CMVi and pMD2.G.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pEXN-MNCX-Fucci</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pMD2.G</div><div>suggested: RRID:Addgene_12259)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell to cell fusion assay: 293T GFP11 cells were transfected with WT full length Spike, and/or with WT Envelope, Membrane, cyclin D3, and empty vector (pCDNA, to ensure equal amount of transfected DNA).</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><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">Harmony (PerkinElmer, Waltham, MA, USA) and ImageJ software were used to measure MFI for each protein in each region.</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">Cell populations positive or negative for SARS-CoV-2 nucleocapsid staining were gated and Cdt1-RFP positive (G1 phase), Geminin-GFP positive (S/G2/M phase), and Cdt1-RFP/ Geminin-GFP positive (early S phase) populations were identified using flow cytometry using LSRFortessa X-20 (BD Biosciences, UK) and FlowJo software (Tree Star, OR, USA).</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>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Reply to the reviewers

1. General Statements [optional]

We would like to thank the reviewers for their helpful and constructive comments.

2. Point-by-point description of the revisions

Reviewer #1

This reviewer thought our findings would be of interest to a broad range of scientists from both the centrosome and mitosis fields, but noted some important aspects for improvements.

Additional Experiments (we number these points for ease of discussion).

1. • Figure 3. The reviewer points out that because our analysis of Ana2-∆CC and Ana2-∆STAN mutant proteins was conducted in the presence of endogenous WT protein, we should be more cautious in our interpretation.* We agree and apologise for overstating these findings. We have now rewritten the title and text of this section to be more cautious (p11, para.2)

2. Figure 5A. The reviewer wonders whether the reduced recruitment of Sas-6 in the presence of Ana2(12A) is due to reduced binding, and they request we test this biochemically. This is our favoured interpretation, but we have been unable to test this biochemically for two reasons. First, although we have successfully purified several recombinant Sas-6 and/or Ana2 fragments (Cottee et al., eLife, 2015), the full-length proteins are poorly behaved (tending to precipitate, likely due to their inherent ability to self-oligomerise). Thus, we have been unable to reconstitute their interaction in vitro*. Second, as we show here, the proteins are normally expressed in embryos at surprisingly low concentrations (~5-20nM), and we can detect no interaction between them in coimmunoprecipitation experiments from embryo extracts (not shown). Indeed, this concentration is so low that Sas-6 does not even appear to form a homo-dimer in the embryo, even though Sas-6 clearly functions as a homo-dimer in centriole assembly (new Figure S4A). We now explain these points, and state that our favoured hypothesis that Ana2(12A) has reduced affinity for Sas-6 (or other core duplication proteins) remains to be tested (p22, para.2).

3. The Reviewer wonders if all 12 of the potential Cdk1 phosphorylation sites that we mutate in Ana2(12A) are important in vivo, and whether we have tested whether mutating fewer sites (e.g. the two sites [S284/T301] that we show are phosphorylated by Cdk1/Cyclin B in vitro) might be sufficient to recapitulate the Ana2(12A) phenotype. *We have now tested this by mutating just the S284/T301 sites to Alanine [Ana2(2A)], but the results were not very informative (Reviewer Figure 1 [RF1]). Whereas Ana2(12A) is recruited to centrioles for a longer period and to higher levels than WT Ana2 (Figure 4A), Ana2(2A) is recruited to centrioles for a normal period but to lower levels (RF1A,B). The interpretation of this result is complicated because western blots show that Ana2(2A) is also present at lower-levels than normal (RF1B). Thus, it is clear that Ana2(2A) does not recapitulate well the behaviour of Ana2(12A). We have decided not to present this data as it is difficult to interpret and it does not change any of our conclusions.

4. Figure 6. The reviewer asks whether the 12A mutations impair the interaction with Plk4, influence Plk4’s kinase activity or the ability of Plk4 to phosphorylate Ana2. These are excellent questions but, for the same reasons described in point 2 above, we cannot address them biochemically as we cannot purify well-behaved recombinant full-length Ana2 or active Plk4 in vitro, and both proteins are present at such low levels in the embryo that we cannot detect any interaction between them in embryo extracts. We are working hard to reconstitute in vitro* systems to probe these important points, but it may be sometime before we are able to do so.

5. Figure 7. The reviewer suggests that the 12D/E phosphomimetic substitutions introduce more negative charge than the putative phosphorylation of Ser/Thr residues and they ask if the Ana2(2D/E) [stated as Ana2(3D/E)] is, like the Ana2(12D/E) mutant, not efficiently recruited to centrioles.* This is a fair comment, but we have not analysed an Ana2(2D/E) mutant because, as described in point 3 above, the Ana2(2A) mutant did not recapitulate well the Ana2(12A) phenotype.

Minor comments

1. • Figure S1. The reviewer requests that we show that the mNG tag on its own is not recruited to centrioles.* We do not show this (as it would create a lot of white space in this Figure), but now state that mNG and dNG do not detectably localise to centrioles (p7, para.1).
2. • Figure S4C.* We have included the missing error bars (now Figure S4B).
3. • Figure S5A. The reviewer asks about the expression levels of the Ana2(12A) mutant, which are not shown in this Figure. They also state that the expression levels of the transgenes shown in Figure 5A are not similar.* The expression level of Ana2(12A) is shown in Figure S9, as this data was analysed independently of the other mutant proteins shown in Figure S5. We agree that it was overly simplifying the situation to state that the expression levels of WT Ana2-mNG, eAna2(∆CC)-mNG and eAna2(∆STAN)-mNG were “similar” (Figure S5), and we now specifically mention the differences between them (p11, para.3). Reviewer #2

This reviewer found this a rigorous study that advances our understanding of the regulation of centriole duplication, but raised some minor points.

Minor Points

The reviewer requests that we mention the literature describing how Ana2/STIL can influence the abundance and centriolar localisation of Plk4. We apologise for this omission, and have amended our description of this literature in the Introduction to include this point (p3, para.2).

The reviewer notes that we interpret the ability of the Ana2(12A) mutant to keep incorporating into the centrioles for a longer period as being consistent with our idea that rising levels of Cdk activity during S-phase normally reduce the ability of WT Ana2 to bind to the centriole. They ask us to show how Cdk activity increases over this time-course, and to test whether dampening Cdk has the same effect on Ana2 recruitment (i.e. allows Ana2 to be recruited for a longer period). The time-course of Cdk activation in these embryos has been reported previously (Deneke et al., Dev. Cell, 2016; we present the relevant data from this paper in RF#2A [black line]). This reveals how Cdk activity rises throughout S-phase, which is crucial for our model. To assess the effect of dampening Cdk activity in these embryos we have now analysed the effect of halving the genetic dose of Cyclin B (RF#2B). This perturbation extends S-phase length, but has a complicated effect on the recruitment dynamics of Ana2 (RF#2B). As we would predict, Ana2 is recruited to centrioles for a longer period in these embryos, but it is also recruited more slowly (so it accumulates to lower levels). This is consistent with our hypothesis that Cdk1 activity might first stimulate and then ultimately inhibit the centriolar recruitment of Ana2. The interpretation of this experiment is not straightforward, however, as dampening Cdk1 activity alters Ana2 recruitment dynamics (and many other processes in the embryo) in complicated ways, so we have decided not to include it in the manuscript.

The reviewer suggests that it would be valuable to show that all 12 of the potential Cdk1 phosphorylation sites in Ana2 can be phosphorylated by Cdk1 in vitro. We think this would not be particularly informative as our hypothesis does not rely on all 12 sites being phosphorylated to generate the Ana2(12A) phenotype. We simply mutate all 12 sites because we don’t know which, if any, are relevant. Thus, showing that some/all of the 12 sites can/cannot be phosphorylated in vitro does not test any hypothesis and would not change any of our conclusions. We now explain our thinking on this in more detail (p12, para.2)

Other points

Figure 3. We have corrected the amino-acid numbering mistakes.

Figure 5Aii. We have changed the x-axis (time) labelling in this and all other Figures.

Figure Legends. We have tried to eliminate the typos from the Figure legends, and apologise that these errors made it through to the final submitted version of our manuscript.

Reviewer #3

This reviewer thought our manuscript would be of great interest to not only the centrosome field but also to cell biologists more generally. Although they had no major concerns, they made a number of suggestions for improvements.

1. As the reviewer suggests, we now explicitly state that although the Ana2(12A) mutant appears to be largely functional, the overall conformation of the protein may be altered, changing its function in ways we do not appreciate (p21, para.2).

2. The reviewer suggests we include a multiple sequence alignment of Ana2/STIL proteins to provide more context about the distribution and conservation of the 12 S/T-P sites mutated in Ana2(12A).* This is an excellent idea, and we now include this in a new Figure S6, where we also provide more information about which of these sites have been shown to be phosphorylated in embryo or S2-cell extracts

3. The reviewer is confused as to why the 12A and 12D/E mutants rescue the ana2-/- mutant flies so well, which suggests that the mechanism we propose here cannot be essential for centriole duplication. We understand this confusion and we now make this point more clearly and explain why we think this occurs in more detail (e.g. p22, para.1). We propose that Cdk normally phosphorylates Ana2 to inhibit its ability to promote centriole duplication, but this phosphorylation does not entirely block this function. So, if all other elements of the system are functional, Ana2(12A) is recruited to centrioles for longer than normal, but this does not dramatically perturb centriole duplication because the many other factors that regulate centriole duplication (such as the pulse of Plk4 recruitment to centrioles [Aydogan et al., Cell, 2020]) still occur normally and are sufficient to ensure that centrioles still duplicate normally. When Ana2 phosphorylation is mimicked [Ana2(12D/E)], the ability of Ana2 to promote centriole duplication is perturbed (but not abolished). This perturbation is lethal in the early embryo—where the centrioles must duplicate in just a few minutes to keep pace with the rapid nuclear divisions. In somatic cells S-phase is much longer, so these cells can still duplicate their centrioles (as we observe) even though Ana2(12D/E) does not function efficiently. As we now explain, this phenotype (being lethal in the early embryo, but not in somatic cells) is a common feature of mutations that influence the efficiency* of centriole and centrosome assembly (p17, para.2).

4A. The reviewer asks us to comment in more detail on why centrioles do not seem to be elongated in the Ana2(12A) mutant wing disc cells (now Figure S8C), even though we show that Ana2(12A) (Figure 4A), and also Sas-6 (Figure 5), are recruited to centrioles for an abnormally long period. This is an excellent question and, although we do not know the answer, we now discuss this interesting point in more detail (p16, para.1). We think this is likely due to the “homeostatic” nature of centriole growth: in our hands, almost any perturbation that makes centrioles grow for a longer/shorter period, also makes them grow more slowly/quickly, so that they tend to grow to a similar size (Aydogan et al., JCB, 2018; Cell, 2020). This is fascinating, but poorly understood. When we perturb the system by expressing Ana2(12A), both Ana2(12A) and Sas-6 incorporate into centrioles for a longer period, as we predict (Figure 4A and 5A). Unexpectedly, however, Sas-6 is also recruited to centrioles much more slowly. Thus, as so often happens, when we perturb the system so the centrioles grow for a longer time, the centrioles “adapt” by growing more slowly. We do not currently understand why this occurs (although we speculate that Ana2 may also be regulated by Cdk/Cyclins to help recruit Sas-6 to centrioles in early S-phase). In the embryo, where S-phase is very short, this homeostatic compensation is not perfect, and the centrioles appear to actually be shorter than normal. In somatic wing-disc cells, where S-phase is much longer, we suspect that there is more scope for homeostatic compensation and so the centrioles grow to the correct size.

4B. In this point (also labelled [4] by the reviewer, so we have retained this numbering but labelled the points A and B) the reviewer asks why levels of Ana2(12A) eventually decline at centrioles once the embryos actually enter mitosis. The reviewer notes our rheostat theory, but suggests a discussion of other mechanisms might be interesting. This is a good point, and we agree that the observation that Ana2(12A) levels ultimately still decline at centrioles during mitosis is likely to be important in explaining why centriole duplication is not more dramatically perturbed by Ana2(12A). We now expand our discussion of this point, highlighting that other mechanisms must help to ensure that Ana2 is not recruited to centrioles during M-phase, and discussing the possibility that the receptors that recruit Ana2 to centrioles are themselves inactivated during mitosis by high levels of Cdk activity (p15, para.1). In such a model, the rapid drop in WT Ana2 centriolar levels is due to a combination of switching off Ana2’s ability to bind to centrioles (as we propose here) and switching off the ability of the centrioles to recruit Ana2. For Ana2(12A), only the latter mechanism would operate, so Ana2(12A) levels would start to drop later in the cycle (as the inflexion point at which Ana2 recruitment and loss balances out would be moved to later in the cycle), and these levels would drop more slowly—as we observe.

• The reviewer is confused to how the Ana2(12D/E) mutant can rescue the mutant phenotype when it is recruited to centrioles so poorly. Ana2(12D/E) is indeed recruited very poorly to centrioles in the experiment shown in Figure 7. However, this experiment had to be conducted in the presence of WT untagged Ana2—as the embryos do not develop in the presence of only Ana2(12D/E). We would predict that WT Ana2 would bind more efficiently to centrioles than Ana2(12D/E) (which appears to behave as if it has been phosphorylated by Cdk/Cyclins, and so cannot be recruited to centrioles efficiently). Thus, in the experiment we show in Figure 7, the Ana2(12D/E) protein is probably being “outcompeted” for binding to the centriole by the WT protein. In somatic cells expressing only* Ana2(12D/E) presumably sufficient mutant protein can be recruited to centrioles to support normal centriole duplication (as it no longer has to compete with the WT protein). We now explain our thinking on this point (p18, para.1).

• The reviewer wonders whether Ana2(12D/E) may be unable to homo-oligomerize, and this may explain why the protein is not recruited to centrioles efficiently even in the presence of WT protein. This is indeed a possibility, but we think it unlikely as it is widely believed that Ana2/STIL proteins must multimerize to be functional (Arquint et al., eLife, 2015; Cottee et al., eLife, 2015; Rogala et al., eLife, 2015; David et al., Sci. Rep., 2016). As Ana2(12D/E) strongly restores centriole duplication in ana2-/-* mutant somatic cells, it seems unlikely that it cannot multimerize. Nevertheless, we now specifically highlight that the 12D/E (and 12A) mutations might alter the ability of Ana2 to multimerise (p21, para.2).

We thank the reviewers again for their thoughtful and constructive comments. We hope they will agree that the revised manuscript is now improved and would be appropriate for publication in The Journal of Cell Biology.

With best wishes,

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Referee #1

Evidence, reproducibility and clarity

Centriole duplication is a conserved pathway that need to be tightly regulated. The key enzyme of centriole assembly is Plk4 which is recruited to the centrioles and undergoes dynamic re-localization from a ring-like pattern around a centriole to a dot-like morphology at the daughter centriole assembly site. This event is central for inducing centriole biogenesis. Plk4 then phosphorylates Ana2/STIL which allows recruitment of Sas-6 to form the cartwheel structure for centriole assembly.

