To concatenate multiple highlights simply add .c<number in sequence>, for the first highlight you would use .c1, for the second, .c2.

Adding keywords isn't just to make finding things easier later on, the act of associating a highlight with a keyword or linking it to a project through a tag elevates your reading to be active rather than simply passive.

Readwise does tag expansion, use a shorthand tag name such as .prob and rename that in Readwise to .probability and from then on Readwise will expand the tag name from there. If slashes are okay in a inline tag name this would make it easy to expand .question to .annotation/question.

https://www.oxfordjournals.org/nar/database/summary/813

Artikel 254 Endverwendung

(1) In der Endverwendung können Waren aufgrund ihres besonderen Zwecks abgabenfrei oder zu einem ermäßigten Abgabensatz zum zollrechtlich freien Verkehr überlassen werden.

(2) Befinden sich Waren auf einer Herstellungsstufe, in der in wirtschaftlicher Hinsicht lediglich die vorgeschriebene Endverwendung erreicht werden kann, so können die Zollbehörden in der Bewilligung die Bedingungen festlegen, unter denen die Waren als zu den Zwecken verwendet gelten, die für die Anwendung der Abgabenfreiheit oder des ermäßigten Einfuhrabgabensatzes festgelegt wurden.

(3) Sind die Waren zur mehrfachen Verwendung geeignet und halten die Zollbehörden es zwecks Vermeidung von Missbrauch für angebracht, so wird die zollamtliche Überwachung fortgesetzt für einen Zeitraum von höchstens zwei Jahren ab dem Tag der ersten Verwendung dieser Waren zu den Zwecken, die maßgeblich für die Anwendung der Abgabenfreiheit oder des ermäßigten Einfuhrabgabensatzes waren.

(4) Die zollamtliche Überwachung in der Endverwendung endet in allen folgenden Fällen:

a) wenn die Waren zu den Zwecken verwendet wurden, die maßgeblich für die Anwendung der Abgabenfreiheit oder des ermäßigten Einfuhrabgabensatzes waren,

b) wenn die Waren aus dem Zollgebiet der Union verbracht, zerstört oder zugunsten der Staatskasse aufgegeben werden,

c) wenn die Waren zu anderen Zwecken als denen, die maßgeblich für die Anwendung der Abgabenfreiheit oder des ermäßigten Einfuhrabgabensatzes waren, verwendet und die anwendbaren Einfuhrabgaben entrichtet wurden.

(5) Wird eine bestimmte Ausbeute verlangt, so gilt Artikel 255 für die Endverwendung.

(6) Abfälle oder Reste, die bei der Be- oder Verarbeitung von Waren im Rahmen der vorgeschriebenen Verwendung anfallen, sowie Verluste aufgrund natürlichen Schwundes gelten als der vorgeschriebenen Endverwendung zugeführt.

(7) Abfälle oder Reste, die bei der Zerstörung von Waren in der Endverwendung anfallen, gelten als in das Zolllagerverfahren übergeführt.

Fußnoten

To add a tag to an annotation simple use a . followed by a single word to create that tag like .productivity or .InProgress.

I need to find out if / characters will break it.

Interesting way to classify different kind of tags, keyword tags are topical while categorical tags that are associated with automated actions.

SciScore for 10.1101/2022.02.22.480950: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A positive selection follows, comprised of incubation with anti-Myc-AlexaFluor647 antibody (1:100 volume:volume) and anti-AlexaFluor647 magnetic beads (1:10 volume:volume) and flowed over a Milltenyi column on a magnet at 4 °C, such that yeast with bound peptide are retained on the column.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Myc-AlexaFluor647</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-AlexaFluor647</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This extended product was assembled in yeast with linearized pYal vector at a 5:1 insert:vector via electroporation with electrocompetent RJY100 yeast.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pYal</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Yeast were then washed into 4 °C acid saline (150mM NaCl, 20mM citric acid, pH5) with 1 μM HLA-DM and incubated at 4 °C overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pH5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Ectodomain sequences of each chain were formatted with a C-terminal poly-histidine purification tag and cloned into pAcGP67a vectors.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pAcGP67a</div><div>suggested: RRID:Addgene_41812)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To examine conservation between viruses, viral proteins are aligned using ClustalOmega (Madeira et al., 2019).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ClustalOmega</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Amplicons were sequenced on an Illumina MiSeq using paired-end MiSeq v2 300bp kits at the MIT BioMicroCenter.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MiSeq</div><div>suggested: (A5-miseq, RRID:SCR_012148)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Paired-end reads were assembled using PandaSeq (Masella et al., 2012).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PandaSeq</div><div>suggested: (PANDAseq, RRID:SCR_002705)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Each vector was individually transfected into SF9 insect cells (Thermo Fisher) with BestBac 2.0 linearized baculovirus DNA (Expression Systems; Davis, CA) and Cellfectin II Reagent (Thermo Fisher), and propagated to high titer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BestBac</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Relative binding curves were then generated and fit in Prism 9.3 to the equation y = 1/(1+[pep]/IC50), where [pep] is the concentration of un-labelled competitor peptide, in order to determine the concentration of half-maximal inhibition, the IC50 value.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>

---

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

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

---

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

Results from rtransparent:

---

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

---

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

---

<footer>

About SciScore

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

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Author Response:

Reviewer #1 (Public Review):

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

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

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

We thank the reviewer for their enthusiasm and support of our work!

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

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

Thank you for the suggestion. We have added a new figure supplement (Figure 6-Figure Supplement 3) with additional example traces for all odorants from Figure 6 for which we found a statistically significant difference between the two genotypes (Ir25a versus wildtype).

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

For these experiments, we followed a standard practice in Drosophila optogenetics to test the same experimental genotype in the presence or absence of the essential cofactor all-trans-retinal. This controls for potential effects from the genetic background. It is possible our description of these experiments was unclear (as also suggested by comments from Reviewer 2). As such, we have clarified our experimental design for the optogenetic experiments in the revised manuscript:

Modified text: “No light-induced responses were found in control flies, which had the same genotype as experimental flies but were not fed all-trans retinal (-ATR), a necessary co-factor for channelrhodopsin function (see Methods).” and “Bottom trace is control animal, which has the same genotype as the experimental animal but was not fed the required all-trans retinal cofactor (-ATR).”

Figure 4-Figure Supplement 1 legend: “In all optogenetic experiments, control animals have the same genotypes as the corresponding experimental animals but have not been fed all-trans retinal.”

Methods: “For all optogenetic experiments, the control flies were of the same genotype as experimental flies but had not been fed all-trans retinal.”

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

We share this reviewer’s excitement regarding the numerous questions our work now raises. While testing additional functional ramifications of chemosensory co-receptor expression is beyond the scope of this work (but will undoubtedly be the focus of future studies), we did expand on what this might mean in the revised Discussion section of the revised manuscript. Previously, we had raised the hypothesis that chemoreceptor co-expression could be an evolutionary relic of Ir25a expression in all chemoreceptor neurons , or a biological mechanism to broaden the response profile of an olfactory neuron without sacrificing its ability to respond to specific odors. We now extend our discussion to raise additional possible ramifications. For example, we suggest that modulating Ir25a coexpression could alter the electrical properties of a neuron, making it more (or possibly less) sensitive to Orco-dependent responses. We also suggest that Ir25a coexpression might be an evolutionary mechanism to allow olfactory neurons to adjust their response activities. That is, that most Orco-positive olfactory neurons are already primed to be able to express a functional Ir receptor if one were to be expressed. Such co-expression in some olfactory neurons might present an evolutionary advantage by ensuring olfactory responses to a complex but crucial biologically relevant odor, like human odors to some mosquitoes.

Reviewer #2 (Public Review):

In the present study, the authors: 1) generated knock-in lines for Orco, Ir8a, Ir25a, and IR7ba, and examined their expression, with a main focus on the adult olfactory organs. 2) confirmed the expression of these receptors using antibody staining. 3) examined the innervation patterns of these knock-in lines in the nervous system. 4) identified a glomerulus, VM6, that is divided into three subdivisions. 5) examined olfactory responses of neurons co-expressing Orco and Ir25a

The results of the first four sets of experiments are well presented and support the conclusions, but the results of the last set of experiments (the electrophysiology part) need some details. Please find my detailed comments below.

We thank the reviewer for their support of our work and appreciating the importance of our findings. In the revised manuscript, we now provide the additional experimental details for the electrophysiology work as requested.

Major points

Line 167-171: I wonder if the authors also compared the Orco-T2A-QF2 knock-in with antibody staining of the antenna.

