SciScore for 10.1101/2021.10.13.464050: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells and expression vectors: HEK293T cells and SiHa cells were procured from the central cell line repository of RGCB (CCL</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SiHa</div><div>suggested: KCB Cat# KCB 2013026YJ, RRID:CVCL_0032)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CaCo-2 cells were obtained from ATCC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CaCo-2</div><div>suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">DLD-1 cells were obtained from Sigma (Merck #90102540).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DLD-1</div><div>suggested: ECACC Cat# 90102540, RRID:CVCL_0248)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were transfected with lenti-ZsGreen (Addgene) and 10 µg of psPAX2 (Addgene) and SARS-CoV-2-S variant (pCDNA 3.1_Spike_Del19, Addgene) at a ratio of 1:2:1 using the transfection reagent (Lipofectamine 3000, Thermo Fisher Scientific #L3000001).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mixture was added to the HEK293T-HA-ACE2 cells for 4 hours, followed by fresh medium, and wells with IgG served as control.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-HA-ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse immunization and serum preparation: BALB/c mice were immunized with purified RBD-His protein expressed in E. coli as per the standard protocol approved by IAEC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: RRID:IMSR_ORNL:BALB/cRl)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">) Spike S1 Gene with C terminal OFP Spark fluorescent tag (pCMV3-C-OFPSpark) was obtained from Sino biological (#VG40591-ACR)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3-C-OFPSpark</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Full-length DNA clone of Human angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 with C terminal GFPSpark tag (pCMV3-hACE2-GFPSpark) was obtained from Sino biological (#HG10108-ACG).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3-hACE2-GFPSpark</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mammalian expression vector for soluble ACE2 pcDNA3-sACE2 (WT)-sfGFP (#145171) was obtained from Addgene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3-sACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The packaging vectors and Lenti ZsGreen were from Addgene (pHIV-Zsgreen #18121</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIV-Zsgreen</div><div>suggested: RRID:Addgene_18121)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immobilization of GFP Nanobody to Agarose beads: The bacterial expression vector for anti-GFP nanobody pGEX-6P-1 was procured from Addgene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEX-6P-1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were transfected with lenti-ZsGreen (Addgene) and 10 µg of psPAX2 (Addgene) and SARS-CoV-2-S variant (pCDNA 3.1_Spike_Del19, Addgene) at a ratio of 1:2:1 using the transfection reagent (Lipofectamine 3000, Thermo Fisher Scientific #L3000001).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>pCDNA 3.1_Spike_Del19</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The images were acquired and analyzed using NIS-Elements software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NIS-Elements</div><div>suggested: (NIS-Elements, RRID:SCR_014329)</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 22. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2021.10.13.464307: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The anti-His tag antibodies, diluted at 50 μg/mL in 10 mM sodium acetate, pH 4.5, were immobilized on both the active and reference flow cells surface of the activated CM5 sensor chip using amine coupling method.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Approximately 200 RU of His-tagged SARS-CoV-2 and SARS-CoV spike trimers and RBDs were captured onto the chip for the active surface, and anti-His antibody alone served as the reference surface.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: HEK293T/17 (cat# CRL-11268) and Vero E6 cells (cat# CRL-1586) were from ATCC, 293T-ACE2 cells were kindly provided by J. Sodroski of Harvard Medical School, and they were cultured in 10% fetal bovine serum (FBS, GIBCO cat# 16140071) supplemented Dulbecco’s Modified Eagle Medium (DMEM, ATCC cat# 30-2002) at 37°C, 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>293T-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK293T cells were grown to 80% confluency before transfection with the spike gene using Lipofectamine 3000 (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The proteins were expressed in HEK293 Freestyle cells (Invitrogen) in suspension culture using serum-free media (Invitrogen) and transfected into HEK293 cells using polyethyleneimine (Polysciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The results were then converted into percentage neutralization at a given sample dilution or mAb concentration, and the averages ± SEM were plotted using a five-parameter dose-response curve in GraphPad Prism v.8.4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data processing was performed using cryoSPARC v2.15 [14].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Model building and refinement: The 2-36-RBD complex model was built starting from template PDB structures 6BE2 (Fab) and 7BZ5 (RBD) using Phenix Sculptor.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phenix Sculptor</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Automated and manual model building were iteratively performed using real space refinement in Phenix [16] and Coot [17].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phenix</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Geometry validation and structure quality assessment were performed using Molprobity [18].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Molprobity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The visualization of sequence entropy was displayed by PyMol version 2.3.2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr></table>

---

Results from OddPub: Thank you for sharing your data.

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2021.10.13.464254: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">In brief, female Syrian hamsters (Mesocricetus auratus) of 6-8 weeks old were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 μL containing 1×104 TCID50 Beta B.1.351 (derived from hCoV-19/Belgium/rega-1920/2021; EPI_ISL_896474, 2021-01-11).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody isolation and recombinant production: Antigen specific IgG+ memory B cells were isolated and cloned from PBMC of SARS-CoV-2 convalescent individuals.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Antigen specific IgG+</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">goat anti-human IgG secondary antibody (Southern Biotech, 2040-04) was added and incubated for 45 min at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>goat anti-human IgG secondary antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (SouthernBiotech Cat# 2040-04, RRID:AB_2795643)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 2 h, infected cells were washed an additional five times with DMEM prior to adding media supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC) to reduce parental background.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-VSV-G</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were fixed with 4% PFA (Electron Microscopy Sciences, #15714S), permeabilized with Triton X-100 (SIGMA, #X100-500ML) and stained with an antibody against the viral nucleocapsid protein (Sino Biologicals, #40143-R001) followed by a staining with the nuclear dye Hoechst 33342 (Fisher Scientific, # H1399) and a goat anti-rabbit Alexa Fluor 647 antibody (Invitrogen, #A-21245).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>#X100-500ML</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: (Molecular Probes Cat# A-21245, RRID:AB_141775)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T-ACE2, Vero-TMPRSS2) (32)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped viruses were prepared using Lenti-X 293 cells seeded in 15-cm dishes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For neutralization experiments, HEK-293T cells expressing hACE2 (Crawford et al. 2020) in DMEM supplemented with 10% FBS and 1% PenStrep were seeded at 20,000 cells per well into clear bottom, white manually poly-D-lysine coated 96 well plates and incubated at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization of authentic SARS-CoV-2 viruses: Vero-TMPRSS2 cells were seeded into black-walled, clear-bottom 96-well plates at 2 × 104 cells/well and cultured overnight at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell-surface mAb-mediated S1 shedding: CHO cells stably expressing the prototypic SARS-CoV-2 Spike protein were harvested, washed in wash buffer (PBS 1% BSA 2 mM EDTA) and resuspended in PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO</div><div>suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral replication fitness assays: Vero E6 cells (ATCC, CRL-1586) were seeded at 1×106 cells per well in 6-well plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After a 15-minute incubation, Jurkat cells stably expressing FcγRIIIa receptor (V158 variant) or FcγRIIa receptor (H131 variant) and NFAT-driven luciferase gene (effector cells) were added at an effector to target ratio of 6:1 for FcγRIIIa and 5:1 for FcγRIIa.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">fold-on trimerization motif, and an 8× His tag in the pCMV vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_20783)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Using the Database IMGT (http://www.imgt.org), the VH and VL gene family and the number of somatic mutations were determined by analyzing the homology of the VH and VL sequences to known human V, D and J genes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>http://www.imgt.org</div><div>suggested: (IMGT - the international ImMunoGeneTics information system, RRID:SCR_012780)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">UCA sequences of heavy and light variable regions were constructed using IMGT/V-QUEST.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IMGT/V-QUEST</div><div>suggested: (IMGT/V-QUEST, RRID:SCR_010749)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 45 min incubation, absorbance at 405 nm was measured by a plate reader (Biotek) and data plotted using Prism GraphPad.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After a 30 sec stabilization step in KB, biosensors were moved in SARS-CoV or SARS-CoV-2 :2 dilution series (starting concentration: 18.5 nM) for the 600 sec association step, and then moved back in KB to record dissociation signals for 540 sec.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: (BioLegend Cat# 946101, RRID:AB_2892515)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data were baseline subtracted, results fitted using the Pall FortéBio/Sartorius analysis software (version 12.0) and plotted using GraphPad Prism (version 9.1.1</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed and visualized with Prism (Version 9.1.1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Micrographs were recorded using the Leginon software on a 120 kV FEI Tecnai G2 Spirit with a Gatan Ultrascan 4000 4k</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Leginon</div><div>suggested: (Leginon, RRID:SCR_016731)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">3D refinements were carried out using non-uniform refinement along with per-particle defocus refinement in CryoSPARC (57).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Model building and refinement: UCSF Chimera (61) and Coot (62) were used to fit atomic models into the cryoEM maps.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This variant was originally isolated in house from nasopharyngeal swabs taken from travelers returning to Belgium (baseline surveillance) and were subjected to sequencing on a MinION platform (Oxford Nanopore) directly from the nasopharyngeal swabs (65).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MinION</div><div>suggested: (MinION, RRID:SCR_017985)</div></div></td></tr></table>

---

Results from OddPub: Thank you for sharing your code and data.

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

---

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 24. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2021.10.16.464644: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cleaned Fasta file was then uploaded to SnapGene (version 5.3.2) for multisequence analysis via the MAFFT tool.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SnapGene</div><div>suggested: (SnapGene, RRID:SCR_015052)</div></div><div style="margin-bottom:8px"><div>MAFFT</div><div>suggested: (MAFFT, RRID:SCR_011811)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">With these factors all considered, an optimal tree was selected from the 21 trees for re-rooting via FigTree (https://github.com/rambaut/figtree/releases/tag/v1.4.4).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FigTree</div><div>suggested: (FigTree, RRID:SCR_008515)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting tree was exported for image processing via Adobe Photoshop and subsequent presentation via Illustrator.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Adobe Photoshop</div><div>suggested: (Adobe Photoshop, RRID:SCR_014199)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PyMol structural modeling: The PyMol molecular graphics system (version 2.4.2, https://pymol.org/2/) from Schrödinger, Inc. was used for downloading structure files from the PDB database for further analysis and image export.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The images were cropped via Adobe Photoshop and further presentation using Illustrator.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Illustrator</div><div>suggested: (Adobe Illustrator, RRID:SCR_010279)</div></div></td></tr></table>

---

Results from OddPub: Thank you for sharing your code.

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2021.10.13.21264916: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Deidentified data from staff testing and patients were obtained from the Infections in Oxfordshire Research Database (IORD) which has generic Research Ethics Committee, Health Research Authority and Confidentiality Advisory Group approvals (19/SC/0403, ECC5-017(A)/2009).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Secondary antibody rabbit anti-human whole IgG conjugated to alkaline phosphatase (Sigma, USA) or a secondary antibody mouse anti-human IgA conjugated to horse radish peroxidase (Sigma, USA) was added at a dilution of 1:1000 in casein–PBS solution and incubated for 1 hour at RT after which a final wash was performed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human whole IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For detection of the IgA secondary antibody, plates were developed by adding Tetramethylbenzidine (TMB) (Thermo scientific USA) to visualize and Sulfuric acid to stop the reaction.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A 1x working concentration of the SULFO-TAG anti-human IgG Detection Antibody was prepared in Diluent 100.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (RevMAb Biosciences Cat# 31-1019-MK, RRID:AB_2783627)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Microplates were coated with RBD in Dulbecco’s Phosphate Buffered Saline (DPBS) to bind corresponding ACE2 or blocking antibodies of the sample.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MSD V-PLEX assay: IgG antibody responses to SARS-CoV-2 spike, RBD, NTD and nucleocapsid and the spike proteins of SARS-CoV-1, HCoV-229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43 were assessed using the Meso Scale Diagnostics (MSD) Multi-Spot Assay System (MSD, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HCoV-NL63</div><div>suggested: RRID:CVCL_RW88)</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<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 10.1101/2021.10.17.464700: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Patient Samples and Collection: Use of human samples for this study was approved by the University of Ottawa Ethics Review Board: Certificates H-04-20-5727, H-04-21-6643 and H-07-20-6009.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In initial experiments, aliquots of each fraction were visualized via immunoblotting (anti-His antibody; Cat: SAB2702218</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In conjunction, titration curves of conformation-dependent monoclonal IgM (Absolute Antibody, Ab01680-15.0), IgA (Absolute Antibody, Ab01680-16.0), and IgG (Absolute Antibody, Ab01680-10.0) CR3022 antibodies were used as reference material to assess protein folding.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>titration curves of conformation-dependent monoclonal IgM ( Absolute Antibody , Ab01680-15.0) , IgA ( Absolute Antibody , Ab01680-16.0)</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG ( Absolute Antibody , Ab01680-10.0 ) CR3022 antibodies</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After blocking, plates were washed thrice with PBS-T, and followed by addition of 100 μL of the respective diluted serum samples and CR3022 antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CR3022</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plates were incubated for two hours on a shaker at room temperature, washed thrice with PBS-T followed by the addition of 50 μL of the respective secondary-HRP antibody at specified dilutions (1:4000 secondary anti-human IgG-HRP (NRC anti-hIgG#5-HRP</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG-HRP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Surrogate Neutralization ELISA (snELISA) assay for evaluation of neutralization in serum samples: The described methodology was adapted from the surrogate neutralization ELISA assay as shown in Abe et al. 2020, for the evaluation of the relative inhibition of neutralizing antibodies to RBD protein from binding to soluble ACE2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD Production in mammalian cells: For comparison, a mammalian RBD was produced in HEK 293F cells and purified.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293F</div><div>suggested: RRID:CVCL_6642)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(Mount Sinai, NYC) encoding the Whuhan-Hu-1 RBD (MN908947) sequence coding for the amino acid 319-541 and fused with the N-terminal SARS-CoV-2 spike secretory signal and a C-terminal hexa-histidine tag was transfected into 293F cells cultivated in Freestyle 293 expression media (Thermo Fisher, #12338018) at 37°C, 7% CO2, while shaking (125rpm).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Whuhan-Hu-1 RBD</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The construct was codon-optimized for N. benthamiana expression and was synthesized by GenScript into the pHREAC vector (Peyret et al., 2019) via BsaI sites</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHREAC</div><div>suggested: RRID:Addgene_134908)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lectin-binding human chaperone calreticulin (NP_004334.1) was codon-optimized for N. benthamiana and synthesized in the pHRE vector (Peyret et al., 2019).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHRE</div><div>suggested: RRID:Addgene_134909)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Transient expression in Nicotiana benthamiana: Prior to infiltration, pHREAC-RBD and pHRE-calreticulin were freshly transformed into Agrobacterium tumefaciens strains AGL1 and Gv2260, respectively, via electroporation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHREAC-RBD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pHRE-calreticulin</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Dissociation constant (KD) was determined using 4-parameter curve fitting with GraphPad Prism 9.1.2 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

I love the verbs chosen for each page/discussion/assignment. It is a great call to action! The structure is outstanding. The only issue I see with this display is that the image raises an equity issue - students who require screen readers will not be able to access this information unless there is a really long alt tag associated with the image.

Go here https://github.com/clojure/tools.build

Reviewer #1 (Public Review): 

In this manuscript, the authors challenge the long-standing conclusion that Orco and IR-dependent olfactory receptor neurons are segregated into subtypes such that Orco and IR expression do not overlap. First, the authors generate new knock-in lines to tag the endogenous loci with an expression reporter system, QF/QUAS. They then compare the observed expression of these knock-ins with the widely used system of enhancer transgenes of the same receptors, namely Orco, IR8a, IR25a, and IR76b. Surprisingly, they observe an expansion of the expression of the individual knock-in reporters as compared to the transgenic reporters in more chemosensory neurons targeting more glomeruli per receptor type than previously reported. They verify the expression of the knock-in reporters with antibody staining, in situ hybridization and by mining RNA sequencing data. 

Finally, they address the question of physiological relevance of such co-expression of receptor systems by combining optogenetic activation with single sensillum recordings and mutant analysis. Their data suggests that IR25a activation can modulate Orco-dependent signaling and activation of olfactory sensory neurons. 

The paper is well written and easy to follow. The data are well presented and very convincing due in part to the combination of complementary methods used to test the same point. Thus, the finding that co-receptors are more broadly and overlappingly expressed than previously thought is very convincing and invites speculation of how this might be relevant for the animal and chemosensory processing in general. In addition, the new method to make knock-ins and the generated knock-ins themselves will be of interest to the fly community. 

The last part of the manuscript, although perhaps the most interesting, is the least developed compared to the other parts. In particular, the following points could be addressed: 

- It would be good to see a few more traces and not just the quantifications. For instance, the trace of ethyl acetate in Fig. 6C, and penthyl acetate for 6G. 

- In Fig. 4D, the authors show the non-retinal fed control, which is great. An additional genetic control fed with retinal would have been nice. 

- It appears that mostly IR25a is strongly co-expressed with other co-receptors. The provided experiments suggest a possible modulation between IR25a and Orco-dependent neuronal activity. However, what does this mean? How could this be relevant? And moreover, is this a feature of Drosophila melanogaster after many generations in laboratories?

Noting here University of Edinburgh's collection of OERs that support SD4 including

• Foundations for All - supporting refugee scholars
• Wikimedia in Education published in cooperation with Wikimedia UK "bringing together a collection of case studies from across the UK in order to provide insight into the use of the Wikimedia projects in education."
• Critical Thinking in Global Challenges - a series of open licensed videos "to better understand what critical thinking is, and to practice and enhance critical thinking skills" and how they were applied in course activities designed around global issues

There's much more here https://open.ed.ac.uk/tag/sdg4/

Here is a way to make better highlights

SciScore for 10.1101/2021.10.12.464150: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Informed consent was obtained from all patients, and all patient samples were collected after a full recovery from illness and under IRB approval.<br>IACUC: Animal work was performed at Lovelace Biomedical Research Institute (LBRI), with approval from the Institutional Animal Care and Use Committee (IACUC) and within Animal Biosafety Level 3 (ABSL3) containment.<br>Euthanasia Agents: All animals were euthanized with an euthanasia solution consisting of 390 mg of sodium pentobarbital and 50 mg of phenytoin per mL.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">The FPF of delivered dose was calculated as the total amount of sugar or sugar alcohol (e.g., trehaose, mannitol) collected with an aerodynamic diameter below 5 µm as a percentage of the total amount of sugar or sugar alcohol deposited on the adapter, the induction port, stages 1–7 and Micro-Orifice Collector. 2.8. Efficacy of AUG-3387 in and in vivo model of SARS-CoV-2 infected Syrian Hamsters: An in vivo efficacy study was performed with male Syrian Hamsters (Mesocricetus auratus) approximately 9 weeks of age with a weight range of 110-134 g, at time of randomization, were sourced from Charles River Laboratory.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The FPF of delivered dose was calculated as the total amount of sugar or sugar alcohol (e.g., trehaose, mannitol) collected with an aerodynamic diameter below 5 µm as a percentage of the total amount of sugar or sugar alcohol deposited on the adapter, the induction port, stages 1–7 and Micro-Orifice Collector. 2.8. Efficacy of AUG-3387 in and in vivo model of SARS-CoV-2 infected Syrian Hamsters: An in vivo efficacy study was performed with male Syrian Hamsters (Mesocricetus auratus) approximately 9 weeks of age with a weight range of 110-134 g, at time of randomization, were sourced from Charles River Laboratory.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A fluorescently labelled anti-human IgG/IgA/IgM secondary antibody was also added to each well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG/IgA/IgM</div><div>suggested: (Sigma-Aldrich Cat# S4893, RRID:AB_10625701)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For identification of surface bound antibodies binding to SARS-CoV-2, SARS-CoV-2 antigens conjugated to Alexa Fluor 488 stained SARS-CoV-2 antigen was used as a staining agent for single cell sorting of antigen reactive memory B cells into 96 well plates on a Sony SH-800 Cell Sorter.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: (Thermo Fisher Scientific Cat# 53-6490-82, RRID:AB_2884046)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">AUG-3705 was attached to the LSA flow cell via interaction with its V5 tag and a surface bound anti-V5 antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-V5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cells were enriched 4 times until 97% of the cells showed signal above the negative control as read out by staining with anti-ACE-2 and secondary antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-ACE-2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Assay: 2.4.1 ACE-2 Expressing HEK293T Cell Line Construction: An ACE2 expressing HEK293T cell line (“LentiX ACE2.S4”) was constructed by packaging pCMV-AC-GFP (Origene) into lentivirus and transducing HEK293T’s (ATCC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infected cell culture supernatant was diluted with 950 µL D10 media, and then serial diluted before 50 µL of each dilution was added to 8 wells of Vero E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 96 hours, 100 µL CellTiterGlo reagent was added to each well of the infected Calu-3 cells to assay for live cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu-3</div><div>suggested: BCRJ Cat# 0264, RRID:CVCL_0609)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Assay: 2.4.1 ACE-2 Expressing HEK293T Cell Line Construction: An ACE2 expressing HEK293T cell line (“LentiX ACE2.S4”) was constructed by packaging pCMV-AC-GFP (Origene) into lentivirus and transducing HEK293T’s (ATCC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-AC-GFP</div><div>suggested: None</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04872231</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Completed</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Single Ascending Dose and Multiple Ascending Dose Study of V…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04576325</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Pharmacokinetic Profile of Voriconazole Inhalation Powder in…</td></tr></table>

---

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

---

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

---

Results from rtransparent:

