SciScore for 10.1101/2021.04.21.440833: (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">Anti-acetyl lysine antibody was purchased from ImmuneChem.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-acetyl</div><div>suggested: (ImmuneChem Cat# ICP0380, RRID:AB_2801477)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-SUPT16H, anti-SSRP1, anti-TIP60, anti-IFI16, anti-MX1, anti-ISG15, anti-GAPDH and normal mouse IgG antibodies were purchased from Santa Cruz Biotechnology.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-SUPT16H,</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-SUPT16H</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SSRP1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-TIP60</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-IFI16</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-MX1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-ISG15</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-GAPDH</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-BRD4 antibody was purchased from Bethyl Laboratories.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-BRD4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-FLAG, anti-V5, and normal rabbit IgG antibodies were purchased from Invitrogen.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-FLAG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-V5</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-K48Ub, anti-histone H3, anti-mouse HRP-linked and anti-rabbit HRP-linked antibodies were purchased from Cell Signaling Technology.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-K48Ub, anti-histone</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-K48Ub, anti-histone H3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-histone</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit HRP-linked antibodies were purchased from Cell Signaling Technology.</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-HDAC1 antibody was purchased from Novus Biologicals.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-HDAC1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-H3K9me3, anti-H3K27me3, anti-H3ac (pan-acetyl), and anti-EZH2 antibodies were purchased from Active Motif.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-H3K9me3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-H3K27me3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-H3ac</div><div>suggested: (Millipore Cat# 06-599, RRID:AB_2115283)</div></div><div style="margin-bottom:8px"><div>anti-EZH2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PE/Cyanine7 anti-human CD107a (LAMP-1), PE anti-human IFNγ and anti-IL-6 antibodies were purchased from BioLegend.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PE/Cyanine7 anti-human CD107a</div><div>suggested: (BioLegend Cat# 328618, RRID:AB_11147955)</div></div><div style="margin-bottom:8px"><div>anti-human CD107a</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IFNγ</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-IL-6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-flavivirus group antigen antibody that probes ZIKV E protein and anti-SARS-CoV-1/2 NP 1C7C7 antibody was purchased from Sigma-Aldrich.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-flavivirus group antigen</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-1/2 NP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-influenza A virus nucleoprotein (NP) antibody was obtained from BEI Resources.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-influenza A virus nucleoprotein (NP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-IL-4, anti-IL-8, and FITC Mouse anti-rat IgG1 antibodies was purchased from BD Biosciences.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-IL-4,</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-IL-4</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-IL-8</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rat IgG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To determine acetylation, ubiquitination, and other protein binders of the targeted proteins, cell lysates were incubated with the specific antibodies recognizing the targeted proteins or control IgG, followed by the incubation with protein A/G magnetic beads.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>control IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells and plasmids: HEK293T, HeLa, Vero E6, and TZM-bl cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)</div></div><div style="margin-bottom:8px"><div>TZM-bl</div><div>suggested: NIH-ARP Cat# 8129-442, RRID:CVCL_B478)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Jurkat cells were maintained in RPMI 1640 medium (Gibco).</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><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HeLa or A549 cells were seeded at the density of 8,000 cells/well on 96-well culture plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, K562 target cells were pre-stained with the CellTrace™ CFSE Cell Proliferation Kit (Invitrogen) according to the manufacturer’s protocols.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K562</div><div>suggested: NCI-DTP Cat# K-562, RRID:CVCL_0004)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">1 × 106 NK-92 cells were co-cultured with the same number of K562 cells (1 : 1 of effector : target [E : T] ratio) for 4 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NK-92</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 were seeded on 96-well plates with 1 × 104 cells/well at 24 h prior to viral infection.</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">Four domains of SUPT16H, NTD, DD, MD and CTD, were cloned in pQCXIP (Clontech) with a N-terminal FLAG tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pQCXIP</div><div>suggested: RRID:Addgene_15714)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Site-specific mutation of K674R was introduced in pQCXIP-FLAG-MD by using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) following the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pQCXIP-FLAG-MD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">TIP60 shRNA (5’-TCG AAT TGT TTG GGC ACT GAT-3’) and firefly luciferase (FLuc) shRNA (5’-CAC AAA CGC TCT CAT CGA CAA G-3’) were cloned in a pAPM lentiviral vector as previously described (Huang et al., 2019).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pAPM</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">cis-reporting vectors of IFN activity, pISRE-Luc and pGAS-Luc, were purchased (Agilent Technologies).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pISRE-Luc</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HDAC1 was cloned in pcDNA-DEST40 with a C-terminal V5 tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA-DEST40</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pLX317-EZH2-V5 expression vector was acquired from Sigma-Aldrich.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLX317-EZH2-V5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">At 72 h post-transfection, cells were further transfected with pISRE-Luc or pGAS-Luc vector along with pRL-TK by using the TurboFect™ Transfection Reagent (Thermo Scientific) for 24 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGAS-Luc</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pRL-TK</div><div>suggested: RRID:Addgene_11313)</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 intensity of protein bands was quantified by using the ImageJ software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Percentages of virus-infected cells was quantified by using the Gen5 Image+ software (BioTek).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gen5 Image+</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Differential expression of genes was analyzed by DESeq2 (Love et al., 2014).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DESeq2</div><div>suggested: (DESeq, RRID:SCR_000154)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">R packages, pheatmap and clusterProfiler, were used for heatmap construction and pathway analysis, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>clusterProfiler</div><div>suggested: (clusterProfiler, RRID:SCR_016884)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MFI was calculated by using the FlowJo V10 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Results were presented as either means ± standard deviation (SD) or means ± standard error of the mean (SEM), and graphed by using the GraphPad Prism 9.0 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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

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

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

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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.

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About SciScore

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

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SciScore for 10.1101/2021.04.18.440302: (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: Study approval: Animal experiments involving SARS-CoV-2 were conducted in a BSL3 facility and treatment of animals was in accordance with regulations outlined in the U.S. Department of Agriculture (USDA) Animal Welfare Act and the conditions specified in the Guide for Care and Use of Laboratory Animals (National Institute of Health, 2011).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Mice: Homozygous female outbred K18-hACE2 transgenic mice (2B6.Cg-Tg(K18-ACE2)2Prlmn/J, Stock No: 034860, Jackson laboratory) 6-8 weeks old were maintained at 20-22°C with relative humidity of 50 ± 10% on a 12hrs light/dark cycle.</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">Mice were randomly assigned to experimental groups of 7-8 mice per group.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">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">Purified anti-DYKDDDDK Tag Antibody (Cat#637302, BioLegend)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-DYKDDDDK</div><div>suggested: (BioLegend Cat# 637302, RRID:AB_1134268)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The following secondary antibodies were used: Alexa Fluor 647-conjugated Goat Anti-Rabbit IgG (Cat#111-606-144, Jackson ImmunoResearch Laboratories)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Rabbit IgG</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 111-606-144, RRID:AB_2338083)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Afterwards, cells were washed in FACS buffer and stained with Alexa Fluor 647-conjugated anti-human IgG secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were then washed, and an Alexa Fluor 647 Anti-Mouse IgG secondary antibody was added.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Mouse IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Generation of 293T-Spike cells: First, 293T cells were grown overnight in 6-well plates (2.2X105 cells/well).</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 293T-ACE2 cells lysate was prepared and 10 ug of it was incubated with RBD-Ig according to the manufacturer instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-ACE2</div><div>suggested: RRID:CVCL_YZ65)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6 cell monolayers were washed once with DMEM and 200µl of each dilution of protein-virus mixture was added in triplicates for 1 hour at 37°C.</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">Sera were diluted to 1:500, 1:1K, 1:5K, 1:10K per well and added to 50,000 293T-Parental cells or 293T-Spike cells in a 96-U-well plate for 1 hour at 4°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-Parental</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blocking with mice sera: Sera from the various immunized mice groups was diluted to 1:100 per well and added to 50K 293T-Parental cells or 293T-Spike cells in a 96-U-well plate for 1 hour at 4°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-Spike</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 flag-tagged ACE2 was cloned into the plasmid pHAGE-DsRED(-) GFP(+).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHAGE-DsRED(-</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As a control we performed the same co-transfection but with the VSV-G envelope plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VSV-G</div><div>suggested: RRID:Addgene_138479)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistics: Statistical analysis were performed using either Prism 8 (GraphPad) or Excel (Microsoft).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>Excel</div><div>suggested: None</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

<footer>

About SciScore

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

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This is defined as a sharp division of things or ideas into two contradictory parts.

One can draw parallels to DuBois telling Washington that he would be considered privileged due to the fcat that he can be considered part of the "Top Tenth" because of his education and connections.

This reminds me of the ongoing metaphor in "Jubilee" of the slaves being cage birds. So with Augustus him having education, him being a free man, it gives him the ability to fly like a bird. Much like the age old saying the "the skies the limit".

Like he said there is not room to build a character, to have a struggle to have redemption arcs, because they are just meant to be a symbol because of the limited representation

The 1992 LA riots were caused because the LAPD had used excessive force in the arrest and beating of Rodney King, because of media it spread like wildfire.

These are all prominent black men, from social activists, to philosophers, to rappers and athletes.

Perhaps this could be because of the fact he was born into slavery which is why he tends to lean more on realism and not idealism much like DuBois

What did DuBois mean by this, what could be the contamination be? Maybe the struggle the rest of black Americans had to face in society

Its name is talented tenth because it is referencing the able ten percent of black Americans that developed leadership capacity from higher education

This is why DuBois had become one of Washington's biggest opponents because he did not believe in a "sitting duck" method, wanting to create change not waiting to be accepted.

This is reminding me after the emancipation proclamation and after the civil war when the south was in a time of reconstruction, they had given freed slaves the ability to have land in the Midwest and begin their agricultural journey. Could this be in reference?

I see Washington's philosophy as a form of assimilation and conformity to American society and to gradually comfort those with a negative view of black people.

This plays a role in why Washington's philosophy is what it is

This just meaning the system of division of black and white Americans that many believed (and still do) believe in

This being to fix the societal gap between black people and white people

The Talented Tenth is a term that designated a leadership class of African descendant Americans in the early 20th century

He urged blacks to accept discrimination for the time being and concentrate on elevating themselves through hard work and material prosperity

Represents how racial injustice continued throughout several generations just taking on various forms and altercations as time passed.

Somewhat represents the idea of double-consciousness and even Washington, when he funded Jim Crow opposing organizations and Black newspapers.

2 other figures with differing views but within the civil rights movement period. MLK advocated for confrontational and peaceful protests such as marches, boycotts, and sit-ins, while Malcom X promoted more violent approaches such as riots.

Just as the poem we had to annotate as well, Washington speaks his views first, and Du Bois follows with his rebuttal.

Since Washington was born a slave, it's easier to understand why he has such views. He was born into an oppressive system and may not see a light at the end of the tunnel that Du Bois saw in which protesting for reform would bring him closer to.

One of the common grounds shared between Washington and Du Bois, both were highly-educated individuals.

My Harlem Renaissance figure, Ida B. Wells, was also a founder of the NAACP.

Washington was tasked with the objective to publicly speak to a majority-white crowd upon race relations and justice.

Washington was born into slavery, which Du Bois was not. This differing factor could contribute to their differing views concerning the strive for racial equality.

Refers to the various approaches to obtain racial equality presented from 2 different advocates. This problem has spread over generations and media outlets upon national coverage.

Introduction to the differing ideologies between activists Washington and Du Bois

I personally think this is commentary on white privilege. Freeman doesn't see how difficult it is for Raquel, because he is born with a privilege that Raquel doesn't have.

This gives the writer an opportunity to talk about different events that have effected the black community.

Black culture became more and more a part of American culture. The social movements highly influenced American culture.

More education would mean more opportunities for black people to get involved in politics and law discussions.

This shows how as people do more research and look from different perspectives, their way of thinking can change.

It has been shown in history time and time again that the only way to get equal opportunities and rights is through protest, unrest, and fighting back.

He believed that in order for white people to get on the bandwagon of giving black people rights, they had to prove their effect on the economy.

He gave a new way of looking and thinking about this discourse.

Uses a different type of medium to confront the discourse between these two leaders.

They had very different ideas of how to solve the racial issues in America.

There were many ways Black Activists went about trying to fix this problem. Such as accommodating, fighting for equal rights, or just straight up leaving.

The Negro Problem refers to the separation of Black People from American Culture.

Educating the best minds of the race disseminates into the rest, allowing the general uplift of all.

It covers law, education, disenfranchisement, and Black Americans' place in American society.

Just like in irl, Raquel just like Dubois wins over people when faced with the truth concerning the problems of always abiding and assimilating in society.

It incorporates the debates between Booker and Dubois but to a more modern audience to interpret

This shows the shift in ideas as Dubois Ideas became the center part for a lot of other black activist that came after him, such as MLK and Malcolm X

This in context helps young black kids understand the history with African involvement in America

Contrast both characters as Superman landed in a happy time without any conflict in Kansas, while In Milestone, Icon lands in a southern planation where the brutal reality of slavery is taking place and mistreatment of blacks is the norm.

Show that young black people can relate to characters with their culture and be able to see themselves with what they might not always get with other super heroes.

At the time many of the times most famous pronounced super heroes were all white. With Captain America, Superman, and Batman to name a few.

That solely writing a character to depict blacks also does no justice to the character as it makes the character bland, so therefore why he creates characters with complexity and with the character being black, it helps the black community in the long run as it does not show stereotypes nor have a white washing effect.

To teach that the black community can do anything no matter there skin religion and or gender

Monolithic meaning not open to new ideas and remaining with the same old stereotypes and depictions of blacks in the past.

This site is here to help the Black community get more involved in poetry and music and other jobs that we may think only white people run

I agree with this not a lot of people go out and say my favorite hero is Wonder woman or my favorite hero is Black panther. I think we need more male black hero's

We need more hero's that are of different origin's I know of a few hero's that are black (Black Panther, Storm, and cyborg just to name a few.