We did perform whole-mount anti-Orco antibody staining on Orco-T2A-QF2 > GFP antennae (example image below). We saw broad overlap between Orco+ and GFP+ cells, similar to the palps. However, we did not include these results since quantification of these tissues is challenging for the following reasons:

1. There are ~1,200 olfactory neurons in each antenna, many of which are Orco+.
2. The thickness of the tissue makes determinations of co-localization difficult in wholemount staining.
3. Co-localization is further complicated by the sub-cellular localization of the signals: Orco antibodies preferentially label dendrites and weakly label cell bodies, while our GFP reporter is cytoplasmic and preferentially labels cell bodies. For these reasons, we focused on the numerically simpler palps for quantification. For the Ir8aT2A-QF2 and Ir76b-T2A-QF2 lines, palp quantification was not an option as neither knock-in drove expression in the palps (and the available antibodies did not work with the whole-mount staining protocol). This is why we performed antennal cryosections to validate these lines. Below is an example image of the antennal whole-mount staining in the Orco-T2A-QF2 knock-in line, illustrating the quantification challenges enumerated above.

*Co-staining of anti-Orco and GFP in Orco-T2A-QF2 > 10xQUAS-6xGFP antenna *

Lines 316-319 (Figure 4D): It would be better if the authors compare the responses of Ir25a>CsChrimson to those of Orco>CsChrimson.

The goal of the optogenetic experiments was to provide experimental support for Ir25a expression in Orco+ neurons in an approach independent to previous methods. Our main question was whether we could activate what was previously considered Orco-only olfactory neurons using the Ir25a knock-in. These experiments were not designed to determine if this optogenetic activation recapitulated the normal activity of these neurons. For these reasons, we did not attempt the optogenetic experiments with Orco>CsChrimson flies.

Line 324-326: Why the authors tested control flies not fed all-trans retinal? They should test Ir25a-T2A-QF2>QUAS-CsChrimson not fed all-trans retinal as a control.

We apologize for the confusion. The “control” flies we used were indeed Ir25a-T2AQF2>QUAS-CsChrimson flies not fed all-trans retinal as suggested by the reviewer. This detail was in the methods, yet likely was not clear. We have amended the main text in multiple locations to state the full genotype of the control fly more clearly:

Modified text: “No light-induced responses were found in control flies, which had the same genotype as experimental flies but were not fed all-trans retinal (-ATR), a necessary co-factor for channelrhodopsin function (see Methods).” and “Bottom trace is control animal, which has the same genotype as the experimental animal but was not fed the required all-trans retinal cofactor (-ATR).”

Figure 4-Figure Supplement 1 legend: “In all optogenetic experiments, control animals have the same genotypes as the corresponding experimental animals but have not been fed all-trans retinal.”

Methods: “For all optogenetic experiments, the control flies were of the same genotype as experimental flies but had not been fed all-trans retinal.”

Line 478-500: I wonder if the observed differences between the wildtype and Ir25a2 mutant lines are due to differences in the genetic background between both lines. Did the authors backcross Ir25a2 mutant line with the used wildtype for at least five generations?

Yes, the mutants are outcrossed into the same genetic background as the wildtypes for at least five generations. Please see Methods, revised manuscript: “Ir25a2 and Orco2 mutant fly lines were outcrossed into the w1118 wildtype genetic background for at least 5 generations.”

Line 1602-1603: Does the identification of ab3 sensilla using fluorescent-guided SSR apply for ab3 sensilla in Orco mutant flies. How does this ab3 fluorescent-guided SSR work?

In fluorescence guided SSR (fgSSR; Lin and Potter, PloS One, 2015), the ab3 sensilla is GFPlabelled (genotype: Or22a-Gal4>UAS-mCD8:GFP), which allows this sensilla to be specifically identified under a microscope and targeted for SSR recordings. We generated fly stocks for fgSSR identification of ab3 in all three genetic backgrounds (wildtype, Orco mutant, Ir25a mutant).

These three genotypes are described in the methods:

“Full genotypes for ab3 fgSSR were:

Pin/CyO; Or22a-Gal4,15XUAS-IVS-mcd8GFP/TM6B (wildtype),

Ir25a2; Or22a-Gal4,15XUAS-IVS-mcd8GFP/TM6B (Ir25a2 mutant),

Or22a-Gal4/10XUAS-IVS-mcd8GFP (attp40); Orco2 (Orco2 mutant).”

Line 1602-1604: There is no mention of how the authors identified ab9 sensilla.

Information on the identification of ab9 sensilla is under the optogenetics section of the methods: “Identification of ab9 sensilla was assisted by fluorescence-guided Single Sensillum Recording (fgSSR) (Lin and Potter, 2015) using Or67b-Gal4 (BDSC #9995) recombined with 15XUAS-IVS-mCD8::GFP (BDSC #32193).”

Line 1648: what are the set of odorants that were used to identify the different coeloconic sensilla?

We have added the specific odorants used for sensillar identification for coeloconic SSR in the Methods. The protocol and odorants used were:

*2,3-butanedione (BUT), 1,4-diaminobutane (DIA), Ammonia (AM), hexanol (HEX), phenethylamine (PHEN), and propanal (PROP) to distinguish coeloconic sensilla:

o Wildtype flies: Strong DIA and BUT responses identify ac2 and rule out ac4. Absence of strong AM response rules out ac1, absence of HEX response rules out ac3, absence of PHEN response further rules out ac4.

o Ir25a mutant flies (amine responses lost, so cannot use PHEN and DIA as diagnostics): Strong BUT response and moderate PROP response identify ac2 and rule out ac4. Absence of strong AM response rules out ac1, absence of HEX response rules out ac3. Ac4 is further ruled out anatomically based on sensillar location compared to ac2.

Revised text: “Different classes of coeloconic sensilla were identified by their known location on the antenna and confirmed with their responses to a small panel of diagnostic odorants: in wildtype flies, ac2 sensilla were identified by their strong responses to 1,4-diaminobutane and 2,3-butanedione. The absence of a strong response to ammonia was used to rule out ac1 sensilla, the absence of a hexanol response was used to rule out ac3 sensilla, and the absence of a phenethylamine response was used to rule out ac4 sensilla. In Ir25a mutant flies in which amine responses were largely abolished, ac2 and ac4 sensilla were distinguished based on anatomical location, as well as the strong response of ac2 to 2,3-butanedione and the moderate response to propanal (both absent in ac4). Ac1 and ac3 sensilla were excluded similarly in the mutant and wildtype flies. No more than 4 sensilla per fly were recorded. Each sensillum was tested with multiple odorants, with a rest time of at least 10s between applications.

SciScore for 10.1101/2022.02.21.481262: (What is this?)

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

Table 1: Rigor

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

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Serological test systems: All samples collected in the 2021/22 hunting season were tested for the presence of anti-SBV antibodies by a glycoprotein Gc-based ELISA performed as described previously [42].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SBV</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The latter allows for the detection of anti-BVDV antibodies as well as for antibodies against the ovine BDV.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-BVDV</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the detection of antibodies directed against SARS-CoV-1, an ELISA system was established in line with the SARS-CoV-2 test.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expi293 cells were grown in suspension in Expi293 expression medium (Thermo Fisher Scientific) at 37 °C, 8 % CO2 and 125 rpm.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For expression of the recombinant protein, the RBD-SD1 domain (aa 306 – 577) of the SARS coronavirus strain Tor2 (NC_004718.3) was ordered as a synthetic DNA string fragment (GeneArt synthesis; Thermo Fisher Scientific, Darmstadt, Germany) and cloned into the pEXPR103 expression vector (IBA Lifesciences, Göttingen, Germany) in-frame with a C-terminal Strep-tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pEXPR103</div><div>suggested: None</div></div></td></tr></table>

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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.

---

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

---

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

Results from rtransparent:

---

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

---

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

---

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About SciScore

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

</footer>

SciScore for 10.1101/2022.02.21.481223: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: This study protocol was approved by the Institutional Review Board of UCSD’s Human Research Protections Program (181180).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The foreground was a bed file of significant IDR peaks; the background was randomly defined peaks within the same annotated region as the foreground peaks.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Authentication: Cell culture and cell line generation: BEAS-2B, HEK293T and Vero E6 cells were purchased from the American Type Culture Collection and were not further authenticated.<br>Contamination: Cells were routinely tested for mycoplasma contamination with a MycoAlert mycoplasma test kit (Lonza) and were found negative for mycoplasma.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The remaining lysates were immunoprecipitated using 15 µl anti-Strep or 10 µl anti-FLAG antibody (depending on the epitope tag of the construct; Supplementary Table 4) on Sheep Anti-Mouse IgG Dynabeads M-280 (ThermoFisher) overnight at 4°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-FLAG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-Mouse IgG</div><div>suggested: (Thermo Fisher Scientific Cat# 11201D, RRID:AB_2783640)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Negative control samples are wild type (WT) BEAS-2B cells, and performed using both anti-Strep and anti-FLAG antibodies (separately).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Strep</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were fixed with 4% paraformaldehyde for 30 min, which inactivates the virus, before transferring from BSL3 to BSL2, and proceeding with immunofluorescence staining using anti-Nucleocapsid antibody (40143-R019, Sino Biological).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BSL2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Nucleocapsid</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Next, cells were incubated with primary antibodies (Supplementary Table 4) at 1:250-2000 dilutions in blocking buffer for 16 h at 4 °C, washed with PBS+0.01% Triton X-100 three times for 5 min each at room temperature, and then incubated with secondary antibody (goat anti-rabbit secondary IgG (H+L)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture and cell line generation: BEAS-2B, HEK293T and Vero E6 cells were purchased from the American Type Culture Collection and were not further authenticated.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The ACE2-overexpressing A549 cell line (A549-ACE2) was clonally generated and a gift from Benjamin tenOever52.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BEAS-2B cells were cultured on Matrigel (Corning) coated plates and maintained in the PneumaCult-Ex Plus Medium (Stem Cell Technologies), supplemented with 33 µg/ml hydrocortisone (Stem Cell Technologies).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BEAS-2B</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T, Vero E6 and A549-ACE2 cells were cultured in DMEM (ThermoFisher) supplemented with 10% FBS (ThermoFisher) and passaged every three days.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>A549-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 virus infection: SARS-CoV-2 isolates USA-WA1/2020 (BEI Resources, #NR-52281), hCoV-19/USA/CA_UCSD_5574/2020 (lineage B.1.1.7) and hCoV-19/South Africa/KRISP-K005325/2020 (lineage B.1.351 BEI Resources NR-54009) were propagated and infectious units quantified by plaque assay using Vero E6 cells for the WA1 variant, and Vero-TMPRSS2 cells for variants B.1.1.7 and B.1.351.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture and cell line generation: BEAS-2B, HEK293T and Vero E6 cells were purchased from the American Type Culture Collection and were not further authenticated.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BEAS-2B</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmid construction: 2xStrep-tagged plasmids in a pLVX vector expressing SARS-CoV-2 proteins were a gift from Nevan Krogan1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX</div><div>suggested: RRID:Addgene_174088)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cloning into the pcDNA3.4 was performed using FastDigest restriction enzymes EcoRI and BshT1 (Invitrogen) and Gibson assembly (NEB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.4</div><div>suggested: RRID:Addgene_131198)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 ORFs were amplified by PCR (KAPA HiFi HotStart ReadyMix, Roche) from the 2xStrep-tagged or 3xFLAG-tagged plasmids with oligonucleotide primers containing attB recombination sites and recombined into pDONR221 using BP clonase II (ThermoFisher) (Supplementary Table 6).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDONR221</div><div>suggested: RRID:Addgene_63743)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This study protocol was approved by the Institutional Review Board of UCSD’s Human Research Protections Program (181180).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Human Research Protections Program</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For reads mapped to the SARS-CoV-2 genome, bedgraph densities were generated using SAM tools v1.9 to obtain read densities at each nucleotide position.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SAM</div><div>suggested: (SAM, RRID:SCR_010951)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For reads mapped to the African Green Monkey genome, peaks were called on the usable reads by CLIPper54 and assigned to gene annotations in Ensembl ChlSab1.1 release 102 and Sars_cov_2 ASM985889 v3.101 were used to annotate peaks mapped to the African Green Monkey and SARS-CoV-2 genome.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Ensembl</div><div>suggested: (Ensembl, RRID:SCR_002344)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Gene Ontology analysis of eCLIP target genes was performed using ENRICHR (https://maayanlab.cloud/Enrichr/https://maayanlab.cloud/Enrichr/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ENRICHR</div><div>suggested: (Enrichr, RRID:SCR_001575)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cluster maps were visualized using Cytoscape version 3.8.1</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Cytoscape</div><div>suggested: (Cytoscape, RRID:SCR_003032)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">GENCODE v19 gene annotations and featureCounts (v.1.5.30) were used to create read count matrices.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>featureCounts</div><div>suggested: (featureCounts, RRID:SCR_012919)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequencing reads are first processed as RNA-seq libraries, where RNA-seq reads were trimmed of adaptor sequences using cutadapt (v1.4.0) and mapped to repetitive elements (RepBase v18.04) using STAR (v2.4.0i).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>STAR</div><div>suggested: (STAR, RRID:SCR_004463)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Multiple sequence alignment was performed using MAFFT v7.453 and default parameters, and sequence alignment was visualized using Jalview (version 1.0).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MAFFT</div><div>suggested: (MAFFT, RRID:SCR_011811)</div></div><div style="margin-bottom:8px"><div>Jalview</div><div>suggested: (Jalview, RRID:SCR_006459)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">De novo motif analysis: HOMER was used to identify de novo motifs using reads from IDR peaks.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HOMER</div><div>suggested: (HOMER, RRID:SCR_010881)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The length of each region within the metagene was then scaled to 8%, 62% and 30%, corresponding to the average length of regions from the most highly expressed transcripts in ENCODE HepG2 RNA-seq control datasets.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ENCODE</div><div>suggested: (Encode, RRID:SCR_015482)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Interval features such as introns and exons were extracted from the Gencode v19 coordinates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gencode</div><div>suggested: (GENCODE, RRID:SCR_014966)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Images were collected via Zeiss ZEN software and converted to tiff for downstream analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Zeiss ZEN</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Images were analyzed using a custom-developed pipeline in CellProfiler (v.3.1.09).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CellProfiler</div><div>suggested: None</div></div></td></tr></table>

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

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

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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: Please consider improving the rainbow (“jet”) colormap(s) used on page 64. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

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

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

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Female BALB/c mice (age 6-8 weeks, Envigo), housed in specific-pathogen free environments, were immunized intramuscularly by injection of 50 μL of vaccine formulated in endotoxin-free PBS (Gibco) into both hind limbs of each animal (100 μL total).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse monoclonal anti-hexon antibody (B025/AD51, Thermo-Fisher) was added at 1:1000 dilution in 1% w/v milk in PBS and incubated for 1 h at 25°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-hexon</div><div>suggested: (DSHB Cat# TC31-27F11.C2, RRID:AB_1553403)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were washed with 1% w/v milk in PBS prior to addition of a secondary goat anti-mouse alkaline phosphatase (ALP) conjugated antibody (STAR117A, BioRad) at 1:1000 dilution in 1% w/v milk in PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse alkaline phosphatase (ALP</div><div>suggested: (CEDARLANE Cat# CLF004ALP, RRID:AB_10060171)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Coupling efficiency was assessed by comparing band intensities of unconjugated hexon-Tag in ligand decorated samples to undecorated (control) samples using Image J: Anti-vector antibody neutralization assay: For assessment of vector neutralization by potent neutralising mouse monoclonal antibody (mAb) 9C12 (24) (Developmental Studies Hybridoma Bank, University of Iowa), Ad5 vectors expressing GFP were incubated with serially diluted mAb 9C12 antibody at a 1:1 ratio in complete media for 1 h at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-vector</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For assessment of vector neutralization by serum containing anti-adenovirus antibodies, serum samples were obtained by immunizing C57BL/6 mice with 1E+8 ifu of an Ad5 vector expressing ovalbumin (vector had an unmodified hexon).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-adenovirus</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Linearized DNA was transfected (Lipofectamine 2000, Invitrogen) into E1-complementing Human Embryonic Kidney (HEK) 293 cell lines; either 293A cells (Invitrogen) for Ad vectors expressing GFP and DogCatcher-NANP18, or 293TREX cells (Invitrogen) for Ad vectors expressing SARS-CoV-2 spike.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>293</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>293A</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>293TREX</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Titration of recombinant adenoviruses: Infectious titer of vector preparations was assessed by single cell infectivity assay on HEK293A cells or 293TREX cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293A</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Diluted serum was incubated with Ad5(GFP) vectors, the mix incubated on HEK293 cells, and bulk GFP fluorescence read 24 h later as described above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Assessment of coagulation Factor X-mediated vector transduction of SKOV3 cells: SKOV3 cells (human ovary adenocarcinoma) were obtained from Public Health England and cultured in McCoy’s 5a media with 2 mM Glutamine and 15% v/v fetal bovine serum (complete McCoy’s).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SKOV3</div><div>suggested: RRID:CVCL_5J11)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 pseudovirus neutralization (pVNT) assay: Pseudotyped HIV-1 viruses incorporating the SARS-CoV-2 full-length spike (Wuhan strain or B.1.1.7, B.1.617.2 or B.1.351 variants of concern) were generated and SARS-CoV-2 pVNT assays performed as previously described using Hela cells stably expressing ACE2 as target cells (28).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Hela</div><div>suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For assessment of vector neutralization by serum containing anti-adenovirus antibodies, serum samples were obtained by immunizing C57BL/6 mice with 1E+8 ifu of an Ad5 vector expressing ovalbumin (vector had an unmodified hexon).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Female BALB/c mice (age 6-8 weeks, Envigo), housed in specific-pathogen free environments, were immunized intramuscularly by injection of 50 μL of vaccine formulated in endotoxin-free PBS (Gibco) into both hind limbs of each animal (100 μL total).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bacterial artificial chromosome (BAC) sequences from pBELOBAC11 (NEB) were amplified using forward (5’-TTAATTAAcgtcgaccaattctcatg) and reverse (5’-TTAATTAAgtcgacagcgacacacttg) primers to introduce PacI sites at either end of the BAC cassette.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pBELOBAC11</div><div>suggested: RRID:Addgene_60342)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The entire Ad5(GFP) genome was subsequently cloned into the BAC with PacI, to generate pBAC-Ad5(GFP).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pBAC-Ad5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant vectors expressing DogCatcher-NANP18 and SARS-CoV-2 spike (residues 1-1208 Wuhan strain, codon-optimized for mammalian expression and including stabilizing mutations K986P and V987P and mutation of the furin cleavage site 682-GSAS-685 (60)) were generated through cloning of gene constructs into pENTR4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pENTR4</div><div>suggested: RRID:Addgene_26366)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein production and purification: DNA sequences for expression of DogCatcher, DogCatcher-NANP9, DogCatcher-NANP18 and SpyCatcher were cloned into expression plasmid pET45(+) (EMD Millipore) for protein production in BL21(DE3) E. coli (NEB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET45</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">DNA sequences for expression of DogCatcher fused to SARS-CoV-2 spike receptor binding domain (Wuhan strain, residues 319-532) (DogCatcher-RBD), were cloned into mammalian protein expression plasmid pcDNA3.4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.4</div><div>suggested: RRID:Addgene_131198)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Coupling efficiency was assessed by comparing band intensities of unconjugated hexon-Tag in ligand decorated samples to undecorated (control) samples using Image J: Anti-vector antibody neutralization assay: For assessment of vector neutralization by potent neutralising mouse monoclonal antibody (mAb) 9C12 (24) (Developmental Studies Hybridoma Bank, University of Iowa), Ad5 vectors expressing GFP were incubated with serially diluted mAb 9C12 antibody at a 1:1 ratio in complete media for 1 h at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Image J</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Automated data-collection was performed with Leginon software (65) at a nominal magnification of 28,000×, corresponding to a pixel size of 5.19 Å.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Leginon</div><div>suggested: (Leginon, RRID:SCR_016731)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistics: Statistical analyses were performed in GraphPad Prism 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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