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

---

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

---

<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 10.1101/2021.10.12.464114: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Description of cohort and ethics statement: Samples were collected from participants enrolled in a prospective cohort study approved by the Biomedical Research Ethics Committee (BREC) at the University of KwaZulu–Natal (reference BREC/00001275/2020).<br>Consent: Written informed consent was obtained from each participant.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Four were males and 5 were females.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For RBD expression experiments, 45 OD units of yeast were labeled in 1:100 diluted chicken-anti-Myc-FITC antibody (Immunology Consultants CMYC45F) to detect the RBD’s C-terminal Myc tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Myc tag .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After the plasma incubations, the libraries were secondarily labeled for 1 hour with 1:100 fluorescein isothiocyanate-conjugated anti-MYC antibody (Immunology Consultants Lab, CYMC-45F) to label for RBD expression and 1:200 Alexa Fluor-647-conjugated goat anti-human-IgA+IgG+IgM (Jackson ImmunoResearch 109-605-064) to label for bound plasma antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-MYC</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human-IgA+IgG+IgM</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, version 0.8.10) to process Illumina sequences into counts of each barcoded RBD variant in each pre-selection and antibody-escape population.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-escape population .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Magnetic Separation Rack, Thermo Fisher Scientific, CS15000) was used to separate antibodies that bind RBD from the supernatant, and the supernatant (the post-RBD antibody depletion sample) was removed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>post-RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Note that these assays were performed in 293T cells over-expressing human ACE2, which may underestimate contributions of non-RBD-binding antibodies to viral neutralization (7, 35, 60).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Dilution series of the synthetic sera comprised of the anti-RBD antibody REGN10987 (72), which binds to both Wuhan-1-like RBD and B.1.351 RBD, and pooled pre-pandemic human serum from 2017-2018 (Gemini Biosciences; nos. 100–110, lot H86W03J; pooled from 75 donors) were performed such that the anti-spike antibody was present at a highest concentration of 0.25 µg/mL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pre-pandemic serum alone, without anti-RBD antibody depletion, was used as a negative control, averaged over 2 replicates</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody binding was detected with TMB/E HRP substrate (Millipore Sigma, ES001) and 1 N HCl was used to stop the reaction.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ES001</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Generation of pseudotyped lentiviral particles: HEK-293T (American Type Culture Collection, CRL-3216) cells were used to generate SARS-CoV-2 spike-pseudotyped lentiviral particles and 293T-ACE2 cells (Biodefense and Emerging Infectious Research Resources Repository (BEI Resources), NR-52511) were used to titer the SARS-CoV-2 spike-pseudotyped lentiviral particles and to perform neutralization assays (see below).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate spike-pseudotyped lentiviral particles (70), 6e105 HEK-293T (ATCC CRL-3216) cells per well were seeded in 6-well plates in 2 mL D10 growth media (Dulbecco’s Modified Eagle Medium with 10% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Note that these assays were performed in 293T cells over-expressing human ACE2, which may underestimate contributions of non-RBD-binding antibodies to viral neutralization (7, 35, 60).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For experiments involving D614G spike, we used spike-pseudotyped lentiviral particles that were generated essentially as described in (70), using a codon-optimized SARS-CoV-2 spike from Wuhan-Hu-1 strain that contains a 21-amino-acid deletion at the end of the cytoplasmic tail (27) and the D614G mutation that is now predominant in human SARS-CoV-2 (30).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Wuhan-Hu-1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">AWY101 yeast containing a negative control (containing an empty vector pETcon plasmid) were grown overnight at 30°C in galactose-containing media.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pETcon</div><div>suggested: RRID:Addgene_41522)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting CCSs are available on the NCBI Sequence Read Archive, BioProject PRJNA770094,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NCBI Sequence Read Archive</div><div>suggested: (NCBI Sequence Read Archive (SRA, RRID:SCR_004891)</div></div><div style="margin-bottom:8px"><div>BioProject</div><div>suggested: (NCBI BioProject, RRID:SCR_004801)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical Analysis: The percent of neutralizing activity of early-2020 and B.1.351-convalescent plasmas due to RBD-binding antibodies is plotted with the plotnine python package, version 0.8.0 (https://plotnine.readthedocs.io/en/stable/index.html), shown as a Tukey boxplot (middle line indicating median, box limits indicating interquartile range) with individual measurements overlaid as points.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>python</div><div>suggested: (IPython, RRID:SCR_001658)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All raw sequencing data are available on the NCBI Short Read Archive at BioProject PRJNA770094, BioSample SAMN22208699, SAMN22208700.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NCBI Short Read Archive</div><div>suggested: None</div></div></td></tr></table>

---

Results from OddPub: Thank you for sharing your code and data.

---

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

Our study has several limitations. The cohorts of individuals infected with early 2020 and B.1.351 viruses are small, and are geographically and temporally distinct. Nevertheless, the two cohorts are relatively well-matched with respect to age, sex, and days-post symptom onset of sample collection (Table 1) and assays were performed under comparable conditions. Our deep mutational scanning measured binding to yeast-displayed RBD, which may not capture all relevant features of full-length spike in the context of virus. Finally, our neutralization assays used pseudotyped lentiviral particles and ACE2-overexpressing cells, and some recent works suggest that the relative importance of different spike epitopes for neutralization can depend on the viral system and target cell line used (7, 35, 36, 60). Although the B.1.351 variant has now been displaced by the Delta variant, our results illustrate the need to understand immunity elicited by different SARS-CoV-2 variants. As population immunity due to infection or vaccination increases, preexisting immunity is becoming an increasingly important driver of SARS-CoV-2 evolution (61), as has shown to be the case for seasonal coronaviruses (62, 63). Moreover, as individuals begin to accumulate more complex SARS-CoV-2 immune histories due to multiple infections and/or vaccinations, the effects of immune imprinting or original antigenic sin (64, 65) may start to interact with the variant-specific immunodominance hierarchies we have describ...

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

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

How to tag and store the "really good sources" for later review? I wouldn't want to have thousands of really good sources because then I wouldn't have any bandwidth for new material. Always pop something out if something goes in?

SciScore for 10.1101/2021.10.11.463956: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">No statistical methods were used to predetermine sample size.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell Lines: 293T (or HEK293T) cells, 293T-ACE2, and Expi293F cells were cultured in DMEM media, supplemented with 10% FBS, 100 U/ml penicillin/ streptomycin, 2 mM L-glutamine, and in the presence of 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Expi293F</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293T-ACE2 cells, derived from 293T, stably express human ACE2 71.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expi293F cells, derived from the 293 cells, were purchased from ThermoFisher Scientific (cat # A14528; RRID: CVCL_D615).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293F</div><div>detected: ( RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral infectivity measurements: The infectivity of lentivirus particles carrying S proteins (including variants) on the surface was evaluated using a vector containing an HIV-1 long terminal repeat (LTR) that expresses a Gaussia luciferase reporter (HIV-1-inGluc) 72,73. 293T cells were transfected at 60–80 % confluency with the plasmid encoding indicated full-length SARS-CoV-2 S glycoproteins, the plasmid encoding an intron-regulated Gluc (HIV-1-inGluc), and a plasmid pCMV delta R8.2 encoding HIV-1 GagPol (Addgene, plasmid # 12263) using FuGENE 6 (Promega, # E2311).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Untagged and double-tagged SARS-CoV-2 spike variants were generated based on a template full-length pCMV3-SARS-CoV-2 Spike (codon-optimized, Sino Biological, cat # VG40589-UT) plasmid that has translated amino acid sequence identical to QHD43416.1 (GenBank).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">D614G point mutation was introduced into both untagged full-length pCMV3 SD614 and double-tagged SD614 Q3/A4 constructs to generate both untagged and tagged SG614 variants.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3</div><div>suggested: RRID:Addgene_161029)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">E484K point mutation was introduced into both untagged and tagged pcDNA3.1 SAlpha by site-specific mutagenesis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral infectivity measurements: The infectivity of lentivirus particles carrying S proteins (including variants) on the surface was evaluated using a vector containing an HIV-1 long terminal repeat (LTR) that expresses a Gaussia luciferase reporter (HIV-1-inGluc) 72,73. 293T cells were transfected at 60–80 % confluency with the plasmid encoding indicated full-length SARS-CoV-2 S glycoproteins, the plasmid encoding an intron-regulated Gluc (HIV-1-inGluc), and a plasmid pCMV delta R8.2 encoding HIV-1 GagPol (Addgene, plasmid # 12263) using FuGENE 6 (Promega, # E2311).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_20783)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, DNA sequence encoding monomeric hACE2 followed by an HRV3C cleavage site, monomeric Fc tag, and 8xHisTag at the 3’-end were synthesized and cloned into the pVRC8400 vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVRC8400</div><div>suggested: RRID:Addgene_63164)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Where indicated, the conformational effects of hACE2 on S proteins were conducted by pre-incubating fluorescently labeled viruses with 200 μg/ml hACE2 for 90 mins at room temperature before imaging and smFRET imaging data were taken in the continued presence of 200 μg/ml hACE2. smFRET quantification and statistical analysis: The analysis of smFRET data was performed using a MATLAB-based customized SPARTAN software package 78.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPARTAN</div><div>suggested: (SPARTAN, RRID:SCR_014901)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Based on visual inspection of fluorescence and FRET traces that revealed direct observations of state-to-state (conformation-to-conformation) transitions, FRET histograms were fitted into the sum of four Gaussian distributions using the least-squares fitting algorithm in MATLAB.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MATLAB</div><div>suggested: (MATLAB, RRID:SCR_001622)</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

---

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 20 and 22. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

Results from rtransparent:

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

---

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

---

<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>

Reviewer #1 (Public Review):

Cytoplasmic germ granules are a common feature of germ cells across species. Great effort has gone into trying to (1) identify proteins that localize to these condensates, (2) gain insights into how these granules assemble, and (3) characterize their functional significance. Pioneering studies on P granules in C. elegans have greatly expanded our understanding of these structures.

In this paper, Price et al. use proximately labeling to identify previously unknown P granule components. They tag several known P granule protein genes with TurboID at their endogenous loci and perform biotinylation reactions in two different ways. Labeled proteins were subsequentially pulled down and identified using mass spectrometry. This approach successfully identified known P granule components and many new potential candidates. The authors focus their efforts on characterizing two related proteins EGGD-1 and EGGD-2 (also known as MIP-1 and MIP-2, based on recently published work). Knockdown of EGGD-1 and EGGD-2 using RNAi results in P granule defects. Cas-9 induced mutations confirm and extend this genetic analysis. The authors also perform structure/function analysis on EGGD-1 and define specific roles for its LOTUS and Intrinsically Disordered Region (IDR) domains in perinuclear P granule formation and function. Epistasis analysis shows that EGGD-1 acts upstream of GLH-1 in P granule assembly, and overexpression of EGGD-1 can drive granule formation outside of germ cells.

Strengths

The P granule proteome data presented here provides a useful resource for the community. The genetic analysis on eggd-1 provides important insights into the function of a new P granule component. The data are convincing, and the experiments are well-controlled.

Weaknesses

There are relatively few weaknesses and the general conclusions are supported by the data.

I just bookmarked this article published today in Current Biology for later reading and annotation. While the article isn't specifically focused on memory, the fact that it touches on visual structures, emotion, music, and movement (dance) which are core to some peoples' memory toolkits, I thought that many here would find it to be of interest.

One of the authors provided the following tl;dr synopsis:

"Across the world, people express emotion through music and dance. But why do music and dance go together?

We tested a deceptively simple hypothesis: Music and movement are represented the same way in the brain."

• Article: Visual and auditory brain areas share a representational structure that supports emotion perception01283-5) (Current Biology, 2021)
• Preprint: https://www.biorxiv.org/content/10.1101/254961v4

For those who haven't integrated song or dance into their practices, searching around for the idea of songlines will give you some background on their possible uses.

cc: @LynneKelly

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

1. General Statements

We want to thank all three reviewers for their positive feedback, constructive comments, and suggestions for clarity and improvement. We are delighted to find their consensus that the manuscript represents a contribution to the field.

Accordingly, we made changes in the text (all highlighted in blue in the revised manuscript) and added a new figure as detailed in the point-by-point response.

2. Point-by-point description of the revisions

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

The authors describe results of the comprehensive analysis of the prevalence and functionality of intrinsically disordered regions of the pathogen-encoded signaling receptor Tir, which serves as an illustrative example of the bacterial effector proteins secreted by Attaching and Effacing (A/E) pathogens. This is an interesting and important study that represents an impressive amount of data generated computationally and using a broad spectrum of biophysical techniques. The work serves as a model of the well-designed and perfectly conducted study, where intriguing conclusions are based on the results of the comprehensive experiments. The manuscript is well-written and concise, and I have a real pleasure reading it. The text and figures are clear and accurate.

We thank the Reviewer for these positive comments on our work.

Although, in general, prior studies are referenced appropriately, the authors should mention that the pre-formed structural elements they found in Tir are in line with the concept of "PreSMos" (pre-structured motifs) previously introduced and described in several important studies from the laboratory of Kyou-Hoon Han.

We thank the Reviewer for this suggestion. We have added a sentence to acknowledge the presence of “PreSMos” in the target-free state of Tir as putative signatures for target-binding, referring to a review article summarizing several local structural elements in unbound IDPs:

“This supports the presence of pre-structured motifs (PreSMos) as pre-existing signatures for target binding and function within target-free Tir (72)**.”

Please, note that we decided to keep this discussion to a minimum, as we cannot rule out the contribution of the induced fit model to the binding mechanism (i.e., disorder-to-order transition upon binding).

Reviewer #1 (Significance (Required)):

Solid evidence is provided that structural disorder and short linear motifs represent common features of A/E pathogen effectors. In fact, using a set of bioinformatics tools, the authors first show that although prokaryotic proteins typically contain significantly less intrinsic disorder than eukaryotic proteins, A/E pathogen effectors are as disordered as eukaryotic proteins. Using the translocated intimin receptor (Tir) as a subject of focused study, the authors then utilized a number of biophysical techniques to draw an impressive picture of disorder-based functionality. This study clearly represents a major advancement in the field of functional intrinsic disorder in general and in disorder-based functionality of proteins expressed by pathogenic bacteria. This was adds significantly to the field and will have a noticeable impact.

Again, reading this manuscript was a real joy. Finally, this work perfectly fits in the area of my expertise, since for the past 25 years or so I am working on the different aspects of intrinsically disordered proteins.

Thank you for this encouraging assessment.

**Referee Cross-commenting**

I agree with the amended recommendation of reviewer #3 to add in the manuscript EPEC O127.

According to the suggestion of Reviewer #3, we have now included EPEC O127:H6 in the manuscript.

I completely agree with comments of reviewer #2 and partially agree with reviewer #3. In my view, comparison of various strains as references for EPEC represents an interesting but independent project. It can be recommended to the authors as one of the potential future developments of their work.

Thanks for the suggestion. We are pursuing that line of research.

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

The general impression is that this is an excellent study that establishes

The C-terminal intracellular region of Tir called C-Tir spanning residues 338 to 550 is largely disordered, however, observe helical structural elements involved with lipid interactions; multi-phosphorylation. The intracellular N-terminal part of Tir called N-Tir spanning residues 1 to 233 is also partially disordered but include a folded domain that is shown to assemble into a dimer

The only major concern is that no SDS-PAGE gels or size exclusion chromatograms have been included to verify purity and monodispersed of the various constructs worked on. In particular, the SAXS and CD measurement is highly sensitive to purity, and the level of degradation as IDPs are notorious for being difficult to handle in solution. it would strengthen the arguments made based that

We produced N-Tir and C-Tir as fusion proteins with a cleavable N-terminal thioredoxin tag (Trx-His6) and C-terminal Strep-tag. The latter allowed us to purify them via Strep-tag affinity chromatography as indicated by SDS-PAGE (please see Fig. S1).

We agree with the Reviewer that even small amounts of impurities (i.e., higher oligomers/degradation) can interfere with the data analysis and make interpretation of the resulting data difficult and potentially misleading. So, to avoid such problems, all samples were purified in monodispersed forms by size-exclusion chromatography (SEC) before any biophysical study.

Following the Reviewer's suggestion, we added a new supplementary figure (Fig. S5) showing the SEC-SAXS chromatogram profiles of C-Tir, N-Tir, and NS-Tir. Briefly, in the inline SEC-SAXS experiment, the sample eluates from an HPLC system directly and continuously into a BioSAXS flow cell for subsequent X-ray interrogation. Under our experimental conditions, C-Tir elutes as a single peak with Rg-values and mass compatible with a disordered monomeric protein, providing an excellent fit to the experimental SAXS curves. For N-Tir and NS-Tir, by SEC-SAXS, we separated the dimer from small amounts of high-order oligomers to yield the experimental SAXS curves of the pure dimers.

“Fig. S5. SEC-SAXS chromatograms of (A) C-Tir, (B) N-Tir, and (C) NS-Tir. Each plane shows normalized total scattering intensity I(s), over the entire s range, from each frame acquired along elution volume and respective Rg-value (black circles). The flat variation of Rg reflects a pure monodisperse sample. The column type for size exclusion chromatography and sample concentrations are on the top left of each panel. For reference, the retention volume for monomeric BSA (66.4 kDa) is displayed by red triangles.”

**Minor Comments**

Read through the manuscript to remove passages with spoken language

We thank the Reviewer for this suggestion. We went through the manuscript and improved the writing to reduce passages with spoken language.

Line 263, "To do so", should be removed

Line 290 "Our data thus" replaced with "this"

We have amended the manuscript accordingly.

Line 292 "lipid bilayers that might potentially fine-tune Tir's activity in the host cell." Weak sentence and the word fine-tune is slang. Rewrite the sentence. The interaction with lipids is fascinating!

Thanks for the suggestion. The sentence has now been changed to “**This shows that C-Tir can undergo multivalent and tunable electrostatic interaction with lipid bilayers via pre-structured elements, suggesting that membrane-protein interplay at the intracellular side might control the activity and interactions of Tir in host cells.**”

We also reinforce this fascinating message in the abstract by adding the sentence: “Membrane affinity is residue-specific and modulated by lipid composition, suggesting a previously unrecognized mechanism for interaction with the host.”

Line 192 "In figure Fig. 3A," remove the Fig

Fixed.

Line 326, "In a similar fashion," is redundant. Rewrite the sentences below.

We have modified the sentence as follows: “We evaluated whether the N-terminal cytosolic region of Tir (N-Tir; Fig S1) was also intrinsically disordered ...”

Line 342 add spaces between digit and SI unit "52kDa" there are more cases of this.

Thank you for pointing this out. This has now been corrected to 52 kDa.

Reviewer #2 (Significance (Required)):

I expect this study to have broad relevance to microbiologists working with the intimin and translocated intimin receptor, in particular the lipid interaction is likely to be followed up by the community.

We thank the reviewer for this comment. Indeed, we believe that further studies on Tir's lipid-binding ability as a novel molecular strategy in host-pathogen interactions, will potentially provide new insights on virulence, transmembrane signaling in general, and disorder-mediated functions.

**Referee Cross-commenting**

What reviewer 3 suggested in the comments sounds like added value and should be included.

I agree with reviewer 1, that the strain comparison potentially is beyond the scope presented in this manuscript.

We have now included EPEC O127:H6 in the manuscript.

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

**Summary:**

This interesting manuscript look at the structure of the Nter and Cter of the effector Tir from enteropathogenic E. coli. The authors confirmed previous study highlighting the "disordered" part of the Cter. However, the extended experimental work (NMR, Small-angle X-ray scattering and CD spectroscopy) from this study also reveals the connection between different area of Tir and its implication during Tir phosphorylation and its interactions with SH2 domain.

We thank the Reviewer for this positive remark. Indeed, in our work, we highlight the structural features of the SH2-mediated interaction between Tir and host SHP-1 protein, and we also show that C-Tir is capable of lipid interaction via pre-structured motifs and that N-Tir is disordered but assembled into a dimer. Overall, we provide an updated and wide picture of Tir's intracellular side that goes beyond the scrutiny of previously described disorder features.

**Major Comments:**

The authors used E2348/69 (O127:H7) strain as a reference for EPEC. However, this strain are the least effectors of all the EPEC sequences and may over estimated the PDR in EPEC. It would be wiser to use a strain like B171 as a reference for EPEC to be able to conclude "Disordered Proteins (PDR) with long disordered regions occur in EPEC effectors similar to the human proteome". I believe that the PDR in EPEC is similar to EHEC and CR. I do not have any major concern for the rest of the work.

We thank the Reviewer for this comment. So, to clarify, we amended “EPEC” with “EPEC O127:H6” in text and figures.

We also added a paragraph at the beginning of the Discussion section to acknowledge that our prediction analysis concerns EPEC O127:H6 and two additional representative A/E bacteria strains:

“Among the enteropathogenic Escherichia coli strains EPEC O127:H6 (E2348/69) is commonly used as a prototype strain to study EPEC biology, genetics, and virulence (69). Here, we have determined the structural disorder propensity of EPEC O127:H6 sequences and two additional representatives of A/E bacteria: EHEC O157:H7 and CR ICC168.”

Finally, the Reviewer suggests to include EPEC strain B171 (serotype O111:NM) in our analysis. We agree that considering additional strains would be of value, however we believe that this is beyond the scope of this manuscript, which mainly focuses on the characterization of the structural features of the E2348/69 Tir effector. We are currently working on a broader comparative analysis among different Escherichia coli pathogenic strains, including B171, and we hope to share our findings with the community in the near future.

**Minor comments**

Statistic problem: Mann Whitney U Test (Wilcoxon Rank Sum Test) is a comparison of two independent samples with the underlying assumption is normally distributed or that the samples were sufficiently large. It is not certain that any of this assumption is correct. In addition, the effector are part of the whole proteome. Can it be then considered that both groups are independent?

We thank the Reviewer for this remark, which allows us to clarify the choice of this particular test. Indeed the Mann Whitney U-test is a non-parametric test to compare two samples with the alternative hypothesis being that one of the two samples is stochastically greater than the other. As it is a nonparametric test samples are not required to be normally distributed, as it is for the Student t-test.

Regarding the independence of the samples, when comparing the effectors collections to their corresponding proteomes, we did exclude the effectors sequences from the latter. We have clarified this point in the Supplementary Material and Methods section.

Line 120 and 442: O127 not H127

Thank you for pointing this out. It has now been corrected to O127.

Line 212: positions 409 or 405?

Yes, it should be 405. Thank you.

Reviewer #3 (Significance (Required)):

**Nature and significance:**

Tir plays a major role during EPEC infection. It is a signalling platform that has been reported to interact with multiple proteins. Whereas the extracellular part has been well characterised and crystallised, the intracellular part has been proven so far to be difficult to study. Over the last decade, no progress has been made to explain how Tir works. This manuscript provides interesting information that shade some light on how the protein could work.