All of these things being prevalent as there was still issues with women rights, gays being shamed , and blacks still being discriminated and stereotyped.

Shows me that black artists are dying down which isn't a good thing

Here I see Cam Newton abiding by the NFL therefore assimilating to the white audience, while I see Colin Kaepernick using his freedom of speech to voice his opinions on black lives

MLK taking the civil approach with civil protest, and Malcom X taking the violent riot approach of demanding rights now

To me you shouldn't do something just to please someone do it because you like to do it

What I don't get is that if he came from a child hood where he was a slave and saw the impossible happen being the freedom of slaves, why not continue the ambitions of the slaves but instead continue to fight for their right stop be actually equal instead of being free, but confined by white society.

Shows me that the whites don't understand where this young poet is coming from and now yawl want to be nice to him just doesn't make sense

I can see why Booker T says this because after all he was a slave, when coming from an upbringing like that, all anybody would want is to just live life like how society already is instead of giving in for hope for change that might never come.

People are gonna hate you for what you do and that's the truth I make music and not everyone likes it but I'm still standing tall

Ironic how Booker T was the first one to set up a school for high education, which eventually led to Dubois getting his education and being Booker T biggest opponents.

This is true through everything like being a song writer don't be someone your not just because a certain crowd doesn't follow you be yourself and your time will come

This shows me that this young poet doesn't believe in his race it shows me he is less confident about his race.

He wants blacks to accommodate yet founded a school for high education for African Americans, that clearly goes against what many whites think of about black people. In my opinion contradicts his own statement.

The order that gave freedom to slaves in 10 states during the civil war.

This is where I would disagree with him, as not seeking for opportunity in itself is already bad advice as given an opportunity one should take it.

This proves how Booker T Washington was living a double consciences, by living his life out but also pertaining to his white counterparts.

pre civil war and post civil war

This pertains with the many different activist with all differing views from one another

With Civil disobedience I think of Martin Luther King

Malcolm X

This being Dubois

This being Booker T. Washington idea

The disputes between ideologies, such as Dubois and Booker T, with Dubois arguing for having his rights and equality now VS Booker wanting to accommodate.

Which elements won't have a closing tag ? A possible answer might be elements that dont have any content. for example, img.

The way she looks at the audience to give the information is so powerful. The FBI took a big step to recognize what has been denied for years

I really love this because it seems very powerful just by the start

I don't think "each" civilian paid for this rover because there has to be at least a few hundred people who did not pay.

Kapolres Kendari Ciptakan Group Facebook untuk Jalin Silaturahmi

Media sosial saat ini benar benar sudah menjadi dunia kedua bagi para penggunanya, dalam sebuah indicator beberapa platform sistem informasi , media sosial sudah menjadi sebuah barometer informasi dan juga sebagai ajang curhat bagi para penggunanya. Seperti halnya Facebook, Semua pengguna media sosial pasti mengenal nama Facebook walaupun tidak semuanya menyukai platform yang satu ini. Facebook dikenal sebagai platform media sosial yang paling banyak digunakan di indonesia dan sangat banyak sekali peminatnya, selain mudah dalam penggunaan , facebook juga dikenal sebagai paltform yang mempunyai banyak manfaat, salah satunya adalah Fanpage dan Facebook Group. Kapolres Kendari yang saat ini dijabat oleh AKBP Didik Erfianto S.IK , juga memanfaatkan Platform Facebook Group, berdasarkan pantauan Group Facebook yang mempunya nama POLISI KENDARI ini sudah mempunyai ribuan member , di dalam group tersebut antara anggota polisi polres kendari saling membagikan informasi berupa tautan link dari sebuah website atau juga sebuah video. sehingga warga kendari dan juga masyarakat wilayah sultra tidak perlu lagi bingung jika ingin mencari informasi seputar kinerja Kepolisian Resort Kendari, karena setiap hari seluruh anggota Polres Kendari selalu membagikan informasi informasi terbaru. Beberapa komentar masyarat pada group tersebut terlihat jelas, bahwa inovasi atau strategi sederhana ini juga bisa digunakan sebagai ajang silaturohmi antara Anggota Polisi Kendari dan Warga Kendari. sehingga bisa mempersempit Hoaks atau berita berita bohong yang ada di wilayah kendari.

Tidak cukup Sampai disitu Saja , Kapolres Kendari AKBP Didik Erfianto S.IK juga membuat sebuah Fanpage atau Halaman Facebook dengan nama Kapolres Kendari, dimana fanpage tersebut digunakan sebagai Admin dari group Facebook Polisi Kendari. Berdasarkan pantauan Fanpage Kapolres Kendari sangat Aktif, bahkan sekali posting kadang bisa mencapai Ratusan Ribu Reach Views atau jangkauan postingan pada fanpage tersebut. Dengan Strategi sederhana ini Kapolres Kendari berharap media sosial ini bisa digunakan sebagai ajang silaturohmi dan juga sebagai Viralisasi berita berita positif Kinerja Polisi Kendari

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The things that are important / worth mentioning to different people. I agree with this one.

annoying

never done; keeps wanting to continue edit/update

helping it gain attention/publicity

SciScore for 10.1101/2021.04.09.439147: (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">PVDF membranes were treated with 6xHis Tag Monoclonal Antibody (3D5) HRP (Invitorogen, USA) for 6xHis-tagged proteins, and ANTI-FLAG M2-peroxidase (HRP)-conjugated</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>M2-peroxidase</div><div>suggested: (Sigma-Aldrich Cat# A8592, RRID:AB_439702)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then, serial diluted recombinant ACE2 (from 1 μg/ml to 0.06 μg/mL) (ab151852, abcam), subsequent rabbit anti ACE2 antibody (HPA000288, Atlas Antibodies) and HRP conjugated anti rabbit IgG (7074S, Cell signaling) were incubated.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>subsequent rabbit anti ACE2 antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti ACE2</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">To detect K-874A binding to immobilized S protein, serial diluted K-874A (1 μg/ml to 0.002 μg/ml) in 1%BSA/PBS was incubated, and K-874A which bound to S protein was detected by HRP-conjugated anti VHH antibody (#128-035-232, Jackson immuno Research).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti VHH</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 binding on and early replication in Vero/TMPRSS2 cells: 2.5×104 TCID50 (MOI=50) of SARS-CoV-2 was incubated with 150 μg/ml of VHHs for 2 hours at 37°C and then 24 hours at 4°C and inoculated to 5.0×104 VeroE6/TMPRSS2 cells for an hour.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6/TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The raw Illumina paired-end reads that passed through the Q30 filter were merged using PEAR software [21].</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></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The VHHs-encoding sequences were translated based on standard genetic code using MEGA X software [22].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MEGA X</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Equal numbers of ZsGreen-HiBiT-expressing cells and LgBiT-expressing cells were plated on a 96-well plate (1603101, ThermoFisher Science).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ThermoFisher Science</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Individual movies were subjected to per-frame drift correction by MotionCor2 [26].</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">The contrast transfer function parameters of each micrograph were estimated using CTFFIND4 [27] and the flowing 2D and 3D classification, 3D refinement, and local resolution calculation were performed with RELION3.1 software [28].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RELION3.1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The extracted density was used to generate the mask using “Mask creation” in 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">The homology models of K-874A, RBD and NTD were rigid-body-fitted into the map using the COOT [31] and then refined using PHENIX [32].</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></table>

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

<footer>

About SciScore

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

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I believed this when I first read it, but changed my mind when I read this good rebuttal: https://hyp.is/f1ndKJ5eEeu_IBtubiLybA/stackoverflow.com/questions/640190/image-width-height-as-an-attribute-or-in-css

claim: that the OP's question and this answer are incorrect

Could we say that this answer (that this comment replies to) missed the point?

I actually believed and thought this answer was spot on ... until I read this comment, and then I reversed my opinion.

With notion i could do a tag, or entry classifier

only cosmetic

The OP wasn't wrong in exactly the way this comment implies: he didn't just ask how to detect whether stdin is a pipe. The OP actaully asked how to detect whether it is a terminal or a pipe. The only mistake he made, then, was in assuming those were the only two possible alternatives, when in fact there is (apparently) a 3rd one: that stdin is redirected from a file (not sure why the OS would need to treat that any differently from a pipe/stream but apparently it does).

This omission is answered/corrected more clearly here:

stdin can be a pipe or redirected from a file. Better to check if it is interactive than to check if it is not.

theory

enhances these qualities

I'm using this tag fo keep notes about the changes that need to be made now that Writefreely exists in its own organization on GitHub. See https://discuss.write.as/t/moving-the-writefreely-repo-on-github/2568

possible but doesn't seem preferred

looking at what the authors themselves use

coroutines message passing

SciScore for 10.1101/2021.04.08.21254348: (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">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Patient selection, samples and Institutional Review Board permits: Experiments were carried out following the ethical principles established in the Declaration of Helsinki. Patients (or their representatives) were informed about the study and gave a written informed consent.<br>IACUC: Implications for therapeutic decision-making” approved by La Princesa Health Research Institute Research Ethics Committee (register # 4070) were used; samples and data from patients with severe vs mild disease were provided by the Biobank Hospital Universitario Puerta de Hierro Majadahonda (HUPHM)/Instituto de Investigación Sanitaria Puerta de Hierro-Segovia de Arana (IDIPHISA) (PT17/0015/0020 in the Spanish National Biobanks Network), they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees.</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">Samples were stratified and randomly spliced into a training and a test set.</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">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">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">Beads were incubated with either rabbit anti-His-tag antibody (Proteintech Group) or plasma from patients or healthy donors in a final volume of 50 μl in 96-well-plates (Nunc™ MicroWell™ 96-Well, Thermo Fisher Scientific) using the dilutions indicated in each experiment.</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">To visualize antibody bound to antigen-coated beads, either PE-conjugated anti-rabbit antibody (0.25 μg/ml, Southern Biotech)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PE-conjugated anti-rabbit antibody ( 0.25 μg/ml , Southern Biotech)</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, PE-conjugated anti-human IgG and IgM, or FITC-conjugated anti-human IgA antibody (Immunostep S.L.) were added (30 μL/well) and incubated for 20 minutes at room temperature under agitation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgM</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human 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">Recombinant cDNAs coding for soluble S (residues 1 to 1208) and RBD (332 to 534) proteins were cloned in the pcDNA3.1 vector for expression in HEK-293F cells using standard transfection methods.</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">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: To assess the prediction capacity of the new methodology, an algorithm was built using Scikit-learn python package [11] (code available on request).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Scikit-learn</div><div>suggested: (scikit-learn, RRID:SCR_002577)</div></div><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">Comparison between severe and mild patients in each variable was performed by multiple t-tests followed by False Discovery Rate (1%) correction by two-stage step-up method in Graph Pad Prism 8 Software (GraphPad Software, USA, www.graphpad.com).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

---

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

In addition to an impact on early classification of patients, current limitations in the availability of vaccine doses suggest a novel possible application for sensitive multi-antigen assays for SARS-CoV-2 seropositivity. It has been shown that the antibody response to the first vaccine dose in individuals with pre-existing immunity is comparable or greater to that observed in naïve individuals who have been immunized twice [17]. Screening of the unvaccinated population with an assay sufficiently sensitive to identify individuals previously infected despite waning of antibody titres over time, would allow these individuals to be given the vaccine as a single booster, sparing them from possible suffering and complications after a second dose, and freeing up many urgently needed vaccine doses to be given to individuals with no protection. The test described here would also provide comprehensive information to support selection of convalescent sera or plasma for therapeutic use. Our data indicate the importance of this multi-antigen, multi-isotype analysis to detect potential SARS-CoV-2 reinfections in vaccinated individuals and suggest a possible use in establishing alternative vaccine administration routes that may elicit more potent IgA responses. This multi-antigen and multi-Ig assay can be easily modified for detection of antibodies in other fluids as saliva and breast milk. It is also highly tunable to different research needs, including detection of different immunoglobul...

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

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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.

---

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About SciScore

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

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SciScore for 10.1101/2021.04.08.438924: (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">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid 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">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">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: All cells were routinely checked to confirm the absence of mycoplasma contamination.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bound proteins were eluted from the beads using the elute buffer [25 mM Tris pH 8.0, 150 mM NaCl, 0.1% (w/v) DDM, 0.01% (w/v) CHS, and 250 ng/mL Flag peptide], and analyzed by immunoblotting using antibodies for the Strep tag (HuaxingBio, HX1816) or His tag (TransGen, HT501)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HT501</div><div>suggested: (Transgen Biotech Cat# HT501, RRID:AB_2801417)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The results were analyzed by immunoblotting using antibodies for the Flag tag (SIGMA, SLCD6338).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Flag tag (SIGMA, SLCD6338</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture: The HEK293T cell line was from EdiGene Inc., and Huh 7.5 cell line was from S.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CRISPRa screening for SARS-CoV-2 entry factors: The HEK293T cells were engineered to stably express the CRISPRa system including lenti dCAS-VP64_Blast and lenti MS2-P65-HSF1_Hygro vectors, termed as HEK293T-CRISPRa cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-CRISPRa</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Strep-tagged S6P spike protein was expressed in the HEK293F cells and purified as described previously (50).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293F</div><div>suggested: RRID:CVCL_6642)</div></div></td></tr><tr><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">GO enrichment and expression pattern analysis: The Gene Ontology (GO) enrichment analysis of identified host factors (RRA score < 0.001) was performed using Metascape Resource (32).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Metascape</div><div>suggested: (Metascape, RRID:SCR_016620)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Statistical analysis of all data apart from CRISPRa screening was performed using GraphPad Prism software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

Results from OddPub: 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.

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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).

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Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 10 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.

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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.