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Further investigation will be required to comprehensively determine limitations for capsid display in terms of size and structure of ligands, but similar sized receptor-binding domains of other viral proteins, including influenza hemagglutinin (∼25 kDa), have been expressed independently as recombinant proteins and displayed on VLPs (56) implying that this could be a generalizable concept. Capsid decoration using our protein superglue technology is simple, requiring only co-incubation of spontaneously and irreversibly reacting components with no chemical modification required. A similar conjugation process has already been scaled under good manufacturing practice (GMP) during development of a VLP-based SARS-CoV-2 vaccine currently in Phase I/II clinical trials (19). The ability of our adenovirus-based platform to induce both robust cellular and humoral immunity and to enhance efficacy of multi-shot regimens could be advantageous for applications beyond prophylactic vaccines, including therapeutic vaccines against chronic viral pathogens and cancer. Methods of rapid and customizable covalent decoration of Ad capsids could also be utilized for development of personalized therapies. In prophylactic settings, adenovirus capsid decoration could be utilized in the design of pan-coronavirus and pan-influenza vaccines; combining broad and conserved T cell immunity from encoded antigens with exchangeable capsid ligands delivering potent neutralizing humoral immunity.

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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: Please consider improving the rainbow (“jet”) colormap(s) used on page 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

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

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

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About SciScore

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

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SciScore for 10.1101/2022.02.19.481107: (What is this?)

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

Table 1: Rigor

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

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The monoclonal antibodies SARS-CoV-1/SARS-CoV-2 Spike Protein S2 (1A9) and SARS-CoV-1/SARS-CoV-2 Nucleocapsid (6H3) were purchased from ThermoFisher Scientific.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-1/SARS-CoV-2 Nucleocapsid ( 6H3 )</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The rabbit polyclonal Anti-GAPDH antibody were purchased from Abcam.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-GAPDH</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The rabbit polyclonal Anti-HIV-1 p24 antibody was purchased from MilliporeSigma.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-HIV-1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mouse anti-N protein antibody (clone 1C7) was purchased from Bioss Antibodies, and the rabbit anti-SARS-CoV-2 spike protein (clone 007) antibody, was purchased from Sino Biological.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-N protein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">protein primary antibody and an anti-mouse IgG HRP secondary antibody in conjunction with SIGMAFAST™ OPD developing solution</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In parallel and after blocking, the second plate was incubated for one hour with a primary antibody solution formulated in PBS + 1% non-fat milk containing both mouse anti-N protein (1 μg/mL, clone 1C7) and rabbit anti-SARS-CoV-2 spike protein (1:500 dilution, clone 007) antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-N protein ( 1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 spike protein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">donkey anti-rabbit IgG Alexa Fluor Plus 594 (2 μg/mL, Invitrogen) antibodies and DAPI (1:1000, Millipore Sigma) in PBS + 0.5% BSA consisting of.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: (Thermo Fisher Scientific Cat# A48284, RRID:AB_2896348)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines, inhibitors, and antibodies: HEK293T (ATCC), HEK293T-ACE2 (kind gift of Hyeryun Choe, Scripps Research), HT1080 cells (ATCC) and Calu3 (ATCC) were cultured in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma), 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.3 mg/mL L-glutamine.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Fusion assays: For the syncytium formation assay HEK293T and HEK293T-Ace2 cells were seeded in 24-well plates and grown to approximately 80% confluency.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-Ace2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell-cell fusion assay with soluble ACE2, effector HEK293T cells were transiently transfected with plasmid DNA encoding mCherry, and SARS-CoV2 spike and target HEK293T cells were transiently transfected with plasmid DNA encoding LTR-GFP, TMPRSS2 or pCAGGS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Gelatin zymography: HEK293T, HEK293T-ACE2, Calu3 and HT1080 cells were analyzed for MMP2 and MMP9 activity through zymographic analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HT1080</div><div>suggested: CLS Cat# 300216/p517_HT-1080, RRID:CVCL_0317)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HT1080-ACE2 cells were cultured in DMEM supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), HEPES, L-Glutamine (0.3 mg/mL), 10% FBS (all from Thermo Fisher Scientific) and puromycin (1 μg/mL, InvivoGen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HT1080-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Twenty-four hours before infection, 2.5×104 HT1080 ACE2 cells were seeded per well of duplicate 96 well plates in puromycin-deficient DMEM and cultured overnight (37°C/5% CO2) for cell monolayer to adhere.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HT1080 ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HT1080 cells stably expressing ACE2 were generated by infection with lentiviral particles generated with psPAX2, pMDG and pLENTI_hACE2_PURO (gift from Raffaele De Francesco</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>pMDG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLENTI_hACE2_PURO</div><div>suggested: RRID:Addgene_155295)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The full gene, untagged or with a N-terminal FLAG tag, was reconstituted by Gibson assembly, amplified by PCR, and cloned in pCAGGS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_127347)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Time-course imaging of the syncytia formation was performed using an Incucyte-Zoom (EssenBioscience), and images were analyzed in imageJ to measure the percentage of green surface area over background.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>imageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analyses were performed with GraphPad Prism 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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

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

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

Results from rtransparent:

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

---

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

---

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About SciScore

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

</footer>

dear fellow citizens. today is a terrible day for ukraine and a gloomy day for europe. we all care and for peace

a name attribute

anchor on same page

This part stuck out to me because its quantifying race is a very tricks subject and trying to "tag" it into categories can be scene as very difficult and touchy.

Not a thesis, just an assertion of how important this discussion is

SciScore for 10.1101/2022.02.15.480546: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Since there was no antibody-hCoV229E spike protein complex in the training set of ScanNet, we used all the 55 networks trained for antibody binding site prediction (including the 11 used elsewhere that were not trained on SARS-CoV-1/2 data).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-hCoV229E</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plate was washed by the washing buffer to remove the unbound hACE2. 1:5,000 diluted Pierce™ High Sensitivity NeutrAvidin™-HRP (Thermo Fisher cat# 31030) or 1:7,500 diluted T7-tag polyclonal antibody-HRP (Thermo Fisher, cat# PA1-31449) were incubated with the plate for 1 hr at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-HRP</div><div>suggested: (Thermo Fisher Scientific Cat# PA1-31449, RRID:AB_1960906)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped SARS-CoV-2 neutralization assay: The 293T-hsACE2 stable cell line (Integral Molecular, cat# C-HA101, Lot# TA060720MC) and pseudotyped SARS-CoV-2 (Wuhan-Hu-1 strain D614G and Omicron) particles with luciferase reporters were purchased from the Integral Molecular.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-hsACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The isotonic regression fit was performed using scikit-learn (sklearn.isotonic.IsotonicRegression, default parameters)32. Mice: 8 weeks old female C57BL/6 mice were ordered from The Jackson Laboratory and housed in pathogen-free conditions at the core animal facility at the University of Pittsburgh Medical Center with the approval from the University of Pittsburgh Institutional Animal Care and Use Committee.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Construction of the MSA: Homologs of the WT RBD were first searched in the UniprotKB using BLAST.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>UniprotKB</div><div>suggested: (UniProtKB, RRID:SCR_004426)</div></div><div style="margin-bottom:8px"><div>BLAST</div><div>suggested: (BLASTX, RRID:SCR_001653)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The isotonic regression fit was performed using scikit-learn (sklearn.isotonic.IsotonicRegression, default parameters)32. Mice: 8 weeks old female C57BL/6 mice were ordered from The Jackson Laboratory and housed in pathogen-free conditions at the core animal facility at the University of Pittsburgh Medical Center with the approval from the University of Pittsburgh Institutional Animal Care and Use Committee.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>scikit-learn</div><div>suggested: (scikit-learn, RRID:SCR_002577)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The raw data were processed by Prism 9 (GraphPad) to fit into a 4PL curve and to calculate IC50/logIC50.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your code.