In the present study, Steinacker, Wong et al. monitor how cytoplasmic concentrations of the key proteins in centriole assembly, Plk4, Asl/Cep152, Ana2/STIL, Sas-6 and Sas-4/CPAP change during the centriole assembly process in the Drosophila embryo by using fluorescence correlation spectroscopy (FCS) and Peak Counting Spectroscopy (PeCoS). They find that their concentrations remain constant with exception of Ana2/STIL of which cytoplasmic diffusion rate increased at the end of S-phase and is dependent on phosphorylation by Cdk1/CyclinB. Phosphorylated Ana2/STIL blocks centriole duplication thus preventing premature initiation of centriole duplication in mitosis.

Major comments

The manuscript is interesting and very well written. Most of the experiments are carefully performed. However, there are some important aspects for improvements that are listed below

Additional experiments:

• Figure 3: the transgenic flies that were generated here, CC and STAN, still contain wild-type Ana2. So, the authors therefore need remove or dampen their claim that the change in Ana2's cytoplasmic diffusion does not depend on its interaction with Sas-6 (page 11).
• Figure 5A: is the observed reduced recruitment of Sas-6 by Ana2(12A) due to a decrease in binding affinity? This should also be shown by analyzing protein-protein interactions between Ana2(12A) and Sas-6 biochemically.
• The authors use an Ana2(12A) mutant which comprises putative Cdk1 phosphorylation sites that have been identified in Mc Lamarrah et al. JCB 2018. However, only three of them were phosphorylated by Cdk1/cyclin B in vitro (Fig. S6). Are all these 12 putative Cdk1 phosphorylation sites important in vivo? Did the authors generate the Ana2(3A) or the S284A/T301A mutants to see whether it can rescue the ana2-/- mutant phenotype similar to the 12A mutant? These might be sufficient to observe the phenotype.
• Figure 6: is the interaction between Plk4 and Ana2(12A) impaired? Similarly, Plk4 activity and phosphorylation of Ana2(12A) by Plk4
• Figure 7: Phosphomimetics, in this case 12 amino acid changes, have the disadvantage of introducing more negative charge than the phosphorylated residue. The Ana2/(12D/E)-mNG is not efficiently recruited to centrioles. Is effect also observed for the Ana2/(3D/E) mutant?

Minor comments

Figure S1: only mNG-tagged centriolar proteins are shown. An empty mNGtag or an mNG-tagged non-centriolar protein should be shown to exclude that the tag by itself shows centriolar localization or somehow affects the localization

S4C: Sas6-mNG CPM error bars are missing for the 10min time point

S5A: What are the expression levels of the Ana2(12A) mutant? The expression levels shown in this Figure are not similar.

Significance

Centriole duplication normally begins at the G1/S phase transition. An important question in the field is how premature centriole duplication in mitosis is prevented. The authors used fluorescence correlation spectroscopy (FCS) and Peak Counting Spectroscopy (PeCoS) to study the major conserved proteins in the centriole assembly pathwayq and found that only Ana2/STIL's cytoplasmic diffusion increases at the end of S-phase. It is known from the literature that Cdk1 prevent Plk4-STIL complex assembly in centriole biogenesis by directly competing with Plk4 for the CC domain of Ana2/STIL (Zitouni et al. Curr Biol 26, 1127-1137 (2016). However, Ana2/STIL can also bind to Plk4 via its conserved C-terminal region of STIL (Ohta et al., Cell Reports 11, 2018; McLamarrah et al., J Cell Biol 2018, 217, 1217-1231). The work by Steinacker, Wong et al. suggest that at least in fly embryos, growth of the daughter centriole is regulated though phosphorylation of Ana2 by Cdk1/CyclinB rather than binding. The findings described in this manuscript are interesting for a broad range of scientists from both the centrosome and mitosis fields

Expertise of the reviewer: centriole biogenesis, structural and numerical centrosomal aberrations in disease

Your task:

What is the topic and the main idea of this article, based on the abstract? TAG the sentences with "topic" and "main idea" - make your annotations public, please.

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Referee #1

Evidence, reproducibility and clarity

In this manuscript, Dantas and colleagues report that confinement is sufficient to restore G2/M transition in cells than can't adhere to their matrix. Exploring further the mechanisms involved, they show that confinement (dynamic cell compression) stimulates nuclear import of cyclin B1 and nuclear envelope permeability using cells in 2D culture. The authors observed that actomyosin contractility increases NE tension in cells preparing for prophase, leading to an increase in nuclear translocation of cyclin B1. However, a few inconsistencies between the data and the conclusion make the current report too preliminary for publication. It may require significant additional work to consolidate the authors' model.

• The specific contribution of Nuclear Envelope tension. The authors conclude that confinement acts through increasing NE tension, although confinement may affect cytoplasmic signaling, which could contribute to G2/M transition. The authors should test whether compressing the nucleus versus compressing the cytoplasm have distinct effects on cyclin B1 nuclear translocation and G2/M, as it has been done by others when addressing nuclear mechanosensitive mechanisms (Elosegui-Artola et al. or Lomakin et al.). To consolidate their model, the authors should also test whether decreasing NE tension (independently of actomyosin tension) has opposite effect on G2/M (for example using LBR overexpression). Increase in nuclear membrane tension has been shown to trigger cPLA2 recruitment to the NE (Enyeidi et al, 2013; Lomakin et al. 2020), although the authors show here that confinement does not induce cPLA2 recruitment (but still increases NE tension figure 4G) in the absence of Rock activity or when the LINC complex is disrupted. This is surprising considering that confinement should increase NE tension independently of actomyosin contractility and should increase cPLA2 recruitment at the NE, unless in this case cPLA2 recruitment is not mediated by an increase in NE tension.
• NPC transport versus NE permeability. The authors suggest that confinement increases cyclin B1 transport via NPC-mediated transport and rule out that confinement may affect NE permeability based on the absence of NE rupture using the INM marker lap2. However, the sample size for this observation is missing and NE permeability could be altered even in the absence of major INM rupture observed by confocal. The authors should use a reporter of nuclear permeability (fluorescent cytoplasmic marker or nuclear marker as previously used by Denais et al or, 2016 or Raab et al., 2016) to make sure that NE permeability is not affected by confinement. In addition, NPC function should be tested in parallel with other fluorescent reporter (such as NLS-GFP constructs) to test whether global NPC-mediated transport is changed during prophase (with or without confinement).
• Effect of confinement on cyclin B transport (NEP) in adherent cells. In figure 1D, we can see that confinement enhances cyclin B1 nuclear translocation in cells adhering on fibronectin. Although it is unclear whether confinement has a significant effect in other figures, for example in figure 2F: DMSO is not significantly different from confiner+CDKi (same thing in 3i and 3j with Rock inhibitor and Kash construct). In these figures the untreated+confiner (or control in 3j) is missing, and the absence of difference between treated+confiner and control is puzzling. Either there is no difference between confiner and CDKi+confiner and it means there is no difference between control and confiner (surprising considering figure 1D); or there is a difference between CDKi+confiner and confiner, indicating that CDK inhibition affects confinement-induced cyclin B import. Both possibilities suggest that the authors should significantly revisit their model. In any case, all control (untreated, treated +/- confiner should be in all figures to avoid any misunderstanding).
• Consequences of cPLA2 recruitment at the NE. The authors state that "Active cPLA2 then stimulates actomyosin contractility creating a positive feedback loop" But the NE is already unfolded and distance between NPR is increased before cPLA2 recruitment. Does PLA2 inhibition affect nuclear irregularity (or distance between NPC)? Or does cPLA2 impact cyclin B1 transport via a distinct mechanism? Did the author analyze CDK1 phosphorylation in presence of PLA2 inhibitor?
• Robustness of the main observation. On page 4, the authors report that cells enter mitosis after 140 sec (+/- 80 sec) of confinement, although in the example showed in figure 1b, the cell enters at least 420 sec min after confinement, as we can see that the cell is already confined -420 sec (compressed shape) and NEP occurs at 0. Did the author showed a cell that was not included in their statistics? This would be very surprising considering the very low sample size used for this experiment (n=6 and 10). In addition, many observations have been made on small sample size (n=6 for figure 1) or/and not from independent experiments. The authors should increase their sample size and compare results from independent experiments to consolidate their model.
• 2h shows nuclear signal (cyclin in grayscale), while 2e does not, why?
• starting point to quantify cyclin entry is the lowest intensity, which may depend on many factors (and could be affected by experimental design). It would be necessary to have synchronized cells to homogenize the starting point of these experiments.
• DN-KASH have been transiently transfected for single cell experiments, how does the authors unsure that cell observed are transfected? Does it have a fluorescent tag, if so which one?
• "requires contact with external stimuli" or "that mechanical confinement is sufficient to overcome the lack of external stimuli." (page 4): external stimuli is vague here and it could be better to replace it with a more specific description

Significance

While the physiological relevance of these findings remain to be determined, the authors report an interesting observation that could have a significant impact in the field. The authors do not comment the potential overlap of their findings with other reports involving the LINC complex (Booth et al., ELife) or CDK-mediated actin remodeling (Ramanathan et al., NCB 2015) during prophase.

Reviewer #3 (Public Review):

Four decades after the seminal work of the Schekman's lab on the genetic identification of the core eukaryotic secretory machinery the molecular roles of the individual components have been largely characterized. Yet our understanding of how these components are organized to define processes is wanting, with notable controversies still hovering over at several levels of the secretory pathway, including the events that take place in the ER/Golgi interface, the transit across the Golgi, the biogenesis of secretory vesicles and the delivery, tethering and docking of these vesicles to the membrane. This manuscript mostly addresses the latest steps of this chain of events and makes some incursions into the biogenesis of vesicles at the TGN. It represents a serious and honest attempt to define the timeline of events that, driven by key components such as the Sec4 ras-in-brain (Rab) GTPase, its effectors myosin-5, Sro7 and the exocyst, its GEF, Sec2 and the prototypic Sec/Munc protein Sec1, a regulator of trans-SNARE complex formation, ultimately result in the tethering, docking and fusion of vesicles with the membrane of the polarized bud of the ascomycete yeast Saccharomyces cerevisiae. Tethering, as defined by light microscopy appears to be a robust process reproducibly lasting for five seconds, before fusion, as defined by the loss of vesicle components, takes place. Important evidence is provided that the exocyst is incorporated as an holo-complex to secretory vesicles. Overall, even though this work will likely suffer modifications and amendments as knowledge and technology progress, it will nevertheless become the reference blueprint around which any future work in the field will pivot.

This work represents a very substantial advance in the field of exocytosis. Besides reporting with unmatched time resolution the tethering of vesicles with the membrane, it describes a herculean effort to gain mechanistic understanding of the process by using a score of genetic perturbations and fluorescent reporters. I feel that evidence that Sec3 travels with the exocyst rather than contributing a milestone for exocyst landing will be disputed, but this referee finds it as convincing as appealing. Nearly as important is the timing of Sec1 action in the fusion step. However, it is the delineation of a timeline that will make this paper a reference in the field.

Understanding the technology for image acquisition is critical to appreciating the strengths of this MS (333 ms/Z-stack time point may be considered super-resolution - in the time dimension. Therefore, its description requires clarification in places. The experimental work is almost exclusively based on live microscopy using fluorescent proteins tagged by allelic replacement. The microscopy routine for single fluorophore analysis provides time series with a resolution of 3-5 fps that enables authors to resolve, using robust statistical tests, events separated by seconds. In this context, it is notable that dual-channel imaging appears to be made by sequential, not simultaneous, acquisition, which deserves a currently missing comment. Moreover, given the weight that image acquisition plays in this project, it might be described and justified better. The Materials and methods lack detail, for example, the laser lines & power used for excitation. This referee could not fully understand the routine of image acquisition, specifically, the continuous movement of the stage in the Z-axis as images are streamed (to the RAM or to the disk? the latter takes time, line 177); does it mean that Z-stepping is solely governed by the exposure time? The CCD camera penalizes pixel size (16 µm) at the expense of achieving outstanding quantum efficiency. The optical path includes a 100x objective and a 2x magnification lens to compensate for the large camera pixel size, thereby achieving 0.085 µm/pixel, but these lenses 'waste' part of the fluorescent signal. One wonders if the CMOS camera (6.5 µm pixel size) coupled with a 63x objective wouldn't be appropriate? A brief discussion on this choice would be helpful for readers.

There is an elephant in the room of in vivo microscopy that no one dares to comment on: reporter proteins are mutant versions carrying a heavy and potentially oligomerising rucksack - the fluorescent protein tag. The authors take the honest approach of acknowledging that some of the tagged proteins such as Sec4 are disfunctional and that certain reporters are incompatible with each other as they give rise to synthetic negative effects. In the end, they conclude that using diploids carrying the GFP-tagged allele in heterozygosis with the wt represents the most physiological approach to track proteins until less intrusive fluorescent tags are developed.

It is remarkable that Sec2 and Sec4 are recruited to membranes even before a vesicle is formed (Fig 6I). I find somewhat weak the evidence that RAB11s 'mark' the TGN, and disturbing the fact that RAB11 reaches the PM (does GFP tagging prevent GAP accession?). I should like to recommend strongly that the authors integrate into the introduction/discussion information on the late steps of exocytosis available for Aspergillus nidulans, another ascomycete that is particularly well suited for studying this process. Here RAB11 is not a late Golgi resident but is transiently (20 s) recruited to TGN cisternae in the late stages of their 120 s maturation cycle to drive the transition between Golgi and post-Golgi (Pantazopoulou MBoC, 2014). Recruitment of RAB11 to the TGN is preceded by the arrival of its TRAPPII GEF (Pinar, PNAS 2015; Pinar PLOS Gen 2019), a huge complex that is incorporated en bloc to the TGN (Pinar JoCS, 2020). Upon RAB11 acquisition RAB11 membranes engage molecular motors (Penalva, MBoC 2017) to undertake a several-micron journey that transports them to a vesicle supply center located underneath the apex (review, Pinar & Penalva, 2021). Here is where Sec4 is located, strongly indicating that there is a division of work between two Rabs each mediating one of the two stages between the TGN and the membrane (Pantazopoulou, 2014, MBoC).