---

<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>

TTY is right there in the name, but this article makes no attempt to clarify what exactly the relationship between a pseudoterminal and a TTY. I feel like a whole paragraph about the relation to TTY would be warranted, including a link to TTY article, of course, which does link [back] to and explain some of the relation to pseudoterminal:

In many computing contexts, "TTY" has become the name for any text terminal, such as an external console device, a user dialing into the system on a modem on a serial port device, a printing or graphical computer terminal on a computer's serial port or the RS-232 port on a USB-to-RS-232 converter attached to a computer's USB port, or even a terminal emulator application in the window system using a pseudoterminal device.

lateral thinking

A note.

new tag?: jailbreaking a device

due to echo adding the spaces, not due to the spaces already being present

Tag: not so much:

whose responsibility is it? but more: what handles this / where does it come from? (how exactly should I word it?)

an intricate piece of a larger system / problem / schema

Was trying to figure out where the canonical repo even is. Hard to figure out. Could be made clearer (like a prominent notice on one saying this is an unofficial clone with a link to the canonical source).

Ended up here via link from https://unix.stackexchange.com/questions/86879/suppress-rsync-warning-some-files-vanished-before-they-could-be-transferred to https://git.samba.org/?p=rsync.git;a=blob_plain;f=support/rsync-no-vanished;hb=HEAD

But then found https://github.com/WayneD/rsync, which I now believe to be canonical based on:

• last change here is Mon, 15 Mar 2021 09:35:39 -0700 (09:35 -0700) but on https://github.com/WayneD/rsync it was April 3.
• https://rsync.samba.org/bug-tracking.html links to: create an issue on GitHub

possibly rename: games: luck: managing/mitigating the luck to games: luck: dealing with/mitigating the luck problem

Kollektivtrafikresenärers rättigheter

Lagen om kollektivtrafikresenärers rättigheter innehåller delar om tillgänglighet för personer med funktionsnedsättning. Lagen om kollektivtrafikresenärers rättigheter innehåller bestämmelser om reseinformation, ersättning och prisavdrag vid förseningar. Den innehåller också delar om att kunna säga upp periodbiljett vid resor i kollektivtrafik med tåg, spårvagn, tunnelbanetåg, buss och personbil. Enligt lagen har transportören en skyldighet att tillhandahålla information om en försening eller annan störning i trafiken och dess orsak, varaktighet och konsekvenser, resenärens rättigheter som finns i lagen transportörens allmänna avtalsvillkor, biljettpriser, tidtabeller och linjesträckningar, tillgänglighet till fordon, stationer och hållplatser, möjligheten att medföra cyklar och villkor för detta, säkerhets- och trygghetsfrågor hur transportören kan kontaktas. Information ska ges i den eller de former som är mest lämpliga för att resenärerna ska kunna ta del av informationen. Vid bedömningen av lämplig form ska särskild vikt läggas vid behoven hos personer med funktionsnedsättning. Konsumentverket har rätt att utfärda föreskrifter enligt lagen och har dessutom tillsynsansvar. Lag (2015:953) om kollektivtrafikresenärers rättigheter på riksdagens webbplats Senast granskad: 19 februari 2020 https://www.mfd.se/verktyg/lagar-och-regler-om-tillganglighet/transporter/kollektivtrafikresenarers-rattigheter/

The strategy in the game is not only what you want to do, but also what you should do. Because there are rules in the game, we should understand what we should do while having our own ideas.

Author Response:

We thank the editors and the reviewers for their positive assessment of our work.

Reviewer #1 (Public Review):

[...] One major concern is that the levels of protein expression and folding are not verified. This is concerning for the Gln118 mutation because lack of fitness could result trivially from misfolding or accelerated degradation that might result from increased flexibility and conformational stability. Moreover, the authors' finding that it was not possible to purify Gln118 mutant proteins for biochemical studies is consistent with this sort of trivial explanation for apparent lack of biological function.

As described in the manuscript, the sidechain of Gln 118 makes hydrogen bonds with the backbone segment leading into an adjacent helix. We had omitted to point out in the original manuscript that Gln 118 is completely buried in thestructure (we now do so in the revised manuscript, on page 29). As shown by Worth and Blundell, buried polar sidechains that form backbone hydrogen bonds (as Gln 118 does) are highly conserved in proteins, and these polar sidechains are important for the stabilization of the protein architecture (Worth and Blundell BMC Evolutionary Biology 2010, 10:161). Thus, we do expect the mutation of Gln118 to destabilize the clamp loader structure. However, we do not find the identification of the importance of Gln118 to be a trivial finding, because the role of polar residues in maintaining structure is quite commonly linked to their functional role, making it difficult to separate the two effects. For example, the proximal histidine that links the F helix in hemoglobin to the iron atom is perhaps the most important residue for allosteric communication in hemoglobin. Mutation of the proximal histidine severely destabilizes hemoglobin, due to loss of heme binding and conversion to a molten globule state (see, for example, Brennan and Matthews, Hemoglobin, 21:393-403, 1997).

It was an oversight for us to have not analyzed the effects of Q118 mutations on stability and function, and we have now rectified this. We now include the results of the following four experiments, in which we compare the expression of the mutant and wild-type forms of the clamp loader, their behavior on gel filtration analysis, and their activities in ATPase assays and DNA replication assays. These experiments demonstrate that the mutation most likely destabilizes the protein, and affects the nature of the assembled complex. These results further emphasize the crucial nature of the hydrogen-bonding interactions made by the Gln 118 sidechain.

1) We created a clamp loader variant in which the ATPase subunit is C-terminally tagged with the fluorescent protein mCherry, allowing the expression levels of the proteins to be monitored by flow cytometry of E. coli cells. This experiment shows that introduction of the Q118N mutation leads to a very substantial reduction in protein expression (Figure 6 supplement 2 in the revised manuscript). An important point is that the proteins are expressed using a strong promoter (T7 RNA polymerase promoter), which was done so as to purify proteins for biochemical experimentation and also enable ready detection of the mCherry fluorescence. The natural T4 promoter that is used in the phage assay results in very low levels of protein expression (no detectable fluorescence signal when mCherry is fused to the ATPase subunit), and we do not know whether the expression defect that we see is also manifested under conditions where the protein expression is low. Nevertheless, the data do indicate that the Q118N mutation destabilizes the clamp loader complex.

2) We purified mCherry tagged variants of the wild-type clamp-loader complex, the Q118N mutant complex, and the Q118N/I141L double mutant that has partial recovery of fitness in the phage propagation assay. SDS-PAGE analysis (not shown) confirms that all complexes have the ATPase and clasp subunits of the clamp loader in the proper 4:1 ratio. Gel filtration analysis shows that the wild-type complex corresponds to a single peak eluting at ~70 ml, which we assume corresponds to correctly assembled clamp loader (see Figure 9 supplement 1 in the revised manuscript). For both mutants, there is a peak at ~70 ml, corresponding to the properly assembled clamp loader, but also an additional peak that is close to the void volume of the column (~45 ml). For the Q118N mutant, the fraction of the protein corresponding to the properly assembled clamp loader is small. This fraction is substantially larger for the double mutant that has increased fitness (Q118N/I141L), indicating that one effect of the second mutation is to recover the ability of the clamp loader to assemble properly.

3) We measured the rates of DNA-stimulated ATP hydrolysis for purified and mCherry-tagged wild-type clamp loader and the Q118N mutant, as we had described in the original manuscript for several other mutants (Figure 9 supplement 2 in the revised manuscript). Addition of the mCherry tag to the wild-type clamp loader results in a slight reduction of the ATPase activity. The Q118N mutation has a very low rate of DNA-stimulated ATPase activity (less than 10% of activity of the wild-type mCherry-tagged clamp loader). These data indicate that even in the fraction of Q118N mutant that can be purified as part of an intact clamp loader complex, the mutation compromises the ability to hydrolyze ATP. This is likely to be due to the failure to assemble into a competent conformation.

4) We measured the extent of plasmid DNA replication by the T4 replisome, using wild-type and mutant clamp loaders, as described in the original manuscript (Figure 9 supplement 3 in the revised manuscript). As for the ATPase assay, addition of the mCherry tag to the wild-type clamp loader results in a slight reduction of replication efficiency. Introduction of the Q118N mutation leads to a near-total loss of replication efficiency, to a level comparable to that seen in the absence of the clamp loader.

The main text of the manuscript now includes a description of these new results, and the new data are included as supplementary figures.

Reviewer #2 (Public Review):

[...] One potential weakness is that among all the questions posed at the beginning of the study not all received a definitive answer. In particular, the question "To what extent does the mutational sensitivity of the system in a particular organism, carrying out the essential function of DNA replication, reflect the sequence diversity seen across the spread of life?" is only partially addressed.

We agree that we have not fully probed the issue of evolutionary sequence diversity versus mutational sensitivity. We find it exciting that the two sets of data show clear divergence in certain positions, pointing to epistasis. This will be an exciting direction for future exploration.

The second question, "The clamp loader subunits respond cooperatively to the clamp, ATP and DNA. How do the mechanisms underlying this cooperativity impose constraints on the sequence?", has not been answered and goes beyond the scope of this study.

Answering this question requires the analysis of second-order mutations. We have demonstrated the feasibility of using the phage propagation assay for such analysis, but we agree with the reviewer that this is beyond the scope of the present study.

using attractive/familiar brand/name to lure customers

welcome

Compartir contenido de biblioteca digital, proceso de uso de información en la Web 2.0

SciScore for 10.1101/2021.04.06.438709: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">B-cell isolation and recombinant mAb production: Discovery and initial characterization of six antibodies in our panel was previously reported (S309 and S3044,17, S2X35, S2H13 and S2H1417, and S2E128), and six new antibodies are first described here (S2H97, S2X16, S2H58, S2D106, S2X58, S2X227).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>S2H1417</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>S2E128</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Epitope classes shown in Fig. 1a are defined as in Piccoli et al.17 Briefly, the classification of these epitope classes results from Octet binning experiments using structurally characterized antibodies, structural insights to define the recognition of open-only RBD and ability of antibodies to interfere with RBD binding to ACE2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were washed, and secondarily labeled with 1:200 PE-conjugated goat anti-human-IgG antibody (Jackson ImmunoResearch 109-115-098) to label for bound antibody, and 1:100 FITC-conjugated chicken anti-Myc-tag (Immunology Consultants Lab, CYMC-45F) to label for RBD surface expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human-IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Myc-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We sorted approximately 7.5e6 RBD+ cells per library on a BD FACSAria II, collecting yeast cells from the antibody-escape sort bin (fractions of library falling into antibody escape bin given in Extended Data Fig.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-escape sort bin</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sorted cells were recovered overnight, plasmids were extracted from the pre-sort and antibody-escape populations, and variant-identifier barcode sequences were PCR amplified and sequenced on an Illumina HiSeq 25003,28.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-escape populations ,</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As previously described3,23, sequencing counts pre- and post-selection were used to estimate the “escape fraction” for each library variant, which reflects the fraction of yeast expressing a variant that fall into the antibody-escape FACS bin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-escape FACS bin .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The interactive visualizations of antibody escape maps (https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_Vir_mAbs) were created using dms-view42.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>dms-view42</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Although we have various follow-up binding data illustrating reduced affinity binding for some “escaped” sarbecovirus RBDs, these follow-up experiments were not conducted systematically for all antibody/RBD combinations, and therefore would bias breadth estimates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody/RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These antibodies and their citations include: S2X259, accompanying paper Tortorici et al. 2021; LY-CoV55521; COV2-2196 and COV2-213033; REGN10933, REGN10987, and LY-CoV01623; and all other COV2 antibodies and CR30223.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CR30223</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">2c incorporate the authentic SARS-CoV-2 neutralization IC50s as reported in this study (Fig. 1a), together with the live SARS-CoV-2 neuralization IC50s for the COV2 antibodies reported by Zost et al.10.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COV2</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 1 h 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><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">Lenti-X 293T cells (Takara, 632180) were seeded in 10-cm dishes at a density of 1e5 cells/cm2 and the following day transfected with 5 μg of spike expression plasmid with TransIT-Lenti (Mirus, 6600) 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: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VeroE6 cells were used for VSV-SARS-CoV-2, VSV-SARS-CoV-1, and VSV-GD-Pangolin-CoV.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BHK-21 cells stably expressing ACE2 were used for VSV-GX-Pangolin-CoV and VSV-WIV1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BHK-21</div><div>suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 HexaPro Spike protein for cryoEM analysis was produced in HEK293F cells grown in suspension using FreeStyle 293 expression medium (Life Technologies) at 37°C in a humidified 8% CO2 incubator rotating at 130 r.p.m.</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">Escape clones were plaque-purified on Vero cells in the presence of mAb, and plaques in agarose plugs were amplified on MA104 cells with the mAb present in the medium.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">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">Percent neutralization data were analyzed and graphed using Prism (GraphPad, v9.0.1)</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><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 geometric mean from at least two independent experiments was calculated using Excel (Microsoft, Version 16.45)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Excel</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The gene sequence phylogeny was inferred using RAxML version 8.2.1249, with the GTRGAMMA substitution model and a partition model with separate parameters for first, second, and third codon positions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RAxML</div><div>suggested: (RAxML, RRID:SCR_006086)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">2 was performed using the Python scikit-learn package.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Python</div><div>suggested: (IPython, RRID:SCR_001658)</div></div><div style="margin-bottom:8px"><div>scikit-learn</div><div>suggested: (scikit-learn, RRID:SCR_002577)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">3D refinements were carried out using non-uniform refinement64 along with per-particle defocus refinement in CryoSPARC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mutant RBD:S309 complexes were constructed from the fully glycosylated WT RBD:S309 complex using PyMOL 2.3.5 (Schrödinger, LLC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The glycosylated proteins were parameterized with the Amber ff14SB74 and GLYCAM_06j-175 force fields.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Amber</div><div>suggested: (AMBER, RRID:SCR_016151)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Non-bonded interactions were treated with the Particle Mesh Ewald method79 using a real-space cutoff of 1.0 nm and the OpenMM default relative error tolerance of 0.0005, with grid spacing selected automatically.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>OpenMM</div><div>suggested: (OpenMM, RRID:SCR_000436)</div></div></td></tr></table>

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

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

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

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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.