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

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

Results from rtransparent:

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• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
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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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A reference to the phrase "useless use of cat". See e.g. https://porkmail.org/era/unix/award.html

in browser

This is a perfect way to distibute this capability just add a script tag for it remove original submit event handler and provide your own

https://dweb.link/ipfs/bafybeic5r5yxjh5xpmeczfp34ysrjcoa66pllnjgffahopzrl5yhex7d7i

Use for add build tag in version

不懂场景，是说硬件的tag么？

• This integration method provides a default Razorpay Pay with Razorpay button that invokes the Checkout form.
• The Checkout form options are passed as data attributes inside a <script> tag.
• You can add any additional, hidden or visible fields to the form, which will be submitted along with the form.

Evaluation Summary:

The authors describe the hei-tag, which, when added to a genome editing enzyme, results in increased editing rates in fish embryos and mammalian cell culture. The hei-tag tool could provide a valuable alternative that can potentially boost genome editing efficiency in different species and systems. The wider applicability of this approach still requires further investigation, since the improvement of editing efficiency is so far supported by experimental data on only a few targets. It would also be important to learn how the authors' design decisions affect activity, especially when benchmarked against current state-of-art genome editing tools.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewer #1 (Public Review):

Improving the efficiency of genome engineering tools is a very important and competitive field. Moreover, the design of improved tools with a focus on models outside the mammalian cell editing dominated space is very welcome, as cross species activity should not be taken for granted. Thomas Thumberger et al focus on improving Cas9 activity in medaka and zebrafish embryos by rethinking about the "bells and whistles" attached to the enzyme in its commonly available variants.

They first develop an assay that links Cas9 activity to a phenotypic readout (retinal pigmentation controlled by oc2) in fish embryos. Using the first Cas9 variant reported to edit zebrafish embryos (Hwang et al 2013) as benchmark, they test an improved Cas9 variant (Zhang et al 2014) and their own heiCas9, and report up to 8-fold "enhanced activity" in medaka and 27-fold in zebrafish, when comparing to the Hwang et al variant. Improvements over the Zhang et al variant, tested only in medaka, were more modest.

Lacking a phenotypic assay in mammalian cells, the authors use genome editing outcomes deconvolution software (ICE and TIDE) on Sanger sequencing data to compare the editing efficiency of the Hwang variant, a commercially available Cas9, and heiCas9. HeiCas9 scores better in both analyses, and also shows a clear improvement at the ICE knockout score.

Finally, the authors move away from DSB inducing methods of genome editing and construct a heiBE4 (C to T base editor) variant. By adopting their phenotypic assay in medaka to introduce a stop codon (CAG>TAG) in oc2, they show stronger pigment loss phenotypes in injected embryos when using heiBE4, compared to the original BE4. They further quantify and confirm the high rate of C>T transitions by sequencing.

Overall, the manuscript is well written and the results are clearly presented. Boosting genome editing without modifying the primary sequence of the enzyme is a very interesting approach, and has been reported before (Liu et al 2021). Such methods could also be compatible with artificially evolved Cas9 variants (e.g. high fidelity, relaxed PAM recognition) or even other Cas enzymes (e.g. Cas12), providing an orthogonal approach to increase their activity.

The authors also provide evidence that the hei-tag is not restricted to the conventional DSB inducing approach, by trying a BaseEditor.

How their height-tag works to improve genome editing is not investigated in detail. Knowing the mechanistic underpinnings can help predict the usefulness or lack thereof across different organisms, developmental stages, or cell states. Moreover, the balance between ON- and OFF-target activity is not considered, an important parameter for cell culture experiments where outcrossing is not possible to segregate non-specific modifications of the genome. As a result, the mammalian cell culture data are interesting, but don't add much to the value of the hei-tag.

Reviewer #2 (Public Review):

The paper focuses on describing a novel tag (named hei-tag) consisting of two optimized NLS sequences and a Myc epitope separated by a linker peptide. This tag fused with Cas9 ORF increases the efficiency of genome editing in fish embryos following mRNA+sgRNA injection. The authors assess these results with a rigorous quantification of pigmentation reduction in fish embryos following the targeting of the pigmentation locus Oca2 in Medaka and zebrafish embryos. The same improved version of Cas9 is tested in mammalian cells in culture comparing the results with older version of Cas9. Finally the authors fuse the hei-tag to the base editor BE4-Gam showing also in this case an increased activity. The data would be more significant for the community if the injection of the Cas9 optimized construct would be tested as protein as this is the most efficient and commonly used approach in fish experiments. Similarly the comparison with the BE4-Gam should be extended to the more recent family of improved Cytidine Base Editors including the ancBE4Max that was optimized for nuclear shuttling among other properties. In the present manuscript the presence of the Myc epitope in the hei-tag is not tested in any of its possible applications and it remains unclear what is the utility of this part of the hei-tag system.

Reviewer #3 (Public Review):

In this manuscript, Thumberger et al. developed a novel high-efficiency tag to be used with existing CRISPR/Cas9 tools that boosted the editing efficiency in fish (medaka, zebrafish) and mammalian cell culture. Compared to the baseline gene editing methods chosen by the authors, hei-tag improved bi-allelic editing efficiency in medaka fish by about 30% and resulted in about 10% more indels in editing mammalian cells. The authors have also shown that hei-tag can be added to other Cas9-based techniques such as base editors to boost editing efficiency.

The authors have shown convincing evidence that hei-tag improves editing efficiency compared to the baseline methods they have chosen (JDS246-Cas9, myc-Cas9) in fish, demonstrating a boost in bi-allelic targeting efficiency of the oca2 gene in making eye pigment in medaka and zebrafish. Applications beyond gene knockout in fish have been carried out in mammalian cell culture, and also in base editing tools in medaka, implicating the potential broad application of the tool.

However, it is not clear under what scenarios hei-tag carries out significant and practical improvement compared to the state-of-art gene editing techniques. Especially concerning its original purpose of editing earlier and more efficiently in early embryos, the common strategy is to inject Cas9 protein rather than mRNA, which the authors did not account for. Even in the realm of RNA-based editing tools, it is questionable whether JDS246-Cas9, a construct originally made for mammalian gene editing, is the best baseline to compare to. Part of the ambiguity originates from a lack of systematic comparison of existing editing tools in the field, but the authors would need to ensure they are comparing to the state-of-art, and demonstrate the universality and limitation of hei-tag in practical use.

Overall, I think hei-tag would be a good addition to the exiting gene editing tools and has a potential to boost editing efficiency in many systems, although its practical improvement is yet to be solidly demonstrated. Further investigation of the impact of N-terminal tags and linker structure on Cas9 specificity and efficacy will be useful to guide future improvement of protein engineering.