SciScore for 10.1101/2022.05.03.22274395: (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: PCR confirmed and clinically suspected severe COVID-19 cases admitted to hospital were recruited into the DISCOVER study at North Bristol NHS Trust for which HRA Approval was granted by the South Yorkshire Research Ethics Committee (20/YH/0121).<br>Consent: All samples were used in accordance with the Human Tissue Act (2004) with appropriate consent and ethical approvals in place.</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">Blinding of validation set: The validation set of samples (n=807) were split into multiple aliquots (n=5) for randomisation and blinding by assigning a new barcode ID for each aliquot.</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">Blinding of validation set: The validation set of samples (n=807) were split into multiple aliquots (n=5) for randomisation and blinding by assigning a new barcode ID for each aliquot.</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">Purified proteins were analysed by SDS-PAGE and by Western-blots assays using an anti-His tag antibody (Sigma).</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">After washing, HRP-conjugated anti-human Pan-Immunoglobulin (Pan) (Sigma), IgG (Southern Biotech), IgA (Sigma) or IgM (Sigma) secondary antibody, in the same dilution buffer as the samples, was added (50 µl per well) and incubated for 1 hour.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG ( Southern Biotech)</div><div>suggested: (SouthernBiotech Cat# 1050-01, RRID:AB_2737431)</div></div><div style="margin-bottom:8px"><div>IgA ( Sigma )</div><div>suggested: (Sigma-Aldrich Cat# I1010, RRID:AB_1163625)</div></div><div style="margin-bottom:8px"><div>IgM ( Sigma ) secondary antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgM</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Roche SARS-CoV-2 anti-nucleocapsid antibody assay: Serum samples from PCR-confirmed cases were analysed using the commercial Elecsys® Anti-SARS-CoV-2 (Roche) in the Department of Microbiology, Infection Sciences, Southmead Hospital, North Bristol NHS Trust, Southmead Road, BS10 5NB, UK following manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-nucleocapsid</div><div>suggested: None</div></div><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">Transfected 293T cells were then infected with VSV*G-FLuc particles for 2 hours, washed with PBS, then incubated with fresh DMEM, supplemented with 10% FBS and 1:2000 (v/v) I1 (anti-VSV-G) antibody (absolute antibody Ab01401-10.3).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>I1</div><div>suggested: None</div></div><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">VSV-G-harbouring BHK21 cells were infected with VSV*ΔG- FLuc particles to generate complemented VSV*G-FLuc particles as previously described (35).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BHK21</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">, 293T cells were seeded and transiently transfected with a plasmid corresponding to the original Wuhan strain Spike protein (pCAGGS-S2-spike) using Turbofect transfection reagent (ThermoFisher R0532) for 16 hours following the manufacturer’s instructions.</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">Optimal pseudotype cell entry was achieved using VeroE6 cells stably expressing the human angiotensin-converting enzyme 2 (ACE2) receptor and the cell surface protease TMPRSS2 (Vero ACE2 TMPRSS2 (VAT) cells, which were a kind gift from Dr Suzannah Rihn, MRC-University of Glasgow Centre for Virus Research (36)</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><div style="margin-bottom:8px"><div>Vero ACE2</div><div>suggested: RRID:CVCL_A7UJ)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequence was synthesized with an NdeI restriction site at the 5’ end and the BamHI site at the 3’ end and cloned into pET28a expression vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET28a</div><div>suggested: RRID:Addgene_139598)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The recombinant plasmids (pET28a-NP-FL) were transformed into E. coli strain BL21 (DE3)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET28a-NP-FL</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, 293T cells were seeded and transiently transfected with a plasmid corresponding to the original Wuhan strain Spike protein (pCAGGS-S2-spike) using Turbofect transfection reagent (ThermoFisher R0532) for 16 hours following the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS-S2-spike</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">Images were acquired on the ImageXpress Pico Automated Cell Imaging System (Molecular Devices) using a 10X objective and infected cells detected and quantified using Cell ReporterXpress software (Molecular Devices).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Cell ReporterXpress</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical Analysis: Data analyses were performed using either R software with R Studio, and GraphPad Prism (version 9) as detailed below.</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">However, sample readouts using other methods including interpolated unit values (from a 4- parameter logistic regression model fit (on Prism or within BMG software) to the 7- point standard pool dilution series) and AUC from sample dilution series were used in the development stage.</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>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

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Strengths of this study include rigorous development of high performance, low blood volume, cost-effective tests which can be easily deployed in a variety of settings, but our approach also has several limitations. Firstly, whilst samples from pre-pandemic children were included, samples from children with COVID-19 were not available to us and as such, assay performance for detecting recent paediatric infections cannot be reported. However, since widespread vaccination of children is not currently common in many countries while asymptomatic/mild paediatric infections are, antibody assays offer a useful tool for monitoring infection in this age group. The antigens used in the in-house assays were generated using the genetic sequence from the parent Wuhan strain of SARS-CoV-2 first described in 2020 (7) from which several new variants of concern (VOC) have evolved and have caused significant waves of infection globally. Some of these variants, especially Omicron, include multiple mutations in these target antigens and as such, may lead to antibody responses with differential binding to the target antigens. Indeed, antibodies responses raised to antigens from one SARS-CoV-2 variant genetic sequence lead to differential ability to neutralise VOC strains. However, whilst others have shown reduced binding to antigens from sequences of VOCs, rates of seropositivity when using different antigens, and/or from people who were infected with non-Wuhan variants, appear to be relatively un...

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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Here's where things get spicy

See also https://hypothes.is/search?q=tag%3A%27%22algorithm%22+vs+%22heuristic%22%27

SciScore for 10.1101/2022.05.04.490614: (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">Staining antibodies are as follows (Hu Fc Block Pure Fc1.3216 (BD, Cat# 564220), APC anti-HLA-ABC (Thermofisher, Cat# 17- 9983-42), APC/Cy7 anti-HLA-DR (BioLegend, Cat# 307618), PE anti- DYKDDDDK Tag (BioLegend, Cat# 637309), AF488 anti-SARS-CoV-2 Spike S1 Subunit (R&D Systems,Cat# FAB105403G), FITC anti-Influenza A NP (Thermofisher, Cat# MA1-7322), PE anti-mouse CD45 (BioLegend, Cat# 109808), BV421 anti-mouse CD31 (BioLegend, Cat# 102423), APC anti-mouse EpCAM (BioLegend, Cat# 118213), PerCP/Cy5.5 anti-H-2Kb/H-2Db (BioLegend,Cat# 114620)).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-HLA-ABC</div><div>suggested: (Thermo Fisher Scientific Cat# 17-9983-41, RRID:AB_10753773)</div></div><div style="margin-bottom:8px"><div>anti-HLA-DR</div><div>suggested: (BioLegend Cat# 307618, RRID:AB_493586)</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-Influenza</div><div>suggested: (Thermo Fisher Scientific Cat# MA1-7322, RRID:AB_1017747)</div></div><div style="margin-bottom:8px"><div>anti-H-2Kb/H-2Db</div><div>suggested: (BioLegend Cat# 114620, RRID:AB_2750200)</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: pDONR207-SARS-CoV-2 E (#141273), pDONR207-SARS-CoV-2 M (#141274), pDONR207-SARS-CoV-2 ORF7a (#141276), pDONR223-SARS-CoV-2 ORF7b (#141277), pDONR223-SARS-CoV-2 ORF8 (#141278) were purchased from addgene (Kim et al., 2020) and used as templates for construction of plasmids expressing SARS-CoV-2 viral proteins.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDONR207-SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pDONR223-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">For HIV Nef expressing plasmid construction, NL4-3-dE-EGFP (kindly provided by Dr. Ya-Chi Ho) was used as a template.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NL4-3-dE-EGFP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For construction of plasmids expressing SARS-CoV viral proteins, oligonucleotides corresponding to both strands of SARS-CoV Tor2 (GenBank accession: NC_004718.3) ORF8a and ORF8b containing XhoI and BamHI sites at the 5’ and 3’ ends were synthesized (IDT) and cloned into XhoI-BamHI site of c-Flag pcDNA3 vector.</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">To investigate the prevalence of amino acid mutations, we downloaded up to 965 sequences of each lineage and aligned the ORF8 nucleotide sequences using Jalview software (http://www.jalview.org/) (Waterhouse et al. Bioinformatics. 2009) by MUSCLE algorithm (Edgar RC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jalview</div><div>suggested: (Jalview, RRID:SCR_006459)</div></div><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">FlowJo software (Tree Star) was used for the data analysis.</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>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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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>

SciScore for 10.1101/2022.05.03.490428: (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: Human convalescent serum samples: Human convalescent serum samples from recovered COVID-19 patients were obtained from Public Health Clinical Center of Chengdu in Chengdu, China, under approved guidelines by the Institutional Review Board (IRB), and all patients had provided written informed consent before serum sample were collected.<br>Consent: Human convalescent serum samples: Human convalescent serum samples from recovered COVID-19 patients were obtained from Public Health Clinical Center of Chengdu in Chengdu, China, under approved guidelines by the Institutional Review Board (IRB), and all patients had provided written informed consent before serum sample 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">Animal studies, facilities and ethics statements: Specific pathogen-free (SPF) BALB/c female mice (6-8 weeks old) for immunogenicity studies were purchased from Charles River Experimental Animals Co.,</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">Serum was collected on D35 (2 weeks PD2), D56 (Day of 3rd dose boost), D85 (1 month post dose 3), D113 (2 months post dose3) and D141 (3 months post dose 3) for pseudovirus neutralizing antibody test.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>D56</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>D85</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>D113</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>D141</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">Pseudovirions were produced by co-transfection HEK 293T cells with psPAX2, pLVX-AcGFP-N1-Fluc, and plasmids encoding various S genes by using Lipofectamine 3000 (Invitrogen, L3000-015).</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">Pseudoviruses stock were titrated by infecting 293T-ACE2 cells and luciferase activity was determined following a 44-48 h incubation period at 37°C and 5% CO2 by addition Bright-Glo Luciferase Assay System (Promega, E2650) using a microplate reader (TECAN, Spark).</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">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 three dose boost study, Balb/c mice, female (n=10/group) prime and boost with SCB-2019 3 μg adjuvanted with 75 μg alum plus 150 μg CpG 1018 twice on Day 0 and Day 21, then boosted with 3 μg SCB-2019, or SCB-2022B or Bivalent adjuvanted with 75 μg alum plus 150 μg CpG 1018 on Day 57 via intramuscular injection.</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 cDNA was subcloned into pTRIMER expression vector (GenHunter Corporation) at Hind III and Bgl II sites to allow in-frame fusion of the soluble S protein to Trimer-Tag (amino acid residue 1156-1406 from human Type I(α) collagen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTRIMER</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus construction and production: The variants of concern of SARS-CoV-2 spike protein genes were optimized using mammalian codon and synthesized by Genscript, then cloned into pcDNA3.1(+) eukaryotic expression 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_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirions were produced by co-transfection HEK 293T cells with psPAX2, pLVX-AcGFP-N1-Fluc, and plasmids encoding various S genes by using Lipofectamine 3000 (Invitrogen, L3000-015).</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>pLVX-AcGFP-N1-Fluc</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: Data arrangement was performed by Excel and statistical analyses were performed using the Prism 9.2.0 (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Excel</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<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">NCT04405908</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Completed</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">SCB-2019 as COVID-19 Vaccine</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04672395</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 Controlled Phase 2/3 Study of Adjuvanted Recombinant SARS-…</td></tr></table>

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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I argue that PHP is not reasonable here. The only appropriate thing for this use case is (unminified) JS—or some other program text encoded as a document resource permitting introspection and that the user agent just happens to be able to execute/simulate.*

• Just like the advocates of "a little jQuery", author here doesn't seem to realize that the use of PHP was the first step towards what is widely acknowledged to be messed up about the "modern" Web. People can pine for the days of simple server-side rendering, but there's no use denying that today's Web is the natural result of an outgrowth that began with abuses of the fundamental mechanisms underpinning the Web—abuses that first took root with PHP.

* Refer to the fourth and sixth laws of "Sane Personal Computing, esp. re "reveals purpose"

Controversial opinion: this is actually closer to doing the Web the way that it should be done, taking the intent of its design into account. NB: this is not exculpatory of minified JS bundles (where "megabyte" is the appropriate unit order of magnitude for measuring their weight) or anything about "modern" SPAs that thumb their nose at graceful degradation.

Most people born since the advent of the filing cabinet and the computer have spent a lifetime using a hierarchical folder-based mental model for their knowledge. For greater value and efficiency one needs to get away from this model and move toward linking individual ideas together in ways that they can more easily be re-used.

To accomplish this many people use an index-based method that uses topical or subject headings which can be useful. However after even a few years of utilizing a generic tag (science for example) it may become overwhelmed and generally useless in a broad search. Even switching to narrower sub-headings (physics, biology, chemistry) may show the same effect. As a result one will increasingly need to spend time and effort to maintain and work at this sort of taxonomical system.

The better option is to directly link related ideas to each other. Each atomic idea will have a much more limited set of links to other ideas which will create a much more valuable set of interlinks for later use. Limiting your links at this level will be incredibly more useful over time.

One of the biggest benefits of the physical system used by Niklas Luhmann was that each card was required to be placed next to at least one card in a branching tree of knowledge (or a whole new branch had to be created.) Though he often noted links to other atomic ideas there was at least a minimum link of one on every idea in the system.

For those who have difficulty deciding where to place a new idea within their system, it can certainly be helpful to add a few broad keywords of the type one might put into an index. This may help you in linking your individual ideas as you can do a search of one or more of your keywords to narrow down the existing ones within your collection. This may help you link your new idea to one or more of those already in your system. This method may be even more useful and helpful for those who are starting out and have fewer than 500-1000 notes in their system and have even less to link their new atomic ideas to.

For those who have graphical systems, it may be helpful to look for one or two individual "tags" in a graph structure to visually see the number of first degree notes that link to them as a means of creating links between atomic ideas.

To have a better idea of a hierarchy of value within these ideas, it may help to have some names and delineate this hierarchy of potential links. Perhaps we might borrow some well ideas from library and information science to guide us? There's a system in library science that uses a hierarchical set up using the phrases: "broader terms", "narrower terms", "related terms", and "used for" (think alias or also known as) for cataloging books and related materials.

We might try using tags or index-like links in each of these levels to become more specific, but let's append "connected atomic ideas" to the bottom of the list.

Here's an example:

• broader terms (BT): [[physics]]
• narrower terms (NT): [[mechanics]], [[dynamics]]
• related terms (RT): [[acceleration]], [[velocity]]
• used for (UF) or aliases:
• connected atomic ideas: [[force = mass * acceleration]], [[$$v^2=v_0^2​+2aΔx$$]]

Chances are that within a particular text, one's notes may connect and interrelate to each other quite easily, but it's important to also link those ideas to other ideas that are already in your pre-existing body of knowledge.

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See also: Thesaurus for Graphic Materials I: Subject Terms (TGM I) https://www.loc.gov/rr/print/tgm1/ic.html

Author Response

*Reviewer #2 (Public Review):

This manuscript describes studies on the structural determinants of activation for the adhesion GPCR (aGPCR) GPR116 both in vitro and in vivo. The authors define key residues for activation on the receptors' N-terminus (the "tethered agonist") and the extracellular loops. Thus, the studies provide novel insights into the structural determinants of GPR116 activation. However, some interpretational issues (detailed below) complicate some of the authors' conclusions. Specific comments are as follows:

1. Results section, first paragraph, last sentence: The authors write, "These results taken together indicate that the H991A mutant is capable of proper trafficking to the membrane, is able to response to exogenous peptide, but is unable to be cleaved and activated by endogenous ligands in vivo." The last part of this sentence represents an over-interpretation, as the data shown in Figure 1 do NOT show that the non-cleavable receptor is unable to be activated by endogenous ligands in vivo. It is entirely conceivable that a non-cleavable aGPCR could still be activated by endogenous adhesive ligands if those ligands were to change the position of the tethered agonist in manner that alters receptor signaling activity.