**Existing literature:**

The last research manuscript trying to highlight the structural function of Tir dates from 2007 (PMC1896257). This study is far more extended and in depth than any other previous work done.

**Audience:**

the Audience may probably limited to researcher working on the field of cellular microbiology and the function associated with bacterial effector in the host. This study could be also a useful tool to identify new effectors base on their "disorder".

We thank the Reviewer for recognizing the importance of this study. We agree that our work highlights the pivotal role of disordered regions in bacterial effectors, thus enabling a better understanding of the molecular mechanisms used by pathogens to subvert the host-cell processes. We indeed believe that our work can stimulate further research on the characterization of intrinsically disordered effectors, and also beyond the cellular microbiology field, in order to gain a broader knowledge on the molecular dialogue at the host-pathogen interface, which is essential to design better therapeutic strategies.

**Expertise:**

I have been working on A/E pathogens for the last 15 years with a particular interest in Tir signalling. My domain of expertise is more in relation to cell signalling than crystallography or structural study.

**Referee Cross-commenting**

I agree with both reviewers. My comment about EPEC is more about the conclusion for some of the figures. I don't think they should conclude for the whole EPEC. The Tir variation among EHEC O157:H7 is low, but it is far more diverse for EPEC. Simply adding in the manuscript EPEC O127 should be enough.

We thank the Reviewer for this comment. As mentioned above, we now state in the manuscript, in both Results and Discussion sections, that we used E2348/69 as a representative strain for EPEC.

SciScore for 10.1101/2021.10.05.21264054: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Study participants and ethics statement: Blood samples were obtained from 143 dialysis patients and 48 healthcare workers under study protocols approved by the local Institutional Review Boards (Canton Ticino Ethics Committee, Switzerland).<br>Consent: All subjects provided written informed consent for the use of blood and blood components (such as PBMCs, sera or plasma).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">Statistical analysis: The study was designed to have 80% power to detect a minimum 25% difference in total incidence of cases with poor neutralizing antibody response (i.e., low or undetectable plasma antibody titers) or in average neutralizing titers between dialysis patients and healthy controls as well as within the HD subgroups.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: Cell lines used in this study were obtained from ATCC (Vero E6 TMPRSS2) or ThermoFisher Scientific (Expi CHO cells, Expi293F™ and FreeStyle 293 cells)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6 TMPRSS2 cells were grown in DMEM supplemented with 10% HyClone (FBS)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6 TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasma pseudovirus neutralization assay: Vero E6-TMPRSS2 were grown in DMEM supplemented with 10% FBS and seeded into white bottom 96 well plates (PerkinElmer, 6005688), as previously described35.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6-TMPRSS2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of recombinant glycoproteins: The SARS-CoV-2 RBD WT construct was synthesized by GenScript into phCMV1, with a sequence encoding an N-terminal mu-phosphatase signal peptide, an ‘ETGT’ linker, SARS-CoV-2 S residues 328-531, a linker sequence, an Avi tag, a twin Strep tag and a 8xHis-tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>phCMV1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 stabilized Spike WT (D614G) construct was synthesized by GenScript into pCDNA with an N-terminal mu-phosphatase signal peptide, 2P stabilizing mutation28,29, a TEV cleavage site and a C-terminal foldon, 8x His-tag, Avi tag and C-tag30 and expressed in FreeStyle 293 cells following manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA</div><div>suggested: RRID:Addgene_105932)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Amino acid substitutions were introduced into the D614G pCDNA_SARS-CoV-2_S plasmid as previously described31.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA_SARS-CoV-2_S</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed and visualized with Prism (Version 9.1.0).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were plotted and analyzed with GraphPad Prism software (version 9.1.0).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

---

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

Our study has some limitations including a missing control group of patients matched by age, gender and comorbidities, without ESKD, while our control group was composed by younger individuals with few or no comorbidities. Another limitation was the low number of PD patients that did not allow to make subgroup analyses of risk factors of defective response. In addition, our patients were unbalanced in terms of type of mRNA vaccine received (BNT162b2 or mRNA-1273), although we had sufficient statistical power to make a comparison, even in a subgroup of patients matched by age, gender and comorbidity index. In conclusion, our study demonstrates, at the functional level, that mRNA vaccines induce a defective neutralizing antibody response against SARS-CoV-2 variants in dialysis patients, in particular in naïve HD patients immunized with BNT162b2. Our findings support the need of an additional boost, preferentially with a high-dose mRNA vaccine, in this population58-60, which, however, need to be continuously monitored with proper serological tests that measure not only the serum antibody levels, but also their neutralizing activity, either directly or indirectly through an avidity test. Finally, our data suggest that some patients may not respond efficiently even after an additional boost and, therefore, in case of SARS-CoV-2 infection, they should be considered for other therapeutic strategies, including early immunotherapy with monoclonal antibodies.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<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>

• about : base tag

base tag HTML

SciScore for 10.1101/2021.10.05.463282: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Written informed consent was obtained from all patients.<br>IRB: This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster, the Hong Kong East Cluster Research Ethics Committee, and the Kowloon West Cluster Research Ethics Committee (UW 13-265, HKECREC-2018-068, KW/EX-20-038[144-26]).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Male and female golden Syrian hamsters (Mesocricetus auratus) (aged 6–10 weeks) were purchased from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Laboratory Animal Unit</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The hamsters were randomized from different litters into experimental groups.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Serially diluted plasma from healthy individuals or previously published monoclonal antibodies against HIV-1 (VRC01) were used as negative controls.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HIV-1</div><div>suggested: (bNAber Cat# bNAberID_1, RRID:AB_2491019)</div></div><div style="margin-bottom:8px"><div>VRC01</div><div>suggested: (bNAber Cat# bNAberID_1, RRID:AB_2491019)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two consecutive staining steps were conducted: the first one used an antibody and RBD cocktail incubation of 30 min at 4 °C; the second staining involved staining with anti-His-APC and anti-His-FITC antibodies (Abcam) at 4 °C for 30 min to detect the His tag of the RBD.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His-APC</div><div>suggested: (Miltenyi Biotec Cat# 130-101-320, RRID:AB_2747411)</div></div><div style="margin-bottom:8px"><div>anti-His-FITC</div><div>suggested: (Miltenyi Biotec Cat# 130-092-675, RRID:AB_1103226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For dIgA antibody production, plasmids of paired heavy chain (IgA1, IgA2) and kappa light chain together with a J chain were co-transfected into Expi293™ expression system (Thermo Fisher Scientific) at the ratio of 1:1:1 following the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgA1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgA2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies produced from cell culture supernatants were purified immediately by affinity chromatography using recombinant Protein G-Agarose (Thermo Fisher Scientific) or CaptureSelect™ IgA Affinity Matrix (Thermo Fisher Scientific) according to the manufacturer’s instructions, to purify IgG and IgA, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">2 mL-fractions were collected, pooled, concentrated and evaluated by western blot using mouse anti-IGJ monoclonal antibody [KT109] (Abcam) and rabbit anti-human IgA alpha chain antibody (Abcam).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-IGJ</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgA alpha chain antibody ( Abcam) .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The differences in response units between ACE2 injection alone and prior antibody incubation reflect the antibodies’ competitive ability against ACE2 binding to the spike protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For identification and localization of SARS-CoV-2 nucleocapsid protein (NP) in organ tissues, immunofluorescence staining was performed on deparaffinized and rehydrated tissue sections using a rabbit anti-SARS-CoV-2-NP protein antibody together with an AF488-conjugated anti-rabbit IgG (Jackson ImmunoResearch, PA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 nucleocapsid protein (NP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-2-NP protein</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After blocking with 0.1% Sudan black B for 15 min and 1% bovine serum albumin (BSA)/PBS at RT for 30 min, the primary rabbit anti-SARS-CoV-2-NP antibody (1:4000 dilution with 1% BSA/PBS) was incubated at 4°C overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2-NP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For identification of DC-SIGN expression, we stained the NT slices with rabbit anti-DC-SIGN primary antibody (Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-DC-SIGN</div><div>suggested: (IMGENEX Cat# DDX0208A488, RRID:AB_1929964)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For identification of ACE2 expression, the goat anti-ACE2 primary antibody (R&D) and Alexa Fluor 568 donkey anti-goat IgG (H+L) secondary antibodies (Invitrogen) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-goat IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The infectious medium was replaced with fresh medium containing respective concentration of antibody after washing 3 times with PBS. 24 h later, the infected cells were imaged under fluorescence microscope after staining with AF488-conjugated anti-SARS-CoV-2 NP antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 NP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were further permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit sera anti-SARS-CoV-2-N for 1 hour at RT before adding Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2-N</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After treatment with B8 antibodies at the dose of 3000 ng/ml/mL for 1 hour, HEK293T cells transfected with SARS-CoV-2 spike-GFP were added into the treated Vero-E6 TMPRSS2 cells and co-cultured for 48 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>B8</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The viral challenge experiments were then conducted in our Biosafety Level-3 animal facility following SOPs strictly, with strict adherence to SOPs Cell lines: HEK293T cells, HEK293T-hACE2 cells Vero-E6 cells, HK2 cells and Vero-E6-TMPRSS2 cells were maintained in DMEM containing 10% FBS, 2 mM L-glutamine, 100 U/mL/mL penicillin and incubated at 37 □ in a 5% CO2 setting 62</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-hACE2</div><div>suggested: RRID:CVCL_A7UK)</div></div><div style="margin-bottom:8px"><div>Vero-E6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Vero-E6-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, The pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively 77</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibody-virus mixtures were subsequently added to pre-seeded HEK 293T-ACE2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293T-ACE2</div><div>suggested: RRID:CVCL_A7UK)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mixtures were then transferred to 96-well plates pre-seeded with 1×104/well Vero E6 cells and incubated at 37°C for 24 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The HEK293T-CD209 cells were pre-treated with 10 ng/ml/mL of B8-dIgA or control dIgA and incubated for 6 h prior SARS-CoV-2 infection (MOI=0.05).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-CD209</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Effects of B8 antibodies on SARS-CoV-2 mediated cell-cell fusion: Vero-E6 TMPRSS2 cells were seeded into 48-well plates and cultured overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6 TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After treatment with B8 antibodies at the dose of 3000 ng/ml/mL for 1 hour, HEK293T cells transfected with SARS-CoV-2 spike-GFP were added into the treated Vero-E6 TMPRSS2 cells and co-cultured for 48 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, The pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively 77</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVax-1-S-COVID19</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pNL4-3Luc_Env_Vpr</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequences were aligned using Clustal W in the BioEdit sequence analysis package (Version 7.2)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BioEdit</div><div>suggested: (BioEdit, RRID:SCR_007361)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Half-maximal (IC50) or 90% (IC90) inhibitory concentrations of the evaluated antibody were determined by inhibitor vs. normalized response -- 4 Variable slope using GraphPad Prism 6 or later (GraphPad Software Inc.)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The defocus value of each image, which was set from −1.0 to −2.0 μm during data collection, was determined by Gctf.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gctf</div><div>suggested: (GCTF, RRID:SCR_016500)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequential data processing was carried out on RELION 3.0 and RELION 3.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RELION</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD-Fab maps were fitted onto the whole structure map using Chimera, then combined using PHENIX combine_focused_maps.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Model building and structure refinement: The spike model (PDB code: 6VSB) and the initial model of the B8 Fab generated by SWISS-Model were fitted into the EM density map, and further manually adjusted with Coot.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The final structures were validated using Phenix.molprobity</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>molprobity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">. UCSF Chimera, ChimeraX and PyMol were used for map segmentation and figure generation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">NP+ cells per field were quantified based on the mean fluorescence intensity (MFI) using the ZEN BLACK 3.0 and ImageJ (NIH).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The fluorescence density of SARS-CoV-2 infected cells was acquired using a Sapphire Biomolecular Imager (Azure Biosystems) and then the MFI of four randomly selected areas of each sample was quantified using Fiji software (NIH).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Fiji</div><div>suggested: (Fiji, RRID:SCR_002285)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Re-analysis of published nasal brushing single-cell data: The preprocessed scRNA-seq data from nasal brushing samples of 2 healthy controls and 4 COVID-19 patients were downloaded from Gene Expression Omnibus (GEO) database with accession numbers GSE171488 and GSE164547.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gene Expression Omnibus</div><div>suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quantification and statistical analysis: Statistical analysis was performed using PRISM 6.0 or later.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PRISM</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>

---

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

---

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

One limitation is the insufficient amounts of HuNAbs distributed on the nasal mucosal surface for protection 47. Other reasons might include alternative entry pathways engaged by SARS-CoV-2 to evade HuNAbs. To this end, Liu et al. reported recently that antibodies against the spike N-terminal domain (NTD) induced an open conformation of the RBD and thus enhanced the binding capacity of the spike to the ACE2 receptor, leading to increased viral infectivity 66. Yeung et al. demonstrated nicely that SARS-CoV-2 could engage soluble ACE2 (sACE2) and then bind alternate receptors for viral entry, through interaction between a spike/sACE2 complex with the angiotensin II AT1 receptor, or interaction between a spike/sACE2/vasopressin complex with the AVPR1B vasopressin receptor, respectively 49. In this study, we found that, in the presence of potent neutralizing B8-dIgA1 or B8-dIgA2 antibodies, SARS-CoV-2 used the cellular receptor CD209 for capture or infection, which likely expanded the use of CD209+ cells as target cells, leading to enhanced NT infection and trans-infection. Interestingly, a preprint report suggests that cells expressing CD209 can be infected directly by SARS-CoV-2 through an interaction of the spike with the NTD instead of the RBD 67. This mode of action, however, was unlikely to explain our findings, because no enhancement of SARS-CoV-2 nasal infection was found in presence of control dIgA1 and dIgA2. Our results rather suggest that the direct binding of virus-b...

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2021.10.03.462919: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: Animal work was performed at Lovelace Biomedical, with approval from the Institutional Animal Care and Use Committee.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following 2-hr sample incubation, the plate was washed and SULFO-TAG (MSD) anti-hamster IgA (Brookwood Biomedical) detection antibody was added.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-hamster IgA</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VeroE6 cell monolayers at ≥ 90% confluency in 96-well plates were rinsed with PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vaccine constructs: r-Ad-S is a rAd5 vector containing full-length SARS-CoV-2 S gene under control of the CMV promoter.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>rAd5</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IgA MSD: S1 protein was biotinylated according to manufacturer’s instructions (EZ-link, Thermofisher), and was conjugated to a U-Plex MSD linker (Mesoscale Diagnostics).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Thermofisher</div><div>suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)</div></div></td></tr></table>

---

Results from OddPub: Thank you for sharing your data.

---

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

Our study does have limitations. Firstly, we do not measure mucosal T cell responses which are shown to play a role in limiting SARS-CoV-2 infection at mucosal sites19. Additionally, the SAR-CoV-2 challenge dose we used was above physiological dose likely to be picked up by an environmental exposure, as evidenced by the high viral RNA load in the nose at 1-day post infection in all index hamsters. It is likely that mucosal immunization would provide greater protection against SARS-CoV-2 transmission when lower doses of challenge virus are used. Our study was meant to be a stringent challenge to clearly identify advantages between vaccine groups. For practical reasons, such as the limitation of the number of aerosol transmission chambers, lower dose levels or evaluation at later time points were not done. Additionally, future work should include challenging mucosally-vaccinated hamsters with the Delta variant and other variants of concern. An orally-delivered, temperature-stable SARS-CoV-2 vaccine is ideal for global vaccination, where adequate storage and qualified health care providers maybe in short supply. IN delivery has some of the same advantages, but translating IN SARS-CoV-2 vaccination efficacy in humans has proven to be more difficult than in animals20,21. Implementing oral vaccine campaigns around the world has been done, as evidenced by the rotavirus and poliovirus vaccination efforts22,23. We have previously demonstrated that our SARS-CoV-2 clinical candidate vac...

---

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04563702</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Safety and Immunogenicity Trial of an Oral SARS-CoV-2 Vaccin…</td></tr></table>

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

wtf is a H5P tag?! If someone knows and reads his, please help a girl out and tell me?

SciScore for 10.1101/2021.10.04.21264522: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Ethical considerations: The Cuban Ministry of Public Health (MINSAP), the Independent Ethics Committee (IEC) for Studies on Human Subjects, at CENATOX and the Cuban National Regulatory Agency (Centre for State Control of Medicines and Medical Devices, CECMED), approved the trial and the procedures (CECMED, Authorization date: 13/10/2020, Reference number: 05.013.20BA).<br>Consent: Written informed consent was obtained from all volunteers.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Sixty Cuban volunteers aged 19-59 years, with body mass index 18.5-29.9 kg/m2, of both sexes, were recruited (Table 1).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Study design and participants: This phase I, randomized, double-blind clinical trial was carried out at the National Centre of Toxicology (CENATOX) in Havana, Cuba.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">Randomization and blinding: Stratified random blinded sampling was used to select the sample of the universe of Cuban citizens aged 19-59 years, which was proportionally divided in two age subgroups: 19-39 and 40-59 years to ensure a proper representation of each age subgroup.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Exclusion criteria were: history of COVID-19, SARS-CoV-2 PCR-positive tests or detection of antibodies anti-SARS-CoV-2, any severe disease or decompensated chronic disease, immunodeficiency, history of serious allergy, pregnancy, breastfeeding, and immunological treatment during the last 30 days.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Seroconversion rates for IgG anti-RBD antibodies (≥4-fold increase in antibody titres over pre-immunization titres) were calculated for each subject.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">sequence: 319-541 amino acid residues with a poly-histidine fusion tag at its C-terminus), expressed in CHO cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO</div><div>suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analyses were done using SPSS version 25·0; EPIDAT version 4.1,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPSS</div><div>suggested: (SPSS, RRID:SCR_002865)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, Prism GraphPad version 6.0.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

This review reflects comments and contributions by Ricardo Carvalho, Joachim Goedhart, Sónia Gomes Pereira, Pratima Gurung, Samuel Lord, Claudia Molina, Arthur Molines, Gregory Redpath, Mugdha Sathe, Sagar Varankar. Review synthesized by Ewa Sitarska.

This preprint introduces a recombinant profilin that has a flexible linker to a genetically encoded fluorescent tag (either mApple or Halo). Fluorescent protein tagging is a popular and accepted method to study the properties of a protein of interest in solution and in cells. A careful analysis of the tagged protein relative to the untagged, native protein is crucial to understand whether the tagged protein faithfully reflects the behavior of the native protein. Therefore, studies like these are very valuable and the current manuscript is a good example of how such a study should be performed. The flexible linker presented here overcomes challenges observed in previous papers that found that linking a fluorescent protein to profilin disrupted some of its actin-related functions in cells and in vitro.

This study is carefully conducted and nicely describes the properties of a fluorescent protein tagged profilin in a detailed manner. In particular, the authors use various in vitro assays as well as rescue experiments to demonstrate that their tagged version of profilin appears to behave similarly to wildtype profilin. The manuscript is written in a clear manner and was an enjoyable read. The comments below cover a couple of experiments, clarifications and questions for consideration to further add to the work. Regardless, this work is likely to contribute to the field, as anyone studying profilin is likely to try this construct in their future experiments.

General comments:

• Why was mApple chosen as a tag (as opposed to the popular and best known fluorescent protein mEGFP)?
• The mApple is prone to photochromicity/photoswitching (https://doi.org/10.1038/nmeth.1209, https://doi.org/10.1038/nmeth.4074). This should be mentioned to warn future users of this fusion protein.
• It would be advisable to be consistent with the naming of ‘untagged profilin’ throughout the manuscript. Currently unlabeled, untagged, wild-type or rescue are used interchangeably.
• In Figures 4B, 5A there appears to be differences between untagged and tagged profilin in the images. Maybe a more representative image would be beneficial, where applicable.
• Depositing the plasmids from this paper at addgene.org would be beneficial for the public (the plasmids can be deposited under condition that these will be released only after publication of this work in a peer-reviewed journal).

Title

‘Functional fluorescently-tagged human profilin’– suggest clarifying in the title and throughout the text that the fluorescent tags are genetically encoded.

Abstract

‘high cellular concentrations (121 µM)’– This is a very precise number for such a general statement. It seems that the number is derived from a specific cell line, so it would be beneficial to present it as a number from this cell line or change it to an approximation (~100 µM).

Introduction

'Some profilin outcompetes actin bound'– suggest some rewording to clarify the fragment, for example, it could be mentioned whether it refers to F-actin or G-actin.

Results- Design of tagged profilin

‘Profilin is considerably smaller than the smallest fluorescent tags’ – clarifying that it is a genetically encoded fluorescent tag would be of advantage. There are no smaller fluorescent proteins (FP) yet, but genetically encoded FP of a similar size exist, for example miRFP670nano is 17kDa. https://doi.org/10.1038/s41467-018-08050-8

‘Traditional direct labeling approaches are cytotoxic and disrupt actin-based functions’ – is there data showing the new fluorescent profilin side-by-side with one without a flexible linker (or other version used previously) to show that the latter disrupts profilin's functions? It’s not essential but it would strengthen this point and confirm the improvement over prior work.

‘Estimates of cellular profilin concentration are very high depending on the cell type’ – would be nice to provide a rough estimate at this point, similarly to the introduction part.

‘with an mApple fluorescent probe or as Halo-tagged single molecules’ - What is the meaning of 'single' here?

‘We cloned mApple- or Halo-tags fused to a ten amino acid flexible linker on the N-terminus of human profilin-1.’ – As in the introduction, it is stated that “Positioning a GFP-derived fluorescent tag on the C- or N-terminus disrupts PLP- and PIP-binding interactions, effectively rendering the fluorescent version flawed for critical measurements in cells”, it would be beneficial to state the rationale for tagging profillin at the N-terminus? Also, how was the linker composition and length determined and how is it related to other linkers used in compromised fusions of profilin?

Figure 1

• In panel A, it would be helpful to indicate the N-terminus, as this is the side where the fluorescent protein is attached.
• In the legend, PFN1 is introduced for the first time and thus, it could be replaced with ‘tagged-profilin (PFN1)’.