SciScore for 10.1101/2022.02.08.479664: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: Stock cells were tested for mycoplasma contamination and authenticated by short-tandem repeat (STR) profiling.<br>Authentication: Stock cells were tested for mycoplasma contamination and authenticated by short-tandem repeat (STR) profiling.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibodies used were: anti-HIV-1 p24 (ARP432, CFAR), anti-GFP (sc-9996, SCBT), anti-HA (C29F4, CST), anti-Strep-tag</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Primary antibodies used were: anti-HIV-1 p24 ( ARP432 , CFAR) , anti-GFP ( sc-9996 , SCBT) , anti-HA ( C29F4 , CST)</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-HIV-1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-GFP</div><div>suggested: (Santa Cruz Biotechnology Cat# sc-9996, RRID:AB_627695)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Secondary antibodies include, anti-mouse (61-6520, Thermo Fisher)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: (Innovative Research Cat# 61-6520, RRID:AB_138451)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were labelled with the following primary antibodies, anti-Nup98 or anti-ORF6 diluted in 4% BSA in PBS for 1 hour at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Nup98</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-ORF6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were then incubated with the following secondary antibodies, donkey anti-rabbit-AF568 (ab175692, Abcam) and donkey anti-sheep AF568 (A-21099, Invitrogen) diluted in 4% BSA in PBS for 1 hour at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit-AF568</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>donkey anti-sheep AF568 (A-21099, Invitrogen) diluted in 4% BSA in PBS for 1 hour at RT.</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: HEK293T, HeLa, Mus dunni tail fibroblast (MDTF) and Vero cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher), supplemented with 10% heat-inactivated foetal bovine serum (FBS, Biosera) and 1% Penicillin/Streptomycin (Sigma).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunoprecipitation: HEK293T cells were grown in 10 cm2 culture plates transfected and incubated for 16 hrs after which media was replaced.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viruses were titrated by plaque assay on Vero E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmids used to generate HIV-1 and MLV VLPs for transduction of ORFs, pCMVΔ8.91 and pHIT60 respectively, and pVSV-G have been described before [38, 39].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMVΔ8.91</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pVSV-G</div><div>suggested: RRID:Addgene_138479)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the initial experiments on Gag expression (Figs 1A-C and S1A and B Figs) and ORF6 co-expression with Rae1 (Fig 2A), a pLVX-IRES-Puro vector encoding an individual ORF was used.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-IRES-Puro</div><div>suggested: RRID:Addgene_140240)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pLVX-StrepII-ORF-IRES-Puro plasmids were then cloned using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB) from the pTriEX-6 plasmids, using the primers listed in Table 1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-StrepII-ORF-IRES-Puro</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pTriEX-6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the remaining assays that required ORF detection or immunoprecipitation, a pLVX-EF1α-SARS-CoV-2-ORF-2xStrep/2xStrep-ORF -IRES-Puro encoding one of the nine ORFs or pLVX-EF1α-eGFP-2xStrep-IRES-Puro plasmid was used, referred to as twin-strep-tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-EF1α-SARS-CoV-2-ORF-2xStrep/2xStrep-ORF -IRES-Puro</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLVX-EF1α-eGFP-2xStrep-IRES-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These plasmids were kindly gifted from Nevan Krogan (Addgene plasmid #141383-4, #141387-90, #141392-95); http://n2t.net/addgene:141383-4, 141387-90, 141392-95; RRID:Addgene_141383-4, 141387-90, 141392-95) [5].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>#141383-4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate the pLVX-EF1α-CoV-1-ORF6-2xStrep-IRES-Puro plasmid, the ORF6(CoV-1)-2XStrep gene was synthesised by GeneArt and cloned in using the EcoRI and BamHI restriction sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-EF1α-CoV-1-ORF6-2xStrep-IRES-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ORF6 mutations were introduced into the pLVX-StrepII-SARS-CoV-2-ORF6-IRES-Puro and pLVX-EF1α-SARS-CoV-2-ORF6-2xStrep-IRES-Puro plasmids using the QuickChange II-XL site-directed mutagenesis kit (Agilent) according to the manufacturer’s instructions using SDM primers listed below.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-StrepII-SARS-CoV-2-ORF6-IRES-Puro</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLVX-EF1α-SARS-CoV-2-ORF6-2xStrep-IRES-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The following plasmids were sourced from Addgene, psfGFP-N1 (Addgene, #54737) and pmApple-N1 (Addgene, #54567).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pmApple-N1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pCMVsport6Rae1 (MGC:117333) was purchased from Horizon Discovery Biosciences Limited.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMVsport6Rae1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Virus-like particles (VLP) production: HIV-1 and MLV virus-like particles were generated by co-transfecting HEK293T cells with plasmids, pCMVΔ8.91 (HIV-1) or pHIT60 (MLV) and pVSV-G using lipofectamine 2000 (11668019, Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIT60</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For generating GFP reporter VLPs, pCSGW (HIV-1) or pczCFG2fEGFPf (MLV) were also added [39, 40].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCSGW</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Subcellular fractionation: HEK293T cells were transfected with psfGFP-N1 and either treated with LMB (Merck) at 5nM; or co-transfected with plasmids expressing pLVX-EF1α-ORF6, ORF6(M58A) or ORF9b [5].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psfGFP-N1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLVX-EF1α-ORF6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The following primers and probes were used; sfGFP: for 5’-GCGCACCATCAGCTTCAAGG, rev 5’-GTGTCGCCCTCGAACTTCAC and probe 5’-FAM-CGGCACCTACAAGACCCGCGC-TAMRA. mRNAseq: The quality of extracted RNA was checked by Agilent TapeStation 42000 on Agilent RNA ScreenTape (Agilent technologies) before proceeding to library preparation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Agilent TapeStation</div><div>suggested: (Agilent TapeStation Laptop, RRID:SCR_019547)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RNA-seq libraries were generated using the NEBNext Ultra Directional RNA Library Prep kit for Illumina with NEBNext® Poly(A) mRNA Magnetic Isolation Module (both New England BioLabs).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NEBNext®</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Poly(A</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Enriched GO terms (Biological process) were filtered and mapped using the REViGO tool available at http://revigo.irb.hr/ [41].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>REViGO</div><div>suggested: (REViGO, RRID:SCR_005825)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Normalisation and differential expression analyses were conducted within DESeq2 [43]: normalisation factors for gene-level read counts were produced using counts for the D. melanogaster spike-in controls and differential gene expression calculations were subsequently performed for Human genes alone using the pre-calculated size factors.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DESeq2</div><div>suggested: (DESeq, RRID:SCR_000154)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistics: Statistical analysis was performed using GraphPad Prism 9 software. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, ns – not significant).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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

Results from rtransparent:

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

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

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About SciScore

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

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SciScore for 10.1101/2022.02.07.479477: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">P43 crystals: Protein expression and purification: The gene encoding SARS-CoV-2 NSP3 Mac1 (residues 3-169) was cloned into a pET-22b(+) expression plasmid with a TEV-cleavable N-terminal 6-His tag (Genscript).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-22b(+)</div><div>suggested: RRID:Addgene_12651)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">crystals: Protein expression and purification: The gene encoding SARS-CoV-2 2-170 NSP3 Mac1 was cloned into a pET-11a plasmid (Bio Basic) and transformed into E. coli (BL21-DE3).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-11a</div><div>suggested: RRID:Addgene_154201)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After five macrocycles of refinement, the 2mFO-DFC (unfilled) and the mFO-DFC NSL density maps were inspected in Coot and modifications made to the coordinates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Phases were obtained by molecular replacement with Phaser using the same search model as the lower resolution X-ray experiment.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phaser</div><div>suggested: (Phaser, RRID:SCR_014219)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The number of contacts made by water molecules was defined as the number of protein nitrogen or oxygen atoms within 3.5 Å of a water, with distances calculated using PyMOL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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

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One caveat to using the neutron-determined orientations is that only orientations of highly ordered water molecules could be determined with high confidence (Fig. 5D/E). Fortunately, these waters are most frequently involved in bridging interactions. Simulation-based methods might yield an improved model of the orientations of disordered water molecules (65). We found that the conformational ensemble of Mac1 was unusually robust to temperature and pH perturbation in the crystal. Room temperature crystallography can reveal low occupancy, functionally relevant conformational states of proteins (51, 66–68). The 1.1 Å room temperature structure of Mac1 reported here, in combination with the previously reported 0.85 Å structure determined using the same crystal form at 100 K (14), allowed a detailed analysis of temperature dependent changes in structure and function. Overall, the structure and flexibility at 100 and 293 K were remarkably similar, indicating that unlike several recently reported examples (68–70), Mac1 displays minimal temperature dependent structural changes in crystallo (Fig. 4C). Similarly, assuming that the crystals have equilibrated to the target pH in the ∼4 hour soaks conducted here (71), the pH shift experiments revealed that the Mac1 active site residues and water networks are remarkably robust (Fig. 6I/J), and this is matched by the invariance in ADPr binding across the entire pH range (Fig. 6G/H). One caveat to the pH-shift experiments is the pH-dependent...