---

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About SciScore

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

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SciScore for 10.1101/2021.04.06.438579: (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">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid 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">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">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">Formaldehyde fixed cells were washed with phosphate buffered saline and permeabilized for immunofluorescence using 0.1% Triton X-100 in PBS with 1% bovine serum albumin (BSA) fraction V (www.emdmillipore.com) and stained for SARS-CoV-2 with a primary anti-Nucleocapsid antibody (www.genetex.com GTX135357) labeled with AlexaFluor 594.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Nucleocapsid</div><div>suggested: (GeneTex Cat# GTX135357, RRID:AB_2868464)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Proteins in VLP and cell lysates were separated on 10% SDS-PAGE gels, transferred to PVDF membranes, and immunoblotted with the following antibodies (Fig 7A): mouse monoclonal anti-V5 tag (www.thermofisher.com, #R960-25), rabbit polyclonal anti-SARS M (generous gift of C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-V5</div><div>suggested: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)</div></div><div style="margin-bottom:8px"><div>anti-SARS</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibodies were detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (www.bio-rad.com) or HRP-donkey anti-rabbit IgG (www.bio-rad.com) and Western Clarity detection reagent (www.bio-rad.com).</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><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 antiviral test of amilorides: Vero E6 and Caco-2 were obtained from ATCC and grown in DMEM (www.corning.com) with 10% FBS, 10mM HEPES, and Penicillin-Streptomycin (www.thermofisher.com).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Caco-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 isolate USA-WA1/2020 (www.beiresources.org) was propagated on Caco-2 cells and infectious units quantified by focus forming assay using Vero E6 (ATCC) 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">HEK293T cells were cultured in complete Dulbecco’s modified Eagle medium containing 10% Fetal Bovine Serum and penicillin-streptomycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IC50 and CC50 were determined using the nonlinear regression analysis in GraphPad Prism 9 with the bottom and top parameters constrained to 0 and 100, respectively.</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">Chemiluminescence was detected using a Bio-Rad Chemi Doc imaging system and analyzed using Bio-Rad Image Lab v5.1 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Bio-Rad Image</div><div>suggested: None</div></div></td></tr></table>

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

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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.

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

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

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Results from rtransparent:

• No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

---

<footer>

About SciScore

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

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This is my annotation.

SciScore for 10.1101/2021.04.06.438614: (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">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid 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">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">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">, acetonitrile (ACN), ammonium bicarbonate (NH4HCO3), anti-FALG antibody, anti-FLAG M2 affinity gel, and neuraminidase were purchased from Sigma-Aldrich (St. Louis, MO, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-FALG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-FLAG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>neuraminidase</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 S protein subunit 1 (His tag) expressed by HEK-293 cells were purchased from Sanyoubio (Shanghai,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The glycosites and glycoforms of S protein purified from HEK-293T cells were confirmed according to the mass spectrometry results of commercial S1 using a ‘match between runs’ analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-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">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">Mass spectrometric data analysis: The LC-MS/MS raw data were analyzed manually for each glycopeptide using Xcalibur 3.0.63 (Thermo Fisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Xcalibur</div><div>suggested: (Thermo Xcalibur, RRID:SCR_014593)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All the MD simulations were performed using the Amber14 package(Case et al., 2014).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Amber14</div><div>suggested: (Assisted Model Building with Energy Refinement (AMBER, RRID:SCR_014230)</div></div></td></tr></table>

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

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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).

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Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 29. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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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.

---

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Reply to the reviewers

Response to Reviewers

Title: "Towards deciphering the Nt17 code: How the sequence and conformation of the first 17 amino acids in Huntingtin regulate the aggregation, cellular properties, and neurotoxicity of mutant Httex1".

Tracking #: RC-2021-00675 Authors: Vieweg et al.

MAJOR COMMENTS from Referees #1, #2, and #3

Referee #1

General comments

« The manuscript by Vieweg, Mahul-Mellier, Ruggeri et al., describes the role of the sequence and conformation of the extreme N-terminus of the Huntingtin protein in terms of aggregation and toxicity together with its relation to the polyglutamine length. The authors use some outstanding methods to ensure that the conclusions are based on good quality data. Overall, this is an excellent study.

We thank the referee for the very positive feedback and for recognizing the quality of our work and his/her appreciation of our systematic approach to dissect the role of the Nt17 domain in regulating the aggregation, cellular properties, and neurotoxicity of mutant Httex1.

“The manuscript is generally well written although it might benefit from reducing the length of the discussion section ».

We thank the referee for his/her valuable comment. We have reduced by 10% the discussion as per requested.

Major comments

1) “For their in vitro data, the authors do not go beyond 42 polyglutamines. Is there any particular reason for that? The authors see a clear difference between 36Q and 42Q, but although not critical, it would have been useful to use longer repeats. In my view, the authors should at least discuss the rationale for this, particularly as in cellular models they do use 72Q constructs.”

We thank the referee for raising this point.

Most HD patients have a polyQ repeat stretch of 40-45 glutamines (1-4).

In vitro, the use of Httex1 constructs consisting of 42 polyglutamine residues is sufficient to induce mutant Httex1 aggregation and fibril formation. Mutant Httex1 proteins with polyQ repeats of 72Q or higher are highly aggregation-prone and difficult to purify, handle, or disaggregates. This is why all of the in vitro aggregation studies are based on mutant Htt proteins with polyQ ranging from 23Q-53Q (5-14). We have reviewed the literature carefully and were unable to identify any in vitro studies with recombinant Htt proteins containing polyQ repeats of 72 or greater.

In cells, induction of mature Htt inclusions requires much longer polyQ repeats. This is clearly reflected by the fact that most cellular studies use mutant Htt with polyQ repeats above 64Q and up to 160Q to induce the formation of cellular aggregates (10, 15-30).

We have recently conducted a systematic study on the effect of the polyQ repeat length on Htt inclusion formation in cells https://www.biorxiv.org/content/10.1101/2020.07.29.226977v1 (21). Characterization of the inclusions by EM revealed that the polyQ tract length dramatically influences the ultrastructure properties and the architecture of the Httex1 inclusions in cells. The dark shell structure that delimitated the core from the periphery of the Httex1 72Q inclusions was absent in the Httex1 39Q inclusions. Also, the Httex1 39Q inclusions appeared less dense compared to that of the Httex1 72Q. Finally, no significant cell death was observed in HEK cells overexpressing 39Q constructs while overexpression of Httex1 72Q was toxic. For these reasons, we and others select to use Httex1 with polyQ repeat of 75 or higher.

2) « The role of the N-terminus 17 aminoacids of huntingtin (Nt17) is addressed by comparing peptides with and without the Nt17 and their relation to the adjacent polyglutamine tract. Using this approach, the peptide without the Nt17 is composed of pure polyglutamines in its N-terminus, followed by the rest of exon 1 in its C-terminus. This is clearly the key comparison to address the role of the Nt17 in the context of an exon1 containing polyQ. »

Yes. In fact, we did perform this experiment and assessed if the addition of the Nt17 would be sufficient to inhibit mutant Httex1 aggregation or make ΔNt17-Httex1 aggregate similar to Httex1. This data is included in the original version of the manuscript as Figure S5 in supporting material. We observed that that __the presence of the Nt17 peptide during the aggregation of ΔNt17-exon1 fibrils did not interfere with the aggregation kinetic of mutant Httex1 or alter the fibril morphology of ΔNt17-exon1,__ indicating that intramolecular interactions between the Nt17 domain and the adjacent polyQ tract are key determinants of mutant HTtex1 fibrillization and fibril morphology.

Referee #2

General comments

This article describes the results from studies into mechanisms of the aggregation and toxicity of Htt Exon1 protein. The authors investigated the role of N17, polyQ length, M9C mutation, and phosphorylation. Multiple approaches were used that included biochemical protein design, biophysical measurements, and cell biological experiments with cultured mammalian cells. The authors demonstrate the effects of protein context on aggregation. Furthermore, the authors were able to visualize the aggregates in mammalian cells and in neurons using multiple methods. These are interesting data.

We greatly appreciate the positive feedbacks on our data and our systematic and integrative approaches.

There are several major weaknesses in the study. First problem is that most of the results related to aggregation mechanisms and toxicity are not original and incremental when compared to many previously published articles.

We respectfully disagree with this assessment and suggestion that our studies are not original and represent only incremental advances.

Originality

Our study provides novel mechanistic insights into the role of not only the sequence but also the conformational properties of the Nt17 domain in regulating the dynamics of Httex1 fibrillization, the kinetic fibril growth, the structure and morphology of Httex1 fibrils. Besides, we also addressed for the first time how the Nt17 domain and phosphorylation at different residues within this domain regulate the cellular uptake, subcellular localization (phosphorylated proteins) and toxicity of extracellular monomeric and fibrillar forms of mutant Httex1 in primary neurons. We are not aware of previous reports that have conducted similar studies addressing these questions and using multiple methods. Also, some of our findings using native Httex1 sequences are not in agreement with previous reports using Httex1 proteins fused to peptide/protein tags, thus underscoring the limitations of previous studies.

1) We demonstrated that the Nt17 domain plays an important role in shaping the surface properties of mutant Httex1 fibrils and regulate their lateral association and cellular uptake. Our findings are not in agreement with previous findings published in eLife by Shen et al. __(10)__, where they reported that removal of the Nt17 domain has the opposite effect of what we observed in our study, i.e., ∆Nt17 promotes the formation of fibrils that exhibit a low tendency to laterally associated and form a “bundled” architecture (10). Careful examination of their constructs revealed that all the proteins they used contained a highly charged 15-mer peptide tag (S-tag: Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His-Met-Asp-Ser) at the C-terminus of Httex1, which we believe would strongly influence the aggregation properties of the mainly uncharged ∆Nt17-Httex1 and Httex1 protein, thus possibly explaining the discrepancy between our findings and those of Shen et al (10). In fact, a previous study has shown that adding short peptide tags such as the HA or the LUM tag to mutant Htt171 changed the toxicity dramatically. In addition, we show that the subcellular localization of Htt171 expressed in cells (e.g: expression of Htt171 carrying the LUM tag was more toxic than untagged Htt171 and induced the formation of nuclear aggregates rather than the classical cytoplasmic aggregates) (31). The reference to this paper is now included and discussed in the main manuscript (page 11).

Our observations __highlight__ the critical importance of using tag-free proteins to investigate the sequence and structural determinants of Httex1 aggregation and structure.

2) Our study is the first to demonstrate that the Nt17 domain influences the relationship between fibril length and polyQ repeat length. This correlation disappears when the Nt17 is removed. This aspect of our work was not explored by Shen et al. (10), who limited their in vitro aggregation study to Httex1 wild-type and mutants (∆Nt17 or ∆PRD for Polyn Rich Domain).

3) This is also the first study to assess the effect of modulating the helicity of Nt17 on fibrils growth and morphology and Httex1 cellular properties.

1. a) Using the helix and membrane-binding disrupting mutation (M8P), we showed that disrupting the Nt17 helix (M8P mutation) slows the aggregation propensity of Httex1 in vitro but does not alter the morphology of the fibrils. In contrast, removing the Nt17 domain leads to a strong lateral association of the fibrillar aggregates with ribbon-like morphology. This demonstrated that the __Nt17 sequence, but not its helical structure, is the key determinant of the quaternary packing of Httex1 fibrils. Shen et al. (10) did not investigate the effect of modulating the helicity of Nt17 on fibrils growth and morphology using M8P mutant__.
2. b) Our cellular studies comparing the membrane association and uptake of extracellularly added Httex1 43Q and M8P Httex1 43Q fibrils in primary rat striatal neurons showed that disrupting the Nt17 helix promotes the internalization of M8P Httex1 while Httex1 stays bound to the plasma membrane. These findings suggest that the Nt17 helical conformation persists in the fibrillar state or that the Nt17 domain regains its helical structure upon interaction with the plasma membrane resulting in the sequestration of Httex1 fibrils at the membrane and impeding their uptake. This aspect of our study has never been explored in previous studies.
3. c) Using the site-specific bona fide phosphorylation on T3, S13, SS16, and both S13/S16, this is the first study that shows that modulation of the overall helicity of Httex1 through site-specific phosphorylation of the Nt17 domain (pT3 stabilizes the alpha-helical conformation of Nt17 while pS13 and/or pS16 disrupts it (9)) enhance the rapid uptake of extracellular Httex1 monomeric species into neurons and their nuclear accumulation. Previous studies relied on phosphomimetic mutations (32), which we have shown do not reproduce the effect of phosphorylation at these residues on the structure of Nt17 (8, 9). Shen et al. __(10) did not investigate the effect of modulating the helicity of Nt17 on fibrils growth and morphology using site-specific __phosphorylation of the Nt17 domain. 4) Our overexpression model in HEK cells showed that removing the Nt17 domain or disrupting its helical structure (M8P mutation) was sufficient to prevent the cell death induced by Httex1 72Q overexpression and reduce the number of cells with inclusions drastically. Our data indicate that the cell death level correlates with the number of cells that contain inclusions or the number of inclusions formed in the cells or/and their subcellular localization. In contrast to our results, Shen et al.,__(10)__ demonstrated that the overexpression of ΔN17-Httex1 induced toxicity at a similar level as the full-length Httex1 in striatal-derived neurons or neurons from cortical rat brain slices culture, although ΔN17-Httex1 led to a significant reduction of punctate structures in these cells. The discrepancy between these studies and our Httex1 overexpression model in HEK cells may be due to the fact that in neurons, Httex1 lacking the Nt17 domain accumulates in the nucleus. In contrast, in HEK, it stayed mostly cytosolic. In line with this hypothesis, it has been recently shown that cytosolic inclusions (Httex1 200Q) and nuclear aggregates (Httex1 90Q) contribute – to various extents – to the onset and the progression of the disease in a transgenic HD mice model (33). Thus, the difference in cellular localization but also the cell type (HEK vs. neurons) could influence the toxic response of the cells to the overexpression of ∆Nt17-Httex1, with toxicity triggered only by the nuclear ∆Nt17-Httex1 species.