Thank you for highlighting this misleading wording. We rephrased the sentence to read as follows: Taken together, these results demonstrate that the H991 residue within the GAIN domain is critical for cleavage of GPR116 into NTF and CTF fragments but dispensable for trafficking of the receptor to the plasma membrane and response to exogenous peptide activation in vitro.

1. The data shown in Fig. 1B (surface expression of non-cleavable H991A mutant) need to be quantified in some way in order to be interpretable.

As the H991A construct does not contain a cell surface epitope tag, it is difficult to directly quantitate surface expression of this protein. The data in transiently transfected HEK293 cells (Figure 1, panels C and D) and in primary alveolar epithelial cells (Figure 2, panels C&D) clearly demonstrate that the H991A mutant is activated to comparable levels as the wild-type receptor in response to exogenous peptide stimulation. In light of these functional data, we are confident that the surface expression of H991A is comparable to that of the WT receptor in vitro and in vivo.

1. Results section, second paragraph, penultimate sentence: The authors write, "These data demonstrate that while the non-cleavable receptor is fully activated in vitro by exogenous peptides corresponding to the tethered agonist sequence, cleavage of the receptor and unmasking of the tethered agonist sequence is critical for GPR116 activation in vivo." However, the non-cleavable GPR116 mutant actually has two key differences from WT: i) lack of full liberation of the tethered agonist sequence, and ii) lack of liberation of a free NTF, which might dissociate from the CTF and have important in vivo physiological actions on its own. Isn't it conceivable that the lack of a freely mobile NTF contributes to the similarity in lung phenotype between the non-cleavable knock-in mutant and the GPR116 knockout? Based on the data shown in Figure 2, how can the authors claim these data demonstrate that unmasking of the tethered agonist is critical for GPR116 activation? The data could equally be interpreted as showing that liberation of a free NTF is critical for the physiological effects of GPR116 in vivo.

We thank the reviewer for this comment and, in retrospect, agree that we may have overstated the interpretation of our results for the H991A transgenic mouse. While it is possible that the free NTF may be responsible for the physiological effects of GPR116 in vivo, in light of recently published data by Mitgau et al. (BioRxiv https://doi.org/10.1101/2021.09.13.460127), we believe this not to be the case for the following reasons. First, the H991A and WT receptors are activated to an identical level by exogenous peptide stimulation in a transformed cell line (HEK293) and in primary alveolar type 2 epithelial cells (Figures 1 and 2), irrespective of if the NTF is free floating in solution in the context of the WT receptor. These data would argue against a role of the free NTF in receptor activity. Second, in a recent publication by Mitgau et al., the authors clearly demonstrate that activation of GPR126, an adhesion GPCR that is also cleaved at the GPS and activated by exogenous peptides corresponding to the tethered agonist, by antibodies that bind and crosslink the NTF is completely dependent on cleavage at the GPS. They further demonstrate that antibody-mediated activation does not lead to liberation of the NTF from the CTF. Rather, they postulate that proper GPS processing, as occurs for the WT receptor, leads to a favorable protein confirmation of the tethered agonist, which is indispensable for GPR126 activity. Given these results, we postulate that cleavage at the GPS of WT GPR116 results in a conformation that is critical for the tethered agonist sequence to reach and bind the ECLs, resulting in activation of the receptor, similar to that observed with GPR126. We have edited our interpretation of these data in the revised manuscript.

1. Figure 3: If the authors' hypothesis is that the tethered agonist must be liberated in order to allow activation of GPR116, then why do ANY of the Flag-tagged mutant constructs exhibit constitutive signaling activity? Doesn't the N-terminal Flag tag prevent the tethered agonist from being exposed? How can these data be reconciled with the authors' model?

It is unlikely that the 27 amino acid N-terminal FLAG epitope tag envelopes the tethered agonistic peptide to the same extent as the tertiary structure of the carboxy terminus of the NTF (based on published structures for other aGPCRs). Additionally, we provided data demonstrating that an untagged version of the CTF protein is activated to a similar extent at FLAG-tagged CTF in response to activating peptides (Supplemental Figure 2A). Based on our data from mutagenesis experiments and modeling of GPR116 with the agonist, we do not believe the tethered agonist dives deeply within the binding pocket but rather interacts with critical amino acids at the surface of ECL2 to induce conformational changes to the receptor and downstream activation.

1. The data shown in Fig. 3D are lacking statistical comparisons, so it is not possible to tell whether any of the differences between the mutants are statistically significant.

Statistical analyses for data in this panel have been added

1. The data shown in Fig. 4D (surface expression of the ECL mutants) need to be quantified in some way.

We have added additional data to this figure (Fig4 F-G-H) using the V5-tagged mFL construct as control. As the tag is C-terminal, we quantified by flow cytometry the total expression using an anti-V5 antibody, to complement to immunocytochemistry data showing membrane expression.

1. In interpreting the results of the ECL mutations on GPR116 signaling activity, it is unclear why the authors so explicitly propose that these data demonstrate that the tethered agonist must be interacting with ECL2. Isn't it possible that ECL2 mutants with impaired receptor signaling activity simply lock the receptor in an inactive state? In this way, the effects of the ECL2 mutations could be explained without invoking a physical interaction between the putative tethered agonist and ECL2.

Yes, this interpretation is also possible. We have rephrased the Results and Discussion sections accordingly to reflect this possibility.

SciScore for 10.1101/2022.04.28.489772: (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">Euthanasia Agents: 2 variant at 100 TCID50/mouse under isoflurane anesthesia.<br>IACUC: All procedures were performed according to the animal study protocols approved by the FDA White Oak Animal Program Animal Care and Use Committee.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">In the ABSL-3 lab, K18-hACE2 mice were randomly grouped and were inoculated intranasally with NY (G614), Delta, Omicron BA.1 or Omicron BA.</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 (58).</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">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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For antibody staining, an Alexa Fluor 647 conjugated donkey anti-human IgG Fc F(ab’)2 fragment (Jackson ImmunoResearch, West Grove, PA) was used as secondary antibody at 5 μg/ml concentration.</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">After washing, plates were probed with 1 μg/ml of inhouse developed rabbit polyclonal antibody specific for SARS-CoV-2 membrane/nucleocapsid (33) at 4°C overnight followed by peroxidase-conjugated goat anti-rabbit secondary antibody (SeraCare #5220-0336, 1:2000) for 2h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: (SeraCare KPL Cat# 5220-0336, RRID:AB_2857917)</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">Murine Leukemia Virus (MLV) particles (plasmids of the MLV components kindly provided by Dr. Gary Whittaker at Cornell University and Drs. Catherine Chen and Wei Zheng at National Center for Advancing Translational Sciences, National Institutes of Health), pseudotyped with various SARS-CoV-2 S protein constructs, were generated in HEK 293T cells, following a protocol described previously for SARS-CoV (59, 60).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 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">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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Seed viruses were amplified in Vero E6 (ATCC CRL-1586) or Vero E6 with TMPRSS2 overexpression (BPS Bioscience #78081)</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">In vitro virus replication and focus-forming assay: Vero-E6 cells were pre-seeded in 12-well tissue culture plates overnight and were infected with authentic viruses (G614, Delta, Omicron BA.1 or BA.2) at MOI of 0.01 in Gibco™ high glucose DMEM containing 3% 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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 10-fold serially diluted postinfection were added at 100 μl/well to Vero E6-TMPRSS2 cells pre-seeded in 96-well tissue culture plates.</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">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 study: Hemizygous B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE2</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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In the ABSL-3 lab, K18-hACE2 mice were randomly grouped and were inoculated intranasally with NY (G614), Delta, Omicron BA.1 or Omicron BA.</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">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,</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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Serially diluted pCMV6-AC-ACE2-GFP plasmid or pCC1-CoV2-F7 plasmid expressing SARS-CoV-2 N (62) was used to construct a standard curve.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV6-AC-ACE2-GFP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCC1-CoV2-F7</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 KD was obtained by fitting Req value and its corresponding concentration to the model: “one site-specific” using GraphPad Prism 8.0.2 according to H.J. Motulsky, Prism 5 Statistics Guide, 2007, 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">Automated data collection was carried out using SerialEM version 3.8.6 (63) 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 13.761 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">Local resolution was also determined using cryoSPARC.</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">Several rounds of manual building were performed in Coot.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Iteratively, refinement was performed in both Phenix (real space refinement) and ISOLDE (66), and the Phenix refinement strategy included minimization_global, local_grid_search, and adp, with rotamer, Ramachandran, and reference-model restraints, using 7KRQ and 7KRR as the reference 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></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

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

Results from rtransparent:

---

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2022.04.22.22274032: (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: Subjects or households with suspected or confirmed SARS-CoV-2 infection were recruited from the Greater New Orleans community under Tulane Biomedical Institutional Review Board (federalwide assurance number FWA00002055, under study number 2020-585).</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">Determination of antigen-specific antibody reactivity by multiplexed Luminex analysis: Recombinant SARS-CoV-2 antigens (full-length spike, RBD, and N) and the recombinant spike protein from OC43, HKU1, 229E, and NL63 (Frederick National Laboratories) were coupled with MagPlex beads (Luminex) via sulfo-NHS coupling chemistry.</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><div style="margin-bottom:8px"><div>HKU1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Spike protein ELISA for IgG antibodies has been validated by testing a standard set of positive and negative samples provided by NCI SeroNet staff.</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">NK92 cells in complete alphaMEM culture medium were added at 5 × 104 cells/well in the presence of 4 µg/ml brefeldin A (Biolegend Cat# 420601), 5 µg/ml GolgiStop (BD Biosciences Cat# 554724) and 0.15µg of anti-CD107a antibody (Clone H4A3 PE-Cy7, Biolegend Cat# 328618) for 5 hours at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-CD107a</div><div>suggested: (BioLegend Cat# 328618, RRID:AB_11147955)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody-dependent neutrophil phagocytosis (ADNP): Protocol was adapted from [72]</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Antibody-dependent neutrophil phagocytosis</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Beads were washed with PBS containing 15 mM EDTA and stained with an FITC-conjugated anti-guinea pig C3 antibody (MP Biomedicals).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-guinea pig C3</div><div>suggested: None</div></div></td></tr><tr><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">Neutralization of SARS CoV-2 in Pseudovirus Assay: CHO cells were generated and stably expressed ACE2 by transfecting CHO cells with an ACE2 expression plasmid containing the blasticidin resistance gene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO</div><div>suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CHO-ACE2 cells were similar in SARS CoV-2 susceptibility to the 293T/ACE2 cell line developed by Dr.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Virus neutralization was measured in CHO/ACE2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO/ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudoviruses were produced by co-transfection of the four plasmids into 293T cells grown in T75 flasks with Fugene 6 as transfection reagent.</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">Unbound antibodies were removed by centrifugation before adding THP-1 cells at 2.5×104 cells/well.</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><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">RBD (aa321-535) was similarly expressed in the phCMV plasmid and purified on Streptactin X affinity columns.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>phCMV</div><div>suggested: RRID:Addgene_15802)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A DNA fragment encoding SARS CoV-2 N protein, including its natural leader sequence was generated by PCR of full-length N protein gene from a lentiviral N Protein expression vector (pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro, which was a gift from Nevan Krogan (Addgene plasmid # 141391 ; http://n2t.net/addgene:141391; RRID:Addgene_141391, [68]).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_141391)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These included an expression plasmid for full-length spike protein of the Wuhan-1 strain containing the D614G amino acid chain (VRC7480.G614) [70], a pCMV ΔR8.2 lentivirus backbone plasmid (VRC5602) [71], the VRC5601 plasmid pHR’ CMV Luc containing the firefly luciferase reporter gene [71], and VRC9260 for TMPRSS2 expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV ΔR8.2 lentivirus</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>VRC5601</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">Neutralization titers were defined as the serum dilution (ID50) at which relative luminescence units (RLU) were reduced by 50% compared to virus control wells after subtraction of background RLUs (determined by GraphPad Prism, version 9 for macOS, GraphPad Software, San Diego, California USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Spike Glycoprotein (stabilized) from SARS-Related Coronavirus 2, Wuhan-Hu-1 with C-Terminal Histidine Tag, Recombinant from Baculovirus), and SARS-CoV-2 specific mega pools at 0.2 μg/well including PepTivator SARS-CoV-2 Prot_S (Miltinyi - 130-126-700), SARS-CoV-2 Prot_M (130-126-702), SARS-CoV-2 Prot_N (130-126-699) in 96-well U bottom tissue culture plate (CytoOne CC7672-7596) in 200 μl RPMI-1640 with 10% FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PepTivator</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">) GraphPad Prism (version 9.0.0, GraphPad Software, San Diego, CA), JMP (version 16.2.0, SAS Institute, 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">, Cary, NC), and SAS (version 9.4, SAS Institute, Inc.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SAS Institute</div><div>suggested: (Statistical Analysis System, RRID:SCR_008567)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

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Study limitations primarily involved using SARS-CoV-2 infection to differentiate subjects rather than pre-pandemic samples. In addition, the assays were limited to peripheral blood samples and not tissue-specific responses, which included only effector functions to spike protein and cytokine secretion instead of T-cell subset analyses. Detection of secreted cytokines allowed a greater number of cytokines to be evaluated but prevented confirmation of cells producing cytokines as would be observed intracellular stained cytokines for specific T-cell populations. However, cytokines between spike or peptide pools were highly correlated (Figure S5), indicating T-cell production. Also, high expression of IL-2 has been routinely observed from CD4+ T-cell and not CD8+ T-cells after SARS-CoV-2 infection [28, 38]. In this study, IL-17A secretion was closely correlated to IL-2 and Th1 cytokine release after stimulation with protein or peptide pools, suggesting that IL-17A may be serving as a proxy for a Th1/Th17 subset, as identified in other post-vaccination studies [61] which should be more closely examined. Finally, while the critical role for age in SARS-CoV-2 immunity was validated, it remains an ongoing question of why children exhibit less severity with infection and how differences in qualitative features of immunity depend on patient age. Our study used samples collected from subjects only shortly after the pandemic which will be difficult to perform as COVID subsides and vaccin...