Results - mApple-profilin binds phosphoinositide lipids with similar affinity as untagged profilin

‘PIP’ - PIP is usually an abbreviation for Phosphatidyl Inositol Phosphate (which is a lipid). Phosphoinositide is the same thing, but is not abbreviated as PIP. Recommend using Phosphatidyl Inositol Phosphate = PIP. These types of lipids can be indicated as PIPs (without the addition of lipids).

‘Profilin also binds PI(3,5)P2 which regulates critical signal transduction events through intracellular vesicles to the early endosome’ - The prevailing consensus now is that PI(3,5)P2 is involved in late endosomal trafficking to the lysosome. This could be different for profilin specific purposes, but this statement could be updated with recent references to PI(3,5)P2.

‘Thus, mApple-tagged profilin retains functional interactions with two important PIP lipids.’ – Testing other phospholipids, including PIP3 (Lu et al., 1996 showed an interaction with profilin) and some negative controls would be beneficial. Covering most phosphoinositide species by using the phospholipid-binding dot blots would make this figure stronger.

Figure 2

• In panel B, it would be easier to compare profilin and mApple-profilin binding affinity to each of the PIP lipids. For that, profilin and mApple-profilin samples could be run side by side in the same blot. The suggestion being that panel B includes profilin and mApple-profilin incubated with PI(3,5)P2, and panel C profilin and mApple-profilin incubated with PI(4,5)P2. For clarity, can it be specified in the figure legend or in the figure that the profilin-1 lane is the negative control pellet lacking the liposomes as well as that S and P stands for supernatant.
• Also, in panel B, what are the loading controls? The quantification and western-blots are unclear. For example, it is indicated that 1µM PFN1was used in this experiment, but the pellet band for mAp-PFN1 is not comparable to PFN1 pellet band. Despite this, quantification in 2D shows that they are similar. Please clarify and add the corresponding loading controls.
• Panel D: Please mention what is quantified here (supernatant, pellet or overall level),
• Panel D and E: the shades to pink may be tricky to distinguish, more contrasting colors and/or shapes might be useful.
• It is highly appreciated that the antibodies and dilutions are mentioned in the figure legend.

Results - Direct visualization of fluorescent-profilin with polymerizing actin filaments

‘We used fluorescence anisotropy to measure the binding affinity between profilin and Oregon-Green (OG)-labeled monomeric actin (Figure S2).‘ - This appears confusing with the 3D results. If a change in fluorescence anisotropy with OG-actin is not detected, then is it okay to use OG-actin for bulk polymerisation assay. Maybe the OG-tag interferes only during fluorescence anisotropy, but not during fluorescence microscopy.

‘Several studies demonstrate that thymosin b4 (Tb4) competes with profilin to bind actin monomers‘ – It is worth mentioning that this refers to untagged/unlabeled actin monomers.

Figure 3

• Please consider if it is relevant to compare the competitive and non-competitive data on the same graph.
• In panel A description, ‘10 nM GFP-thymosin b4 (GFP-Tb4) mixed with increasing concentrations of unlabeled actin.’ – Would ‘10nM unlabelled actin monomers in presence of increasing concentration of Τβ4’ be more appropriate?
• In panel D, the curves would be better visible when the y-axis runs from 0-30.
• In panel E, the mApple-Profilin samples show longer filaments, maybe quantification of the filament length could be performed?
• In panel G, are the errors bars based on the means of the technical replicates or on all aggregated data? The first option is preferred, as this plotting strategy was also used in 3F.
• In the panel description, ‘Data were quantified from four separate reactions (FOV) each.’ – could clarify whether the data derive from 4 totally independent reactions, or from the analysis of 4 different fields of view (FOV) from the same experimental procedure?
• In the panel description, ‘ns, not significantly different from 1 µM actin alone control; a, compared with control (p <0.05)‘ – such labeling may be confusing for the reader, it would be beneficial to state that the first ‘ns/a’ are related to the actin alone, and the second ‘ns’ are comparing labelled and unlabeled profilin or omit showing the statistical test in the plot, and show it in a table instead.

Results - Fluorescent profilin stimulates formin-based actin filament assembly

General: Based on the competition and interaction experiments it seems important to generate a dose-dependent inducible construct for profilin to govern the stoichiometry of interactions and study their relevance in the cells.

‘Similar to experiments in assessing only profilin-actin interactions (Figure 3F), we counted significantly fewer actin filaments in reactions containing actin and either profilin (Figure 4D)’ - In Figure 3F the plot shows around 45 actin filaments per FOV with 1 uM actin (20% oregon green actin). In Figure 4D the plot shows more than 100 actin filaments per FOV with 1 uM actin (10% alexa 647 actin). Surprisingly, the elongation rate are similar in Fig 3F and Fig 4D. Is the Oregon-Green actin known to be less efficient at nucleating filaments while retaining the same polymerization ability? If it is the case, it would be worth making a mention.

‘Thus, fluorescent profilin stimulates formin-mediated actin filament nucleation similar to the untagged version.’ - The data seem to suggest that the presence of profilin inhibits actin filament nucleation and polymerization, a clarification would be appreciated.

‘The ac-celerated rate of actin filament’ – Please change to ‘accelerated’.

Results - Profilin directly binds tubulin dimers and enhances the growth rate of microtubules in vitro

‘microtubule stability index‘ – The values indicated on the y-axis for the plot in Fig 5E are confusing (0 / 25/ 50 / 75 / 1). Is the index expressed as a percentage and the max value supposed to be 100? Or is the index supposed to be the number of rescues per catastrophe?

’This suggests a mechanism where profilin accelerates microtubule polymerization by directly binding to tubulin dimers to promote microtubule assembly and then diffusing along the sides of the microtubule lattice to further stabilize microtubule growth.‘ - Microtubules are more stable the faster they grow (catastrophe frequency scales inversely with polymerization rate). In this condition where profilin increases polymerization rate by around 5x, it is unclear how much of the increased stability is due to the lattice binding. The fragment could be softened regarding the role of the transient lattice binding in microtubule stabilization.

Figure 5

• In panel B, intensities are quite different, would it be possible to comment on this?
• In panels H an I, a black/magenta merge is tricky to see. Although it breaks consistency, a green/magenta or cyan/magenta merge may be more informative visually.

Results - Profilin regulates the morphology of N2a cells through actin and microtubule crosstalk

‘We used quantitative western blots to determine the level of endogenous profilin as well as levels of profilin in CRISPR knockout cells following transfection with plasmids containing untagged profilin, mApple-profilin, or Halo-profilin.’ - The levels of profilin are quantified from a blot, which is a bulk measurement. The transfection of profilin will show substantial cell-to-cell variation (some cells may have much higher or lower levels than the measured average). Mentioning it and discussing its implications would be advisable.

‘We chose this parameter because N2a cells have unique actin filament and microtubule cytoskeletal features but do not efficiently perform other classic cell processes that require intact cytoskeletal crosstalk (i.e., migration or division).‘ – While looking at cell shape is one strategy, an additional experiment looking into a migration phenotype may strengthen this point. Another interesting experiment to strengthen this point and providing a direct measure of profilin function could be performing a pulse chase experiment using drugs to depolymerise actin/microtubules. In such an experiment, a distinct change in depolymerisation should be noted between WT, KO, and the profilin rescue cells. This could show that the mApple-profilin can substitute for WT profilin.

‘super resolution confocal microscopy to image fixed cells.‘ – how is super-resolution achieved here? Or is ‘super’ unnecessary here?

‘the ratio of endogenous cell area to other cell conditions’ - This metric is a ratio of areas and it is only valid as an assessment for shape if the cells from each condition cover similar areas. If it is not the case, then the two parameters (shape and area) are convoluted and the ratio measures both the difference in shape and in area covered. It would be good to provide the average area of the cells in each condition for clarification.

‘We also stained these cells for actin filaments (Figures 6H and 6I) and micro-tubules (Figure 6J and 6K) and used a similar morphology parameter to detect broad differences in cytoskeletal architecture.’ – Please clarify the reasons for using the cell area ratio metric for quantification of cell morphology. How is the quantification metric (area) used for sub-cellular network like actin and microtubule? F-actin stained with phalloidin looks different in endogenous PFN1 vs mApple-PFN1, but by using area metric there is no morphological difference. Microtubule, on the other hand, appear similar in all the cells.

Figure 6

• In panel A, it does not seem like mApple-PFN1 and Halo-PFN1 reach endogenous levels. Maybe a quantification would be beneficial.
• In panel B, please report the number of independent replicates. Also, may be worth commenting on why tagged PFN1 rescue cells were not included?
• For panel E, please provide the error bars and the legend that contains the information from where the data derived or from how many independent experiments.
• In panel F, it may be advantageous to use red green and blue as colors for the overlay. This will generate unique colors for the different combinations of the three images.
• For the context of panel F, unprocessed images of the tagged profilin in living cells could be presented somewhere in the main text. They could be larger than the small panels here, and not be segmented into binary images. The point of the fluorescent profilin is that it can be used for live-cell imaging without substantially disrupting the typical profilin interactions. This should be confirmed by presenting live-cell images of the profilin construct. To avoid problems with high cytoplasmic concentrations of profilin drowning out any localization signal, maybe the fluorescent version could be expressed at a very low level or the Halo version could be used with a low concentration of fluorophore.
• In panel G, it is clear that the dots are from different cover slips, how many cells were analyzed per coverslip? Data could be shown from individual cells (not just their average). Also, please clarify if this quantification was made 24 hours after transfection as well?
• For panel I, actin morphology was calculated from actin filament signals similar to the cell morphology index. These calculations could be explained further in the methods. Does this mean that the actin morphology index is the ratio of actin area between the two conditions? Is the actin image somehow thresholded before taking the ratio?
• Panel L, is really appreciated and helpful to understand the "competition" between actin and microtubules for profilin. It would be also nice to represent the plasma membrane and the lipid-binding activity of profilin as well as binding to nucleation promoting factors (the proline-rich motifs of VASP and WASP), as this is mentioned throughout the paper.

Discussion

The fact that mApple and HaloTag both are entirely different and non-disturbing gives confidence that profilin can be fused with other tags, without losing functionality. Mentioning this in the discussion could give new insights for the readers.

‘Our genetic analyses in mammalian cells indicate that mApple-profilin and Halo-profilin are fully inter-changeable with the endogenous version.‘ - Authors have given the field an excelled tool which will be quite useful to study cellular functions of PFN1, its interaction with its several binding partners. Currently, cell shape is the metric used to determine if the tagged versions are fully inter-changeable with the endogenous version. Whether the tagged PFN1 can replace untagged PFN1 for other cellular functions will require further exploration. Also, high concentration of PFN1 will remain an issue even with the mApple-PFN1 developed here. Do the authors suggest mild over-expression as a strategy to go around the high concentration issue?

‘Based on localization experiments using the pan-formin inhibitor, SMIFH2, some interactions between profilin and the sides of micro-tubules are thought to be indirect.’ – suggest clarifying how SMIFH2 treatment leads to conclude that interactions between PFN1 and microtubules is indirect.

Methods

Statistical significance tests do not demonstrate that conditions are identical. That is, when two conditions are not statistically different, it is not possible to say that these are equal (https://doi.org/10.1053/j.seminhematol.2008.04.003). Suggest avoiding the use of "n.s." in graphs to indicate that the data are similar. It is clear from the data that (in many cases) the tagged and untagged profilin show similar properties. If equality needs to be demonstrated, recommend carrying out an equivalence test.

‘Different shades of data points show technical replicates.’ – Please rephrase to clarify, for example “Different colors represent biological replicates. Similar colored dots reflect technical replicates“. Does "technical replicate," mean repeated measurements within each independent experimental run? Or different experimental runs? If the shading is supposed to denote paired experiments (e.g. darkest shading in different conditions are from the same experimental run), that can be stated in the caption.

Il caso del problema di #minivation è quello in cui il prodotto non raggiunge né il massimo del potenziale di mercato né il massimo del potenziale di prezzo. Le aziende che cadono in questa trappola sono aziende che danno al proprio prodotto un prezzo più basso di quanto sia effettivamente il suo valore ed in questo modo rinunciano a dei guadagni.

I read your blog post with interest, as a researcher in literature and cultural studies I was looking for this kind of entry to… maybe end a feeling of loneliness as I’m going to switch for a tool mainly used by programmers? :) I do some programming (mostly copy/paste language for the moment tbh) but I felt the need to find a testimony from someone in my field to give me this last bit of force to make the jump and you gave it to me! I want more now :) would you have some ressources to share about knowledge management taking advantage of org-mode and computational thinking?

I’ve been managing my personal archaic databases with a tag system and relational databases for 20 years including ten years of an overly complicated handwriting mode before years of Excel that ended in a frustrating stateless vagabondage. I came across Org-mode as I was engaging myself in databases programming and would love to have some keystone to help me getting started. Thanx a lot!

SciScore for 10.1101/2021.09.30.462449: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Mouse studies Ethical approval: All the experimental procedures were performed in accordance with the guide for the use of laboratory animals of the University of Sao Paulo and approved by the institutional ethics committee under the protocol number 105/2021.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">SARS-CoV-2 experimental infection and treatments: Female K18-hACE2 mice, 8-week-old, were infected with 2×104 PFU of SARS-CoV-2 (in 40 µL) by intranasal route.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">A total of 10 photomicrographs in 40X magnification per animal were randomly obtained using a microscope Novel (Novel L3000 LED, China) coupled to a HDI camera for images capture.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The virus was propagated and titrated in Vero E6 cells in a biosafety level 3 laboratory (BSL3) at the Ribeirão Preto Medical school</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The viral inoculum was added to Vero cells in DMEM 2% FBS and incubated at 37 °C with 5% CO2 for 48 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For expression, BL21 cells transformed with plasmid were grown in ZYM-5052 to an OD600 of 0.6-0.8 at 37°C and 200 RPM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BL21</div><div>suggested: RRID:CVCL_M639)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">K18-hACE2 mice: To evaluate the effects of Pyronaridine in vivo, we infected the K18-hACE2 humanized mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J)(21, 65, 66).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE2</div><div>suggested: RRID:IMSR_GPT:T037657)</div></div><div style="margin-bottom:8px"><div>B6.Cg-Tg(K18-ACE2)2Prlmn/J</div><div>suggested: RRID:IMSR_JAX:034860)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PCR product was digested with NcoI and XhoI and cloned into pET28a (Novagen) in frame with a C-terminal his-tag coding sequence E. coli BL21 transformed with plasmids were grown in LB to an optical density (OD600) of 0.6 at 37 °C and 200 RPM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET28a</div><div>suggested: RRID:Addgene_114156)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The total septal area and total area were analyzed with the aid of the Pro Plus 7 software (Media Cybernetics, Inc., MD, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Pro Plus</div><div>suggested: (Image-Pro Plus, RRID:SCR_016879)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Prior to docking, ligand and protein were prepared with LigPrep (Schrödinger, 2017, USA) and Protein Preparation Wizard (Schrödinger, 2017, USA) using default parameters.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>LigPrep</div><div>suggested: (Ligprep, RRID:SCR_016746)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Figures were generated with ChimeraX.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ChimeraX</div><div>suggested: (UCSF ChimeraX, RRID:SCR_015872)</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<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 10.1101/2021.09.30.462420: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Patient recruitment: All donors have given written informed consent and analyses were approved by the Institutional Review Board of Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin (study protocol number EA1/258/18), the Institutional Review Board of the Faculty of Medicine at Ludwig-Maximilians-Universität (LMU) Munich, Germany (20-371), as well as the ethics committee of the Innsbruck Medical University (1167/2020).<br>IRB: Patient recruitment: All donors have given written informed consent and analyses were approved by the Institutional Review Board of Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin (study protocol number EA1/258/18), the Institutional Review Board of the Faculty of Medicine at Ludwig-Maximilians-Universität (LMU) Munich, Germany (20-371), as well as the ethics committee of the Innsbruck Medical University (1167/2020).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Diagnostic antibody testing: Initial serological testing of patient samples was performed using a solid phase immunoassay (SeraSpot®Anti-SARS-CoV-2 IgG, Seramun Diagnostica GmbH, Heidesee, Germany).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SeraSpot®Anti-SARS-CoV-2 IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing, bound antibodies were detected by incubation with horseradish peroxidase (HRP)-labeled anti-human IgG for 30 minutes at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Second, the presence of SARS-CoV-2 S1-specific antibodies was analyzed using a commercially available anti-SARS-CoV-2-S1 IgG ELISA (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2-S1 IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After His-tagged RBD Beta was immobilized on anti-Penta His BLI sensors, sensors were first dipped into CS82 IgG (50 μg/ml), and then dipped into indicated IgG antibodies (12.5 μg/ml).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Penta</div><div>suggested: (Bio-Rad Cat# MCA5995P, RRID:AB_2888689)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">His-tagged recombinant RBD Beta protein was produced in HEK cells (ACROBiosystems, SPD-C52Hp) and covalently labeled using CruzFluor488 (Santa Cruz Biotechnology, sc-362617) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK293T cell-secreted RBD-Fc fusion proteins composed of the RBD-SD1 component of the SARS-CoV-2 spike S1 subunit (amino acids 319-591) and the constant region of rabbit IgG1 heavy chain (Fc) were immobilized onto 96-well plates via anti-rabbit IgG (Dianova, 711-005-152)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, Vero E6 cells (1.6 x105 cells/well) were seeded in 24-well plates and incubated overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Crystallization and structural determination: The RBD (residues 333-529) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1) and the Beta variant that carries three mutations on the RBD (K417N, E484K, and N501Y) were cloned into a customized pFastBac vector (37), and fused with an N-terminal gp67 signal peptide and C-terminal His6 tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFastBac</div><div>suggested: RRID:Addgene_1925)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For expression and purification of the Fabs, heavy and light chains were cloned into phCMV3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>phCMV3</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cloning was considered successful when sequence identity was >99.5% as verified by the cBASE module of BASE software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BASE</div><div>suggested: (BASE, RRID:SCR_010937)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After identification of public clonotypes, they were plotted in a Circos plot using the R package circlize (33).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Circos</div><div>suggested: (Circos, RRID:SCR_011798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As the CoV-AbDab includes SARS-CoV-2 mAbs from other sources than humans, and against other epitopes than the RBD, the following selection criteria were used (nomenclature like in CoV-AbDab): Binds to: SARS-CoV-2,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: (BioLegend Cat# 946101, RRID:AB_2892515)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Iterative model building and refinement were carried out in COOT (41) and PHENIX (42), respectively (table S4).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COOT</div><div>suggested: (Coot, RRID:SCR_014222)</div></div><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">1 and all statistical analyses were performed using GraphPad Prism (9.2.0).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

Results from OddPub: Thank you for sharing your data.

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 34. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

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

2) Articles are clearly separated - this is positive for the majority of users that do not have accessibility needs. Font size throughout is large, both of article title and article preview. This is positive for those with difficulty seeing/minor-major eyesight trouble. This is also positive for people that have ADHD, as a lack of clutter will make it an easier read.

3) Image does not have an <alt> tag - this is negative regarding accessibility for the blind. *I have included 3) here along with 2) because when I highlight just the picture the functions that hypothes.is offers do not appear.

SciScore for 10.1101/2021.09.28.21264207: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Written informed consent was obtained from all participants, and the trial was done in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice.<br>IRB: Study approval in the UK was done by the Medicines and Healthcare products Regulatory Agency (reference 21584/0424/001-0001) and the South Central Berkshire Research Ethics Committee (reference 20/SC/0145).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">For some assays and where sample availability allowed, comparison was made with age- and sex-matched participants who were HIV negative, aged 18–55 years, enrolled into the main COV002 phase 2/3 randomised clinical trial, and randomly assigned (5:1) to receive either ChAdOx1 nCoV-19 or MenACWY by intramuscular vaccination.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 2-hour incubation and a washing step, detection antibody (MSD SULFO-TAG™ Anti-Human IgG Antibody, 1/200) was added.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Assays were performed using Multiscreen IP ELISpot plates (Merck Millipore, Watford, UK) coated with 10 μg/mL human anti-IFNγ antibody and developed using SA-ALP antibody conjugate kits (Mabtech, Stockholm, Sweden) and BCIP NBT-plus chromogenic substrate (Moss Inc., Pasadena, MA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-IFNγ</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>SA-ALP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After stimulation cells were stained with the anti-human antibodies contained in supplementary table 6.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A MULTI-SPOT® 96-well, 10 spot plate was coated with three SARS CoV-2 antigens (S, RBD, N), SARS-CoV-1 and MERS-CoV spike trimers, as well as spike proteins from seasonal human coronaviruses, HCoV-OC43, HCoV-HKU1, HCoV-229E and HCoV-NL63, and bovine serum albumin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HCoV-229E</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HCoV-NL63</div><div>suggested: RRID:CVCL_RW88)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mixtures were then transferred to 96-well, cell culture-treated, flat-bottom microplate containing confluent Vero cell monolayers in duplicate and incubated for further 2 hours, followed by the addition of 1.5% semi-solid carboxymethyl cellulose (CMC) overlay medium to each well to limit virus diffusion.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Participants with a history of laboratory-confirmed SARS-CoV-2 infection by anti-N protein IgG immunoassay (Abbott Architect, Abbott Park, IL, USA) at screening were excluded.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Abbott Architect</div><div>suggested: (Abbott ARCHITECT i1000sr System, RRID:SCR_019328)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Standardised EUs were determined from a single dilution of each sample against the standard curve which was plotted using the 4-Parameter logistic model (Gen5 v3.09, BioTek).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gen5</div><div>suggested: (Gen5, RRID:SCR_017317)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The percentage of focus reduction was calculated and IC50 (reported as FRNT50) was determined using the probit program from the SPSS package.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPSS</div><div>suggested: (SPSS, RRID:SCR_002865)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data was analysed using FlowJo version 10 and Prism version 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Gating strategies are as shown in supplementary figure 1a and b Phylogenetic analysis: We used protein BLAST to download all human coronavirus S protein sequences from NCBI database.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BLAST</div><div>suggested: (BLASTX, RRID:SCR_001653)</div></div><div style="margin-bottom:8px"><div>NCBI</div><div>suggested: (NCBI, RRID:SCR_006472)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We used MAFFT to align all chosen human corona viruses, SARS-CoV, MERS-CoV and SARS-CoV-2 S protein sequences.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MAFFT</div><div>suggested: (MAFFT, RRID:SCR_011811)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We then calculated the pairwise distances between the sequences and built a neighbour joining tree using MATLAB.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MATLAB</div><div>suggested: (MATLAB, RRID:SCR_001622)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We did all analyses using R (version 3.6.1 or later), and Prism 9 (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04400838</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Investigating a Vaccine Against COVID-19</td></tr></table>

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

Don't do that.