5) This is also the first study to investigate the role of the Nt17 domain and Nt17 PTMs in influencing the uptake, the subcellular localization, and the toxicity of extracellular Httex1 species (monomers and fibrils) in primary neurons. We showed that the helical propensity of Nt17 strongly influences the uptake of Httex1 fibrils into primary striatal neurons. At the same time, phosphorylation (at T3 or S13/S16) or removal of the Nt17 domain increased the uptake and accumulation of Httex1 fibrils into the nucleus and induced neuronal cell death. Our findings suggest that the Nt17 domain is exposed in the fibrillar state and is sufficiently dynamic to mediate fibril-membrane interactions and internalization.

Altogether our results, combined with previous findings from our groups and others demonstrating the role of Nt17 in regulating Htt degradation (34-36), suggest that this domain serves as one of the key master regulators of Htt aggregation subcellular localization of the pathological aggregates, and their toxicity. They further demonstrate that targeting Nt17 represents a viable strategy for developing disease-modifying therapies to treat HD.

Limitations of previous studies:

Although the effects of the Nt17 domain in regulating Httex1 aggregation and cellular properties have been studied and reported on by other groups, we would like to stress that most of the published studies had major limitations and used protein constructs that do not share the sequence of native Httex1 and exhibit biophysical and cellular properties that differ from those of native Httex1 sequences.

1) Many of the studies used Httex1-like model peptides (13, 37), which do not contain the complete sequence of Httex1 (e.g., Nt17 peptide (37)), contain additional solubilizing amino acids such as lysine residues(38-43) or are fused to large proteins (e.g., GST, YFP) (37).

2) Other studies relied on artificial fusion constructs whereby the polyQ domain (44-46) or Httex1 itself (12, 47-61) are fused to large solubilizing protein tags, such as glutathione-S-transferase (GST), maltose-binding protein (MBP) or thioredoxin (TRX) or C-terminal S-tag (10, 62, 63) or fluorescent proteins (e.g., GFP or YFP) (10, 15, 49, 64, 65) for the cellular studies.

One of the major limitations of using fusion constructs as precursors for the generation of Httex1 (12, 47-61) is the requirement to cleave the fusion protein in situ by adding a protease to release and initiate the aggregation of Httex1. Enzyme-mediated cleavage of Httex1-fusion proteins often results in the incorporation of additional amino acids at the N- or C-terminus of the protein. This could alter the biophysical and biochemical properties of Httex1 because of the important role of the Nt17 domain and the proline-rich domain in regulating the conformational and aggregation properties of the protein (38, 40, 43, 65, 66). Moreover, it has been shown that commonly used enzymes such as trypsin and thrombin may lead to cleavages within the Nt17 domain and result in the generation of undesired Httex1 fragments (7, 42, 60). The net effect of incomplete and/or unspecific enzymatic cleavage of Httex1 fusion proteins is the generation of heterogeneous protein mixtures, which precludes accurate interpretation and comparison of aggregation and structural data across different laboratories.

Moreover, several studies have shown that the fusion of small peptide tags or large fusion protein alters the aggregation of mutant Httex1 in vitro and in cells.

1. We have previously shown that the presence of such tags (e.g., GST) alters the ultrastructural and biochemical properties of Httex1 as well as its aggregation properties in vitro (11).

We have also recently completed a comprehensive assessment of the GFP tag's impact on the aggregation, inclusion formation, and cellular properties of Httex1 (preprint paper available in BioRxiv https://www.biorxiv.org/content/10.1101/2020.07.29.226977v1 (21)). In this paper, we show that inclusions produced by mutant Httex1 72Q-GFP exhibit striking differences in terms of organization, ultrastructural properties, composition, and their impact on mitochondria functions as compared to the inclusions formed by the tag-free mutant Httex1 72Q. These findings highlight the critical importance of developing new tools that minimize the impact of large fluorescent proteins and/or label-free imaging methods and monitoring Htt aggregation in inclusion formation in cells.

From Riguet et al., __(21)__. Influence of GFP on the ultrastructural properties of Httex1 cellular inclusions by Correlative light electron microscopy (CLEM). CLEM of Httex1 72Q (+/-GFP) transfected in HEK 293 cells after 48h. Confocal images of A. Httex1 72Q and. B Httex1 72Q GFP, 48h after transfection. Httex1 expression (red) was detected using a specific primary antibody against the N-terminal part of Htt (amino acids 50-64) and the nucleus was stained with DAPI (blue). Electron micrographs of C. Httex1 72Q and D. Httex1 72Q GFP inclusions corresponding to confocal images panel A, and B (white square), respectively. Add-in binary images generated from electron micrographs by median filtering and Otsu intensity threshold. E. **Schematic depictions and original electron micrographs of cytoplasmic inclusions formed by native (tag-free) mutant Huntington exon1 proteins (Httex1 72Q, left) and the corresponding GFP fusion protein (Httex1 72Q-GFP).

A recent study by Chongtham et al. (31) also supports our findings and shows that adding short peptide tags such as the HA or the LUM tag to mutant Htt171 changed dramatically the toxic properties of Htt171 as well as its subcellular localization and the compactness of the aggregates formed in cells (e.g.: expression of Htt171 carrying the LUM tag was more toxic than untagged Htt171 and induce the formation of nuclear aggregates rather than the classical cytoplasmic aggregates, See Figure 4).

Figure 4 from Chongtham et al., __(31)__. The influence of peptide modifications on HTT171 fragment behavior. (A) When expressed ubiquitously with da>Gal4, the HTT171-120Q fragment exhibits little or no lethality, but appending either an HA ( ... YPYDVPDYA∗)oraLUMtag ( ... GCCPGCCGG∗) to the C-terminus dramatically increases the toxicity of the fragment. (B) Surviving adult flies expressing an HA-tagged HTT171 transgene exhibit about half the life span of those expressing untagged 171. Flies expressingLUM-tagged HTT171 do not survive to adulthood. (C) Flies expressing HA- or LUM-tagged 171 in tracheal cells show only modest increases in lethality that do not rise to the level of significance (P=0.12; 0.09), but the inclusion of tags changes the subcellular behavior significantly. (D) In contrast, in the prothoracic gland, expression of LUM-tagged 171 shows a significant increase in toxicity compared to 171 alone, while the HA-tagged 171 borders on significance (P=0.051). (E) In trachea, pure 171 forms cytoplasmic aggregates, while the inclusion of HA causes some HTT to become nuclear diffuse, and inclusion of the LUM tag causes the bulk of the HTT to appear as diffuse nuclear material with some cytoplasmic aggregates remaining when expressed with btl>Gal4 at 29◦C. (F) In the prothoracic gland, addition of the LUM tag causes aggregated- **cytoplasmic HTT to become weakly staining diffuse-cytoplasmic material while HTT171HA remains as extensive aggregates in the cytoplasm with a haze of diffuse staining as well. Scale bars are 10 μm

Therefore, in this study, we aimed to investigate for the first time the role of the sequence and the conformational properties of the Nt17 domain in regulating the dynamics of Httex1 fibrillization, the structure and morphology of Httex1 fibrils using a tag-free Httex1 constructs. In our studies, we used multiple methods to examine the structural and cellular properties of these proteins under the same conditions and in the same cellular systems, thus making it possible to correlate the sequence, structural and cellular properties of the different Httex1 proteins (monomers and fibrils). We are happy to see that this was nicely recognized and appreciated by the referee.

Referee #1 “The authors use some outstanding methods to ensure that the conclusions are based on good quality data. Overall, this is an excellent study.” Referee #3 “Their findings provided the precise information for the role of tag-free Nt17. The paper advanced our knowledge of Nt17, especially in the Huntington disease field.”

Major comments

Referee #2 raised the following concerns:

1) « The main hypothesis of this study solely depends on the ability of N17 domain to enhance aggregation (Fig 1 and Fig 2). According to the method for the protein solubilization 1mM TCEP was added to ∆Htt-Ex1, but not to Htt-Ex1 proteins. It is necessary to rule out the potential effects of TCEP on aggregation assay. »

We thank the referee for raising this important point. Indeed, we were also concerned about the potential effect of TCEP and conducted experiments to address this point. Our data show that TCEP does not affect our aggregation assay. This new panel is now included in supporting information as Figure S2A-B and mentioned in the corresponding section of the Material and method (page 29).

2) « The author needs to provide biophysical data of the mutation and phosphorylated proteins with/without Tag. »

All the proteins used in this study have been extensively characterized in recent publications from our lab __(9, 11, 21)__. All these papers are cited throughout our manuscript as well as in the material and method section.

The expression, purification and characterization of native tag-free Httex1 with polyQ repeats ranging from 7 to 49Q has been fully described in Vieweg et al., 2016 __(11)__. In this paper, the aggregation properties of tag-free Httex1 and Httex1 fused to GST or MBP tags were compared by sedimentation assay, while the morphology and length of the resulting fibrils were compared by EM.

The semisynthesis, purification, and characterization of Httex1 42Q phosphorylated at Ser-13 and/or Ser-16 or at T3 was described respectively in Deguire et al., 2018 __(9) and Chiki et al., 2017 (8)__. These studies include kinetics of aggregation and morphological assessment (i.e: heights and lengths) by EM and AFM of the fibrils formed by phosphorylated or unphosphorylated mutants Httex1.

The Httex1 mutants carrying the GFP tag were not used in the in vitro studies but were studied in our overexpression-based cellular model. The direct comparison characterization of inclusion formation by tag-free and GFP-tagged mutant Httex1 and their impact on cellular homeostasis are fully described in a preprint paper available in BioRxiv https://www.biorxiv.org/content/10.1101/2020.07.29.226977v1 (21). In this paper, we show that inclusions produced by mutant Httex1 72Q-GFP exhibit striking differences in terms of organization, ultrastructural properties, composition, and their impact on mitochondria functions as compared to the inclusions formed by the tag-free mutant Httex1 72Q. These findings highlight the critical importance of developing new tools that minimize the impact of large fluorescent proteins and/or label-free imaging methods and monitoring Htt aggregation in inclusion formation in cells.

Referee #3

General comments

“Their findings provided the precise information for the role of tag-free Nt17.__ The paper advanced our knowledge of Nt17, especially in the Huntington disease field.”__

We thank referee #3 for the very positive feedback and for recognizing the quality, depth and significance of our work and its potential impact in the field of Huntingtin disease.

“However, the conceptual advance is limited.”

We respectfully disagree with this assessment that the conceptual advance of our study is limited.

Please see our detailed response to Referee #2 regarding our work's originality and novelty (pages 3-8, in our referees' letter).

Major comments

Referee #3 raised the following concerns:

1) Finding of lateral association (bundling) of __Δ__Nt17-Httex1 fibrils is interesting.

We agree and thank the referee for further highlighting this point.

However, pathological significance is not clear

We agree that the significance for delta 17 is not clear as we do not know whether this cleavage occurs in vivo or not. This is why we decided to extend our studies beyond the removal of Nt17 and investigated the effect of natural PTMs that are known to alter the sequence of Nt17 and modulate its helicity. One additional distinguishing feature of our work is that we used proteins (monomers and fibrils) that bear site-specific bona fide phosphorylation on T3, S13, SS16, and both S13/S16.

1. a) Does even non-truncated form also increase this kind of bundling when polyQ is expanded? We have addressed this specific question in a previous study (11) in which we have compared the morphology and length of fibrils formed from Httex1 with polyQ tract from 23Q to 43Q. The increased lateral association was not observed for the fibrils generated from Httex1 43Q or Httex1 23Q, 29Q, or 37Q (Figure 5F) (11). Besides, in this paper, we were the first to show an inverse correlation between the polyQ-length and fibril length, which suggests structural differences between Httex1 proteins with different polyQ repeat lengths. Others have investigated Httex1 with different polyQ repeat, but not using tag-free Httex1 proteins, and they did not observe this inverse relationship between polyQ lenth and fibril length, as we did here and in our previous studies (11).

2. b) When fibrils are added to striatal neurons like in Fig.5, is this structural feature preserved on the membrane or inside of the cells? We agree with the referee that this is an important point to address. However, deciphering the structural properties of the membrane-bound and internalized fibrils is not trivial, especially given the limited amount of unlabelled fibrils that are taken up by the cells. This would require extensive optimization of the CLEM technique or the use of an alternative approach such as tomography. Due to the resources and time required to address this important question, this part of the project will be included as part of future projects aimed at investigating the mechanisms of Htt seeding and propagation. We are not aware of any reports by other groups that monitor the structural changes of exogenous fibril after internalization into cells.

3. c) When Httex1 fibrils species are expressed, is this bundling also observed? In fact, we have recently completed a comprehensive analysis of the comparison of the inclusions formed by mutant Httex1-72Q and ΔNt17 Httex1.

In this study, we have shown that the expression of Httex1 72Q and the truncated form ΔNt17 Httex1 72Q form cytosolic inclusions of similar size and shape in HEK 293 (Figure 4). We have further characterized the architecture and organization of these inclusions at the ultrastructural level in the context of another project. Our findings are now available online (see Riguet et al., 2021, BioRxiv (21)).

Using correlative light electron microscopy, we showed that the inclusions formed by Httex1 full length or lacking the Nt17 domain exhibited similar architecture and a ring-like organization. Interestingly, we showed the inclusions are composed of highly organized fibrillar network at the core and periphery of the inclusions. In cells inclusion formation is a multiphasic process driven by different phases of polyQ dependent aggregation processes and complex interactions with lipids, proteins and organelles (ER).