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

---

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2022.04.21.489021: (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 cells were incubated with 100μM of the respective peptide and 1:200 dilution of rabbit anti-HA tag antibody (Sigma; catalogue no. Cat # H6908) at 4°C for 1hr.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-HA</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 1:200 dilution of goat anti-rabbit Alexa fluor-647 antibody (Invitrogen) and 1:100 dilution of neutravidin fluorescein conjugate (Invitrogen; FITC, catalogue no. A2662, used for the first FACS) at 4°C for 30min.</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>antibody (Invitrogen)</div><div>suggested: (Rockland Cat# 00-8844-25, RRID:AB_2610705)</div></div><div style="margin-bottom:8px"><div>30min</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 1:500 dilution of mouse anti-FLAG monoclonal antibody (Merck, catalogue no. F3165) 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></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The blots were probed with 1:5000 anti Flag antibody (Sigma, F3165) overnight at 4□C followed by 1:10000 anti-mouse secondary at room temperature for 1hr.</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</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The surface of the well was washed twice with blocking buffer and incubated with HRP-conjugated rabbit anti-6xHis tag antibody (Abcam; catalogue no. AB1187), 1:10,000 dilution at 4°C overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-6xHis tag</div><div>suggested: (Abcam Cat# ab1187, RRID:AB_298652)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">eGFP-ACE2/HEK293T immunostained with FLAG antibody was imaged at 60x oil objective of FV3000 confocal microscope (Figure 2B) and 100x oil objective of H-TIRF microscope (Supplementary Figure 2A) using 405nm, 488nm and 647nm laser lines for DAPI, eGFP, and Alexa fluor-647 fluorophores.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>eGFP</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 experiments: Wild type mammalian HEK293T cells and LentiX-293T cells (Takara Bio, catalogue no. 632180) were used in this study for pseudotyped spike virus production and viral transduction assay.</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>LentiX-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Caco2 cells were lysed for total RNA purification using the Trizol method.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Caco2</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">Pseudoviral transduction assay: The GFP/HEK293T cells or eGFP-ACE2/HEK293T cells were grown up to 60-70% confluency in complete media before viral transduction.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GFP/HEK293T</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>eGFP-ACE2/HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In this assay, the viral titre was used in a concentration such that to obtain more than 70% transduction efficiency in the eGFP-ACE2/HEK293T or eGFP/HEK293T cell line.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>eGFP/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">Cloning and protein purification: The nanobody gene was amplified from isolated yeast colonies and cloned between HindIII and XhoI sites in a pET-22b(+) plasmid containing a C-terminal 6x histidine tag.</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">For viral particle production, 5 µg pHR lentiviral vector cloned with mCherry fluorescent protein, 3.75 µg packaging plasmid psPAX2 (Addgene; #12260), and 2.5 µg envelope plasmid for the expression of Spike glycoprotein (obtained as a kind gift from Prof. Nevan Krogan, UCSF, USA) of SARS-CoV-2 were mixed in 500 µl OptiMEM media and 20 µl PLUS reagent (Invitrogen; LTX transfection reagent, catalogue no. L15338100) and kept for incubation at room temperature for 5min.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHR</div><div>suggested: RRID:Addgene_16514)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We also generated control lentiviral particles by replacing Spike plasmid with VSV-G envelope protein, pmDG2 (Addgene; #12259) plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pmDG2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Omicron pseudotyped virus production: Omicron pseudotyped viruses were produced similarly as described above for spike pseudoviruses but instead used omicron envelope plasmid along with packaging plasmid (psPAX2) and lentiviral plasmid (pHR mCherry) in the following ratio: psPAX2 (1.3pmol), pHR mCherry: 1.64pmol, SARS-CoV-2 Omicron Strain S gene (Genscript, Cat # MC_0101274): 0.72pmol.</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></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This construct contains amino-terminus EGFP followed by self-cleaving 2A peptide sequence followed by ACE2 and carboxy-termini SNAP-tag and FLAG tags (eGFP-ACE2/HEK293T) Generation of stable HEK293T cell line for over-expression of ACE2: The lentiviral pTRIP vector cloned with eGFP-ACE2/HEK293T under CMV enhancer and chicken β-actin promoter (CAG promoter) flanked with 5′and 3′ long terminal repeat (LTR) sequences (39), were used to produce lentiviral particles as per the method described before (35).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTRIP</div><div>suggested: RRID:Addgene_127663)</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 peptide sequences from the receptor-binding domain (RBD) of the spike were synthesized from LifeTein with a biotin tag: Peptide-1: [FNCYFPLQS]S-K-Biotin Peptide-2: Biotin-[GFQPTNGVGY] Sequence Alignment for Covid Variants The hCoV19 spike (Wuhan/WIV04/2019), GISAID (EPI_ISL_402124) construct is a kind gift from Prof. Nevan Krogan, UCSF, USA (38).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>LifeTein</div><div>suggested: (LifeTein, RRID:SCR_012626)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For viral particle production, 5 µg pHR lentiviral vector cloned with mCherry fluorescent protein, 3.75 µg packaging plasmid psPAX2 (Addgene; #12260), and 2.5 µg envelope plasmid for the expression of Spike glycoprotein (obtained as a kind gift from Prof. Nevan Krogan, UCSF, USA) of SARS-CoV-2 were mixed in 500 µl OptiMEM media and 20 µl PLUS reagent (Invitrogen; LTX transfection reagent, catalogue no. L15338100) and kept for incubation at room temperature for 5min.</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">Images were analysed on Fiji software to calculate mean fluorescence intensity (MFI) for eGFP (ACE2 expression) and mCherry (viral transduction) channel from the z-projected stacks.</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></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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Author Response:

Reviewer #2:

The authors investigated changes in the unstressed and stressed oligomeric states of the mammalian endoplasmic reticulum (ER) stress sensor, IRE1a. Previous biochemical and microscopy studies in mammalian cells and studies of the related protein Ire1 in yeast, describe an increase in oligomerization of the stress sensor upon treatment of cells with chemical agents that impair the ER protein folding environment. The general view has been that IRE1 in unstressed cells is a monomer and varying degrees of misfolded protein stress stimulate dimerization, activation, and higher order oligomerization. Distinguishing between monomers and dimers, as well as tetramers or other small oligomers is technically challenging, especially for integral membrane proteins. To address this challenge, the authors turned to single particle tracking fluorescence microscopy of Halo-tagged endogenous IRE1. Using a clever combination of random labeling with two fluorescent dyes and oblique angle illumination to visualize single molecules, as well as dimers, the authors surprisingly find that their endogenous IRE1 reporter appears to be dimeric in homeostatic cells. This observation challenges the predominant model in which IRE1 is monomeric in unstressed cells and that even dimerization represents a switch into an active state. The authors claim to detect evidence for higher order oligomers following treatment with stressors. The authors then use a series of IRE1 mutants to identify how oligomerization is regulated and present a new model to reconcile the different models of IRE1 activation in the literature.

The authors have extensively characterized their novel experimental system in terms of protein expression levels, functionality, and ability to distinguish monomers and dimers. The data are well presented and the authors are clearly familiar with the arguments that have surrounded the IRE1 oligomer question. That the authors observe the characteristic XBP1 mRNA splicing activity in the absence of visible large IRE1 clusters may suggest that the large clusters reported by others may have distinct roles, perhaps in more permissive mRNA cleavage.

The present study is undermined by two major weaknesses. First, while the authors persuasively demonstrate that they can detect IRE1a dimers, a major claim of the manuscript rests upon detection of tetramers and possibly higher order oligomers. Unfortunately, the authors provide no independent controls to show what tetramer or higher order oligomer data would look like. Thus, the authors can only infer that higher order oligomers are detected, based on modest shifts in the percent of correlated particle trajectories observed in some cells. More robust evidence is needed to make claims of oligomerization. Tools have been developed by others that can induce reversible oligomerization of proteins. Application of these tools would provide powerful controls for tetramers or even higher order oligomers in this study.

The second, deeper concern, is the discrepancy between the Halo Tag clustering results in this study and studies by this lab and several other labs that report a distinct stress phenotype. In mammalian cells and yeast, IRE1 and Ire1, tagged with different fluorescent proteins or even a small HA peptide epitope tag, undergo quantitative visible formation of puncta or clusters upon treatment with stressors. The small number of bright clusters that form effectively deplete the rest of the ER of IRE1 signal. In the present study, the authors observe no visible change in IRE1-Halo localization in stress cells. The authors do not investigate the cause of this difference. While one might argue that the presence of stress-inducible IRE1 activity is sufficient to argue that the reporter in this study is functional, IRE1 reporters (that do cluster) described in previous studies by the Walter lab and other groups are also demonstrably functional. Does IRE1 normally cluster? Is it cell-type dependent? Tag-dependent? Notably, the Pincus et al. PLoS Biology paper from the Walter lab used two different fluorescent protein tags that do not heterozygously dimerize. Robust colocalization and FRET signals were detected upon treatment of cells with stressors and clustering was subsequently observed. A 2007 Journal of Cell Biology study from Kimata et al. reported clustering in yeast with an Ire1 tagged with an HA epitope peptide. The HA peptide seems unlikely to be prone to any oligomerization propensities that GFP tagged reporters might experience. Importantly, a 2020 PNAS paper from the Walter lab (Belyy et al.) studied clustering of a robustly monomeric mNeonGreen-tagged IRE1 in U2-OS cells and mouse embryonic fibroblasts and this construct readily clustered following stress induction.

When evaluated against the backdrop of the extensive literature describing the visual behavior of IRE1a in live cells, the absence of stress-induced clustering is both puzzling and disconcerting. Given the focus of this study is to use visual techniques to study IRE1a interactions, the burden of proof is on the authors to resolve this significant discrepancy with the rest of the IRE1a literature. One can easily imagine that incorporation of the majority of the pool of IRE1a into 10-100 clusters could produce very different correlated trajectory behavior. Until the authors can determine why their reporters behave differently from other IRE1a reporters and establish which version accurately reflects physiologic IRE1a behavior, the potential impact of the findings of this manuscript are of unknown value.

We thank the reviewer for this detailed assessment of our work. We agree that the question of apparent discrepancy in the formation of observable IRE1 clusters between this manuscript and earlier work is important. We have now addressed this issue both in the revised version of the manuscript and in specific point-by-point responses to reviewers’ comments. As a brief summary, we addressed the reviewer’s first concern (lack of controls larger than dimers) by cloning and validating a tetrameric HaloTag construct, the measurements from which were entirely consistent with the model we presented in the original version of the manuscript. To address the reviewer’s second concern, we present several lines of evidence showing that the discrepancy between the formation of microscopically visible IRE1 clusters in earlier studies and the absence of such clusters in the present work almost certainly results from differences in expression levels. First, our IRE1-HaloTag construct is perfectly capable of forming stress- induced clusters, as we show in the new Figure 1 – Figure Supplement 3. Second, we point to a parallel study by Gómez-Puerta et al., who demonstrate that a more “conventional” IRE1-GFP construct does not form visible stress-dependent puncta when it is expressed at a low level comparable to that of untagged IRE1 in HeLa cells, despite being fully active. Third, our earlier work in the 2020 PNAS paper referenced by the reviewer actually showed that even in the overexpression context, IRE1-mNeonGreen only forms visible puncta in just over half of all cells, despite the fact that XBP1 processing is nearly 100% effective in bulk assays. Furthermore, in the same paper we show that, rather than all IRE1 molecules being sequestered in clusters, only a small fraction (~5%) of IRE1-mNeonGreen assembles into large puncta while the remaining 95% of IRE1 stays uniformly distributed throughout the ER. Taken together, we believe that IRE1 does have the propensity to assemble into larger clusters when its expression levels are high (regardless of the tag used), but that these clusters are not strictly required for its activation. We have made significant changes to the discussion section of the manuscript to clarify the above points and directly address the apparent discrepancy between the present work and earlier studies.

Reviewer #3:

In this paper, the authors' aim was to test how IRE1's oligomerization state relates to its activation status without relying on ectopic overexpression. The principle underlying the work is a rather simple one, which is that, if the population of IRE1 can be labeled stochastically with either of two different fluorescent probes, then if the protein dimerizes, presuming single molecules can be visualized, correlated migration of a spot of each fluorophore should be observed for some of those dimers. Any correlated migration, maintained for long enough, will by necessity by some sort of dimer or multimer. In principle, if my math is right, the correlation should be 50% of spots of each color, assuming all the molecules are in a dimer, all molecules are labeled with one fluorophore or the other, and the koff of the fluorophores is very low. In practice, the correlation appears closer to 10%, which the authors establish using a control molecule that should not dimerize except by chance, and another for which pseudo-dimerization is enforced due to the two HALO domains used to bind the fluorophores being conjugated to the same molecule in cis. Much of the paper is devoted to establishing the fundamentals of the system. For these experiments, the authors replaced endogenous IRE1 with the HALO-tagged version to generate near-normal expression and show that the IRE1-HALO behaves similarly to endogenous. They also show that correlated migration is observed in the dimer control to a much greater extent than in the monomer.

Using these findings, they demonstrate, in my mind quite conclusively, that IRE1 exists as a dimer even in the unstimulated state. During ER stress, the authors observe a state that is more highly ordered. Mathematical modeling suggests a transition from predominantly dimers to a mix of dimers and something more highly ordered, with tetramers being the simplest explanation. Satisfyingly, a mutation that breaks the known dimer interface causes the protein to exist solely in monomers, as does deletion of the IRE1 lumenal domain, while disrupting the oligomerization interface keeps the protein as dimers. Mutation or deletion of the kinase and RNase domains does not affect higher order status, suggesting that activation of these domains is not a prerequisite for assembly. It is clear from this that the central claims of the paper, which is that IRE1 exists in a dimer in the basal state and transitions to a higher ordered structure in the activated state, are supported. Moreover, the general approach is likely to be appealing to the study of other molecules activated by multimerization.

We thank the reviewer for this thoughtful and helpful analysis of our work.

The principal advance of the paper is the technological approach for tracking IRE1 (and, presumably, other molecules whose activity is regulated by dimerization). The approach is quite elegant for that purpose. Its impact in terms of conclusions about IRE1 is perhaps less clear. The authors rationalize their endogenous-replacement approach by describing how their previous efforts and those of others relied on ectopic overexpression of GFP-tagged IRE1. The authors take great pains to claim that the observed multimerization status of the IRE1-HALO constructs is not a function of expression level, which would imply then that expression level alone is not responsible for the previously observed IRE1 oligomeric puncta. It is not clear why exactly the authors' results differ from this group's previous studies on the topic nor where the truth lies, including whether something inherent to the GFP-tagged overexpression approach favors non-physiologic structures, whether the difference is fundamentally one of cell type, or whether multimerization and activation are correlated but not causally related, with multimer-breaking mutations killing IRE1 by some other mechanism.

The question of reconciling our present data with earlier work (including work from our group) is clearly and understandably a central question for all three reviewers. As we detailed above in our responses to reviewers 1 and 2, we are convinced that the formation of large IRE1 clusters is largely dependent on expression level rather than the differences between fluorescent protein tags and the HaloTag. We added new supplementary figures and substantially revised the text of the manuscript to address this question directly.