Water is something that humans cannot live without, but at the same time something that humans can get for nearly free. Putting a price tag on water effectively capitalizes on humans' need to live.

Hello! this is an annotation! i promise!

My father was talking about how Lewis Carroll’s concrete poetry was related to his experience of suffering from migraine headaches.

The way to export Instapaper annotations to Roam without using Readwise as a middleware

SciScore for 10.1101/2021.09.27.21264163: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The S3 study, the COSCA study and the RECoVERED study were approved by the medical ethical review board of the Amsterdam University Medical Centers (<br>Consent: All participants provided written informed consent.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All S constructs were verified by Sanger sequencing and subsequently produced in HEK293F cells (ThermoFisher) and purified as previously described22.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293F</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudoviruses were produced by co-transfecting the SARS-CoV-2-S expression plasmid with the pHIV-1NL43 ΔEnv-NanoLuc reporter virus plasmid in HEK293T cells (ATCC, CRL-11268), as previously described48.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Shortly, HEK293T/ACE2 cells, kindly provided by Dr. Paul Bieniasz48, were seeded at a density of 20,000 cells/well in a 96-well plate coated with 50 μg/mL poly-L-lysine one day prior to the start of the neutralization assay.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T/ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">They were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned in a pPPI4 expression vector containing a hexahistidine (his) tag with Gibson Assembly (ThermoFisher)22.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pPPI4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus construction: The WT, D614G, Alpha, Alpha E484K, Beta and Gamma pseudovirus S constructs were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned using SacI and ApaI in the pCR3 SARS-CoV-2-SΔ19 expression plasmid48 using Gibson Assembly (ThermoFisher)c</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCR3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudoviruses were produced by co-transfecting the SARS-CoV-2-S expression plasmid with the pHIV-1NL43 ΔEnv-NanoLuc reporter virus plasmid in HEK293T cells (ATCC, CRL-11268), as previously described48.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIV-1NL43 ΔEnv-NanoLuc</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The inhibitory concentration (IC50) and neutralization titers (ID50) were determined as the NAb concentration and serum dilution at which infectivity was inhibited by 50%, respectively, using a non-linear regression curve fit (GraphPad Prism software version 8.3).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

---

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

There are several limitations of our study. First, our study includes substantially more female than male participants, reflecting the gender distribution among HCW at our institute. Second, the age distribution in the four groups is not identical. In particular, the AZD1222 group is, on average, considerably older as a consequence of restrictive use of the AZD1222 vaccine in individuals aged 60-64 years in the Netherlands. As immune responses tend to become weaker with higher age, this is a relevant factor when considering the weaker responses in the AZD1222 group. Finally, the samples we tested were taken at the expected peak of immunity. It will be relevant to study the durability of the neutralizing antibody responses after vaccination with each of these vaccines. Some studies suggest that immunity induced by adenovirus vaccines might be more durable than immunity from mRNA vaccines8,45. We have only analyzed known VOCs and VOIs and cannot predict how our results apply to future variants. One consideration is that current VOCs were probably selected based on increased fitness and/or transmissibility, while future variants may very well be selected based on escape from immunity when more and more people are vaccinated or have experienced COVD-19. Such escape variants may be more resistant to neutralizing antibodies induced by current vaccines than the VOCs and VOIs studied here and may render vaccines even less effective at preventing infection. However, while circulating ...

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<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>

An artist's depictions of Bath in the early 19th century in context of Austen's life and work

The OEGlobal 2021 conference was themed around the five action areas. For "raw material" to anchor here, you can find sessions related to each action area via these links:

• Building Capacity
• Developing Supportive Policy
• Inclusive and Equitable OER
• Sustainable OER
• Facilitating international cooperation

SciScore for 10.1101/2021.09.24.21263853: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: All subjects were recruited following protocols approved by local Institutional Review Boards (IRBs) or from NIH NIAID IRB, as indicated below: Chile: IRB: Comité Ético Científico Facultad de Medicina Clínica Alemana Universidad del Desarrollo, Santiago, Chile Protocol: 2020-41 Title: Infección por Sars CoV-2 y enfermedad Covid-19: caracterización epidemiológica, clínica, virológica e inmunológica (SARS-CoV-2 infection and COVID-19: Epidemiologic, clinical, virologic and immunologic characterization) Pavia, Italy: IRB: Ethics Committee of the Fondazione IRCCS Policlinico San Matteo, Pavia Protocol: 20200037677 Title: Analisi comprensiva della risposta immunitaria innata ed adattativa durante</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 antibody testing: SARS-CoV-2 anti-spike and anti-nucleocapsid antibody testing was performed via luciferase immunoprecipitation systems assay, as previously described [6].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-spike</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-nucleocapsid</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PBMC pools were Fc blocked (Human TruStain FcX, BioLegend) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human ‘hashtag’</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The fluorescence-labeled antibody cocktail against human CD45 (APC/Cyanine7), CD3 (AF488), CD19 (APC), CCR7 (BV786), CD95 (BV650), IgD (PerCP-Cy5.5) and CD27(PE/Cyanine7; all antibodies obtained from Biolegend) were added at the end of blocking and incubated for 20 minutes at 4°C in the dark.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human CD45</div><div>suggested: (BD Biosciences Cat# 563716, RRID:AB_2716864)</div></div><div style="margin-bottom:8px"><div>CD3</div><div>suggested: (SouthernBiotech Cat# 8200-30, RRID:AB_2796425)</div></div><div style="margin-bottom:8px"><div>CD19</div><div>suggested: (BD Biosciences Cat# 563333, RRID:AB_2738141)</div></div><div style="margin-bottom:8px"><div>CCR7</div><div>suggested: (Creative Diagnostics Cat# CPBT-66983GM, RRID:AB_2528776)</div></div><div style="margin-bottom:8px"><div>BV786</div><div>suggested: (BD Biosciences Cat# 740991, RRID:AB_2740614)</div></div><div style="margin-bottom:8px"><div>CD95</div><div>suggested: (BioLegend Cat# 305642, RRID:AB_2632622)</div></div><div style="margin-bottom:8px"><div>BV650</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>PerCP-Cy5.5</div><div>suggested: (BD Biosciences Cat# 560612, RRID:AB_1727457)</div></div><div style="margin-bottom:8px"><div>CD27</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Because multiple samples from different timepoints for each donor were collected and could not be demultiplexed by this method alone, ‘hashtag’ antibodies (Biolegend) were used to uniquely label the different time points.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>‘hashtag’</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data of several proteins that directly bind with secondary antibodies detected through buffer incubation without any serum were excluded (such as IGHG1, IGHG3 and so on) alongside the controls (such as Rhodamine+IgG64, Anti-human IgG, GST 10ng/ul etc.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IGHG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IGHG3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-human IgG</div><div>suggested: (LSBio (LifeSpan Cat# LS-C23907-500, RRID:AB_900919)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Multiplex particle-based anticytokine autoantibody screening assay and functional evaluation: Plasma samples were screened for autoantibodies against IFN-α, IFN-β, IFN-ω and IFN-γ in a multiplex particle-based assay [31], in which differentially fluorescent magnetic beads were covalently coupled to recombinant human proteins (2.5 ug/reaction).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IFN-α, IFN-β</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IFN-ω</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the comparison of pHC with pCOVID-19, the classification was then repeated after the exclusion of allergic pHC, with similar results.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCOVID-19</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These analyses were completed with IBM SPSS Statistics v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPSS</div><div>suggested: (SPSS, RRID:SCR_002865)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">27 and GraphPad Prism version 9 [7].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Trained with Python sklearn library’s RandomForestClassifier object, using parameters: n_estimator=2000, random_state=42 for data set.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Python</div><div>suggested: (IPython, RRID:SCR_001658)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The statistical analysis of SOMAscan® results was performed using R Studio (R Core Team, 2020) [11], also using a specifically developed webtool for basic data plotting and analysis [12].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SOMAscan®</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To assess the difference in the overall predictive importance (derived by GENIE3) of each variable with and without IVIG treatment, we summed the interaction strengths associated with each predictor-target pair for a given predictor variable in either treatment condition (steroid treatment applied in both conditions).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GENIE3</div><div>suggested: (GENIE3, RRID:SCR_000217)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting values were visualized using the Complexheatmap package in R.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Complexheatmap</div><div>suggested: (ComplexHeatmap, RRID:SCR_017270)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All sequencing data were then processed with Burrows–Wheeler Aligner (BWA) and the Genome Analysis Toolkit (GATK</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BWA</div><div>suggested: (BWA, RRID:SCR_010910)</div></div><div style="margin-bottom:8px"><div>Genome Analysis Toolkit</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>GATK</div><div>suggested: (GATK, RRID:SCR_001876)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Raw fastq files were trimmed using Trimmomatic v0.39 [15] and mapped to the hg38 human reference genome using BWA-MEM v07.17.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Trimmomatic</div><div>suggested: (Trimmomatic, RRID:SCR_011848)</div></div><div style="margin-bottom:8px"><div>BWA-MEM</div><div>suggested: (Sniffles, RRID:SCR_017619)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PCR Duplicates were marked using Samblaster v0.1.2.5 [16] and GATK4 v4.1.9.0 was used to perform BAM recalibration, and HLA*LA [17] was used to call HLA genotypes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Samblaster</div><div>suggested: (SAMBLASTER, RRID:SCR_000468)</div></div><div style="margin-bottom:8px"><div>GATK4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting CDR3 sequences were collapsed and filtered to quantify the absolute abundance and frequency of each unique CDR3 region with Adaptive Biotechnologies’ pipeline [19]</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Biotechnologies’</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. d) Bulk RNA sequencing and single cell sample demultiplexing: For each sample, 100,000-500,000 cells were processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD) and standard RNA sequencing libraries were generated using Illumina Truseq library preparation kits.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Genomics</div><div>suggested: (UTHSCSA Genomics Core, RRID:SCR_012239)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol [23].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>STAR</div><div>suggested: (STAR, RRID:SCR_004463)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lowly expressed genes were removed for each cell type individually using the filterByExpr function from edgeR [26]</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>edgeR</div><div>suggested: (edgeR, RRID:SCR_012802)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Differentially expressed genes were identified using the limma voom [27] workflow which models the log of the cpm (counts per million) of each gene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>limma</div><div>suggested: (LIMMA, RRID:SCR_010943)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Enriched gene sets were identified using the pre-ranked GSEA algorithm implemented in the FGSEA R package [https://www.biorxiv.org/content/10.1101/060012v3].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FGSEA</div><div>suggested: (fgsea, RRID:SCR_020938)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pseudobulk gene counts were normalized with the varianceStabilizingTransformation function from DEseq2 (doi:10.1186/s13059-014-0550-8) for the score calculation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DEseq2</div><div>suggested: (DESeq2, RRID:SCR_015687)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For BCR data, V(D)J sequencing contigs from 10x CellRanger output was processed using Immcantation v2.7.0 toolbox (https://immcantation.readthedocs.io/en/latest/index.html).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Immcantation</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BCR sequence genotype inference and mutation load quantification were performed with reference to the pipeline from Mathew et al. [29] using the TIgGER package [30] and SHazaM package [28].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>TIgGER</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>SHazaM</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Autoantibody analysis: Autoantibody analysis was performed using HuProt™ v4.0 human protein microarrays and processed by CDI Laboratories (Baltimore, MD)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HuProt™</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Beads were washed again, resuspended in assay buffer, and analyzed on a BioPlex X200 instrument.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BioPlex</div><div>suggested: (BioPlex, RRID:SCR_016144)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were acquired on a BD LSRFortessa cytometer, gated on CD14+ monocytes, and analyzed with FlowJo software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr></table>

---

Results from OddPub: Thank you for sharing your code and data.

---

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

This study has some limitations. Only a few children with severe pCOVID-19 were included in the study, and therefore the results obtained may not apply to rare cases of life-threatening disease in children. We did not have longitudinal samples available for all MIS-C patients. However, the number of patients included in the study was sufficient to detect early and late signatures of the disease. Finally, while an increasing number of cases of pCOVID-19 due to the delta variant have been recently reported [62], almost all samples were collected prior to the emergence of this variant. Therefore, the impact of the delta variant on innate and adaptive immune responses in children with pCOVID-19 and MIS-C remains to be studied. Despite these limitations, this study has helped identify novel age-, time- and treatment-related immunopathological signatures that characterize MIS-C and pCOVID-19.

---

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT03394053</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">The Mechanistic Biology of Primary Immunodeficiency Disorder…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT03610802</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Send-In Sample Collection to Achieve Genetic and Immunologic…</td></tr></table>

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<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 article initially looked compelling and true. The name is catchy and so I assumed The Root was a community organizer for black justice but after scrolling over the check I learned it is a digital magazine platform that shares thought provoking articles from various black perspectives. I used the consensus/check for other coverage and found out there is no actual news on this and the link in the tweet shared is directly to the blog post on their page.

SciScore for 10.1101/2021.09.24.461616: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: Animal studies were carried out at Galveston National Laboratory at University of Texas Medical Branch at Galveston, Texas, an AAALAC accredited (November 24, 2020) and PHS OLAW approved (February 26, 2021) high-containment National Laboratory, based on a protocol approved by the Institutional Animal Care and Use Committee at UTMB at Galveston.<br>IACUC: Animal studies were carried out at Galveston National Laboratory at University of Texas Medical Branch at Galveston, Texas, an AAALAC accredited (November 24, 2020) and PHS OLAW approved (February 26, 2021) high-containment National Laboratory, based on a protocol approved by the Institutional Animal Care and Use Committee at UTMB at Galveston.<br>IRB: All animal procedures and operations were approved by the ethical committee of Wuhan Institute of Virology, Chinese Academy of Sciences.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Rhesus macaques were randomly divided into control group, low-dose (10 mg/kg of 2G1) and high-dose (50 mg/kg of 2G1) groups with one male and one female in each.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Rhesus macaques were randomly divided into control group, low-dose (10 mg/kg of 2G1) and high-dose (50 mg/kg of 2G1) groups with one male and one female in each.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Biotin labeled RBD (Sino Biological), PE labeled streptavidin (ThermoFisher) and 7AAD (BD) Single memory B cells with potential SARS-CoV-2 antibody secretion were sorted out by gating 7AAD-, CD19+, CD27+, IgG+, and RBD+ using a BD Aria III cell sorter with fluorescence-activated cell sorting modules.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD27+ , IgG+</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The transfection efficiency was examined by flow cytometry using S1-mFc recombinant protein (Sino Biological) as primary antibody and FITC-AffiniPure Goat Anti-Mouse IgG (Jackson) as secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antigen-binding ELISA: Enzyme-linked immunosorbent assays (ELISA) were applied to study the binding ability of antibodies with SARS-CoV-2 RBDs (Sino Biological) and S trimers (AcroBiosystems).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Antigen-binding ELISA</div><div>suggested: (Ling-Chu Hung / Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan Cat# 12-Hung-03 m& 20 ORF3 7D3, RRID:AB_2827541)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After pipetting off the unbound antibodies, plates were washed 4 times with PBST and further incubated with 100 μL per well of goat anti-human IgG (Fc specific)-Peroxidase antibody (1: 5000 dilution, Sigma) for 1 h at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG ( Fc specific)-Peroxidase</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 competition ELISA: For experiments involving the competitive binding of antibodies to SARS-CoV-2 RBD or S trimer, recombinant hACE2-Fc protein was first biotinylated using EZ-Link Sulfo-NHS-Biotin (ThermoFisher) as the instruction described.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Sulfo-NHS-Biotin</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody-Dependent Cellular Phagocytosis (ADCP): In ADCP experiment, CD14+ monocytes (Allcells) were cultured and differentiated for 7 days to obtain macrophage cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Antibody-Dependent Cellular Phagocytosis ( ADCP)</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing, a Goat Anti-human Fc HRP (Sigma) was used as secondary antibody with 1:6000 dilutions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-human Fc HRP</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, HEK-293T cells (ATCC) with 70% - 80% confluence in a 10 cm dish were co-transfected with 12 μg of plasmid pHIV-puro encoding RRE and ACE2 genes, 8 μg of plasmid psPAX2 encoding gag and pol, and 4 μg of plasmid VSV-G encoding G glycoprotein of vesicular stomatitis virus(VSVG) using Lipofectamine 3000 Reagent (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">500 μL of filtered lentivirus supernatant was added in a 24-well plate with Jurkat cells (ATCC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat</div><div>suggested: TKG Cat# TKG 0209, RRID:CVCL_0065)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The culture medium of ACE2-293T cells was removed and then replaced by the antibody-pseudovirus mixture.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mixture was then added into a 96-well plate covered with Vero cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This recombinant S protein was overexpressed using the HEK 293F mammalian cells (Invitrogen) at 37°C under 5% CO2 in a Multitron-Pro shaker (Infors, 130</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293F</div><div>suggested: RRID:CVCL_6642)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pharmacokinetic study and toxicity test: For the pharmacokinetic study, BALB/c mice were tail intravenously injected with 2G1 (15, 30, or 60 mg/kg), or equivalent volume of PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">mAb preparation: Heavy chains and light chain genes were inserted separately into pcDNA3.4 and amplified in E. coli DH5α.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.4</div><div>suggested: RRID:Addgene_131198)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, HEK-293T cells (ATCC) with 70% - 80% confluence in a 10 cm dish were co-transfected with 12 μg of plasmid pHIV-puro encoding RRE and ACE2 genes, 8 μg of plasmid psPAX2 encoding gag and pol, and 4 μg of plasmid VSV-G encoding G glycoprotein of vesicular stomatitis virus(VSVG) using Lipofectamine 3000 Reagent (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIV-puro</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>VSV-G</div><div>suggested: RRID:Addgene_138479)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">S protein over-expression cells: The coding sequence for full-length wild-type S protein (GenBank: QHD43416.1) from Met1 to Thr1273 was inserted into plasmid pHIV-puro1.0, followed by an internal ribosome entry site (IRES) and puromycin resistance gene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIV-puro1.0</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of S protein: The prefusion S extracellular domain (1-1208 a.a) (Genbank ID: QHD43416.1) was cloned into the pCAG vector (Invitrogen) with six proline substitutions at residues 817, 892, 899, 942, 986 and 98739, a “GSAS” substitution (instead of “RRAR”) at residues 682 to 685 and a C-terminal T4 fibritin trimerization motif followed by one Flag tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAG</div><div>suggested: RRID:Addgene_74288)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting bulk transfected population was sorted on a BD FACSJazz Cell Sorter (BD) with the BD FACS™ Sortware.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD FACS™</div><div>suggested: (BD FACS Aria II, RRID:SCR_018091)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed with GraphPad Prism Version 9.0.0 and EC50 values were determined using a four-parameter nonlinear regression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 RBD (Sino Biological), S trimer (AcroBiosystems), mutated RBDs (Sino Biological), and mutated S trimers (AcroBiosystems) were coated onto High Binding ELISA 96-Well Plate (BEAVER).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>AcroBiosystems</div><div>suggested: (ACRObiosystems, RRID:SCR_012550)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The defocus values were estimated with Gctf43. Particles for S in complex with 2G1 were automatically picked using Relion 3.0.644–47 from manually selected micrographs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Relion</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 17. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

.

SciScore for 10.1101/2021.09.16.21263693: (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">Using a R code script, 50 randomly selected values from the extended sera sample cohort (n=345) were used to set quartile thresholds according to the mean values derived from the bootstrap iterations, a process which was then repeated 10,000 times.</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">Serum anti-SARS-CoV-2 RBD antibodies assay: RBD+ Ig levels in sera were determined using 96 well ELISA plates that were coated overnight at 4°C with 2μg/ml RBD in PBS (pH 7.4).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-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">ELISA plates were then washed three times with PBST and 50μl of horseradish peroxidase (HRP) conjugated goat anti-human IgG (Jackson ImmunoResearch, #109035003) / anti-human IgM (Jackson ImmunoResearch, #109035129) / anti-human IgA (Jackson ImmunoResearch, #109035011) secondary antibodies were added to each plate at the detection phase (50μl, 1:5000 ratio in 3% w/v skim milk in PBS) and incubated for 1 hour at room temperature (RT), followed by three washing cycles with 0.05% PBST.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 109-035-003, RRID:AB_2337577)</div></div><div style="margin-bottom:8px"><div>anti-human IgM</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 109-035-129, RRID:AB_2337588)</div></div><div style="margin-bottom:8px"><div>anti-human IgA</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 109-035-011, RRID:AB_2337580)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Computational and statistical analysis: Bootstrapping & quartile establishment: We stratified the RBD+ antibody levels to quartiles by musing the bootstrap method.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RBD+</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cloned region encodes amino acids 1-740 of hACE2 followed by 8xHis tag and a Strep Tag at the 3’ end, cloned in a pCDNA3.1 backbone.</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">All statistics were performed with GraphPad Prism 9.0.2 (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

Results from OddPub: 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>

Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

Here we are in 2021 at the OE Global Conference reflecting back on this declaration that was a key point in the journey to develop the UNESCO OER Recommendation.

How does this declaration stand up over time? What was unanticipated? What has it achieved? Were you there? Do you remember this?

We are using this as a practice annotation activity for activities that will take place after the conference.

Please annotate in abundance, reply others and tag them #OEGlobal21 Then tune in to find out what we will annotate next.

just my opinion

unique way of saying it

many different solutions to a problem

derivative project that removes proprietary software

little-known facts better known as

Indeed, I'd heard of hosts.allowed but would have never known that they were part of a package/system called tcpwrappers (which I don't think I've ever heard of).

what does this mean exactly?