Although CLEM approach in neurons provides very good contrast of cytosolic or nuclear inclusions, the resolution of this method is not sufficient to allow imaging at the level of individual fibrils and assessing their morphology. Differences between CLEM and EM resolution can be explained as the slices of the cellular objects are much thicker (~ 50 nm) than the fibrils prepared in vitro and directly deposited on the EM grids (the height of Httex1 pre-formed fibrils is between 5 and 7 nm). To improve the imaging and get a stronger contrast of de novo fibrils in our CLEM samples, we used a double-contrast method based on uranyl acetate and lead citrate stains. Nevertheless, the complex cellular environment and the presence of various cellular objects (e.g: organelles and proteins) surrounding the de novo fibrils might prevent the optimal stain penetration from allowing imaging at the level of individual fibrils. Finally, the preparation of the neuronal samples for CLEM imaging includes ethanol and detergents incubation and resin embedding. These steps can limit the ultrastructure detection of the de novo fibrils at the level of individual fibrils and therefore does not allow to determine their organization and their lateral association.

1. d) What function (cell death, membrane integrity or others) is most correlated with this structural feature? In our extracellular model, we have shown that the conformation and sequence properties of the Nt17 domain are key determinants of the internalization and the subcellular localization of Httex1 fibrils in primary striatal neurons. Httex1 43Q fibrils mostly accumulate at the outer side of the neuronal plasma membrane, Httex1-ΔNt17 43Q fibrils were detected primarily in the nucleus and the M8P-Httex1 43Q fibrils were equally distributed in the cytosol and nucleus.

Despite exhibiting completely different subcellular distribution and internalization levels, the 3 types of fibrils induced neuronal cell death with the highest toxicity observed for ∆Nt17-Httex1 (Figure 7).

Our data suggest that the neurotoxic response is primarily dependent on the subcellular localization of the Httex1 species: 1) accumulation of the Httex1 43Q fibrils on the plasma membrane is likely to induce loss of membrane integrity, based on previous observation with aSyn fibrils; 2) the nuclear accumulation of ∆Nt17-Httex1 aggregates has been previously shown to be highly toxic in several cellular and animal models (10, 67, 68).

Nevertheless, we could not rule out that the high toxicity of ΔNt17-Httex1 fibrils could also be due to their distinct biophysical and structural properties. ΔNt17-Httex1 forms broad fibrils characterized by lateral association, which could provide a surface for the sequestration of intracellular proteins.

2) The authors claimed, “we investigated for the first time, the role of the Nt17 sequence, PTMs and conformation in regulating the internalization and cell-to-cell propagation of monomeric and fibrillar forms of mutant Httex1”. However, so far this reviewer understands that the authors studied the internalization but not cell-to-cell propagation.

We agree and apologize for this mistake as we indeed only limited our study to the uptake, subcellular localization, and toxicity of extracellular Httex1 species in our primary neuronal model. The text has been amended, and cell-to-cell propagation has been removed from the abstract as well as in pages 2, 4, 12, and 17.

Minor points for Referees #1, #2 and #3

Referee #1

1. « On page 6, the data on how the Nt17 domain affects Httex1 aggregation, the information on which figure it is referring to is missing. Done. The information regarding the Figure related to this data has now been added page 6.

In Figure 1A, it is difficult to compare the data on Nt17 and DNt17, particularly for 36Q and 42Q, as the time axis are different. I understand that the kinetics are different, but particularly for the 42Q peptides (Nt17 and DNt17) as their kinetics are not that different, it may be useful to show them in the same panel. »

Done. The new panel that combined the data on Nt17 and DNt17 has now been added as Figure S1B.

Referee #2

1) “Fig 8 the color codes for PolyQ and PolyP need to be corrected. »

Done.

2) “It is a challenging technical problem to produce proteins which are rich in Pro and Gln content. But there is not enough experimental details provided in the methods. Please add detailed procedures for expression and purification of these proteins. »

We thank referee #2 for recognizing the technical challenges to express and produce Httex1 proteins and mutants. The expression, purification and characterization methods of all the proteins used in this manuscript have been extensively detailed in our previous studies (8, 9, 11, 69-71). We have now added the relevant references in the method section (page 29).

Referee #3

1) « Fig.3B arrowhead could not be seen. »

Done. Arrowheads are now added to Fig. 3B.

2) « Fig.4A: what do arrows mean? No scale bars? »

The arrows indicate the aggregates formed in HEK cells overexpressing Httex1 39Q and 72Q. This now added to the legend section of the Figure 4.

The scale bars are already present in both the main and the insets images.

3) « Fig.5A:no scale bars? »

Done. Scale bars were added in the 4 images where they were missing.

4) « Fig.S3. Height and length seem to be wrong. »

The measurement of height and length are performed as in literature (72), and are consistent with previous studies (8, 9, 11).

5) « Fig.S6C: hard to compare. D: What is Htt2-90? Also in Fig.S13. »

We thank the referee for bringing this to our attention and apologize for the lack of consistency in the names used for the proteins studied in Figures S6 and S13. We realized that in Figures S6 and S13 the names of the proteins have been either mislabelled due to the dash that was misplaced or the same proteins have been named in different ways. We agree that this makes it difficult to compare the data between the different panels. We have now corrected our mistakes and Figures S6B and S13 have been updated accordingly.

The name Htt2-90 corresponds to Httex1 expressed from amino acid 2 to amino acid 90, with the first N-terminal methionine removed.

6) « There are many abbreviations difficult to understand in supplement. » Fig.S1 Htt18-90(Q18C) etc.

His6-Intein Ssp stands for the Intein tagged with Histidine amino acid (6 units)

Htt18-90(Q18C) means Httex1 expressed from amino acid 18 to amino acid 90 with the Glutamine (Q) in position 18 mutated in a Cysteine (C).

Htt2-17 means Httex1 synthesized from amino acid 2 to amino acid 17, with the first N-terminal methionine removed.

Htt18-90(Q18A) corresponds to Httex1 expressed from amino acid 18 to amino acid 90 with the Q in position 18 mutated in an Alanine (A).

References

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Referee #3

Evidence, reproducibility and clarity

The authors investigated the structural feature of N-terminal amino acid (Nt17) of Huntingtin, the gene product of Huntington disease. Nt17 was reported to play roles in modulating Huntingtin's aggregation, its life cycle, membrane binding and toxicity, however, those reports used tagged Nt17 and the authors thought the tags have the potential influence to the aggregation process and others and used tag-free Nt17-huntingtin exon1(Httex1) protein. Using Nt17 deleted Httex1 and mutant which disrupt helix conformation such as M8P, and phosphorylated Nt17, they found Nt17 sequence but not its helical conformation determined the morphology and growth of Httex1 fibrils in vitro. In cells, Nt17 sequence and its helical conformation influenced on aggregation propensity and toxic properties. Furthermore, the uptake o Httex1 into primary striatal neurons is influenced by the helical propensity of Nt17. They concluded Nt17 domain serves as the master regulator of Htt aggregation and toxicity. Their findings provided the precise information for the role of tag-free Nt17.

Major concerns:

1) Finding of lateral association(bundling) of ΔNt17-Httex1 fibrils is interesting. However, pathological significance is not clear. a) Does even non-truncated form also increase this kind of bundling when polyQ is expanded? b) When fibrils are added to striatal neurons like in Fig.5, is this structural feature preserved on the membrane or inside of the cells? c) When Httex1 fibrils species are expressed, is this bundling also observed? d) What function (cell death, membrane integrity or others) is most correlated with this structural feature?

2) The authors claimed < we investigated for the first time, the role of the Nt17 sequence, PTMs and conformation in regulating the internalization and cell-to-cell propagation of monomeric and fibrillar forms of mutant Httex1.>. However, so far this reviewer understand, the authors studied the internalization but not cell-to-cell propagation.

Minor points

1) Fig.3B arrowhead could not be seen.

2) Fig.4A: what do arrows mean? The insets are hard to identify. No scale bars?

3) Fig.5A:no scale bars?

4) Fig.S3. Height and length seem to be wrong.

5) Fig.S6C: hard to compare. D:What is Htt2-90? Also in Fig.S13.

6) There are many abbreviations difficult to understand in supplement. Fig.S1 Htt18-90(Q18C) etc.

Significance

The paper advanced our knowledge of Nt17, especially in the Huntington disease field. However, the conceptual advance is limited.

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Referee #2

Evidence, reproducibility and clarity

This article describes the results from studies into mechanisms of the aggregation and toxicity of Htt Exon1 protein. The authors investigated role of N17, polyQ length, M9C mutation, and phosphorylation. Multiple approaches were used that included biochemical protein design, biophysical measurements, and cell biological experiments with cultured mammalian cells. The authors demonstrates effects of protein context on aggregation. Furthermore, the authors were able to visualize the aggregates in mammalian cells and in neurons using multiple methods. These are interesting data, but there are several major weaknesses in the study. First problem is that most of the results related to aggregation mechanisms and toxicity are not original and incremental when compared to many previously published articles. Moreover, there are several problems in interpretation of obtained data and in making conclusions. Some of the most critical problems are listed below.

• The main hypothesis of this study solely depends on the ability of N17 domain to enhance aggregation (Fig 1 and Fig 2). According to the method for the protein solubilization 1mM TCEP was added to ∆Htt-Ex1, but not to Htt-Ex1 proteins. It is necessary to rule out potential effects of TCEP on aggregation assay.
• The author needs to provide biophysical data of the mutation and phosphorylated proteins with/without Tag. As stated by the authors, even the slight change in a protein context could lead to unexpected changes in structural behavior of a protein. Thus, importance of Tag needs to be evaluated.
• It is a challenging technical problem to produce proteins which are rich in Pro and Gln content. But there is not enough experimental details provided in the methods. Please add detailed procedures for expression and purification of these proteins.
• Fig 8 the color codes for PolyQ and PolyP need to be corrected.

Significance

This article describes the results from studies into mechanisms of the aggregation and toxicity of Htt Exon1 protein. The authors investigated role of N17, polyQ length, M9C mutation, and phosphorylation. Multiple approaches were used that included biochemical protein design, biophysical measurements, and cell biological experiments with cultured mammalian cells. The authors demonstrates effects of protein context on aggregation. Furthermore, the authors were able to visualize the aggregates in mammalian cells and in neurons using multiple methods. These are interesting data, but there are several major weaknesses in the study. First problem is that most of the results related to aggregation mechanisms and toxicity are not original and incremental when compared to many previously published articles. Moreover, there are several problems in interpretation of obtained data and in making conclusions. Some of the most critical problems are listed below.

• The main hypothesis of this study solely depends on the ability of N17 domain to enhance aggregation (Fig 1 and Fig 2). According to the method for the protein solubilization 1mM TCEP was added to ∆Htt-Ex1, but not to Htt-Ex1 proteins. It is necessary to rule out potential effects of TCEP on aggregation assay.
• The author needs to provide biophysical data of the mutation and phosphorylated proteins with/without Tag. As stated by the authors, even the slight change in a protein context could lead to unexpected changes in structural behavior of a protein. Thus, importance of Tag needs to be evaluated.
• It is a challenging technical problem to produce proteins which are rich in Pro and Gln content. But there is not enough experimental details provided in the methods. Please add detailed procedures for expression and purification of these proteins.
• Fig 8 the color codes for PolyQ and PolyP need to be corrected.

SciScore for 10.1101/2021.04.06.438630: (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">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Use of healthy volunteer PBMC for this project, including those from WBS, was ethically approved by the Cardiff University School of Medicine Research Ethics Committee (SMREC) nos. 20/55 and 20/101.</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">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">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies used were against HLA-ABC (W632; AbD Serotec), NCR3LG1/B7-H6 (MAB7144, Biotechne R&D Systems), Nectin 1 (R1.302; Biolegend), MICA (AMO1-100; BAMOMAB), MICB (BMO2-100; BAMOMAB), ULBP2 (BUMO1; BAMOMAB), Spike (1A9; Insight), Nucleocapsid (1C7; Stratech), anti-mouse IgG AF647 (Thermofisher).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HLA-ABC</div><div>suggested: (Leica Biosystems Cat# NCL-HLA-ABC, RRID:AB_563879)</div></div><div style="margin-bottom:8px"><div>MAB7144</div><div>suggested: (R and D Systems Cat# MAB7144, RRID:AB_2636810)</div></div><div style="margin-bottom:8px"><div>BMO2-100</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>BAMOMAB</div><div>suggested: (BAMoMAB Cat# BM02-100, RRID:AB_2636812)</div></div><div style="margin-bottom:8px"><div>ULBP2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: (SouthernBiotech Cat# 1030-31, RRID:AB_2794301)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Target cells were harvested using TrypLE Express (Gibco), preincubated for 30 min with the relevant antibody or serum preparations, then mixed with PBMCs at an effector:target (E:T) ratio of 10:1 in the presence of GolgiStop (0.7 μl/ml, BD Biosciences), Brefeldin-A (1:1000, Biolegend) and anti-CD107a–FITC (clone H4A3, BioLegend).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Brefeldin-A</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-CD107a–FITC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibody (anti-nucleocapsid 1C7, Stratech, 1:500 dilution) was added in PBST containing 1% non-fast milk and incubated for 1h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-nucleocapsid</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing in PBST, secondary antibody (anti-mouse IgG-HRP, Pierce, 1:3,000 dilution) was added in PBST containing 1% non-fat milk and incubated for 1h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG-HRP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody depletions: Anti-spike antibody was depleted from sera using magnetic bead conjugated spike trimer protein (Acrobiosystems).</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><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells and viruses: A549 were transduced with lentiviruses expressing human ACE2, and TMPRSS2 (AAT cells), as previously described 27.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The England2 strain of SARS-CoV2 was obtained from Public Health England (PHE), grown on VeroE6 cells, and titrated by plaque assay on both VeroE6 and AAT, as previously described27.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmids were midiprepped (Nucleobond Xtra Midi; Machery-Nagel), and transfected into 293T cells using GeneJuice (Merck) according to manufacturers’ instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Samples were loaded onto a trapping column (300μm x 5mm PepMap cartridge trap (Thermo Fisher)) at 10μL/min for 5 minutes at 60 degrees.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PepMap</div><div>suggested: (BioWorks, RRID:SCR_014594)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">During the gradient elution, mass spectra were acquired with the parameters detailed in Fig S4 using Tune v3.3 and Xcalibur v4.3 (Thermo Fisher).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Xcalibur</div><div>suggested: (Thermo Xcalibur, RRID:SCR_014593)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository74 with the dataset identifier PXD025000 and 10.6019/PXD025000.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PRIDE</div><div>suggested: (Pride-asap, RRID:SCR_012052)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Functional Annotation Clustering: Assessment of enriched gene annotation terms in temporal cluster one was carried out using the Functional Annotation Clustering tool at DAVID (david.ncifcrf.gov) v6.875, using the default clustering settings for medium stringency and the following libraries: Uniprot UP_Keyword, GOTERM:MF_ALL, BIOCARTA,, KEGG_PATHWAY and REACTOME_PATHWAY.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DAVID</div><div>suggested: (DAVID, RRID:SCR_001881)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmids and transfections: Lentivirus plasmids encoding each SARS-CoV2 ORF individually, with a C-terminal twin-strep tag, were obtained from Addgene76.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Addgene76</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Target cells were harvested using TrypLE Express (Gibco), preincubated for 30 min with the relevant antibody or serum preparations, then mixed with PBMCs at an effector:target (E:T) ratio of 10:1 in the presence of GolgiStop (0.7 μl/ml, BD Biosciences), Brefeldin-A (1:1000, Biolegend) and anti-CD107a–FITC (clone H4A3, BioLegend).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD Biosciences</div><div>suggested: (BD Biosciences, RRID:SCR_013311)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were acquired using an AttuneNxT (Thermo Fisher) and analyzed with Attune NxT software or FlowJo software version 10 (Tree Star).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Where sera were tested at a range of dilutions, the area under the curve (AUC) was calculated using Graphpad Prism 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

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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.