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

Results from rtransparent:

---

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2022.02.07.479468: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: Animal procedures were performed under the approvals of the Institutional Animal Care and Use Committee (IACUC) of University of Washington, Seattle, WA.<br>Euthanasia Agents: Mice were injected intramuscularly into the quadriceps muscle of each hind leg using a 27-gauge needle (BD, San Diego, CA) with 50 μL per injection site (100 μL total) of immunogen under isoflurane anesthesia.<br>IRB: This study was approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00010350)</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">BALB/c mice for RBD-NP, S ‘2P’, and HexaPro immunizations: Female BALB/c mice (Stock # 000651, BALB/c cByJ mice) four weeks old were obtained from Jackson Laboratory, Bar Harbor, Maine, and maintained at the Comparative Medicine Facility at the University of Washington, Seattle, WA, accredited by the American Association for the Accreditation of Laboratory Animal Care International (AAALAC).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">One day post-transfection, cells were infected with VSV(G*ΔG-luciferase)29 and after 2 h were washed five times with DMEM before adding medium supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant, CRL-2700, ATCC)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-VSV-G</div><div>suggested: (LSBio (LifeSpan Cat# LS-C51761-50, RRID:AB_1277784)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK293T cells in DMEM supplemented with 10% FBS, 1% PenStrep seeded in 10-cm dishes were transfected with the plasmid encoding for the corresponding S glycoprotein using lipofectamine 2000 (Life Technologies) following manufacturer’s indications.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus Neutralization: HEK293-hACE2 cells15 or VeroE6-TMPRSS214 were cultured in DMEM with 10% FBS (Hyclone) and 1% PenStrep and 8ug/mL puromycin for TMPRSS2 maintenance with 5% CO2 in a 37°C incubator (ThermoFisher).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS214</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate VSVΔG-based SARS-CoV-2 pseudovirus, BHK-21/WI-2 cells were transfected with the spike expression plasmid and infected by VSVΔG-firefly-luciferase as previously described30.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BHK-21/WI-2</div><div>suggested: RRID:CVCL_HB78)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentivirus encoding hACE2-P2A-TMPRSS2 was made to generate A549-hACE2-TMPRSS2 cells which were maintained in DMEM supplemented with 10% fetal bovine serum and 1µg/mL puromycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549-hACE2-TMPRSS2</div><div>suggested: RRID:CVCL_A5KB)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Both viruses were propagated on Vero-TMPRSS2 cells and subjected to deep sequencing to confirm the presence of expected substitutions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immune complexes were added to VeroE6-TMPRSS2 cell monolayers and incubated for 1 h at 37°C prior to the addition of 1% (w/v) methylcellulose in MEM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BALB/c mice for mRNA-1273 immunizations: Female BALB/c mice (6 to 8 weeks old) were obtained from Charles River Laboratories.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">1292S mice for mRNA-1273 immunizations: 129S2 mice (strain: 129S2/SvPasCrl, Cat # 287) were obtained from Charles River Laboratories and housed in a pathogen-free animal facility at Washington University in St. Louis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>129S2</div><div>suggested: RRID:IMSR_CRL:287)</div></div><div style="margin-bottom:8px"><div>129S2/SvPasCrl</div><div>suggested: RRID:IMSR_CRL:287)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmid construction: The SARS-CoV-2-RBD-Avi construct was synthesized by GenScript into pcDNA3.1-with an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag, flexible linker, and avi tag (GHHHHHHHHGGSSGLNDIFEAQKIEWHE).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-with</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 S ‘2P’ ectodomain trimer (GenBank: YP_009724390.1, BEI NR-52420) was synthesized by GenScript into pCMV with an N-terminal mu-phosphatase signal peptide and a C-terminal TEV cleavage site (GSGRENLYPQG)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_16459)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The RBD-16GS-I53-50A fusion was cloned into pCMV/R using the Xba1 and AvrII restriction sites and Gibson assembly25.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV/R</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus Neutralization Assay for BALB/c mRNA-1273 samples: Codon-optimized full-length spike genes (Wuhan-Hu-1 with G614, Beta, or Gamma) were cloned into pCAGGS vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_127347)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Animals were housed and maintained at the New Iberia Research Center (NIRC) of the University of Louisiana at Lafayette, an AAALAC International accredited institution, in accordance with the rules and regulations of the Guide for the Care and Use of Laboratory Animal Resources.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NIRC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Relative luciferase units were plotted and normalized in Prism (GraphPad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody-dose response curves were analyzed using non-linear regression analysis (with a variable slope) (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

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

---

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 10. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

---

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

---

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

---

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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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Say something.

D

SciScore for 10.1101/2022.02.07.22270617: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Study population, study design and recruitment: 51 SARS-CoV-2 naïve healthy volunteers (Supplementary Table 1) were recruited for the study under informed consent.<br>IRB: This study was approved (IRB No 2021-0898) by the Institutional Review Board (IRB) of Asan medical center in Korea.<br>Field Sample Permit: All methods were carried out in accordance with relevant guidelines and regulations.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">COVID-19 serology and ACE2-neutralization assay: A multiplexed solid-phase chemiluminescence assay (Meso Scale Discovery, MD) was evaluated for the detection of IgG binding to various SARS-CoV-2–derived antigens (V-PLEX SARS-CoV-2 Panel 17 (IgG) Kit, K15524U, and Panel 23 (IgG) Kit, K15567U) and the quantification of antibody-induced ACE-2 binding inhibition to various variants’ spike antigens (pseudo-neutralization assay) (V-PLEX SARS-CoV-2 Panel 18 (ACE2) Kit, K15535U, and Panel 23 (ACE2) Kit, K15570U).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>V-PLEX SARS-CoV-2 Panel 17 (IgG) Kit, K15524U,</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Panel 23 (IgG) Kit, K15567U</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were coated with the specific antigen on spots in the 96 well plate and the bound antibodies in the samples were then detected by anti-human IgG antibodies or ACE2 conjugated with the MSD SULPHO-TAG which is then read on the MSD instrument which measures the light emitted from the tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The raw data were subjected to QC analyses using the FastQC tool (version 0.11.9) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FastQC</div><div>suggested: (FastQC, RRID:SCR_014583)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">mRNA-seq read quality control was done using Trimmomatic37 (version 0.36) and STAR RNA-seq38 (version STAR 2.5.4a) using 150 bp paired-end mode was used to align the reads (hg19).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>STAR</div><div>suggested: (STAR, RRID:SCR_004463)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HTSeq39 (version 0.9.1) was to retrieve the raw counts and subsequently, Bioconductor package DESeq240 in R (https://www.R-project.org/) was used to normalize the counts across samples and perform differential expression gene analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Bioconductor</div><div>suggested: (Bioconductor, RRID:SCR_006442)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Differential expression gene (DEG) identification used Bioconductor package DESeq2 in R.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DESeq2</div><div>suggested: (DESeq, RRID:SCR_000154)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">P-values of cytokines were calculated using two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli on GraphPad Prism software (version 9.0.0).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

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

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Limitations of the study: There are several limitations to this study. First, our study population was limited to a specific geographic area (South Korea) and a specific genetic population. Second, most study subjects were healthy females of normal weight. Third, the homologous ChAd-ChAd cohort was smaller than the heterologous ChAd-BNT cohort. Fourth, our study did not investigate neutralizing antibodies using live or pseudo-SARS-CoV-2 virus and variants.

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

Results from rtransparent:

---

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2022.02.06.479332: (What is this?)

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

Table 1: Rigor

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

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 96 well plates were coated with His-Tag capture antibody and incubated at 4°C overnight then blocked with 1% BSA in PBS for 1 hour at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>His-Tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Serial dilutions of the selected antibodies were loaded in duplicates and incubated for 1 hour at room temperature, followed by the addition of biotinylated human ACE2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bead-based multiplex assay method for SARS-CoV-2 spike protein binding: A custom assay was designed using Magplex-Avidin microspheres (Luminex), which were coated in anti-HIS antibody [Biotin] (GenScript A00613, mouse IgG1k clone 6G2A9) at 2.5 μg/mL, washed, and then each microsphere was coated with 2.5 μg/mL of either SARS-CoV-1 S1-HIS, MERS S1-HIS, and SARS-CoV-2 S1-HIS or SARS-CoV-2 RBD-HIS variant proteins (Sino Biological) to create bead stocks.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-HIS</div><div>suggested: (GenScript Cat# A00613, RRID:AB_915572)</div></div><div style="margin-bottom:8px"><div>mouse IgG1k</div><div>suggested: (GenScript Cat# A00186, RRID:AB_914704)</div></div><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>S1-HIS</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies were incubated with microspheres for 30 minutes at RT with shaking at 300 rpm, washed 3x with PBS, then incubated with Goat anti-Human IgG Fc Secondary Antibody PE, eBioscience (minimal cross-reactivity to bovine/horse/mouse serum proteins)(Thermo Fisher Scientific 12-4998-82) diluted 1:200 in PBS/1% BSAfor 30 minutes.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Human IgG</div><div>suggested: (Thermo Fisher Scientific Cat# 12-4998-82, RRID:AB_465926)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the recommended amount of particles/well was incubated in DMEM + 10% FBS with varying amounts of serially-diluted antibody for 1 hour at 37°C, and then 20,000 ACE2-HEK293T cells (Integral Molecular, see Extended Table 5) were added.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2-HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mutant library was arrayed in 384-well microplates and transiently transfected into HEK-293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Results were analyzed in GraphPad PRISM Version 9.3.0.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad PRISM</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Structural analysis: The figures were rendered using PyMOL Version 2.4.1 (The PyMOL Molecular GraphicsSystem; http://www.pymol.Org).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Multiple alignment of related lineage B betacoronavirus spike protein sequences: Multiple alignment was made using Clustal Omega (Madeira, 2019) and image rendered using Geneious software (Kearse, 2012).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Clustal Omega</div><div>suggested: (Clustal Omega, RRID:SCR_001591)</div></div><div style="margin-bottom:8px"><div>Geneious</div><div>suggested: (Geneious, RRID:SCR_010519)</div></div></td></tr></table>

---

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

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

---

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

Results from rtransparent:

---

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

---

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

---

<footer>

About SciScore

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

</footer>

formal stuff annoys you

prefer practical vs. prefer theoretical/academic

First Annotations on the Hypothes.is site

One thing I find interesting here is how intimidating I find it to consider a proper controlled term / hierarchical system. Maybe the improvements here need to be around UX to curate tags toward proper controlled vocabularies -- do many PKM systems have the concept of a tag redirect, such that my "vampire" can be bucketed in with "vampires" (but unbucketed if necessary)?