Interpreting the data is also complicated by the fact that, while the authors point out that the percent of correlated trajectories (i.e., the measurement of multimerization state) does not itself correlate with expression level (using trajectories-per-movie as a proxy), the proper conclusion from that lack of correlation is not that variance in expression level does not account for the changes in apparent multimerization status, but instead that it cannot be the only factor. In some sense, the authors are attempting to play the argument both ways, by arguing that expression level matters for IRE1 activation (from previous studies) and that it doesn't (from this study). I think to address this the authors will need to better account, one way or another, for why the findings presented here differ from their previous findings and why these are the more salient (if in fact they are).

This is a very important point, and we thank the reviewer for raising it. We are not arguing that expression levels do not matter for the formation of oligomers; quite the contrary, as detailed above and in the revised version of the text, we believe that the formation of massive IRE1 oligomers observed in previous studies and in the new Figure 1 – Figure Supplement 3 is mainly a function of elevated concentration. What we do claim is that our approach can reliably pick out oligomeric differences within the relatively narrow range of concentrations used for single-particle tracking experiments in this paper. We are using the very weak truncated CMVd3 promoter in all transient transfection experiments, and we are only analyzing data from cells that have a comparable density of single-molecule spots to the density we observe in endogenously tagged IRE1-HaloTag cells. In fact, the metric of “trajectories per movie” used as a proxy for expression levels in Figure 5 – Figure Supplement 1 is an overestimation of the true variability of expression levels, since each movie only covers a small fraction of each cell’s area and the number of observed molecules varies depending on cell morphology. Practically speaking, all cells that we image have expression levels that are clustered together rather narrowly, roughly within differences of no more than a factor of 3. These levels, in turn, are significantly lower than the expression levels used in earlier papers by our group and others.

The other somewhat substantial issue is that there is no control for what higher order structures look like. The authors give no sense for the dynamic range of the multimerization assay. I would presume that tetramers would show a higher percentage of correlated trajectories than dimers, and octamers higher still, and that the mathematical model accounts for this theoretical possibility in calculating an average protomer number of 2.7 in the stress condition, but it would be better to see that in practice; at first glance it would seem that engineering a tetrameric and/or higher order control and validating it would be straightforward.

This is another great point raised by all reviewers. In the revised version of the manuscript, we engineered a new tetrameric control construct (See Figure 2 – Figure Supplement 1), the results from which agree remarkably well with the mathematical model we developed in the original version of the manuscript (see Figure 2 – Figure Supplement 3)

Lastly, the data analysis lacks statistical justification for its conclusions. I presume given the high number of readings that the observed changes are all statistically significant, but that should be indicated, as in most cases the 95% confidence intervals shown are overlapping.

This is another excellent point. The reviewer is correct that all relevant conclusions are statistically supported by the data, and our analysis code immediately calculates pairwise p- values for every plot using one of several relevant tests. Our preferred test is the permutation test, since it makes no assumptions about the underlying distributions being compared. To avoid cluttering the main plots, we have included tables of pairwise p-values for each plot in the revised version of the manuscript.

Reviewer #1 (Public Review): 

In this manuscript, the authors sought to define the early events associated with activation of the ER stress-responsive membrane protein IRE1. Towards that aim, they used CRISPR to integrate a HALO tag into the genomic locus of IRE1 at the C-terminus of the protein. The authors then adapted a single molecule fluorescence microscopy approach where the HALO tag is liganded with two different fluorophores to define the oligomeric state of membrane proteins in cellular models. They validated this approach using ER membrane proteins containing defined number of HALO tags (single or double) and imaged with oblique angle illumination microscopy to confirm their ability to detect effect monomer and dimers of these tags. Using this approach with IRE1, they showed that in the absence of stress, there is a high fraction of apparent IRE1 dimers in the membrane. In response to ER stress, this oligomer size (calculated by correlated trajectories) increased, suggesting that ER stress promotes IRE1 oligomerization, eventually returning to dimers at longer treatment times. Intriguingly, using the ER stressor thapsigargin, the authors indicate that oligomerization precedes auto-phosphorylation of IRE1, suggesting that oligomerization is a key step in the activation of this enzyme. Extending this, the authors then transition to an overexpression model where they incorporate IRE1 constructs containing mutant that disrupt specific parts of the protein or prevent dimeric or oligomeric interactions to probe their importance in this early oligomerization observed in response to ER stress. This demonstrated that the oligomerization was primarily dictated by the ER luminal domain and involved two distinct interfaces specifically required for IRE1 dimer formation (in the absence of stress) and oligomer formation (following ER stress). Ultimately, with these results, the authors propose a model whereby IRE1 exists primarily as a autophosphorylation-deficient, back-to-back dimer that upon ER stress oligomerizes to a phosphorylation competent oligomer that allow autophosphorylations and IRE1 activation. 

Overall this is an interesting approach and study to define early stages of IRE1 activation. Notably, it reveals a different model of these early stages of IRE1 activation than those previously reported by this group and others using GFP-tagged IRE1 overexpression constructs (something that was enabled by the integration of HALO tags into the genomic locus). The experiments are well performed and the data appear to all be interpreted correctly, although there are a few remaining questions that should still be addressed.

Reviewer #2 (Public Review): 

The authors investigated changes in the unstressed and stressed oligomeric states of the mammalian endoplasmic reticulum (ER) stress sensor, IRE1a. Previous biochemical and microscopy studies in mammalian cells and studies of the related protein Ire1 in yeast, describe an increase in oligomerization of the stress sensor upon treatment of cells with chemical agents that impair the ER protein folding environment. The general view has been that IRE1 in unstressed cells is a monomer and varying degrees of misfolded protein stress stimulate dimerization, activation, and higher order oligomerization. Distinguishing between monomers and dimers, as well as tetramers or other small oligomers is technically challenging, especially for integral membrane proteins. To address this challenge, the authors turned to single particle tracking fluorescence microscopy of Halo-tagged endogenous IRE1. Using a clever combination of random labeling with two fluorescent dyes and oblique angle illumination to visualize single molecules, as well as dimers, the authors surprisingly find that their endogenous IRE1 reporter appears to be dimeric in homeostatic cells. This observation challenges the predominant model in which IRE1 is monomeric in unstressed cells and that even dimerization represents a switch into an active state. The authors claim to detect evidence for higher order oligomers following treatment with stressors. The authors then use a series of IRE1 mutants to identify how oligomerization is regulated and present a new model to reconcile the different models of IRE1 activation in the literature. 

The authors have extensively characterized their novel experimental system in terms of protein expression levels, functionality, and ability to distinguish monomers and dimers. The data are well presented and the authors are clearly familiar with the arguments that have surrounded the IRE1 oligomer question. That the authors observe the characteristic XBP1 mRNA splicing activity in the absence of visible large IRE1 clusters may suggest that the large clusters reported by others may have distinct roles, perhaps in more permissive mRNA cleavage. 

The present study is undermined by two major weaknesses. First, while the authors persuasively demonstrate that they can detect IRE1a dimers, a major claim of the manuscript rests upon detection of tetramers and possibly higher order oligomers. Unfortunately, the authors provide no independent controls to show what tetramer or higher order oligomer data would look like. Thus, the authors can only infer that higher order oligomers are detected, based on modest shifts in the percent of correlated particle trajectories observed in some cells. More robust evidence is needed to make claims of oligomerization. Tools have been developed by others that can induce reversible oligomerization of proteins. Application of these tools would provide powerful controls for tetramers or even higher order oligomers in this study. 

The second, deeper concern, is the discrepancy between the Halo Tag clustering results in this study and studies by this lab and several other labs that report a distinct stress phenotype. In mammalian cells and yeast, IRE1 and Ire1, tagged with different fluorescent proteins or even a small HA peptide epitope tag, undergo quantitative visible formation of puncta or clusters upon treatment with stressors. The small number of bright clusters that form effectively deplete the rest of the ER of IRE1 signal. In the present study, the authors observe no visible change in IRE1-Halo localization in stress cells. The authors do not investigate the cause of this difference. While one might argue that the presence of stress-inducible IRE1 activity is sufficient to argue that the reporter in this study is functional, IRE1 reporters (that do cluster) described in previous studies by the Walter lab and other groups are also demonstrably functional. Does IRE1 normally cluster? Is it cell-type dependent? Tag-dependent? Notably, the Pincus et al. PLoS Biology paper from the Walter lab used two different fluorescent protein tags that do not heterozygously dimerize. Robust colocalization and FRET signals were detected upon treatment of cells with stressors and clustering was subsequently observed. A 2007 Journal of Cell Biology study from Kimata et al. reported clustering in yeast with an Ire1 tagged with an HA epitope peptide. The HA peptide seems unlikely to be prone to any oligomerization propensities that GFP tagged reporters might experience. Importantly, a 2020 PNAS paper from the Walter lab (Belyy et al.) studied clustering of a robustly monomeric mNeonGreen-tagged IRE1 in U2-OS cells and mouse embryonic fibroblasts and this construct readily clustered following stress induction. 

When evaluated against the backdrop of the extensive literature describing the visual behavior of IRE1a in live cells, the absence of stress-induced clustering is both puzzling and disconcerting. Given the focus of this study is to use visual techniques to study IRE1a interactions, the burden of proof is on the authors to resolve this significant discrepancy with the rest of the IRE1a literature. One can easily imagine that incorporation of the majority of the pool of IRE1a into 10-100 clusters could produce very different correlated trajectory behavior. Until the authors can determine why their reporters behave differently from other IRE1a reporters and establish which version accurately reflects physiologic IRE1a behavior, the potential impact of the findings of this manuscript are of unknown value.

SciScore for 10.1101/2022.04.12.22273675: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: This study was approved by the institutional review board at Emory University under protocols STUDY00000260, 00022371, and 00045821.</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">anti-SARS monoclonal antibody CR302240 was generously provided by Jens Wrammert</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">Spike Trimer Capture ELISA: The following ELISA was adapted from previously published methods17: 96-well half area, high binding plates (Corning #3690) were coated with anti-6x-His-tag monoclonal antibody (#MA1-21315MG, ThermoFisher) at 2 µg /mL in PBS at 4°C overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-6x-His-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Approximately one million viable PBMCs were stained with Zombie aqua fixable cell viability dye (BioLegend) to exclude dead cells; washed with PBS containing 2% FBS, referred to as FACS buffer; surface-stained with the following fluorescent monoclonal antibodies: CD3 (clone SK7, BioLegend),</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing with FACS buffer and fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences), the cells were stained intracellularly with the following fluorescent monoclonal antibodies: CD154 (clone CD40L 24-31, BioLegend),</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD154</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing with FACS buffer and fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences), the cells were stained intracellularly with the following fluorescent monoclonal antibodies: CD154 (clone CD40L 24-31, BioLegend), IL-2 (clone MQ1-17H12, BD Biosciences), IFN-γ (clone 4S.B3, eBioscience), TNF (clone Mab11, BD Biosciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IL-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IFN-γ</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>TNF</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IFN-γ spots were detected with biotinylated murine anti-human IFN-γ antibody (clone 7-B6-1, Mabtech), followed by incubation with streptavidin-HRP (BD) and then developed using AEC substrate (EMD Millipore).</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><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 HeLa cell line transduced to stably express the human ACE2 receptor (ACE2-HeLa) was generously provided by David Nemazee17.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Wuhan-Hu-1 spike trimer protein expression: Spike trimer plasmids were transiently transfected into Expi293 cells (ThermoFisher) with 5 mM kifunensine (Mfr), purified with His-Trap columns (Cytiva), trimers selected with a Superdex 200 gel filtration column (Mfr), and finished product dialyzed into 20 mM Tris pH 8.0, 200 mM sodium chloride, 0.02% sodium azide by the BioExpression and Fermentation Facility at the University of Georgia.</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">Pseudovirus production: Pseudoviruses were produced by seeding 16 million 293T cells (ATCC CRL-3216) into DMEM with 10% heat-inactivated FBS and 1% GlutaMAX (ThermoFisher) (DMEM-10) in a T-150 flask the night prior to transfection and incubating at 37°C in a humidified 5% CO2 incubator.</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">DMEM-10 media was then removed from plates with cells and 50 µl pseudovirus dilutions added onto ACE2-HeLa cells and incubated for two hours at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2-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">Plasmids pCMV ΔR8.2 (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV ΔR8.2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmid nCoV-2P-F3CH2S43 expressing a His-tagged, pre-fusion stabilized SARS-CoV-2 spike trimer from Wuhan-Hu-1 isolate was generously provided by Jason McLellan.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>nCoV-2P-F3CH2S43</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">On the day of transfection, the HIV-1 lentiviral packaging plasmid, pCMV R8.2 (17.5</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><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequences from immunocompromised patients were aligned with 301 reference sequences collected from patients within the Emory Healthcare System between 1/1/2021 and 4/30/2021 using MAFFT as implemented in geneious (geneious.com).</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">A maximum-likelihood tree was constructed using a general time reversible model with empirical base frequencies and a 3 rate model in IQ-TREE version 2.0 with 1,000 ultrafast boostraps38 and visualized in FigTree (http://tree.bio.ed.ac.uk/software/figtree).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IQ-TREE</div><div>suggested: (IQ-TREE, RRID:SCR_017254)</div></div><div style="margin-bottom:8px"><div>FigTree</div><div>suggested: (FigTree, RRID:SCR_008515)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To identify iSNVs, reads were mapped to reference sequence NC_045512.1 using minimap2, variants were called using vphaser2 with maximum strand bias of 5, and variants annotated with SNPeff, all as implemented in viral-ngs version 2.1.19.0-rc119.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SNPeff</div><div>suggested: (SnpEff, RRID:SCR_005191)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To ascertain a precise endpoint titer (ET), curve data (best fit values for the bottom, top, logEC50, and hill slope) were processed by a MATLAB program designed to determine the sample dilution at which each regression curve intersected the healthy control cutoff value.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MATLAB</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing with FACS buffer and fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences), the cells were stained intracellularly with the following fluorescent monoclonal antibodies: CD154 (clone CD40L 24-31, BioLegend), IL-2 (clone MQ1-17H12, BD Biosciences), IFN-γ (clone 4S.B3, eBioscience), TNF (clone Mab11, BD Biosciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD Biosciences</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry data were collected on an LSR Fortessa (BD Biosciences) and analyzed using FlowJo software V10 (Tree Star).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