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About SciScore

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

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DOI: 10.1523/JNEUROSCI.2121-20.2021

Resource: (Cell Signaling Technology Cat# 2368, RRID:AB_2217020)

Curator: @Naa003

SciCrunch record: RRID:AB_2217020

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What is this?

SciScore for 10.1101/2021.04.02.437747: (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">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid 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">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">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">Intermediate R&D preparations of swine glyco-humanized polyclonal antibody against SARS-CoV-2 have been prepared, presenting variable anti-SARS-CoV-2 binding activities 17.</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-SARS-CoV-2 binding activities 17</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Spike/ACE-2 neutralization assay: An assay was developed to assess the properties of anti-SARS-CoV-2 spike antibodies to inhibit binding of ACE-2 to immobilized spike.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-Spike RBD antibodies diluted in PBS-Tween-0.05%-1% skimmed milk (dilution range between 50 and 0.39 µg/mL) were then added and incubated for 30 min.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Spike RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 1h incubation at room temperature and 3 washes, the mouse Fc tag was revealed with a specific HRP-conjugated anti-mouse IgG secondary antibody (diluted in in PBS-Tween-0.05%-1% skimmed milk powder at 1:1000, incubated 1h at RT and washed 3 times).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral stocks were generated using one passage of isolates on 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">CPE reduction assay was performed as follows: Vero E6 cells were seeded in 96-well clusters at a density of 5,000 cells/well 2 days before infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IC50 were analyzed by nonlinear regression using a four-parameter dosage-response variable slope model with the GraphPad Prism 8.0.2 software (GraphPad Software, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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

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

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Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04453384</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Study to Evaluate the Safety and Efficacy of XAV-19 in Patie…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04469179</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, Tolerability, and Pharmacokinetics of SAB-185 in Amb…</td></tr></table>

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

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

---

Results from rtransparent:

• 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.

---

<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>

That's a tough one. I can see it both ways.

From a programmer's perspective:

• It shouldn't be in an <aside>, if it is actually directly about what is in <main>
• An <aside> should be able to be evaluated on its own, (almost entirely) in isolation from, and not dependent on anything in, the <main> content. This could be especially important/relevant for screen readers.

It is truly sad that our country will dump so much money into infrastructure and politics but not conservation and ecosystem management. I know everything has a cost, but with so many billionaires and overall wealthy people in this country, conservation efforts should be one of the lead measures to consider. Putting a price tag on our ecosystems is just so disturbing. Without our biodiversity and ecosystems, money, politics and every other issue is completely irrelevant.

in the case of

briefly mentioning a subject but not going into it in detail the topic/subject need not be related at all (it sounds like).

What about in the case fo:

is defined as going off in a different direction. Does the fact that it's going off in a different direction imply that it at least starts out connected/related to the original (starting point) subject (as it does in the geometry sense of tangential)? Or does it permit "jumping" to another topic (in another direction) without being related/connected at all??

I don't think I like this definition very much. It doesn't quite fit the sense I'm trying to use it for in my tag:

tangentially related content (aside)

Ah, here's a definition that matches what I thought it meant (one of the senses anyway): https://hyp.is/3Bn2bpZ7Eeu3Ok8vg03AVA/www.merriam-webster.com/dictionary/tangential

This is a valid point, but it also creates an issue of equitability for students too. The digital world comes with a price tag that often has to be footed by underfunded school districts in order to make sure that all students have access to the tools.

Not only am I unsurprised by this, but I'd be surprised if it were otherwise. The logic is that schizophrenia is a sleep disorder, and exercise enhances sleep. Additionally, lack of movement is one of the negative symptoms of schizophrenia. Therefore, this poverty of movement may play a role in the pathogenesis of schizophrenia symptoms.

I need to start a google search document with predictions prior to actually searching. It will slow down my research speed, but it is necessary in order to provide unbiased data on my intuitive understanding of diseases. It seems like the majority of my strong intuitions are true. Edit: I'll just record the search phrase in my hypothes.is notes. This one was "exercise schizophrenia"

1742

20.10. Johann Gottlob Klotzsche, Benjamin Heinrich Klotzsche, 4 Jahre, 12 Wochen, 1 Tag

having more eyes is better

DOI: 10.1016/j.celrep.2021.108914

Resource: (IBA GmbH Cat# 2-1507-001, RRID:AB_513133)

Curator: @Naa003

SciCrunch record: RRID:AB_513133

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What is this?

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

Learn more at Review Commons

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Reply to the reviewers

Overall, we were pleased that the reviewers found our study carefully designed and interesting. We have addressed their comments below.

Reviewer #1 (Evidence, reproducibility and clarity)

The manuscript by Kern, et al., demonstrates that phagocytosis in macrophages is regulated in part by the intermolecular distance of phagocytosis-promoting receptors engaging phagocytic targets. Cells expressing chimeric receptors containing cytosolic domains of Fc receptors (FcR) and defined ligand-binding DNA domains were used to drive phagocytosis of opsonized glass beads coated with complementary DNA ligands of defined spacing and number. These so-called origami ligands allowed manipulation of receptor spacing following engagement, which allowed the demonstration that tight spacing of ligands (7 nm or 3.5 nm) optimized signaling for phagocytosis. The study is carefully performed and convincing. I have a few technical concerns and minor suggestions.

1. It is assumed that the origami preparations were entirely uniform. How much variation was there? Is that supported by TIRF microscopy of origami preparations? Was the TIRF microscopy calibrated for uniformity of fluorescence (ie., shade correction)?

Our laboratory, Dong et al., has extensively characterized the origami uniformity and robustness of these exact pegboards. This paper was just posted on bioRxiv (Dong et. al, 2021). We have also cited this paper in our revised manuscript in reference to the characterization of the DNA origami (Line 117).

We did not use any shade correction. Instead we only collected data from a central ROI in our TIRF field. To check for uniformity of illumination, we plotted the origami pegboard fluorescent intensity along the x and y axis. We observed very modest drop off in signal - the average signal intensity of origamis within 100 pixels of the edge is 76 ± 6% the intensity of origamis in a 100 pixel square in the center of the ROI. Fitting this data with a Gaussian model resulted in very poor R values. While this may account for some of the variation in signal intensity at individual points, we expect the normalized averages of each condition to be unaffected. We have amended the methods to describe this strategy (Lines 851-854).

[[images cannot be shown]]

2. Likewise, how much variation was there in the expression of the chimeric receptors? Large variation in receptor numbers per cell could significantly alter the quantitative studies. Aside from the flow sorting for cells expressing two different molecules, how were cells selected for analysis?

We thank the reviewer for bringing up this point. We confirmed comparable receptor expression levels at the cell cortex of the DNA CAR-𝛾 and the DNA CAR-adhesion used throughout the paper. We also have confirmed that receptor levels at the cell cortex were similar for the large DNA CAR constructs used in Figure 6C-D. This data is now included in Figures S5 and S7. We have also altered the text to include this (lines 169-172):

Expression of the various DNA CARs at the cell cortex was comparable, and engulfment of beads functionalized with both the 4T and the 4S origami platforms was dependent on the Fc__𝛾R signaling domain (Figure S5).

When quantifying bead engulfment, cells were selected for analysis based on a threshold of GFP fluorescence, which was held constant throughout analysis for each individual experiment. We have amended the “Quantification of engulfment” methods section to convey this (lines 921-923).

3. The scale of the origami relative to the cells is difficult to discern in Figures 2C and D. Additional text would be helpful to indicate, for example, that the spots on the Fig. 2D inset indicate entire origami rather than ligand spots on individual origami particles.

Thank you for pointing this out, we see how the legend was unclear and have corrected it (lines 453-454), including specifically noting “Each diffraction limited magenta spot represents an origami pegboard.” We have also outlined the cell boundary in yellow to make the cell size more clear.

4. Figure 5 legend, line 482: How was macrophage membrane visualized for these measurements?

We have added the following clarification (line 535-536): “The macrophage membrane was visualized using the DNA CAR__𝛾, which was present throughout the cell cortex.”

5. line 265: "our data suggest that there may be a local density-dependent trigger for receptor phosphorylation and downstream signaling". This threshold-dependent trigger response was also indicated in the study of Zhang, et al. 2010. PNAS.

The Zhang et al. study was influential in our study design, and we wish to give the appropriate credit. Zhang et al. found that a sufficient amount of IgG is necessary to activate late (but not early) steps in the phagocytic signaling pathway. In contrast, our study addresses IgG concentration in small nanoclusters. We find that this nanoscale density affects receptor phosphorylation. Thus, we think these two studies are distinct and complementary.

Lines 283-287 now read:

While this model has largely fallen out of favor, more recent studies have found that a critical IgG threshold is needed to activate the final stages of phagocytosis (Zhang et al., 2010). Our data suggest that there may also be a nanoscale density-dependent trigger for receptor phosphorylation and downstream signaling.

6. line 55: Rephrase, “we found that a minimum threshold of 8 ligands per cluster maximized FcgR-driven engulfment.” It is difficult to picture how a minimum threshold maximizes something.

We now state “we found that 8 or more ligands per cluster maximized FcgR-driven engulfment.”

7. line 184: Rephrase, "we created... pegboards with very high-affinity DNA ligands that are predicted not to dissociate on a time scale of >7 hr". Remove "not".

Thank you for pointing this out, it is now correct.

Reviewer #1 (Significance):

This study provides a significant advance in understanding about the molecular mechanisms of signaling for particle ingestion by phagocytosis.

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Reviewer #2 (Evidence, reproducibility and clarity):

The manuscript on “Tight nanoscale clustering of Fcg-receptors using DNA origami promotes phagocytosis" studies how clustering and nanoscale spacing of ligand molecules for a chimeric Fcg-receptors influence the phagocytosis of functionalized silicon beads by macrophage cell lines. The basis of this study is the design of a chimeric Fc-receptor (DNA-CARg) comprising an extracellular SNAP-tag domain that can be loaded with single-stranded (ss) DNA, the transmembrane part of CD86 and the cytosolic part of the Fc-receptor g-chain containing an immunoreceptor tyrosine-based activation motif (ITAM) as well as a C-terminal green fluorescent protein (GFP). As control the authors used a similar designed DNA-CAR that is lacking the intracellular ITAM-containing FCg tail. The chosen target for this chimeric DNA-CAR, are silicon beads covered by a lipid bilayer that contains biotin-labelled lipids that, via Neutravidin, can be loaded with a biotinylated DNA origami pegboard displaying complimentary ss-DNA as ligand for the DNA-CAR. The DNA origami pegboard contains four ATTO647N fluorescence for visualization and the ssDNA ligand in different quantities and spacing.

Using these principles, the authors study how ligand affinity, concentration and spacing influence the activation of the DNA-CARg and the engulfment of the loaded beads.

The authors show that bead engulfment is increased between 2 till 8 ssDNA ligands on the pegboard. After this, ligand numbers do not play a role anymore in the engulfment. They then study the role of the ligand spacing using pegboards that either contain 4 single strand DNA ligands in close (7nm/3,5nm) proximity or a more spaced version using 21/17,5 nm or 35/38,5 nm. The authors find that the bead engulfment is maximally and positively affected by the close spacing of the ssDNA ligands. In their final experiments the authors vary the design of the DNA-CARs by tetramerization of the ITAM-containing Fcg-signaling subunit. In their discussion the authors mention different possibilities for the effect of spacing on the engulfment process.

I think that, in general, this is an interesting study. However, it has some caveats and open issues that should be clarified before its publication.

Major comments

1. As a general comment, it is somewhat a pity that the authors did not use the endogenous FcR as a control. It would have been quite easy for the authors to place the SNAP-tag domain on the Fcg extracellular domain which would allow to do all their experiments in parallel, not only with the DNA-CAR, but also with a DNA-containing wild type receptor. Such a control would be important because, by using a CD86 transmembrane domain, the authors do not know whether the nanoscale localization of their chimeric receptors is reflecting that of the endogenous Fcg receptor.**

We agree with the reviewer completely. We have repeated experiments shown in Figure 4A with a DNA-CAR containing the Fc𝛾 transmembrane domain instead of CD86 as the reviewer suggests. We also included a DNA-CAR version of the Fc𝛾R1 alpha chain, although this construct was not expressed as well as the others. These data are now included in Figure S5, and referenced in lines 167-168.