How easy is this to do? Is this an example that shows a negative? Is it good for an issue like this to show the negatives and positives? Are there more positives than negatives---if so, what does this show?

Author Response:

Reviewer #3:

Maintaining the balance between stem cell proliferation and cell differentiation is an essential challenge of all stem cell niches. In the shoot apical meristem of plants, these functions are spatially separated into the central zone and peripheral zone, respectively. How these zones communicate to give rise to proper stem cell behavior has been a research focus for many years.

In this manuscript, the authors suggest that the small secreted peptide CLE40 and the receptor kinase like protein BAM1 form a novel pathway that contributes to meristem homeostasis by stimulating the expression of the central stem cell inducer WUSCHEL primarily from the meristem periphery. Importantly, this pathway acts antagonistically to the well-studied CLV pathway, which is only active in the center of the meristem and is molecularly highly similar to the CLE40/BAM1 system. This model is experimentally supported mainly by analysis of spatial localization patterns in the meristem using transcriptional and translational reporters and by the analysis of genetic interactions.

The findings of the authors are novel, highly relevant and would certainly be of great interest for the plant community. However, the manuscript could be substantially improved to provide better support for the conclusions laid out.

Of major concern are the reporter genes and imaging data: Partial colocalization and exclusion from CZ and OC are one of the main arguments of the authors to claim that CLE40/BAM1 function together and antagonistically to CLV3/CLV1 in controlling WUS expression.

Working with reporters as proxies for endogenous gene expression needs to be backed up by proper controls. Given the central importance of the reporters for the conclusions it is essential to show that the regulatory sequences used for the CLE40 reporter are sufficient to rescue a cle40 mutant.

We show now in a new supplemental figure (1) the expression patterns of two different CLE40 reporter lines (differening in length of the promoter region) in the root, which are identical, and (2) that expression CLE40 from the CLE40 promoter rescues the cle40 mutant root phenotypes, which were described in earlier work. See Fig2-SupplFig. 1

It is essential to show that ... the observed expression of the reporter is consistent across the majority of different T1 lines and, most importantly, that the pattern reported here is consistent with in situ data for endogenous CLE40 mRNA.

RNA in situ analysis is difficult due to the low expression level of CLE40, and the small size of the CLE40 transcript. We show in Fig2-SupplFig2 expression data for 4 independent transgenic CLE40 reporter lines, confirming the general conclusions that we present in this manuscript.

The authors have previously published in situs for CLE40 that do not show the exclusion from the CZ and OC (Hobe et al., 2003, Figure 2a,c), which urgently needs clarification.

The RNA expression data from Hobe et al. are displayed at low mag and low resolution, and might have suffered from high background.

Figures 2, 4 and 5 show imaged meristems in great detail but each focus only on a single sample. I strongly recommend to also include quantitative data on multiple samples to substantiate the claims. This could be likely be done with standard software, such as MorphographX.

The data we showed before represented typical examples from a wide range of data that we analysed. All original data are being made publicly available for reanalysis. We have now added multiple examples from multiple samples, and also added quantitative data from fluorescence analysis. See new Supplementary Fig2-SupplFig. 2, Fig.4-SupplFig. 1, Fig.4-SupplFig. 2, Fig.4-SupplFig. 3, Fig.5-SupplFig. 1, Fig.5-SupplFig. 2, Fig.5-SupplFig. 3, Fig.5-SupplFig. 4

Whereas the inhibitory effect of WUS on CLE40 is convincingly shown using ectopic WUS expression and the hypomorphic wus7 allele (Figure 2) the quantification of WUS positive cells in Figure 7 is problematic. Although it was done over multiple samples it heavily relies on manual scoring, which is prone to bias. The same is true for the width/height measurements of different meristems. An unbiased computational image analysis would certainly give more reliable results.

We are grateful for this suggestion. We normally analyse samples in an anonymised manner. We have now also quantified the number of WUS positive cells using the Imaris software, as suggested, see Fig.7-SupplFig.1, and found that this analysis supported our previous conclusions. We also added a figure showing multiple samples from this experiment. See new Supplementary Fig7-SupplFig. 2

One major point that the authors try to establish is that the CLE40 signal that eventually leads to reduction in meristem size is transduced via the BAM1 receptor. However, only genetic interactions, which are complicated by intricate feedbacks, are show to substantiate this claim. For a strong statement on CLE40/BAM1 ligand/receptor interactions, advanced imaging technologies available to the authors or biochemical experiments would be necessary.

We are currently not aware of a reliable and applicable experimental approach that would allow us to show direct interaction of the CLE40 peptide with its receptors in vivo. Biochemical experiments using purified peptides and/or receptors are, so far, contradictory: Shinohara et al. (2015) used chemically synthesized arabinosylated CLV3 peptide and photoaffinity labelling to show binding of CLV3 to a BAM1-Halo-TAG fusion protein expressed in BY-2 cells. However, using BAM1 protein purified from insect cell lines which was biotinylated in the Creoptix WAVE system, Crook et al. (2020) found no significant binding activity for synthetic CLV3 peptide. Our preliminary conclusion from these data sets is that binding of peptides to receptors should be best evaluated in vivo, since important posttransciptional and posttranslational modifications, as well as coreceptors, can strongly modify peptide-receptor interactions.

We have here added data showing that in the root, BAM1 receptor but not CLV1 is required for CLE40 dependent regulation of root meristem development, indicating again that CLE40 and BAM1 are likely to act in the same signaling pathway throughout development. See new Supplementary Fig6-SupplFig. 1

Similarly, the genetic studies need some clarification: The authors show that cle40 and bam1 single mutants as well as cle40/bam1 double mutants all show a comparable reduction in meristem size, suggesting epistasis. In contrast, a reduction in meristem size can not be observed if cle40 is combined with clv1, which according to the proposed model appears to be unexpected. The interpretation of the genetic experiments is complicated by the well-known fact that BAM1 expression is regulated by the CLV pathway and loss of CLV signaling leads to ectopic expression of BAM1 in the OC which can partially compensate for the loss of CLV1, due to the molecular similarity of the two receptors. The shift of BAM1 expression from the PZ towards the OC could explain why there is no significant reduction in meristem size since CLE40 induced signaling at the PZ would be inhibited by the lack of the BAM1 receptor. To clarify the specific interaction of CLE40 with BAM1 and/or CLV1 the authors could try to restore BAM1 levels in the PZ of cle40/clv1 mutants by expressing BAM1-GFP from an appropriate promoter (e.g. RPS5 or UBQ10). This experiment would allow to distinguish between the genetic interaction of CLE40 with CLV1 from the feedback between CLV1 and BAM1 expression.

The suggested experiment, to misexpress BAM1 from the RPS5 or UBQ10 promoter, is not feasible, since this results generally in a much higher expression level, which, in our hands, is not "tolerated" by CLV-family receptors. We found that higher level expression of RLKs generally causes mislocalisation of nonfunctional proteins.

Overall, the manuscript could be strengthened by inclusion of additional molecular data probing the directness of WUS inhibiting CLE40 and/or BAM1 expression.

We are planning to set-up experiments for detailed studies on the transciptional regulation of genes in the stem cell control pathways, and will in the future also investigate the feedback regulation of WUS onto CLE40 and BAM1. However, such analysis goes far beyond the scope of our current manuscript.

Hypothesis: Businesses can use emotional branding to keep up with the times.

NOTE: Colourette Cosmetics is a Filipino makeup brand that also promotes inclusivity. It's cosmetics is geared towards the younger generation no matter the gender.

the world is through the will, connects back to the idea that mother believes the world is run by will not love

similar to harrison believing that she can will herself to nto care about the world

SciScore for 10.1101/2021.09.14.21263603: (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 study participants provided written informed consent and all study protocols were approved by the Cedars-Sinai Medical Center institutional</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">Coupling efficiency was confirmed by incubation of 625 beads from each coupled region with a phycoerythrin-conjugated anti-6×□HisTag antibody (Abcam, Cambridge, UK) at a concentration of 10 μg/mL for 45 min shaking at 900 rpm and room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-6×□HisTag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The bound AABs were detected by addition of R-phycoerythrin-labelled goat anti-human IgG detection antibody (Ab) (5□µg/ml, Dianova, Hamburg, Germany) for 1 hr at RT, after several PBS washes.</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">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">Other antigens were produced in-house using E.coli SCS1 carrying plasmid pSE111, which contains an N-terminally located hexa-histidine-tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSE111</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 all HCW participants, EDTA plasma specimens were transported within 1 hour of phlebotomy to the Cedars-Sinai Department of Pathology and Laboratory Medicine and underwent serology testing using the Abbott Diagnostics SARS-CoV-2 IgG chemiluminescent microparticle immunoassay (Abbott Diagnostics, Abbott Park, Illinois) against the nucleocapsid (N) antigen of the SARS-CoV-2 virus (12, 13).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Abbott</div><div>suggested: (Abbott, RRID:SCR_010477)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analyses: Data processing and analysis were performed using R v3.5.1 and KNIME 2.12 (https://www.knime.org/) (21, 22).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>KNIME</div><div>suggested: (Knime, RRID:SCR_006164)</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 10.1101/2021.09.17.460782: (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">Detection was achieved using horseradish peroxidase-conjugate secondary antibody anti-rabbit and anti-mouse (Bio Rad #1706516, #1706515) and visualized with ECL (Cytiva RPN2232).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: (Bio-Rad Cat# 170-6516, RRID:AB_11125547)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies: The primary antibodies used in this study are: rabbit anti-SARS-CoV-2 Spike S1 Subunit (Sino Biological, 40150-T62)</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">Secondary antibody used are: horseradish peroxidase-conjugate anti-rabbit and anti-mouse (Bio Rad #1706516, #1706515).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: (Bio-Rad Cat# 170-6515, RRID:AB_11125142)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing, AP-conjugated goat anti-rat IgG antibody (SIGMA A8438) or AP-conjugated goat anti-rabbit IgG antibody (SIGMA A8025) was added, and the plates were further incubated for 1 hour at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rat IgG</div><div>suggested: (Sigma-Aldrich Cat# A8438, RRID:AB_258391)</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: (Sigma-Aldrich Cat# A8025, RRID:AB_258372)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FACS: Vero E6 cells were incubated with RBD protein (0.45 μg/mL, final concentration) followed by incubation with human anti-RBD antibody (primary antibody) (40150-D003, Sino Biological) and goat anti-human IgG AF488-conjugated antibody (secondary antibody) (A-11013, Thermo Fisher Scientific)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (Molecular Probes Cat# A-11013, RRID:AB_141360)</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">Hi-5 cells (BTI-TN-5B1-4) (Gibco #B85502) were cultured in Express Five™ SFM (Serum-Free Media) medium (Gibco #B85502 Expression Systems) at a cell density of 0.5 x 106 cells/mL and infected with recombinant virus.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Hi-5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FACS: Vero E6 cells were incubated with RBD protein (0.45 μg/mL, final concentration) followed by incubation with human anti-RBD antibody (primary antibody) (40150-D003, Sino Biological) and goat anti-human IgG AF488-conjugated antibody (secondary antibody) (A-11013, Thermo Fisher Scientific)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After a wash step in 1× kinetic buffer for 120 s, the ACE2-Fc-captured biosensor tips were then submerged for 300 s in wells containing different concentrations of antigen (RBD E. coli, insect, and HEK-293) to evaluate association curves, followed by 900 s of dissociation time in kinetic buffer.</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><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 protein production in E. coli: The SARS-CoV-2 Spike Receptor Binding Domain sequence (aa 319-541, Uniprot ID P0DTC2) was cloned with a C-terminal 6x-His tag into a pET-21a(+) plasmid. E. coli BL21 Star™ (DE3) (genotype: F−ompT hsdSB (rB−, mB−) galdcmrne131) competent cells, and E. coli Lemo21 (DE3) (genotype: fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS/ pLemo(CamR)) competent cells were transformed with 100 ng of plasmid of interest.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-21a(+</div><div>suggested: RRID:Addgene_12669)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD protein production in insect cells: The SARS-CoV-2 Spike Receptor Binding Domain sequence (aa 319-541, Uniprot ID P0DTC2) was cloned into a pFAST-bac1 plasmid downstream of the gp64 signal sequence to promote secretion, along with a C-terminal 8x-His tag for affinity purification.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFAST-bac1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Raw data were analyzed using the Biopharma Finder 2.1 software from ThermoFisher Scientific.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Biopharma Finder</div><div>suggested: None</div></div></td></tr></table>

---

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

---

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

However, the high costs (resources), the time-consuming production, the requirement of specific equipment, and access to dedicated facilities could be a limitation for many laboratories or for the industrial production. By contrast, the bacterial-derived RBD offers a low production cost, a broader availability, and easy handling as main advantages, which make it more accessible. However, limitations in the quality of the produced sample include the absence of glycosylation that partially affects protein stability and efficiency, the presence of heterogeneous folded populations, and the relative low production yields which may result in a final product not eligible for some clinical and medical applications. Overall, all the recombinantly produced RBDs represent valuable tools for research purposes against the pandemic. Recently, expression and purification strategies described in this article have been also proved to be successful in the production of mutants of RBD corresponding to the variants of concern.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2021.09.15.460454: (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: All cell lines have tested negative for mycoplasma contamination.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines used in this study: 293FT cells were obtained from Dr. Kosuke Yusa’s Lab. 293FT.Cas9 cell lines were generated through lentiviral integration of an EF1a-Cas9-T2A-BlastR construct at low MOI to achieve single-copy integration.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293FT</div><div>suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate cell lines permissive to Spike-Pseudotyped lentiviral infection, 293FT.Cas9 cells were engineered to stably express SARS-CoV-2 receptors ACE2 and TMPRSS2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293FT.Cas9</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For in vitro transcribed mRNA, 293T cells were transfected for functional testing using Lipofectamine messengerMAX (Invitrogen) according to 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: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293FT.Cas9.ACE2/TMPRSS2 clonal cell lines were harvested by trypsinization and resuspend at a density of 70.000 cells per 30 μL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293FT.Cas9.ACE2/TMPRSS2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In vitro transcription of S protein mRNA: Templates for in vitro transcription were generated by cloning P1 and P13 between NcoI and NotI sites of pTNT-B18R-6His (addgene plasmid 58979, a kind gift from Steven Dowdy (30)).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTNT-B18R-6His</div><div>suggested: RRID:Addgene_58979)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For transfection, 1 μg of lentiviral transfer vector (pCSGW-GFP), were mixed with 0.72 μg of gag-pol expressing plasmid p8.9 and 68.33 fmol of S protein expressing construct in 500 μL of optiMEM media followed by the addition of 2 μL of PLUS reagent and incubation for 5 minutes at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCSGW-GFP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>p8.9</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These PCR products were both visualized on an agarose gel as well as TA-cloned using ‘TA Cloning Kit with pCR2.1 vector and OneShot TOP10 Chemically Competent E.coli’ (ThermoFisher) according to kit instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCR2.1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The template used to align the ALKBH5 reads to was constructed manually, by performing in silico Gateway cloning, inserting the ALKBH5 CDS and mRuby3 CDS into the pLIX_403 vector (Addgene plasmid #41395).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLIX_403</div><div>suggested: RRID:Addgene_41395)</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">Full DNA sequences of these plasmids are found at a Zenodo provided doi: 10.5281/zenodo.5470001. “Wuhan” in plasmid names refers to the S protein DNA sequence from the Wuhan-Hu-1 isolate (Genbank: MN908947.3) while “18F” refers to the removal of the last 18 amino acids of the S protein C terminus (ER retention sequence) and the addition of a FLAG tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Zenodo</div><div>suggested: (ZENODO, RRID:SCR_004129)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data was analysed using FlowJo software (BD Biosciences) and displayed as % cells infected at 1:500 dilution of pseudotyped virus, normalized to the intronless construct infection rates (Figure 7).</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">Staining was developed using 20X LumiGLO® Reagent and 20X Peroxide reagents according to manufacturer’s recommendations (Cell Signaling Technology, #7003). cDNA analysis: RNA was extracted from the frozen cell pellets using RNeasy Mini Kit (Qiagen) and treated with ezDNase (ThermoFisher) before applying oligo(dT) guided 1st strand cDNA synthesis using SuperScript IV reverse transcriptase (ThermoFisher), all according to manufactures’ recommendations.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ThermoFisher</div><div>suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All reads were mapped back to the original construct DNA sequence using SnapGene software to assess individual mRNA splicing events.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SnapGene</div><div>suggested: (SnapGene, RRID:SCR_015052)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After another round of bead purification, samples were pooled and submitted to Edinburgh Genomics, where they were further processed, barcoded and run on PromethION platform.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PromethION</div><div>suggested: (PromethION, RRID:SCR_017987)</div></div></td></tr></table>

---

Results from OddPub: Thank you for sharing your data.

---

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

The main limitation lies in the quantification of the observed events in comparison to the reads without events which can be influenced by size dependency of some of the sample preparation steps as the full-size S protein cDNA is over 4kb long and many of the observed splicing events can be shorter than 200 bp and depleted by many sample preparation methods. Reported here are distributions based on the full length read population alone within each sample, but care should be applied before comparing these frequencies to other datasets with different sample preparation methods, such as the ChAdOx1 Nanopore RNA direct data (Sup Figure 8). Hundreds of randomly spaced splicing events characterized by the cDNA direct sequencing approach for the S protein and similar events seen in other proteins highlight the fact that even when the transgene’s CDS is apparently well designed and expression of full-length protein can be detected, the status quo design is just not optimal for RNA expression as transcript heterogeneity will inevitably impact both product levels (yield) and homogeneity. The impact of this will be dependent on the nature of the product, whether it is expressed in vitro or in vivo. In vitro expression offers opportunities to improve homogeneity by application of purification methods. Two thirds of cryptically spliced RNA molecules would be out-of-frame and would not just impact protein yield but any translation in vivo would generate novel peptides with potential immuno...

---

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>

"what's it called?"

his tag can be labeled in lysate

small tag may not alter the conformation and function of the protein

2 points:

1. binding Kd is about 1nM
2. Kd for different targets could be slightly different

SciScore for 10.1101/2021.09.14.460356: (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">g, anti-Human antibody was added to the wells and incubated for 1 hour followed by six washes as before.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Human</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD-SD1, wild type RBD and mutant RBD domains were subcloned into pcDNA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RBD-SD1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">One day before transfection, CHO-S cells were seeded at a density of 1 x 106 cells/mL in 45 mL culture flask.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO-S</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lipofectamine® transient transfection of RBD constructs: For transient expression of RBD-SD1, RBD wild-type and RBD mutants, 293T cells were cultured and and incubated at at 37℃ with 5% CO2.</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">Vero E6 neutralization assay: All aspects of the assay utilizing virus were performed in a BSL3 containment facility according to the ISMMS Conventional Biocontainment Facility SOPs for SARS-CoV-2 cell culture studies.</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 incubation with 293TACE2 cells for 48 hours at 37°C, cells were washed twice with PBS, lysed with Luciferase Cell Culture Lysis 5x reagent (Promega), and NanoLuc Luciferase activity in lysates was measured using the Nano-Glo Luciferase Assay System (Promega)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293TACE2</div><div>suggested: RRID:CVCL_YZ65)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 Spike ECD 1-1208 (682-GSAS-685; 986-PP-987) fused to the T4 fibritin trimerization domain with C-terminal Avi- and His-tag were synthesized with gene block (IDT) and cloned into pcDNA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA</div><div>suggested: RRID:Addgene_66792)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">On the day of transfection, 75 µL of FectoPRO® transfection reagent (PolyPlus-transfection®) was mixed with 5 mL of 15 µg/mL pcDNA3 plasmid DNA harboring antibody encoding sequence in CD-CHO media and incubated for 10 min at room temperature.</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">The atomic models and cryo-EM maps generated for the N-612-017, N-612-014, and N-612-004 Fabs complexed with SARS-CoV-2 S have been deposited at the PDB (http://www.rcsb.org/) and the Electron Microscopy Databank (EMDB) (http://www.emdataresource.org/) under accession codes 7S0C, 7S0D, 7S0E and EMD-24786, EMD-24787, EMD-24788, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>http://www.emdataresource.org/</div><div>suggested: (EMDataResource.org, RRID:SCR_003207)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Half-maximal inhibitory concentrations (IC50 values) for mAbs were determined using 4-parameter nonlinear regression (Prism, GraphPad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div><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">Movies were collected using a 3x3 beam image shift pattern with SerialEM automated data collection software (Mastronarde, 2005) at a nominal magnification of 45,000x (super-resolution 0.4345 Å/pixel) using a defocus range of −0.7 to −2.0 µm.</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">For all datasets, movies were patch motion corrected for beam-induced motion including dose-weighting within cryoSPARC v3.1 (Punjani et al.,</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">Coordinates were rigid body and B-factor refined in PHENIX v1.19 (Adams et al., 2010) followed by sequence matching and repeated cycles of 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">refine and manual building in Coot (v0.9.3) (Emsley et al., 2010) (Table S6)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr></table>

---

Results from OddPub: 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>

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Full Revision

Manuscript number: RC-2021-00785

Corresponding author: Christian, G. Specht

1. General Statements

Dear Editor,

We greatly appreciate the reviewers’ constructive comments on our manuscript ‘Identification of a stereotypic molecular arrangement of glycine receptors at native spinal cord synapses’. We were particularly pleased that all four reviewers agreed that our data yield new insights into the structure of inhibitory glycinergic synapses, and represent both a technical and conceptual advance the field of synaptic neuroscience.

The reviewers have consistently raised one main criticism, namely the use of endogenously expressed GlyRs tagged with the fluorescent protein mEos4b, which could potentially have an impact on receptor expression, trafficking and function. We have addressed this point by performing whole-cell recordings of GlyR currents in cultured neurons that show that glycinergic transmission and therefore function is preserved. We have also addressed all other comments of the reviewers in the revised manuscript, including a thorough revision of the text and the addition of new data and figures as detailed in the point-by-point response.