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Limitations to our study include a small number of patients and the use of convenience samples. Larger clinical studies in immunocompromised populations are needed, including serial sampling to further elucidate therapies that promote immune evasion. Our work and others’ emphasize the need to both protect immunocompromised patients from acquiring infection, and to prevent the forward spread of viruses with immune escape mutations. Such needs might be met with broad spectrum monoclonal antibodies and next generation SARS-CoV-2 vaccines that induce potent neutralizing antibody responses to prevent infection and memory CD8+ T cell responses to control breakthrough.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2022.04.19.488806: (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">16 Anti-His tag and microtubule-associated protein 1 light chain 3 beta (LC3) Antibodies were purchased from Millipore Sigma (Burlington, MA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>16 Anti-His tag</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>microtubule-associated protein 1 light chain 3 beta (LC3) Antibodies</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-Adipose Differentiation-Related Protein (ADRP, or Perilipin-2, PLIN2), Nrf2, prostaglandin E synthase 2 (PTGS2), and PI3K-beta antibodies were obtained from ProteinTech (Rosemont, IL, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Adipose Differentiation-Related Protein ( ADRP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>PLIN2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Nrf2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>PTGS2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>PI3K-beta</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-ATG7 antibody was purchased from Abcam (Waltham, MA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-ATG7</div><div>suggested: (Abcam Cat# 2054-1, RRID:AB_991677)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-SRB1 antibody was obtained from Novus (Centennial, CO, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-SRB1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-Fth1, HRP-anti-rabbit or mouse secondary antibodies, and RIPA lysis buffer were obtained from Santa Cruz Biotech (Dallas, TX, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Fth1 , HRP-anti-rabbit or mouse secondary antibodies</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-Fth1 ,</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HRP-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">Palmitic acid (PA)-induced lipotoxicity assay: The HEK293, HEK_pcDNA and HEK_Spike cells were cultured in a 96-well plate and reached 80% confluence on the next day before treatment.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HEK_Spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">H9C2 cells (ATCC, Manassas, VA, USA) were cultured in DMEM (10% FBS) medium in a 96 well-plate with 80% confluence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>H9C2</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">16 The sequence was cloned into a pcDNA3.1 vector to obtain pcDNA-Spike.</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 individual colonies with stable integration of the pcDNA-Spike (HEK_Spike) or pcDNA vector (HEK_pcDNA) were selected and expanded.</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</div><div>suggested: RRID:Addgene_66792)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral production and H9C2 cell culture: The Spike gene was cleaved from pcDNA-Spike plasmid and cloned into lentiviral vector pLV-mCherry (Addgene, Watertown, MA, USA) with removal of mCherry gene to generate pLV-Spike plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLV-Spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The control virus with VSV-G as the tropism and expression of mCherry was generated by co-transfection of pLV-mCherry and pMD2.G vector (Addgene, Watertown, MA, USA) into the Phoenix cells, which was referred to as VSV-G virus.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLV-mCherry</div><div>suggested: RRID:Addgene_36084)</div></div><div style="margin-bottom:8px"><div>pMD2.G</div><div>suggested: RRID:Addgene_12259)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

---

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• No funding statement was detected.
• No protocol registration statement was detected.

---

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

---

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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 10.1101/2022.04.19.488067: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Each Newick file is parsed by a python script which generates a CSV file of edges in the TAG.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>python</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Another option is to use Gephi, but to use this tool, users need to start listening for data streams in Gephi before executing an APOC graph streaming query to push the data to the app.[6] Alternatively, users can connect using the Neo4j plugin for Cytoscape.[7] We found this option to be the most intuitive and sustainable for ad-hoc visualization since you can remotely connect to the graph using a read only user account on the database.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gephi</div><div>suggested: (Gephi, RRID:SCR_004293)</div></div></td></tr></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• No funding statement was detected.
• No protocol registration statement was detected.

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

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<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 10.1101/2022.04.19.488843: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: All animal experiments were approved by the Academia Sinica Institutional Animal Care and Use Committee (IACUC protocol No.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Four- to six-week-old female BALB/c mice were immunized with 5 μg of the Kappa spike and RBD mRNA-LNP by intramuscular (I.M.) injection.</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">Anti-RBD and control antibodies were added to the plates 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-RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then, the cells were washed and horseradish peroxidase-conjugated anti-human antibody (1:2000) was added for 1 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human antibody</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 four inoculations with the same concentration of mRNA-LNP, the splenocytes from immunized mice were harvested and fused with mouse myeloma NS-1 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NS-1</div><div>suggested: RRID:CVCL_IV58)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pseudovirus neutralization assays were performed using HEK293T cells that expressed human ACE2 (HEK293T/hACE2)</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 mixtures were then added to pre-seeded HEK293T/hACE2 cells for 24 h at 37°C.</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><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">Four- to six-week-old female BALB/c mice were immunized with 5 μg of the Kappa spike and RBD mRNA-LNP by intramuscular (I.M.) injection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: RRID:IMSR_ORNL:BALB/cRl)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The PCR products were cloned using the pGEM-T Easy Vector System (Promega) and analyzed by DNA sequencing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEM-T Easy</div><div>suggested: RRID:Addgene_86229)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting VH was cloned into a modified pcDNA5-FRT-Gamma1 expression vector with human IgG1 constant region.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA5-FRT-Gamma1</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">Recombinant protein-based ELISA: Recombinant RBD and spike-His tag proteins for different SARS-CoV-2 variants were purchased from ACROBiosystems.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACROBiosystems</div><div>suggested: (ACRObiosystems, RRID:SCR_012550)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The half maximal inhibitory concentration (IC50) was calculated by nonlinear regression using Prism software version 8.1.0 (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">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">From the sequences, the framework regions (FRs) and complementarity determining regions (CDRs) were defined by searching with the NCBI IgBLAST program (https://www.ncbi.nlm.nih.gov/igblast/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgBLAST</div><div>suggested: (IgBLAST, RRID:SCR_002873)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After analyzing the structure with PyMOL software, we identified the key amino acid residues at which mutations may impact the original conformation of the CDRs.</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>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

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Results from TrialIdentifier: 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>

central to intentional software

reserve roles

sounds like intent marks

reserved roles

peridata

Peridata

Transitional is Queen

https://www.anchormodeling.com/tag/transitional/

don't say that

that's a fun way to say this...

check if the bullet symbol has been used, or it is ul li tag I had replaced the bullets. I hope the content has not been overwritten

SciScore for 10.1101/2022.04.12.488087: (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: Immunization of alpaca, construction of yeast display VHH library, and isolation of VHH yeasts specific for SARS-CoV-2 and SARS-CoV-1 spikes: The animal experiment protocol involving immunization, collection of blood samples, and construction of VHH library was approved by IACUC at NBbiolab, Inc. in Chengdu, China.<br>IACUC: Immunization of alpaca, construction of yeast display VHH library, and isolation of VHH yeasts specific for SARS-CoV-2 and SARS-CoV-1 spikes: The animal experiment protocol involving immunization, collection of blood samples, and construction of VHH library was approved by IACUC at NBbiolab, Inc. in Chengdu, China.</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">Eight-week-old female K18-hACE2 transgenic mice (InVivos Ptd Ltd, Lim Chu Kang, Singapore) were used for this 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">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 extensive wash with cold PBS+1%FBS, the yeast clones were incubated with HA-Tag (6E2) mouse monoclonal antibody conjugated with Alexa Fluor® 488 (1:100 dilution) and eBioscience™ streptavidin conjugated with PE Conjugate (1:200 dilution) on ice for 30 min.</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# 2350, RRID:AB_491023)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cells were then fixed, permeabilized, and incubated with cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological, Inc., China) for 1 h at room temperature before adding an HRP-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-N IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sections were then covered with rabbit anti-SARS-CoV-2 N protein monoclonal antibody (Abcam; 1:1000) for 1 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 N protein</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: HEK293T cells (ATCC, CRL-3216) and HeLa cells expressing hACE2 were kindly provided by Dr. Qiang Ding at Tsinghua University.</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>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">Sf9 cells (ATCC) were maintained at 27°C in Sf-900 II SFM medium.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Sf9</div><div>suggested: RRID:CVCL_4U10)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and production of nanobodies were conducted by transfecting the expression vectors into the HEK293F cells using polyethyleneimine (PEI) (Polysciences).</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">Specifically, human immunodeficiency virus backbones expressing firefly luciferase (pNL4-3-R-E-luciferase) and pcDNA3.1 vector encoding either SARS-CoV-2 or sarbecovirus spike proteins were co-transfected into the HEK-293T cells (ATCC).</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">HeLa-ACE2 cells were then added to the mixture of nanobody-pseudovirus, incubated at 37°C for additional 48 h, and lysed for measuring luciferase-activity.</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">Tissues were homogenized with 0.5 mL DMEM supplemented with antibiotic and antimycotic (Gibco, Waltham, MA, USA) and titrated in Vero E6 cells using plaque assays.</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">Eight-week-old female K18-hACE2 transgenic mice (InVivos Ptd Ltd, Lim Chu Kang, Singapore) were used for this study.</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">Cell lines: HEK293T cells (ATCC, CRL-3216) and HeLa cells expressing hACE2 were kindly provided by Dr. Qiang Ding at Tsinghua University.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>hACE2</div><div>suggested: RRID:Addgene_1786)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VHH sequences were amplified by PCR, cloned into a yeast surface display vector pYD1, and introduced into the electrocompetent EBY100 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pYD1</div><div>suggested: RRID:Addgene_73447)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the former, VHH genes were cloned into the multiple cloning sites of pMD18T containing the upstream CMV promoter, the secretory signal sequence from the mouse Ig heavy chain, and the downstream human IgG1 Fc gene fragment and SV40 poly (A) signal sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pMD18T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the latter, selected VHH genes were cloned into pVRC8400 vector with a 6xHis tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVRC8400</div><div>suggested: RRID:Addgene_63163)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Specifically, human immunodeficiency virus backbones expressing firefly luciferase (pNL4-3-R-E-luciferase) and pcDNA3.1 vector encoding either SARS-CoV-2 or sarbecovirus spike proteins were co-transfected into the HEK-293T cells (ATCC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pNL4-3-R-E-luciferase</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><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 IC50 values were calculated based on the reduction of 50% relative light units (Bright-Glo Luciferase Assay Vector System, Promega, USA) compared to the virus-only control, using Prism 8.0 (GraphPad Software Inc., 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><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">Phylogenetic tree and genetic analysis of nanobodies: Neighbor-joining phylogenetic trees were generated using MEGA version 10.1.8 with 1000 bootstrap replicates 68.</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><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Chord diagrams showing the germline gene usages and V/J gene pairing were analyzed and presented by the R package circlize version 0.4.13 69.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>circlize</div><div>suggested: (circlize, RRID:SCR_002141)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequence logo were plotted using Python package Logomaker 70</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">Subsequent model building and refinement were performed using COOT (PMID: 15572765) and PHENIX (PMID: 12393927), 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">All structure figures were generated with ChimeraX and Pymol (PMID: 28158668)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ChimeraX</div><div>suggested: (UCSF ChimeraX, RRID:SCR_015872)</div></div><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">Half-maximal inhibitory concentration (IC50) of nanobodies was calculated by the equation of four-parameter dose inhibition response using Graphpad Prism 8.0.</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>

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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.

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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:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

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

---

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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• EXAMPLE

SciScore for 10.1101/2022.04.09.487739: (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 membrane was incubated with ACE-2 Antibody (1:2,000, Novus Biologicals, CO, USA), Myc-Tag (9B11</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">The membrane was then incubated with secondary HRP-linked, Anti-rabbit IgG (1:10,000, Cell Signaling Technology, MA, USA) and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (1:2,000, Thermo Fisher Scientific, MA, USA) for 1 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-rabbit IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Mouse IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were cultured in FP medium (DMEM containing 10% FBS, 2 mM GlutaMAX™ Supplement, 0.1 mM MEM Non-Essential Amino Acids, 50 U/mL and 50 μg/mL Penicillin-Streptomycin).</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 establish ACE2-expressed cell line (ACE2-HEK293T cells), HEK293T cells were infected with ACE2-expressing lentivirus and ACE2-positive cells were selected by 2 ug/mL of puromycin.</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><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 protein expression, the cell membrane penetrating peptide (TAT), red fluorescence protein (DsRed) and NK-NT or NKN1 fragments were cloned into pET6xHN-N Vector (Takara, CA, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET6xHN-N</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For ACE2-expressing lentivirus packaging, pscALPSpuro-HsACE2 (human) (Addgene, MA, USA) were co-transfected with psPAX2 and pCMV-VSV-G packaging plasmids into HEK293T cells using FuGENE 6 (Promega, WI, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-VSV-G</div><div>suggested: RRID:Addgene_8454)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For doxycycline (Dox) inducible, Spike protein pseudotyped luciferase-expressing lentivirus preparation, HEK293T cells were transfected with FUW-RLuc-T2A-PuroR(Kanarek et al., 2018) (Addgene, MA), psPAX2 and pUNO1-SARS2-S (D614G) (InvivoGen, CA) packaging plasmids using FuGENE 6.</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></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In competition BiFC Assay, HEK293T cells were co-transfected with 0.5 μg of each construct expressed in pBiFC-VN155 (I152L) and 0.5 μg pBiFC-VC155 vectors, together with and 5 μg competitor constructs with stop codon in pBiFC-VN155 (I152L) vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pBiFC-VC155</div><div>suggested: RRID:Addgene_22011)</div></div><div style="margin-bottom:8px"><div>pBiFC-VN155</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">Fluorescence images were taken at 24 h and 48 h after transfection using a Nikon fluorescence microscope and fluorescence intensity was quantified by Image J.</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></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.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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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>

SciScore for 10.1101/2022.04.11.487660: (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">C5 affinity maturation library construction: The sequence of C5 in phagemid pComb3XSS were used as template for library construction.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pComb3XSS</div><div>suggested: RRID:Addgene_63890)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Nanobody expression and purification: The cDNA encoding the nanobodies in the pComb3XSS vector were PCR amplified and subcloned into vector pET22b to express 6×His tagged proteins.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET22b</div><div>suggested: RRID:Addgene_84863)</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 coding cDNA of 1ZVH was chemically synthesized and subcloned into a modified phage display vector of pComb3XSS, in which the amber stop codon (TAG) were mutated to CAG to facilitate nanobody display in E. coli without gene supE, e.g. SS320(Genentech).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Genentech</div><div>suggested: (Genentech, RRID:SCR_003997)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Standard variation values were calculated using a 3-parameter logistic regression fit 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><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 [61] 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">Contrast transfer function (CTF) fitting and phase-shift estimation were conducted with Gctf [62].</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">Local map resolution was estimated with ResMap [65].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ResMap</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We initially fitted the templates models into the corresponding final cryo-EM map using Chimera [67], and further corrected and adjusted them manually by real-space refinement in Coot [68].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Chimera</div><div>suggested: (Chimera, RRID:SCR_002959)</div></div><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting models were then refined with phenix.real_space_refine in PHENIX [69].</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 [70, 71].</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 figures were generated with Chimera or ChimeraX [72, 73].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ChimeraX</div><div>suggested: (UCSF ChimeraX, RRID:SCR_015872)</div></div></td></tr></table>

---

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

---

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

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

Results from rtransparent:

---

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• 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.