2. An important issue that is discussed by the authors but not addressed in this manuscript is whether the different amount and spacing of the ligand is only impacting on signaling or also on the mechanical stress of the cells. Indeed, mechanical stress on the cytoskeleton arrangement could influence the engulfment process. For this, it would be very important to test that the different bead engulfment, for example, those shown in Fig. 4, is strictly dependent on signaling kinases. The authors should repeat the experiment of Fig. 4 a and b in the presence or absence of kinase inhibitors such as the Syk inhibitor R406 or the Src inhibitor PP2 to show whether the different phase of engulfment is dependent on the signaling function of these kinases. This crucial experiment is clearly missing from their study.

We agree this is an interesting point. We find that ligand spacing affects receptor phosphorylation; however this does not preclude effects on downstream aspects of the signaling pathway. We will clarify this by adding the following comment to the manuscript (line 299-301):

While our data pinpoints a role for ligand spacing in regulating receptor phosphorylation, it is possible that later steps in the phagocytic signaling pathway are also directly affected by ligand spacing.

The DNA-CAR-adhesion in Figure 1 strongly suggests that intracellular signaling is essential for phagocytosis. We have now included additional controls using this construct as detailed in our response to point 3 below. Unfortunately, Src and Syk inhibitors or knockout abrogate Fc𝛾R mediated phagocytosis (for example, PMIDs 11698501, 9632805, 12176909, 15136586) and thus would eliminate phagocytosis in both the 4T and 4S conditions. This precludes analysis of downstream steps in the phagocytic signaling pathway.

3. Another problem of this study is that the authors show in Fig. 1A the control DNA-CAR-adhesion but then hardly use it in their study. For example, the crucial experiments shown in Fig. 4 should be conducted in parallel with DNA-CAR-adhesion expressing macrophage cells. This study could provide another indication whether or not ITAM signaling is important for the engulfment process.

We have added this control. It is now included in Figure S5 and S7. Figure 3D also shows that the DNA-CAR-adhesion combined with the 4T origami pegboards does not activate phagocytosis and we have amended the text to make this more clear (line 152).

4. Another important aspect is how the concentration of the loaded origami pegboard is influencing the engulfment process. In particular, it would be interesting to show the padlocks with different spacings such as the 4T closed spacing versus 4s large spacing show a different dependency on the concentration of this padlock loading on the beads. This would be another important experiment to add to their study.**

We agree that this is an interesting question. We suspect that at a very high origami density, 4S signaling would improve, and potentially approach the 4T. However, we are currently coating the beads in saturating levels of origami pegboards. Thus we cannot increase origami pegboard density and address this directly.

Minor comments:

1. The definition of the ITAM is Immunoreceptor Tyrosine-based Activation Motif and not "Immune Tyrosine Activation Motif" as stated by the authors.

We have corrected this.

2. The authors discuss that it is the segregation of the inhibitory phosphatase CD45 from the clustered Fc receptors is the major mechanism explaining their finding that 4T closed spacing is more effective than 4s large spacing. With the event of the CRISPR/Cas9 technology it is trivial to delete the CD45 gene in the genome of the RAW264.7 macrophage cell line used in this study and I am puzzled why they author are not conducting such a simple but for their study very important experiment (it takes only 1-2 month to get the results).

This experiment may be informative but we have two concerns about its feasibility. First, CD45 is a phosphatase with many different roles in macrophage biology, including activating Src family kinases by dephosphorylating inhibitory phosphorylation sites (PMID 8175795, 18249142, 12414720). Second, CD45 is not the only bulky phosphatase segregated from receptor nanoclusters. For example, CD148 is also excluded from the phagocytic synapse (PMID 21525931). CD45 and CD148 double knockout macrophages show hyperphosphorylation of the inhibitory tyrosine on Src family kinases, severe inhibition of phagocytosis, and an overall decrease in tyrosine phosphorylation (PMID 18249142). CD45 knockout alone showed mild phenotypes in macrophages. We anticipate that knocking out CD45 alone would have little effect, and knocking out both of these phosphatases would preclude analysis of phagocytosis. Because of our feasibility concerns and the lengthy timeline for this experiment, we believe this is outside of the scope of our study.

In our discussion, we simplistically described our possible models in terms of CD45 exclusion, as the mechanisms of CD45 exclusion have been well characterized. This was an error and we have amended our discussion to read (lines 335-343):

As an alternative model, a denser cluster of ligated receptors may enhance the steric exclusion of the bulky transmembrane proteins like the phosphatases CD45 and CD148 (Bakalar et al., 2018; Goodridge et al., 2012; Zhu, Brdicka, Katsumoto, Lin, & Weiss, 2008).

Reviewer #2 (Significance):

The innovative part of this study is the combination of SNAP-tag attached, chimeric Fc-receptor with the DNA origami pegboard technology to address important open question on receptor function.

Referees cross-commenting

I find most of my three reviewing colleagues reasonable I also agrée to Reviewer #1 comments 2

Likewise, how much variation was there in the expression of the chimeric receptors?

Large variation in receptor numbers per cell could significantly alter the quantitative studies. Aside from the flow sorting for cells expressing two different molecules, how were cells selected for analysis?

But I want to add it is not only the amount of receptors but ils the nanoscale location that is key to receptor function

We have ensured that all receptors are trafficked to the cell surface. We have also measured their intensity at the cell cortex as discussed in response to Reviewer 1.

Reviewer #3 (Evidence, reproducibility and clarity):

This is a very nicely done synthetic biology/biophysics study on the effect of ligands spacing on phagocytosis. They use a DNA based recognition system that the group has previously use to investigate T cell signaling, but express the SNAP tag linked transmembrane receptor in a macrophage cell line and present the ligands using DNA origami mats to control the number and spacing of complementary ligands that are designed to be in the typical range for low or high affinity FcR, a receptor that can trigger phagocytosis. The study offers some very nice quantitative data sets that will be of immediate interest to groups working in this area and, in the future, for design of synthetic receptors for immunotherapy applications. Other groups are working on similar platform for TCR. I don't feel there is any need for more experiments, but I have some questions and suggestions. Answering and considering these could clarify the new biological knowledge gained.

We thank the reviewer for their support of our manuscript. Given the reviewer’s statement that no new experiments are required, we have answered their questions to the best of our ability given the current data. Should the editor decide that any of these topics require experimental data to enhance the significance of the paper, we are happy to discuss new experiments.

Reviewer #3 (Significance):

I think the significance would be increased by addressing these questions, that would help understand how the synthesis system described related to other system directed as similar questions and more natural settings.

1.The densities of the freely mobile DNA ligands required to trigger phagocytosis is quite high. Was the length of the DNA duplexes optimized? The entire complex for both the intermediate and high affinity duplexes seems quite short, perhaps <10 nm. Might the stimulation be more efficient if a short stretch of DS DNA is added to increase the length to 12-13 nm?

The extracellular domain of the DNA-CAR (SNAP tag and ssDNA strand) are approximately 10 nm (PMID 28340336). The biotinylated ligand ssDNA is attached to the bilayer via neutravidin, resulting in a predicted 14 nm intermembrane spacing. The endogenous IgG FcR complex is 11.5 nm. Bakalar et al (PMID 29958103) tested the effect of antigen height on phagocytosis and found that the shortest intermembrane distance tested (approximately 15 nm) was the most effective. As the reviewer notes, the optimal distance between macrophage and target may be larger than our DNA-CAR. However we think the intermembrane spacing in our system is within the biologically relevant range.

We saw robust phagocytosis at 300 molecules/micron of ssDNA, which is similar to the IgG density used on supported lipid bilayer-coated beads in other phagocytosis studies (PMID 29958103, 32768386). As the reviewer noticed, this is significantly higher than ligand density necessary to activate T cells (PMID 28340336). We have added a comment on ligand density to lines 96-97.

2. Are the origami mats generally laterally mobile on the bilayers. If so, what is the diffusion coefficient? Can one detect the mats accumulating in the initial interface between the bead and cell, particularly in cased where there is no phagocytosis? Would immobility of the mats make them more efficient at mediating phagocytosis compared to the monodispersed ligands, which I assume are highly mobile and might even be "slippery".

We have confirmed that our bead protocol generally produces mobile bilayers, where his-tagged proteins can freely diffuse to the cell-bead interface (see accumulation of a his-tagged FRB binding to a transmembrane FKBP receptor at the cell-bead synapse below). We can qualitatively say that the origamis appear mobile on a planar lipid bilayer (see Dong et. al 2021 and images below). Directly measuring the diffusion coefficient on the beads is extremely difficult because the beads themselves are mobile (both diffusing and rotating), and cannot be imaged via TIRF. We do not see much accumulation of the origami at cell-bead synapses. This could reflect lower mobility of the origamis, or could be because the relative enrichment of origamis is difficult to detect over the signal from unligated origamis.

Overall, we expect the origami pegboards (tethered by 12 neutravidins) are less mobile than single strand DNA (tethered by a single neutravidin, supported by qualitative images below). We are uncertain whether this promotes phagocytosis. At least one study suggests that increased IgG mobility promotes phagocytosis (PMID 25771017). However, the zipper model would suggest that tethered ligands may provide a better foothold for the macrophage as it zippers the phagosome closed (PMID 14732161). Hypothetically, ligand mobility could affect signaling in two ways - first by promoting nanocluster formation, and second by serving as a stable platform for signaling as the phagosome closes. Since our system has pre-formed nanoclusters, the effect of ligand mobility may be quite different than in the endogenous setting.

[[image cannot be shown]]

In the above images, a 10xHis-FRB labeled with AlexaFluor647 was conjugated to Ni-chelating lipids in the bead supported lipid bilayer. The macrophages express a synthetic receptor containing an extracellular FKBP and an intracellular GFP. Upon addition of rapamycin, FRB and FKBP form a high affinity dimer, and FRB accumulates at the bead-macrophage contact sites.

[[image cannot be shown]]

In the above images, single molecules were imaged for 3 sec. The tracks of each molecule are depicted by lines, colored to distinguish between individual molecules. The scale bar represents 5 microns in both panels.

3. Breaking down the analysis into initiation and completion is interesting. When using the non-signalling adhesion constructs, would they get to the initiation stage or would that attachment be less extensive than the initiation phase.

This is an interesting question. While we did not include the DNA-CAR-adhesion in our kinetic experiments, we have now quantified the frequency of cups that would match our ‘initiation’ criteria in 3 representative data sets where macrophages were fixed after 45 minutes of interaction with origami pegboard-coated beads. We found that an average of 16/125 of 4T beads touching DNA-CAR-adhesion macrophages met the ‘initiation’ criteria and an average of 2/125 were eaten (14% total). In comparison, we examined 4T beads touching DNA CAR𝛾 macrophages and found that on average 23/125 met the ‘initiation’ criteria, and 45/125 were already engulfed (54%). This suggests that the DNA-CAR-adhesion alone may induce enough interaction to meet our initiation criteria, but without active signaling from the FcR this extensive interaction is rare. We have added this data in a new Figure S6 and commented on this in lines 213-215.

4. It would be interesting to put these results in perspective of earier work on spacing with planar nanoarrays, although these can't be applied to beads. For integrin mediated adhesion there was a very distinct threshold for RGD ligand spacing that could be related to the size of some integrin-cytoskeletal linkers (PMID: 15067875). On the other hand, T cell activation seemed more continuous with changes in spacing over a wide range with no discrete threshold (PMID: 24117051, 24125583) unless the spacing was increased to allow access to CD45, in which case a more discrete threshold was generated (PMID: 29713075). The results here for phagocytosis with the very small ligands that would likely exclude CD45 seems to be more of a continuum without a discrete threshold, although high densities of ligand are needed. This issue of continuous sensing vs sharp threshold is biologically interesting so would be good assess this by as consistent standards are possible across systems.**

We agree that this is an interesting body of literature worth adding to our discussion. We have added a paragraph that puts our study in the context of prior work on related systems, including these nanolithography studies (Line 364-382):

How does the spacing requirements for Fc__𝛾R nanoclusters compare to other signaling systems? Engineered multivalent Fc oligomers revealed that IgE ligand geometry alters Fcε receptor signaling in mast cells (Sil, Lee, Luo, Holowka, & Baird, 2007). DNA origami nanoparticles and planar nanolithography arrays have previously examined optimal inter-ligand distance for the T cell receptor, B cell receptor, NK cell receptor CD16, death receptor Fas, and integrins (Arnold et al., 2004; Berger et al., 2020; Cai et al., 2018; Deeg et al., 2013; Delcassian et al., 2013; Dong et al., 2021; Veneziano et al., 2020). Some systems, like integrin-mediated cell adhesion, appear to have very discrete threshold requirements for ligand spacing while others, like T cell activation, appear to continuously improve with reduced intermolecular spacing (Arnold et al., 2004; Cai et al., 2018). Our system may be more similar to the continuous improvement observed in T cell activation, as our most spaced ligands (36.5 nm) are capable of activating some phagocytosis, albeit not as potently as the 4T. Interestingly, as the intermembrane distance between T cell and target increases, the requirement for tight ligand spacing becomes more stringent (Cai et al., 2018). This suggests that IgG bound to tall antigens may be more dependent on tight nanocluster spacing than short antigens. Planar arrays have also been used to vary inter-cluster spacing, in addition to inter-ligand spacing (Cai et al., 2018; Freeman et al., 2016). Examining the optimal inter-cluster spacing during phagosome closure may be an interesting direction for future studies.

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Additional experiments performed in revision

In addition to these reviewer comments, we have added additional controls validating the DNA-CAR-4x𝛾 used in Figure 6c,d. We compared the DNA-CAR-4x𝛾 to versions of the DNA-CAR-1x𝛾-3x𝛥ITAM construct with the functional ITAM in the second and fourth positions (see the schematics now included Figure S7). We found that four individual receptors with a single ITAM each were able to induce phagocytosis regardless of which position the ITAM was in. However the DNA-CAR-4x𝛾 construct, which also contains 4 ITAMs, was not. This further validates the experiment presented in 6c,d. We also fixed minor errors we discovered in the presentation of data for Figures 1C and S1A.