Point-by-point description of the revisions

Reviewer 1:

Summary:

In this manuscript Maynard et al describe a newly generated knockin mouse to study the endogenous distribution of Gly receptors in the spinal cord. Using quantitative confocal imaging and SMLM the distribution and levels of GlyRs at spinal cord synapses is compared between dorsal and ventral horn. They found that levels of synaptic GlyR are higher in dorsal than ventral spinal cord synapses. Nevertheless, the ratio to gephyrin seems constant, except for synapses in superficial layers of the dorsal horn, where gephyrin levels exceeded the levels of GlyRs. There are also fewer, but larger synapses in the ventral horn than in the dorsal horn. These findings are further corroborated by an SR-CLEM approach. Furthermore, it is shown that in a mouse model for hyperekplexia GlyR levels are lower, but still enriched at synapses, and the dorsal-ventral gradient in GlyR expression was maintained. The difference in size of ventral and dorsal synapses observed in WT animals was also lost in the oscillator mouse, suggesting that particularly the ventral synapses are affected. Despite these differences, the density of GlyRs per synapse remained similar.

Major comments:

Line 113: "labeling the_ _b__-subunit has proven difficult". This statement is unclear and it would be informative for readers to grasp what exactly has been difficult, and why the approach described here overcomes that? Related to that, the authors state "KI animals reach adulthood and display no overt phenotype, suggesting that the presence of the N-terminal fluorophore does not affect receptor expression and function". That is indeed reassuring, but it does not exclude that receptor numbers, function and distribution are altered. As it seems there is no prior literature on tagging the beta subunit, additional evidence that the tag does not interfere with receptor trafficking or functioning would be desirable

We have clarified why it has been difficult to label the GlyR beta subunit until now, lines 113-115 _“To date, labeling of GlyRβ in situ using immunocytochemistry has proven difficult due to a lack of reliable antibodies that recognize the native β-subunit (only antibodies for Western blotting recognizing the denatured protein are available), which has severely limited the study of the receptor.”_ Hence it was important to us to generate this knock-in mouse in order to study the endogenous GlyR at synapses, which is the least well studied receptor mediating fast synaptic transmission.

The reviewer makes an important point regarding the labeling of the GlyRβ-subunit with a fluorescent protein that has also been raised by the other reviewers. We have now verified receptor function by patch clamp recordings of glycine currents in whole-cell configuration in spinal cord neuron cultures from the mEos4b KI mouse (new Supplementary Fig. S2C). At saturating glycine concentrations of 300 μM we found no difference in chloride influx between mEos4 KI and WT mice. Since glycine concentrations in the synaptic cleft are in the millimolar range during synaptic transmission, these data strongly suggest that glycinergic transmission is not affected by the presence of the mEos4b under physiological conditions, despite a minor shift in the EC50.

There are several other strong arguments that suggest that mEos4b-GlyRb expression, subcellular localization and function are the same as those of the native subunit. Firstly, the mEos4b sequence was inserted after the signal peptide and before the beginning of the coding sequence of the mature β-subunit (Fig. S1). Since the mEos4b sequence does not interrupt the coding sequence it is less likely to affect the receptor conformation. Secondly, we did not notice any behavioural phenotypes in animals carrying the GlrbEos allele. At the time of weaning, the genotypes of the pups corresponded to the expected Mendelian frequency (new Fig. S2A). Moreover, we did not observe a reduction in live expectancy of GlrbEos/Eos animals (new Fig. S2B), demonstrating that the mEos4b-GlyRb does not cause pathology in older animals.

Most importantly, our imaging data (Fig. 1-3) provide exhaustive evidence that mEos4b-GlyRb assembles with GlyR alpha subunits as heteropentameric receptor complexes that are trafficked to the plasma membrane and inserted into the synaptic membrane due to their interaction with the gephyrin scaffold at functional synapses. Using quantitative imaging, we have also shown that homozygous GlrbEos/Eos KI mice have exactly twice the number of receptors at synapses as heterozygous animals, strongly suggesting no interference in receptor trafficking to the plasma membrane and gephyrin binding. As the mEos4b mice were also bred with the oscillator mouse model of hyperekplexia, which is lethal when homozygous, we could further test the combined effect of GlrbEos and GlyRa1spt-ot. The presence of both alleles did not lead to any noticeable phenotypes in heterozygous oscillator mice. On the contrary, both synaptic targeting and the packing density of the receptors were not altered in this model, despite a region-specific reduction in synapse size due to the reduced availability of the intact GlyRa1 subunit.

We believe that these data overwhelmingly support our conclusion that the presence of the mEos4b tag does not alter the structure and function of the receptor, making this mouse model uniquely suited to study the dynamics and regulation of glycinergic synapses in a quantitative manner and at the molecular level.

In the Discussion the authors conclude that "Our quantitative SR-CLEM data lend support to the first model, whereby inhibitory PSDs in the spinal cord are composed of sub-domains that shape the distribution of the GlyRs". This conclusion seems however based on one example image in Fig 3G that is not very convincing. The EM image seems to show two clearly separated PSDs opposed by two distinct active zones. So, although this conclusion is of high interest, more support should be given to substantiate this conclusion. More general, these subsynaptic domains (SSDs) are hardly further explored, but seem relevant for transmission, particularly given that the synaptic pool of GlyRs at these synapses is not saturated by single release events. How general are these SSDs at these synapses?

The representative image in Fig. 3G shows two SSDs within the same postsynaptic site with a continuous presynaptic active zone. It should be noted that the PALM/SRRF images were taken of the entire 2 µm thick slice, whereas the electron micrograph shows only a single 70 nm section. We verified throughout the full 3D stack of serial sections that the presynaptic site remains continuous, which it does. We would also like to point out the scale of the image showing that the two SSDs are only around 170 nm apart, i.e. spatially very close. Our conclusions are however not based on this single image but the whole dataset. The graph in Fig. 3I shows 3 synapses (out of N = 36), in which the GlyR density at separate SSDs could be quantified, demonstrating that the receptor density is not different between SSDs. The reviewer is correct that we do not further analyse the SSDs beyond their density and the analysis of the segmentation of the postsynaptic sites (Fig. 3E-G). Further work on the functional role of SSDs in synaptic transmission is outside the scope of this manuscript and would indeed merit future study.

The approach for counting molecules based on the PALM acquisition has been developed in prior publications and seems robust. It would however be worth to present the reader with a bit more background and explain the assumptions of this approach in more detail. Particularly, since counting of mEos4b can be problematic, as there are multiple dark and fluorescent states of this fluorophore that could be influenced by the illumination scheme, see for instance De Zitter et al., Nat Methods 2019. Since the preceding SRRF acquisition already exposes the fluorophore to high and continuous 561-nm laser power this could skew the counting due to unaccounted conversion and perhaps bleaching of mEos4b. In line with this, although throughout the manuscript the term 'absolute copy numbers' is used the reported numbers are at best an estimate based on a number of assumptions. I think the wording 'absolute numbers' is therefore deceiving and should be nuanced.

We have clarified how the molecule conversion is calculated (Fig. S7 legend), to provide a more complete description of the way in which the values were obtained. Further we have explained how we calculated the probability of detection. Since the probability of detection accounts for any unconverted or non-functional mEos4b molecules, our molecule counting approach is relatively resistant to potential pre-bleaching of fluorophores. It should be noted, that 561 nm illumination had no obvious effect on the non-converted (green) mEos4b fluorophores, as judged by the fact that the intensity of receptor puncta was unaffected by the SRRF recordings. We appreciate the reviewers point regarding the term ‘absolute copy number’ and we have adjusted our wording throughout the manuscript accordingly.

Related, most of the quantifications are in estimating the number of receptors, and not so much the distribution with the PSD. The term "molecular arrangement" - also used in the title - might therefore be misleading, there is in fact little characterization of how GlyRs are placed within the PSD. More focused analysis quantifying the distribution of receptors within the PSD and/or SSDs would strengthen the manuscript.

By estimating the number of receptors and the exact size of synapses, the main conclusion of our study is that receptor density at dorsal and ventral synapses is identical, independent of synapse size, subdomains, or in fact loss of GlyRs in a mouse model of hyperekplexia. This observation clearly relates to how receptors are packed within synapses, and thus describes their molecular arrangement.

The reported N is confusing and makes it hard to judge the reproducibility of the data. Sometimes it refers to number of images, sometimes number of synapses, but it is unclear from how many experiments these are drawn. This should be reported more completely (number of animals should be reported at least) and consistently. In figure 1, the N numbers (N=3-5 images) are particularly low and question how consistent these findings are across multiple animals.

We have clarified the N in the figure legends, to reflect the full size of the datasets that have been analysed.

The levels of mRFP-Gephyrin seem to differ between the different mouse lines, is this a significant difference?

No significant differences in mRFP-gephyrin levels were found in animals with different mEos4b-GlyRb genotype (Fig. 1B). However, expression of mRFP-gephyrin in heterozygous animals is 50% of that in homozygous mRFP-gephyrin KI animals (not shown).

The ICQ analysis for co-localization is hardly explained. How do we interpret this parameter? What does an average value of ~0.3 mean? A comparison with sets of proteins that do not overlap as a negative control would strengthen the conclusion.

We have clarified that an ICQ value of 0.3 is indicative of a very high spatial correlation between pixels, and provided a corresponding reference for ICQ analysis (lines 209-210). We would like to point out that the scale of the ICQ is between -0.5 to 0.5, meaning that a value of 0.3 comes close to complete correlation.

Minor comments:

Very little fluorescence was detected in the forebrain, despite the high reported expression of the Glrb transcript". Can the authors expand on this? What would explain this discrepancy?

We have clarified the text to include “suggesting that protein levels are controlled by post-translational mechanisms in a region-specific manner, as previously proposed (Weltzien et al., 2012)” (Lines 152-153). The reason for this discrepancy is not known. However, the distribution of mEos4b expression throughout the brain is as expected, based on the literature.

"What region is quantified in Fig 1B? is the same region in all conditions? This should be specified more clearly as the manuscripts presents a clear gradient in expression levels in the spinal cord and thus the location will influence the intensity measurements.

We have explained in the text that this is the region at the centre of the ventral horn identified by the white square in Fig. 1A, and that the same region was analysed for all images across all animals. Page 5, lines 160-161 “The same region of the ventral horn, indicated by the white square in Fig. 1A was taken for quantification of mEos4b-GlyRβ and mRFP-gephyrin expression in all conditions.”

The labeling approach does not differentiate between surface and internal receptors, this should be made more explicit in the text.

Whilst this is correct, we have only analysed mEos4b-positive synapses that had corresponding gephyrin clusters, meaning synapses where receptors are located in the postsynaptic membrane. Indeed we found that all mEos4b clusters imaged colocalised with mRFP-gephyrin clusters. We have adjusted the text accordingly, page 6, line 205-206 “All mEos4b-GlyR clusters closely matched the mRFP-gephyrin clusters, confirming the localization of the receptors in the postsynaptic membrane.”

Significance:

The presented data are interesting and the experiments are technically advanced and carefully performed. Particularly the SR-CLEM approach is technically advanced. The datasets present a quantitatively detailed characterization of spinal cord synapses and will be of interest for researchers working in the field of spinal cord circuitry, as well as super-resolution imaging. The conceptual advance for the field is however somewhat limited. It seems that the presented data confirm the general notion that receptor numbers and synapse size are highly correlated. So, although this manuscript describes very interesting observations, in its present form the manuscript does not provide any new mechanistic insight or significant advance in our understanding of how these synapses operate.

We thank the reviewer for his/her comments relating to the technicality of our manuscript. However we think that the statement “The conceptual advance for the field is however somewhat limited” is unfair, as this level of organisation of inhibitory synapses at the molecular scale has never been achieved before, as pointed out by the other reviewers, and especially not as regards different ages of animals and a disease model that directly affects receptor numbers in a region-specific manner. We therefore believe that our study will have a substantial impact within the fields of synaptic neuroscience as well as quantitative neurobiology.

Referee cross-commenting:

I agree with the other reviewers that this study is technically advanced, but I remain critical towards the extent of conceptual advancement this study brings and there are some important concerns with the presented data that need to be addressed. Nevertheless, indeed many of these concerns can be addressed without additional experiments. As pointed out also by other reviewers additional validation that the fusion proteins are not disrupting their function or organization would be important.

Reviewer 2:

Summary:

Maynard et al. investigate (inhibitory) glycinergic synapses in mouse spinal cord, which regulate motor and sensory processes. The authors analyse the molecular architecture and ultra-structure of these synapses in native spinal cord tissue using quantitative super-resolution correlative light and electron microscopy. The major finding is that GlyRs exhibit equal receptor-scaffold occupancy and constant absolute packing densities across the spinal cord and throughout adulthood, although ventral and dorsal inhibitory synapses differ in size. Moreover, what the authors call a „stereotypic arrangement" is even maintained in a hypomorphic mutant (oscillator), which is deficient in the adult GlyR a1 subunit.

Specific comments:

To reach their conclusions the authors generate two knock-in mouse lines, one with mEOS-labelled GlyR ß-subunit and one with mRFP-labelled gephyrin, a subsynaptic scaffolding protein of inhibitory synapses, which are subsequently crossed. Both changes are not unproblematic, as mutations in the N-terminal end of the GlyR ß subunit polypeptide chain might interfere with the assembly of functional GlyR (consisting of a und ß subunits) and and mutations at the N-terminal end of gephyrin interfere with it's homo-oligomerization into higher molecular assemblies.

We have demonstrated that the function of mEos4b-GlyRb does not differ significantly from WT GlyRs, by carrying out electrophysiological experiments (new Fig. S2C). For a detailed response, please see the response to the first comment of reviewer 1. The mRFP-gephyrin KI strain has been validated and published previously (see Machado et al., 2011, J Neurosci; Specht et al. 2013 , Neuron) and was not specifically generated for this study. The experiments with the oscillator mutant did not include the mRFP-gephyrin allele. In these experiments, the wildtype GlrbEos/Eos (Fig. 4, 5) behaves exactly as the GlrbEos/Eos in the double knock-in (Fig. 1, 2), further validating the mouse models used.

However, in this experimental design both labelled proteins reach postsynaptic membrane specialisations. In case of the ß-subunit quantitative evaluation confirms that heterozygous animals contain only half of the labelled protein as homozygous, which is an indication but not a proof that the correct stoichometry of adult GlyR is maintained. Likewise, mRFP-labelled gephyrin assembles with WT-gephyrin in subsynaptic domains, but it is not clear, if the size and density of the synapses is changed by the knock-in procedure as compared to WT-synapses.

An effect of the mRFP tag on gephyrin clustering can be ruled out, since we observed no difference in synapse size and receptor density in GlrbEos/Eos animals with (Fig. 1, 2) and without the GphnmRFP allele (Fig. 4, 5, oscillator wild-type controls). Similarly, the synaptic mEos4b-GlyRb levels in heterozygous animals were precisely half those of the homozygous animals, strongly suggesting that the expression and trafficking of the tagged receptor subunit is unchanged, as the reviewer acknowledges. In the absence of any obvious behavioural and/or functional phenotypes (Fig. S2) this KI model is in our view is an exceptional tool to study GlyRs expressed at endogenous levels in a cell-type specific manner.

Accepting these constraints, which to the knowledge of this reviewer have never been addressed to satisfaction, the authors provide a technically excellent, comprehensive analysis of glycinergic synapses in the spinal cord of double knock-in mice. Therefore, it should be stated in the title, that the investigations were performed with double knock-in instead of „native" spinal cord. Text and figures are clear and accurate and represent the state of the art.

We thank the reviewer for the positive comments regarding the techniques used in the study, and the clarity of the text and figures. We have adjusted the title as requested.

Finally, the reviewer would like to raise a minor point: the term postsynaptic density is derived from electron microscopical studies of synapses, where asymmetrical synapses display a „postsynaptic density" but symmetrical synapses do not. The latter were identified as inhibitory synapses and therefore, by definition, inhibitory synapses do not have a postsynaptic density, but rather a postsynaptic membrane specialisation. The use of the term „postsynaptic density" should, therefore, be restricted to excitatory synapses.

We are conscious of the importance of correct definitions and have revised the terminology, referring to “postsynaptic sites”, “postsynaptic domains”, and “postsynaptic specializations” as appropriate throughout the manuscript.

Significance:

The authors provide a state of the art advanced light and electron microscopical analysis of glycinergic synapses in the mouse spinal cord. They suggest a robust "stereotypical" mechanism in place, which guarantees a fixed stoichiometry of relevant components, which is even maintained in a hypomorphic mutant, which is believed to represent a mouse model of human hyperekplexia (startle disease).

Referee cross-commenting:

I would like to corroborate the arguments of the previous reviewer: it is not clear to which extent the fusion proteins influence the measurements, which are technically very advanced and well done, however. The authors do definitely not investigate "native spinal cord" as stated in the title.

The argument concerning fusion proteins must be taken especially serious as the fusions were induced in regions known to be responsible for assembly of glycine receptors and oligomerization of gephyrin.

We have verified the receptor function with electrophysiological recordings and clarified exactly where the fluorescent protein was inserted (see reviewer 1 response). Given the similarity in synapse size, fluorescence intensities and molecule densities observed in neurons expressing different combinations of tagged and native receptors and scaffold proteins, we strongly believe that all animal models used are well suited to the experimental aims of our study.

Reviewer 3:

Summary:

Glycinergic synapses are the least well understood of synapses that mediate fast synaptic transmission. The manuscript by Maynard et al. adds new information about the structural aspects of these synapses, using PALM and EM imaging of spinal cord synapses from mice at 2 and 10 months. The authors created a knock-in mouse that expresses a tagged GlyRbeta subunit, allowing synaptic localization of glycine receptors; all synaptically localized glycine receptors are thought to require the beta subunit to be tethered by gephyrin. The authors compare synaptic profiles from: 2 month old vs. 10 month old mice; dorsal vs. ventral horn; and GlyR1-reduced vs. wild type mice. Strikingly, they find a tight relationship across all of these variables between glycine receptor puncta and gephyrin puncta, as well as an apparently constant "packing density" of glycine receptors. They conclude that synaptic extent is likely to be the most important determinant of synaptic strength, as the density of receptors within the postsynaptic density is constant. These results use cutting-edge imaging and are analyzed with care, and add new information to our understanding of these relatively less well characterized synapses._

Major comments:

The key conclusions are convincing and the claims appear solid. Additional experiments are not needed to support these claims. The data and the methods are largely presented in such a way that they can be reproduced, although there are minor suggestions for improvement below.

We thank the reviewer for his/her positive comments.

Minor comments:

Do the authors have any comment on the requirement during, e.g. LTP, for insertion of a gephyrin-GlyR unit? The lead author has speculated that gephyrin creates "slots" for GlyRs; yet apparently each slot is already filled in the snapshots taken here. How might postsynaptic LTP occur (Kandler group, Kauer group papers)?

Given the reciprocity of GlyR and gephyrin clustering at synapses, the occupancy of binding sites (and in turn the number of available ‘slots’) is dependent on the strength of receptor-scaffold interactions, as discussed previously (Specht 2020, Neuropharmacol). In this study we demonstrate that the density of GlyRs at synapses is constant, which implies that the receptor occupancy is also the same, with the possible exception of mixed inhibitory synapses in the superficial dorsal horn that contain a majority of GABAARs. The PALM/SRRF data are represented as rendered image reconstructions and not as pointillist representations, and the detection of unoccupied binding sites is below the spatial resolution of our approach. However, the high spatial correlation of the signal intensities (ICQ ≈ 0.3) suggests that receptor occupancy is equal between and within synapses. It has previously been established that there are more scaffold proteins than receptors at synapses (Specht et al. 2013, Neuron; Patrizio et al. 2017, Sci Rep). Based on these studies we report that approximately half the gephyrin binding sites are occupied by receptors (lines 262-655). We have also expanded the discussion, describing how shape and size of synapses may affect synaptic transmission, as well as the possible role of receptor-gephyrin interactions in synaptic plasticity at glycinergic synapses.

It would be very interesting in the discussion to contrast the present observations with what is known about excitatory synapses (NMDA and AMPAR distributions) and GABAergic synapses. Are the authors at all surprised that receptor packing is constant across conditions? Can the authors speculate on how non-gephyrin binding receptors (homomeric alpha receptors, which are found in recordings) may function and be tethered to the membrane.

We have included additional information about receptor numbers and distributions at excitatory (lines 428-438) and GABAergic (lines 389-393) synapses in the discussion. So far, homomeric GlyRs composed of alpha subunits have been found to be exclusively extrasynaptic. As stated on page 4, lines 111-112 the beta subunit is required for binding of the GlyR to gephyrin and subsequent anchoring at the synapse. Previous studies have shown exocytosis of receptors to occur at extrasynaptic sites followed by lateral diffusion to synapses. Homomeric GlyRs are therefore most likely targeted to the extrasynaptic plasma membrane where they remain due to the lack of the beta subunit.

Figure S1. It would be most helpful to quantify this; at the least to include an atlas-like drawing to allow identification of the structures illustrated and containing Glrb; better yet would be quantification of staining in regions where this is strongest.

We have added an atlas indicating the different brain regions expressing mEos4b-GlyRb protein as a new Supplementary Fig. S3. The regional expression pattern agrees with the available literature about protein expression of the GlyRb subunit in different brain regions and hence provides further evidence that mEos4b-GlyRb is expressed like the native receptor. Due to the relatively low resolution of the tiled image no accurate quantification was possible. We have however added higher magnification confocal images of representative brain regions expressing varying amounts of GlyRb.

The fact that the lower panel in B is labeled as +/+ across all groups is initially confusing; perhaps relabel as mEos4 -/-, +/- and +/+?

We assume that the reviewer is referring to Fig1B. The genotype of both the GlrbEos and the GphnmRFP allele is now indicated on the x-axes, and the legend has been modified to clarify that all these animals were homozygous for GphnmRFP/mRFP. We have strived to remain consistent throughout the manuscript when referring to genotypes and protein levels.

Do gephyrin levels drop in WT mice as well as in the mEosr-GlyRb mouse between 2 and 10 months? Do the authors have any thoughts on this (Supp figure S2)?

We found no differences in gephyrin levels between 2 and 10 months. Fig. S2 (now Fig. S4C) shows the number of synaptic gephyrin clusters, which was the same at different ages and genotypes.