</footer>

SciScore for 10.1101/2022.04.11.487879: (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: All participants in the convalescent cohort provided informed consent for their blood products to be used for research purposes by signing the standard New York Blood Center (NYBC<br>IRB: For participants who received the SARS-CoV-2 mRNA-1273 vaccine (Moderna), whole blood, plasma and serum samples were obtained at the NIH Clinical Research Center in Bethesda, MD under protocols approved by the NIH Institutional Review Board, ClinicalTrials<br>IACUC: Animal ethics statement: Animal research was conducted under an IACUC approved protocols at the Integrated Research Facility, Frederick, Maryland, in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals.<br>Field Sample Permit: Animal ethics statement: Animal research was conducted under an IACUC approved protocols at the Integrated Research Facility, Frederick, Maryland, in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals.</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">Hamsterization of human monoclonal antibodies: Genomes corresponding to the mouse IgG2a heavy and light chains were aligned to the genome assembly MesAur1.0 (GCA_000349665.1) for a female Syrian golden hamster downloaded from Genbank.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">No randomization or blinding was applied to the analysis of participants’ plasma, serum or PBMC samples, but all samples were anonymized before being used in this study.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">No randomization or blinding was applied to the analysis of participants’ plasma, serum or PBMC samples, but all samples were anonymized before being used 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">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 of secreted antibody to the beads was detected in the CY5 or TRED channels by capturing images at 6 min intervals over a 30 min time course.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CY5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Shotgun mutagenesis epitope mapping of antibodies by alanine scanning: Epitope mapping was performed essentially as previously described (43), using a SARS-CoV-2 (Wuhan Hu-1 strain) S2 subunit shotgun mutagenesis mutation library, made using a full-length expression construct for the SARS-CoV-2 spike glycoprotein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 spike glycoprotein .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vaccinee and convalescent plasma binding to peptides: Polyclonal IgG antibodies from plasma or sera of vaccinated, convalescent, or naïve donors were purified using the Pierce Protein G Spin Plate (Thermo Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Polyclonal 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">Baculoviruses were produced by transfection of bacmid DNA into Sf9 cells and used to infect High Five cells (Life Technologies) at high (5 to 10) multiplicity of infection (MOI).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Sf9</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasma IgG reactivity to human coronaviruses and donor selection: Multiplexed beads for SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-OC43, HCoV-HKU1, HCoV-229E and HCoV-NL63 spike proteins, as well as CD4 as a negative control, were incubated with donor plasma diluted at 1/50, 1/250 or 1/1250 for 30 min at room temperature, then washed and stained with 2.5 μg/mL goat anti-human IgG Alexa Fluor 647 (Jackson ImmunoResearch, 109-606-170).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HCoV-229E</div><div>suggested: JCRB Cat# JCRB1838, RRID:CVCL_B3M4)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">mAbs were also expressed in-house by transient transfection of Expi293 cells (Gibco, A14527) using the ExpiFectamine 293 Transfection Kit (Gibco, A14524) according to manufacturer’s instructions.</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">To generate green fluorescent protein (GFP)-tagged receptor cell lines, HeLa-ACE2 cells were transduced with lentivirus encoding GFP and sorted to collect the GFPhigh/ACE2high population.</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">mAbs were added to the wells at a final concentration of 200 μg/mL and cultures were further incubated at 37 °C for 1h. 8,000 GFP+/ACE2+ HeLa cells were then added to each well and the co-cultures were maintained overnight to allow for syncytia development.</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">A plasmid encoding cDNA for each spike protein mutant was transfected into HEK-293T cells and allowed to express for 22 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">For neutralization assays, 5 × 104 RD cells were inoculated at TCID75% OC43-GFP virus and incubated for 1h at 35°C. 4-fold serial dilutions (73 ng/mL - 300 μg/mL) of each mAb were incubated with TCID75 OC43-GFP virus for 1h at 35°C. 60 μL of mAb- virus mixture was used to inoculate each well containing 5 × 104 RD cells and cultures were incubated for 24 h at 35°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">transducing plasmid pHR’ CMV-Luc, a TMPRSS2 plasmid and full-length spike plasmids from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-NL63 and HCoV-229E into 293T cells using Lipofectamine 3000 transfection reagent (ThermoFisher Scientific, Asheville, NC, L3000-001) (49). 293 flpin-TMPRSS2-ACE2 cells (provided by Dr. Adrian Creanga, VRC/NIH) were used for SARS-CoV-2, SARS-CoV and hCoV-NL63 while HuH7.5 cells were used for MERS-CoV and hCoV-229E neutralization assay.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HCoV-NL63</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HuH7.5</div><div>suggested: RRID:CVCL_7927)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS: Addgene #170447; SARS2 #170442; MERS #170448; NL63 #172666; alpha strain #170451; beta #170449; gamma #170450; delta #172320; omicron 180375) were co-transfected in HEK293T with Lipofectamine 2000 (ThermoFisher Scientific, 11668019) to produce single-round infection-competent pseudoviruses.</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">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: Addgene #170447; SARS2 #170442; MERS #170448; NL63 #172666; alpha strain #170451; beta #170449; gamma #170450; delta #172320; omicron 180375) were co-transfected in HEK293T with Lipofectamine 2000 (ThermoFisher Scientific, 11668019) to produce single-round infection-competent pseudoviruses.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>#172666</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">Pre-fusion stabilized constructs for CCoV HuPn-2018 (Accession # QVL91811.1, aa1-1384 with E1140P and E1141P mutations) and PdCoV0081-4 ( Accession # MW685622.1, aa1-1092 with E854P and V855P mutations) were synthesized and cloned into pCDNA3.1- vectors (Genscript) with the following C-terminal modifications: T4 fibritin trimerization motif, HRV3C protease cleavage site, poly-GS linker, Avi-tag, and 8× His 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_52535)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the SARS-CoV-2 NTD and RBD were cloned into an in-house pFastBac vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFastBac</div><div>suggested: RRID:Addgene_1925)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The spike S2 domain (699 to 1207 with F817P, A892P, A899P, A942P, K986P, V987P) was constructed into phCMV3 vector which contained an N-terminal secreting signal peptide, and C-terminal thrombin cleavage site and His6 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">SARS-CoV-2 RBD, SARS- CoV-2 NTD, SARS-CoV-1 spike and SARS-CoV-1 RBD, MERS-CoV spike, OC43-CoV spike, CCoV-HuPn-2018 spike, pPDCoV-0081-4 spike, HCoV-NL63 spike, HCoV-229E spike, HCoV- HKU1 spike, H1 HA and recombinant CD4 (gifted by Gavin Wright, (35)).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pPDCoV-0081-4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Spike-containing lentiviral pseudovirions were produced by co-transfection of packaging plasmid pCMVdR8.2,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMVdR8.2</div><div>suggested: RRID:Addgene_8455)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">transducing plasmid pHR’ CMV-Luc, a TMPRSS2 plasmid and full-length spike plasmids from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-NL63 and HCoV-229E into 293T cells using Lipofectamine 3000 transfection reagent (ThermoFisher Scientific, Asheville, NC, L3000-001) (49). 293 flpin-TMPRSS2-ACE2 cells (provided by Dr. Adrian Creanga, VRC/NIH) were used for SARS-CoV-2, SARS-CoV and hCoV-NL63 while HuH7.5 cells were used for MERS-CoV and hCoV-229E neutralization assay.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHR’</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>TMPRSS2</div><div>suggested: RRID:Addgene_53887)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">2.5μg 2nd generation lentivirus backbone plasmid pCMV-dR8.2 dvpr (Addgene, 8455), 2μg pBOBI-FLuc (Addgene, 170674) and 1μg truncated coronavirus spike expressing plasmids (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-dR8.2</div><div>suggested: RRID:Addgene_8455)</div></div><div style="margin-bottom:8px"><div>pBOBI-FLuc</div><div>suggested: RRID:Addgene_170674)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Hamster genes with the highest homology to the mouse IgG2a heavy chain, lambda and kappa light chains genes were cloned into a pCDNA3.4 vector (Genscript) and expressed in Expi293 cells as described above.</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">) high-throughput flow cytometer and FACS data were analysed with FlowJo (Version 10.8.1</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><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analyses of the VH and Vλ/Vκ genes, CDR3 sequences, and percentage of somatic mutations were carried out using Geneious Prime (Version 2021.0.3, https://www.geneious.com) and the International Immunogenetics Information System database (IMGT, http://www.imgt.org/) (40).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>https://www.geneious.com</div><div>suggested: (Geneious, RRID:SCR_010519)</div></div><div style="margin-bottom:8px"><div>http://www.imgt.org/</div><div>suggested: (IMGT - the international ImMunoGeneTics information system, RRID:SCR_012780)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant IgG mAbs were purified using HiTrap Protein A columns (Cytiva/GE Healthcare Life Sciences, 17040303).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Cytiva/GE Healthcare</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Phylogenetic tree generation: Full-length amino acid sequences of SARS-CoV-2 (accession #NC_045512.2), SARS-CoV (accession # AY278741.1), MERS-CoV (accession # NC_019843) , HCoV-NL63 (accession #NC_005831.2), HCoV-229E (accession #NC_002645.1), CCoV HuPn-2018 (accession #MW591993.2) and PDCov-0081-4 (accession #MW685622) were aligned using the L-INS-i method of MAFFT version 7.</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">The sequence alignment was used to generate a sequence logo plot using the Weblogo 3.0 server and to color conserved amino acid residues on a pre-fusion stabilized spike protein (PDB 6VSB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Weblogo</div><div>suggested: (WEBLOGO, RRID:SCR_010236)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Images were acquired in A488, A568 and DAPI channels using a BZ-X fluorescence microscope (KEYENCE) and processed using Fiji ImageJ (42)</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">Iterative model building and refinement were carried out in Coot (46) and PHENIX (47), 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">Authentic OC43-CoV-GFP virus propagation and neutralization assay: Rhabdomyosarcoma cells (RD, ATCC CCL-136) were maintained at 37°C and 5% CO2 in No-glucose DMEM (Gibco, 11966-025), supplemented with 10% HI-FBS, 4500 mg/mL glucose, 1 mM sodium pyruvate (Gibco, 11360-070), 1 mM HEPES (Gibco, 15630-080) and 50 μg/mL gentamycin (Quality Biological, 120-098-661)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Quality Biological</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">50% neutralization titers (NT50) were calculated using the dose- response-inhibition model with 5-parameter Hill slope equation 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><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Descriptive statistics (mean ± SEM or mean ± SD) and statistical analyses were performed using Prism version 9.3.1 (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>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<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">NCT00001281</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">Studies of Blood and Reproductive Fluids in HIV-Infected and…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT05078905</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">Vaccine Responses to SARS-CoV-2 and Other Emerging Infectiou…</td></tr></table>

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• 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.

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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Hello friends, colleagues, and all others that I hope become part of the first two categories. I am Alan Levine, coming to you from central Canada. Welcome to my unorthodox workshop. I am so web old I can remember annotations in the first Mosaic browser.

Please reply with your own introduction, and share your experience, interest in web annotation.

Do not forget to tag all responses oer22 in this workshop !

At least I think this is talking about this as limitation/problem.

The limitation/problem being that it's not good/useful for performing after-transaction code only for specific actions.

But the next sentence "This is beneficial..." seems contradictory, so I'm a bit confused/unclear of what the intention is...

Looking at this project more, it doesn't appear to solve the "after-transaction code only for specific actions" problem like I initially thought it did (and like https://github.com/grosser/ar_after_transaction does), so I believe I was mistaken. Still not sure what is meant by "instead of specific model actions". Are they claiming that "before_commit_on_create" for example is a "specific model action"? (hardly!) That seems almost identical to the (not specific enough) callbacks provided natively by Rails. Oh yeah, I guess they do point out that Rails 3 adds this functionality, so this gem is only needed for Rails 2.

Maybe include a tag/link here to your Machine-Ethics page as the concepts of 'mannerism' and 'ethnics' can be interrelated.

SciScore for 10.1101/2022.04.11.487828: (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">For detection of S protein, the membrane was incubated with anti-HA tag mouse monoclonal antibody (bimake, USA,1:2000), and the bound antibodies were detected by Horseradish Peroxidase (HRP)-conjugated goat anti-mouse IgG (Abbkine, China, 1:5,000).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-HA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For detection of HIV-1 p24 in supernatants, monoclonal antibody against HIV p24 (p24 MAb) was used as the primary antibody at a dilution of 1:8,000, followed by incubation with HRP-conjugated goat anti-mouse IgG at the same dilution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HIV-1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HIV</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 lines and plasmid construction: HEK293T and Hela cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units of penicillin and 0.1 mg/ml of streptomycin in 5% CO2 at 37 °C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Hela</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, one day prior to transfection for virus production, HEK293T cells were digested and adjusted to an amount of 7×106 cells in a 10cm culture medium and incubated overnight in an incubator at 37 °C with 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></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">pcDNA3.1-S2 plasmid was used as the template to generate the plasmid with mutagenesis in S gene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-S2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">When cells reached 80%-90% confluence, HEK293T cells were co-transfected with a luciferase-expressing HIV-1 plasmid (pNL4-3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HIV-1</div><div>suggested: RRID:Addgene_115809)</div></div><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">The P24 gene of HIV virus was cloned into the vector pCDNA3.1(+) as a plasmid standard, with the viral copy number calculated accordingly.</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">Phylogenetic analysis and sequence alignment: The ACE2 aa sequences were aligned by MAFFT v7.149 in BioAider.</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>BioAider</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then we constructed the maximum likelihood phylogenetic tree of ACE2 by IQ-tree v1.6.10 program with 10,000 ultrafast bootstraps (https://academic.oup.com/mbe/article/32/1/268/2925592), and the most appropriate evolutionary model was JTTDCMut+G4 which calculated using ModelFinder according to the bayesian information criterions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IQ-tree</div><div>suggested: (IQ-TREE, RRID:SCR_017254)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mutations in the models were aligned, and the interactions between the SARS-CoV-2 S and ACE2 proteins were compared 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></table>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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hashtag metoo, FFF, Feministischer Kampftag, Tag der Arbeiter*innen, weitere Beispiele?

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).

• 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).

• 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).

• 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.

• 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.

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.

</footer>

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.

</footer>

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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如何判断要读取的 w 在 cache line 里面？

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>

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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.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

---

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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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:

1. 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.

2. 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.

3. 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.

4. 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.

5. 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:

1. "CyVerse" is misspelled as "CyCVerse" in multiple places in the manuscript.

2. Reference Figure 2 in Line 92.

3. Line 154: "Ratios between tools were calculated by dividing the number of gRNA identified.."

4. In Supplementary Figure 3 "wit haVX2" should be "with aVX2".

5. GitHub link in Line 336 does not work.

6. 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.

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>

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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.

---

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Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

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

Results from rtransparent:

---

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

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

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SciScore for 10.1101/2022.04.03.486854: (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>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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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>

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Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<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>

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Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

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.

---

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Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

---

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

Results from rtransparent:

---

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

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

---

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About SciScore

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>

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.

---

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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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.

---

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>