Significance:

Glycinergic synapses are the least well understood of synapses that mediate fast synaptic transmission. The manuscript by Maynard et al. adds new information about the structural aspects of these synapses, using PALM and EM imaging of spinal cord synapses from mice at 2 and 10 months. The authors created a knock-in mouse that expresses a tagged GlyRbeta subunit, allowing synaptic localization of glycine receptors.

This will be of interest to those studying inhibitory synapses, and more broadly to synaptic morphologists, physiologists and imagers for comparison with other synapse types.

My own expertise is NOT in these techniques, but I am a synaptic physiologist with a standing interest in glycinergic synapses; thus I am not providing serious technical critiques.

Referee cross-commenting:

Hi all, I agree with the other two reviewers, and do not have anything else to add.

Reviewer 4:

Summary:

The authors used a correlative approach and combined photo-activated localization microscopy with electron microscopy to characterise Glycinergic synapses in spinal cord tissue. Some of the major findings are:

• The receptor-scaffold occupancy and packing densities of glycinergic synapses in different regions of the spinal cord are the same.
• Gephyrin clusters in the spinal cord are composed of sub-domains that shape the GlyR clusters.
• Ventral horn synapses are generally larger, more complex (containing a number of gaps) and contain more GlyRs. -In a mouse model of Hyperekplexia, the number of GlyRs is reduced resulting in smaller synapses in the ventral spinal cord.

Major comments:

Are the key conclusions convincing? Yes

Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? No

Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. No

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. N/A

Are the data and the methods presented in such a way that they can be reproduced? Yes

Are the experiments adequately replicated and statistical analysis adequate? Yes

Minor comments:

Specific experimental issues that are easily addressable. Please see below

Are prior studies referenced appropriately? Yes

Are the text and figures clear and accurate? Yes

Do you have suggestions that would help the authors improve the presentation of their data and conclusions? Please see below.

As the authors pointed out, fusing mEos to the extrasynaptic terminal of GlyRb has been difficult and therefore this construct would benefit the larger scientific community. Fig 1C is a nice imaging control for expression efficiency, however, it is in stark contrast with the lack of functional control. Do authors have any electrophysiological evidence showing that the insertion of mEos4b doesn't modulate channel function? I would assume that the construct would be tested in cell lines before the KI mouse line was created. Was any functional analysis done? If yes, it would be very useful to show it. I do appreciate that the authors used a standard insertion between the 4th and 5th AA in the extracellular domain, which in most cases does not abolish channel function. Given the lack of an obvious phenotype in the KI mouse model, I believe that this is also the case here. However, I disagree with the statement in lines 120-121: "the presence of the N-terminal fluorophore does not affect receptor expression and function." I believe that if there are no electrophysiological measurements of GlyR function, this statement remains speculative. As the authors pointed out in their previous publication: "receptor function and gephyrin binding are not independent properties. Instead, we think that conformational changes triggered at extracellular or intracellular protein domains have downstream consequences on channel opening as well as receptor clustering." In line with this, my concern is that the modulation of channel function by mEos4b could result in an altered cluster size at synapses. There is a large body of literature showing that just one missense mutation in the extracellular domain of ion channel subunits can lead to synaptopathies because the channel function gets modulated, and there is an abundance of similar examples involving mutations of GlyR and GABAAR subunits. In my view, comparing the function of GlyRs incorporating wt-GlyRb and mEos4b-GlyRb subunits is important for the correct interpretation of the main findings of this work and would strengthen the publications.

As the reviewer points out, the insertion of the mEos4b sequence was considered carefully in order to have the least impact on receptor function. GlyR channelopathies are often caused by point mutations within the coding sequence, which is not the case in the GlrbEos allele. Instead, the mEos4b sequence was inserted after the single peptide of GlyRb, duplicating several amino acid residues in order to maintain the correct cleavage site and N-terminus of the mature receptor, and to not interrupt the GlyRb coding sequence (Fig. S1B). In order to verify that the mEos4b-tag does not affect GlyR function, we have now carried out electrophysiological experiments (new Fig. 2C). For a detailed description please see the response to the first comment of reviewer 1.

Line 189: Are the authors making conclusions based on intensity comparison of red mEos4b and mRFP? The title of this section implies that the red form of mEos was compared to mRFP(?) But mEos converts from green to red only partially. Was the probability for conversion taken into account at this point? Please clarify which version of mEos was compared to mRFP._

In line 189 (now 218) we compared the intensities of mRFP-gephyrin with those of converted (red) mEos4b in SRRF / PALM super-resolution images of the synapses (Fig. 2D). Since the absolute intensities are altered by the process of image reconstruction, the probability that mEos4b is photoconverted does not have to be taken into account. The constant ratio of the SRRF and PALM image intensities confirms the data in Fig. 1D showing that GlyR and gephyrin amounts are highly correlated throughout the spinal cord (with the exception of the superficial layers of the dorsal horn). We have clarified in the text that this analysis was carried out on reconstructed SRRF images of mRFP-gephyrin and PALM images of mEos4, line 202.

Line 192: Please clarify how the density threshold was calculated/determined? This is important for the replication of the experiments, and it also has implications for the calculated probability of detection of mEos4b. I am not aware that this probability was calculated before for mEos4b and therefore other researchers may decide to rely on the value calculated here.

We have now clarified in more detail how the probability of detection was calculated (new Supplementary Fig. S7 legend).

In Fig. 2 Gephyrin clusters look consistently smaller than GlyR clusters, which is inconsistent with the published work. I assume that the difference in size is a consequence of different image reconstruction methods(?) However, I would assume that SRRF would have lower resolution than your PALM measurements and that would result in wider Gephyrin clusters. Could you please explain this discrepancy? Also, could you provide an estimate for the image resolution in SRRF and PALM techniques? For SMLM, localization precision would suffice.

We have provided an estimate of the resolution of the two techniques using Fourier ring correlation, which gave 46 nm for SRRF and 21 nm for PALM. Additionally we have precised the discrepancy between reconstruction methods, page 6, lines 194-200 “The spatial resolution was estimated using Fourier ring correlation (FRC), which measures the similarity of two images as a function of spatial frequency by comparing the odd and even frames of the raw image sequence. According to this analysis, the spatial resolution of SRRF was 46 nm and that of PALM 21 nm. It should be noted that the synaptic puncta in the SRRF images appear somewhat smaller and brighter due to differences in the reconstruction methods that result in differences in the dynamic intensity range.”

Why is the data in Fig. 5D and E represented as Detections/Synapse instead of GlyRs/Synapse? Could you please re-plot this so that a comparison with Fig. 2H and I is straightforward?

We have converted the detections to receptor copy numbers as requested (Fig. 5D,E).

Figure S5C: for P=0.5, 2=0.25. Please correct. Also, I assume that the second graph is what would be observed experimentally for dimers and P=0.5. Please clarify in the figure caption.

This was a mistake and has been corrected. We have also clarified which parts of the calculations are theoretical and which values were derived from our experimental data. We have provided a more detailed description in the figure legend of Supplementary Fig. S7.

Line 606: Please provide a complete derivation of this formula.

We have provided a full derivation of this formula (new Fig. S7C).

Significance:

The work described here seem to be a natural progression of a publication by Patrizio et al., 2017 that came out from the same laboratory. This study uses advanced methodologies in the imaging space to visualise and characterise Glycinergic synapses in spinal cord tissue. The experiments described here are technically demanding as evidenced by the relatively small number of publications describing super-resolution measurements in tissue samples. Even more rare are studies that attempt to do single protein counting in neuronal culture and tissue sections. Therefore, I believe that this work brings significant technical advancement in the field of super-resolution and corelative microscopy. The findings are also highly significant for all fields of neuroscience in which the structure of inhibitory Glycinergic synapse is relevant, ranging from the fundamental understanding of inhibitory synapse function to pathologies involving Glycinergic signalling._

I have substantial experience in different microscopy methods, including quantitative super-resolution microscopy based on single molecule counting. My background also covers the structure and function of GABAA and Glycine receptors using electrophysiology. I am familiar with the methods used in electron microscopy and the process of creating KI mouse lines, however I don't have hands-on experience in these fields._

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary:

In this manuscript Maynard et al describe a newly generated knockin mouse to study the endogenous distribution of Gly receptors in the spinal cord. Using quantitative confocal imaging and SMLM the distribution and levels of GlyRs at spinal cord synapses is compared between dorsal and ventral horn. They found that levels of synaptic GlyR are higher in dorsal than ventral spinal cord synapses. Nevertheless, the ratio to gephyrin seems constant, except for synapses in superficial layers of the dorsal horn, where gephyrin levels exceeded the levels of GlyRs. There are also fewer, but larger synapses in the ventral horn than in the dorsal horn. These findings are further corroborated by an SR-CLEM approach. Furthermore, it is shown that in a mouse model for hyperekplexia GlyR levels are lower, but still enriched at synapses, and the dorsal-ventral gradient in GlyR expression was maintained. The difference in size of ventral and dorsal synapses observed in WT animals was also lost in the oscillator mouse, suggesting that particularly the ventral synapses are affected. Despite these differences, the density of GlyRs per synapse remained similar.

Major comments:

• Line 113: "labeling the -subunit has proven difficult". This statement is unclear and it would be informative for readers to grasp what exactly has been difficult, and why the approach described here overcomes that? Related to that, the authors state "KI animals reach adulthood and display no overt phenotype, suggesting that the presence of the N-terminal fluorophore does not affect receptor expression and function". That is indeed reassuring, but it does not exclude that receptor numbers, function and distribution are altered. As it seems there is no prior literature on tagging the beta subunit, additional evidence that the tag does not interfere with receptor trafficking or functioning would be desirable
• In the Discussion the authors conclude that "Our quantitative SR-CLEM data lend support to the first model, whereby inhibitory PSDs in the spinal cord are composed of sub-domains that shape the distribution of the GlyRs". This conclusion seems however based on one example image in Fig 3G that is not very convincing. The EM image seems to show two clearly separated PSDs opposed by two distinct active zones. So, although this conclusion is of high interest, more support should be given to substantiate this conclusion. More general, these subsynaptic domains (SSDs) are hardly further explored, but seem relevant for transmission, particularly given that the synaptic pool of GlyRs at these synapses is not saturated by single release events. How general are these SSDs at these synapses?
• The approach for counting molecules based on the PALM acquisition has been developed in prior publications and seems robust. It would however be worth to present the reader with a bit more background and explain the assumptions of this approach in more detail. Particularly, since counting of mEos4b can be problematic, as there are multiple dark and fluorescent states of this fluorophore that could be influenced by the illumination scheme, see for instance De Zitter et al., Nat Methods 2019. Since the preceding SRRF acquisition already exposes the fluorophore to high and continuous 561-nm laser power this could skew the counting due to unaccounted conversion and perhaps bleaching of mEos4b. In line with this, although throughout the manuscript the term 'absolute copy numbers' is used the reported numbers are at best an estimate based on a number of assumptions. I think the wording 'absolute numbers' is therefore deceiving and should be nuanced.
• Related, most of the quantifications are in estimating the number of receptors, and not so much the distribution with the PSD. The term "molecular arrangement" - also used in the title - might therefore be misleading, there is in fact little characterization of how GlyRs are placed within the PSD. More focused analysis quantifying the distribution of receptors within the PSD and/or SSDs would strengthen the manuscript.
• The reported N is confusing and makes it hard to judge the reproducibility of the data. Sometimes it refers to number of images, sometimes number of synapses, but it is unclear from how many experiments these are drawn. This should be reported more completely (number of animals should be reported at least) and consistently. In figure 1, the N numbers (N=3-5 images) are particularly low and question how consistent these findings are across multiple animals.
• The levels of mRFP-Gephyrin seem to differ between the different mouse lines, is this a significant difference?
• The ICQ analysis for co-localization is hardly explained. How do we interpret this parameter? What does an average value of ~0.3 mean? A comparison with sets of proteins that do not overlap as a negative control would strengthen the conclusion.

Minor comments:

• "Very little fluorescence was detected in the forebrain, despite the high reported expression of the Glrb transcript". Can the authors expand on this? What would explain this discrepancy?
• What region is quantified in Fig 1B? is the same region in all conditions? This should be specified more clearly as the manuscripts presents a clear gradient in expression levels in the spinal cord and thus the location will influence the intensity measurements.
• The labeling approach does not differentiate between surface and internal receptors, this should be made more explicit in the text.

Significance

The presented data are interesting and the experiments are technically advanced and carefully performed. Particularly the SR-CLEM approach is technically advanced. The datasets present a quantitatively detailed characterization of spinal cord synapses and will be of interest for researchers working in the field of spinal cord circuitry, as well as super-resolution imaging. The conceptual advance for the field is however somewhat limited. It seems that the presented data confirm the general notion that receptor numbers and synapse size are highly correlated. So, although this manuscript describes very interesting observations, in its present form the manuscript does not provide any new mechanistic insight or significant advance in our understanding of how these synapses operate.

Referee Cross-commenting

I agree with the other reviewers that this study is technically advanced, but I remain critical towards the extent of conceptual advancement this study brings and there are some important concerns with the presented data that need to be addressed. Nevertheless, indeed many of these concerns can be addressed without additional experiments. As pointed out also by other reviewers additional validation that the fusion proteins are not disrupting their function or organization would be important.

SciScore for 10.1101/2021.09.03.21263105: (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 provided written informed consent.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Study participants and sample collection: The study participants were enrolled in the ACTIV-2/AIDS Clinical Trials Group (ACTG) A5401 phase 2 randomized, placebo-controlled trial of bamlanivimab 7000mg and 700mg mAb therapy.</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">IgG was detected by incubation with MSD SULFO-TAG anti-IgG antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-IgG</div><div>suggested: None</div></div></td></tr><tr><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">SARS-CoV-2 quantitative Laboratory Developed Test (LDT) was developed utilizing open mode functionality on m2000sp/rt (Abbott, Chicago, IL) by using EUA Abbott SARS-CoV-2 qualitative reagents30.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Abbott</div><div>suggested: (Abbott, RRID:SCR_010477)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Raw sequence data were analyzed using PASeq v1.4 (https://www.paseq.org).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PASeq</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, data were quality filtered using Trimmomatic (v0.30), using a Q25/5 bp sliding window and a 70 bp minimum length.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Trimmomatic</div><div>suggested: (Trimmomatic, RRID:SCR_011848)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Filtered reads were then merged with pear v0.9.6 aligned to the reference sequence using Bowtie2 v2.1.0)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pear</div><div>suggested: (PEAR, RRID:SCR_003776)</div></div><div style="margin-bottom:8px"><div>Bowtie2</div><div>suggested: (Bowtie 2, RRID:SCR_016368)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical analyses were performed in GraphPad Prism (Version 9.1.1)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

---

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

One limitation to this study is that bamlanivimab is no longer used clinically as a single agent. These results, though, provide important proof of principle for the role that drug resistance may have on virologic and clinical efficacy of SARS-CoV-2 antiviral therapies. These lessons have implications for the development of novel COVID-19 antiviral therapies and have continued relevance for other clinically-approved single agent monoclonal antibody treatments29 and for combination therapies where one agent may be ineffective due to circulating variants13. Another limitation of this study is the limited sample size of this phase 2 study, especially in the bamlanivimab 7000mg cohort. While treatment-emergent mutations were not found in ACTIV-2 participants receiving the higher 7000mg dose of bamlanivimab, they were frequently detected in the larger BLAZE-1 phase 2 trial of the 7000mg dose7. One difference between these studies was the longer duration since symptom onset for the ACTIV-2 participants, who enrolled a median of 6 days since symptom onset versus 4 days for the BLAZE-1 participants. This likely led to higher pretreatment viral loads, which we found to be a risk factor for resistance emergence. Unfortunately, baseline viral loads could not be compared between studies as the BLAZE-1 study did not use a quantitative SARS-CoV-2 viral load assay. These disparate results highlight the importance of incorporating quantitative viral load testing and resistance testing for CO...

---

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">NCT04518410</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">ACTIV-2: A Study for Outpatients With COVID-19</td></tr></table>

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2021.09.07.21262911: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The study was approved by the ethics and research committees of the Instituto Nacional de Medicina Genómica (CEI/1479/20 and CEI 2020/21).<br>Field Sample Permit: Effective reproduction number estimation for variants B.1.1.222 and B.1.1.519: We grouped all sequenced samples based on the epidemiological week as the date of sample collection.<br>Consent: Verbal consent and identification were the first steps when calling each subject included in the final analysis.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequencing was performed on the MinION platform, and the final library (15 ng) was loaded onto the flow cell R.9 according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MinION</div><div>suggested: (MinION, RRID:SCR_017985)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A final consensus FASTA file was generated by first marking positions not covered by at least 20 reads from either group as low coverage and building a pre-consensus FASTA with BCFtools consensus, which was subsequently aligned against the reference sequence using muscle (v.3.8.1551).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BCFtools</div><div>suggested: (SAMtools/BCFtools, RRID:SCR_005227)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Phylogenetic Analysis: The sequences were aligned with MAFFT (version 7.475) using the FFT-NS-2 algorithm20,21.</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 phylogeny was calculated with FastTree (version 2.1.11) compiled with the double precision tag using a generalized time-reversible model (GTR)22,23.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FastTree</div><div>suggested: (FastTree, RRID:SCR_015501)</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>

Hypo.thesis apparently can't handle page 96, so here are my notes on it. New note: "'Or complex,' volleyed Dunraven. 'Remember the universe.' Funny banter isn't something I've seen in many other Borges stories, but he's good at it. New note: "volleyed Dunraven." Borges breaks the rules a bit by using a descriptive dialogue tag. I think he does it really well, though. "Volleyed" communicates more than, say, "retorted." It tells us a bit about Dunraven's tone and what kind of comment he's making, which is already clear because Borges' dialogue is sharp and clear, but it also tells us that Dunraven might think of conversation as a game or a competition. Originality helps here, too. I don't think I've seen "volleyed" used as a dialogue tag anywhere else. New note: "Climbing up steep sandy hills...." Their walk needs a little more specificity, in my opinion. Maybe it would be more clear with a description of these characters coming over a hill past Dunraven's family shack and seeing the labyrinth in the distance, or something like that. The beginning of this paragraph was pretty disorienting at first. The disorientation definitely could be intentional, though. Borges could be setting something up with that vagueness.

SciScore for 10.1101/2021.09.12.459978: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmids of all S variants were transiently expressed in HEK293 Freestyle cells with polyethylenimine.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, transient expression of recombinant ACE2 was achieved in Expi293 cells.</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 neutralization assay: The pseudovirus neutralization assays were performed using 293T cells overexpressing ACE2 as described previously32.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of S-Beta, Gamma, Delta, and Kappa variants: The codon-optimized DNA sequences corresponding to residues 1-1209 of S-Beta, Gamma, Delta, and Kappa variants were individually cloned into the mammalian expression vector pcDNA3.4-TOPO (Invitrogen, U. S. A.), which contains a foldon trimerization domain based on phage T4 fibritin followed by a c-Myc epitope and a hexa-repeat histidine tag as previously described32,42.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.4-TOPO</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data processing was accomplished by using cryoSPARC v2.14.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data acquisition was performed on a 300 keV Titan Krios microscope equipped with a Gatan K3 direct electron detector (Gatan, U. S. A.) in a super-resolution mode using EPU v2.10 software (ThermoFisher Scientific, U. S. A.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>EPU</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing and 3D reconstruction: All 2x binned super-resolution movie files were analyzed by Relion-3.044 with dose-weighting and 5×5 patch-based alignment using MotionCor2 (v1.2.6)45.</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">Structural visualization and rendering of structural representations were accomplished by using UCSF-ChimeraX and Pymol 2.4.1 (Schrodinger Inc. U. S. A.)</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">IC50 was determined by a four-parameter logistic regression using GraphPad Prism 9 (GraphPad, U. S. A.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

Results from OddPub: Thank you for sharing your data.

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

.readwise-whatis

.tag-why .read-howto

.c1 .tag-whyto Use both Keyword and Categorical tags.

.c2

.tag-howto

.c2

SciScore for 10.1101/2021.09.08.21263027: (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 local Ethical Review Board (41/21) and conducted according to the Declaration of Helsinki.<br>Consent: Blood Plasma samples of patients with PIMS/MIS-C according were taken after written informed consent in the department of Pediatric Cardiology of the Saarland University Hospital (Homburg/Saar, Germany), the Department of Pediatrics, Klinikum Saarbrücken (Germany), the Department of Pediatric Rheumatology and Immunology of the University Children’s Hospital Muenster (Germany), the Department of Pediatrics, Hospital Sant Joan de</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">All patients with PIMS/MIS-C fulfilled the WHO criteria, in addition all patients were seropositive for antibodies against SARS-CoV-2 or had positive PCR, with the exception of one patient who had only reported contact to SARS-CoV2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>SARS-CoV2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ELISA for autoantibodies against PGRN, IL-1-Ra: The ELISA for autoantibodies was performed as previously described (19).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PGRN</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant IL-1-Ra at 40ng/mL (Biozol, #PPT-AF-2000-01RA) alone or with either anti-IL-1-Ra antibody at 5 µg/mL (antibodies-online#ABIN2856394), recombinant SLP-antibody at 5µg/mL (abcam, #ab191883), plasma diluted 1:20 of a patient with acute PIMS/MIS-C and high-titered IL-1-Ra-antibodies (PIMS-I), or plasma diluted 1:20 of the same patient 7 months after PIMS/MIS-C without anymore detectable IL-1-Ra-Abs were preincubated for 2h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>#PPT-AF-2000-01RA</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-IL-1-Ra</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>antibodies-online#ABIN2856394</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 short, the antigens were obtained using the coding sequences of the GRN gene encoding PGRN, isoform 1 precursor of IL1RN were recombinantly expressed with a C-terminal FLAG-tag in HEK293 cells under the control of a cytomegalovirus promoter (pSFI).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr></table>

---

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

---

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

The relatively small number of cases included in the present study, due to the rarity of the disease, is a limitation. Nonetheless, these antibodies suggest to be pathogenetically relevant and should be further investigated in PIMS/MIS-C and other hyperinflammatory diseases.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

manual action time to think