SciScore for 10.1101/2022.02.03.479007: (What is this?)

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

Table 1: Rigor

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

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">AlexaFluor-647-conjugated goat anti-human IgG (H+L) Ab (Invitrogen) and AlexaFluor-conjugated donkey anti-goat IgG (H+L) Ab (Invitrogen) was used as secondary antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>AlexaFluor-conjugated donkey anti-goat IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">An anti-mouse SARS-CoV-2 nucleocapsid protein (Clone 1C7, Bioss Antibodies)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse SARS-CoV-2 nucleocapsid protein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following extensive washing (3×) with PBS, an anti-mouse IgG HRP secondary antibody solution was formulated in PBS + 1% non-fat milk.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 293T-ACE2 cell line was previously reported (Prevost et al., 2020)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 293T cells were transfected by the calcium phosphate method with the lentiviral vector pNL4.3 R-E-Luc (NIH AIDS Reagent Program) and a plasmid encoding for SARS-CoV-2 Spike at a ratio of 5:4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All virus-compounds supernatant was removed from wells without disrupting the Vero E6 monolayer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell viability test: To measure the cytotoxicity of VE607 and its stereoisomers on 293T-ACE2 or Vero-E6 cells, a cell viability assay using CellTiter-Glo® One Solution Assay (Promega) was performed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The codon-optimized RBD sequence (encoding residues 319-541) fused to a C-terminal hexahistidine tag was cloned into the pcDNA3.1(+) expression vector and was reported elsewhere (Beaudoin-Bussieres et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1(+)</div><div>suggested: RRID:Addgene_129020)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmid encoding the Delta (B.1.617.2) and Omicron (B.1.1.529) Spikes were generated by overlapping PCR using a codon-optimized wild-type SARS-CoV-2 Spike gene (GeneArt, ThermoFisher) that was synthesized (Biobasic) and cloned in pCAGGS as a template (Chatterjee et al., 2021; Gong et al., 2021b; Tauzin et al., 2022).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_127347)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The vesicular stomatitis virus G (VSV-G)-encoding plasmid (pSVCMV-IN-VSV-G) was previously described (Emi et al., 1991).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VSV-G)-encoding</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pSVCMV-IN-VSV-G</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 293T cells were transfected by the calcium phosphate method with the lentiviral vector pNL4.3 R-E-Luc (NIH AIDS Reagent Program) and a plasmid encoding for SARS-CoV-2 Spike at a ratio of 5:4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pNL4.3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell surface staining and flow cytometry analysis: Using the standard calcium phosphate method, 10 μg of Spike expressor and 2.5 μg of a green fluorescent protein (GFP) expressor (pIRES-GFP) were transfected into 2 × 106 293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pIRES-GFP</div><div>suggested: RRID:Addgene_78264)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VE607 compound was structurally preprocessed using LigPrep (Schrödinger, 2020) to generate multiple states for stereoisomers, tautomers, ring conformations, and protonation states at a selected pH range.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>LigPrep</div><div>suggested: (Ligprep, RRID:SCR_016746)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Samples were acquired on a LSRII cytometer (BD Biosciences, Mississauga, ON, Canada) and data analysis was performed using FlowJo vX.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Densitometry data were acquired with a Typhoon Trio Variable Mode Imager (Amersham Biosciences) in storage phosphor acquisition mode and analyzed using ImageQuant 5.2 (Molecular Dynamics).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageQuant</div><div>suggested: (ImageQuant, RRID:SCR_014246)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quantification and statistical analysis: Statistics were analyzed using GraphPad Prism version 8.0.2 (GraphPad, San Diego, CA, (USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

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

---

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Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

Results from rtransparent:

---

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

---

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

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About SciScore

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

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Author Response:

Reviewer #2 (Public Review):

This work aimed to advance knowledge of the roles of polycystin-1 and polycystin-2 (PC-1, PC-2) in the vascular endothelium. For this, the authors developed tamoxifen-inducible Cre-lox models to delete PC-1, PC-2 or both specifically in endothelial cells of mice. Evidence is presented that flow or sheer stress activates PC-1-dependent current in endothelial cells, which is associated with NOS and KCa channel activation, smooth muscle hyperpolarization, and flow-dependent vasodilation. The Jaggar laboratory has recently reported that deletion of endothelial PC-2, a member of the TRP family, leads to loss of flow-induced Ca2+ influx, NOS and SK/IK activation, reduced vasodilation, and higher blood pressure. Thus, the novelty of the current work is the finding that PC-1 is similarly critical for activation of this pathway by flow, and that it is a physical interaction between membrane-localized PC-1 and PC-2 that underlies complex activation by flow.

Strengths of the current study include the use of powerful inducible knockout models in combination with a wide array of in vivo and ex vivo methods to test hypotheses. Thus, conclusions are based on multiple approaches and are mostly well supported. However, there are some concerns, specifically related to a lack of clarity on the interactions and purported interdependence between PC-1 and PC-2 that warrant further consideration.

1.The prospective impact of the current study is based on the suggestion that interactions between PC-1 and PC-2 via coiled-coil domains are required for activation of inward current by flow. However, the authors did not show evidence, via fluorescence imaging or otherwise (e.g., coIP), that peptides generated to disrupt this interaction actually do so. Does treatment with the coiled-coil domain peptides cause a shift in the PC-1-to-PC-2 distance (using TIRF-SMLM as in Fig 5)?

We have performed new experiments and now show that scrambled peptides of either the PC-1 or PC-2 coiled-coil domains do not alter flow-activated I_Cat in endothelial cells (Figure 6E-G, Figure 6 – figure supplement 2). In contrast, peptides corresponding to the coiled-coil domains present in PC-1 or PC-2 similarly inhibit flow-activated cation currents in endothelial cells.

Multiple different domains in PC-1 and PC-2 physically interact to form the heteromeric complex. Several groups have demonstrated that PC-1 and PC-2 couple via their C-terminal coiled-coils (Qian et al, Nat. Genet. 1997; Zhu et al., PNAS 2011; Yu et al., PNAS 2009; Tsiokas et al., PNAS 1997). Recombinant PC-1 and PC-2 lacking coiled-coils also interact via N-terminal loops (Babich et al, JBC 2004; Feng et al., JBC 2008). The structure of a PC-1/PC-2 heterotetramer that lacked N- and Ctermini was resolved using cryo-EM and indicated that a region between TM6 and TM11 of PC-1 interdigitates with PC-2 (Su et al, Science 2018). As such, it is unlikely that that the coiled-coil domain peptides physically separate PC-1 and PC-2 subunits. Rather, these data suggest that coiled-coil domain coupling in PC-1 and PC-2 is required for flow to activate non-selective cation currents in endothelial cells. In response to your comment, we have expanded discussion of this point in the manuscript.

2.The use of immunoFRET to test for PC-1/PC-2 proximity is not ideal. At minimum, proper negative controls (e.g., use of cells from KO models) should be provided to demonstrate the specificity of this technique for PC-1/PC-2 interactions in endothelial cells.

We agree. As suggested, we have performed new experiments and now provide immunoFRET data for Pkd1 ecKO and Pkd2 ecKO endothelial cells (Figure 4B, C; Figure 4 - figure supplement 1A, B). These data show that N-FRET between PC-1 and PC-2 antibodies is extremely low in Pkd1 ecKO and Pkd2 ecKO endothelial cells.

3.The authors conclude that PC-1/PC-2 clusters in KO cells in SMLM experiments are likely due to non-specific antibody binding. While I agree with this, it raises a question as to the meaning of cluster size data. Considering that the approach relies on fluorophore-tagged antibodies, which cannot be assumed to be in 1:1 stoichiometry with proteins of interest, how relevant is cluster size?

This is an interesting question. All immunofluorescence techniques rely on the use of antibodies to tag proteins. We recognize that the size of clusters reflects the size of both the proteins and antibodies. We now include text in the Discussion stating that the size of the PC-1 and PC-2 clusters reported is the size of both the proteins and the antibodies.

4.Based on data shown in Figure 1, the authors conclude that there is a reduction in inward current with flow. Since the applied technique measures total current, couldn't this result also reflect an increase in outward current (e.g., K+) due to flow that depends on the presence of Ca2+? Also related to these data, the magnitude of initial flow-induced transient current was quite variable (~8 - ~45 pA). Was this due to differences in cell size? The authors should consider expressing data from current recordings in terms of density (pA/pF).

We agree that presenting data as current density is useful and now do so throughout the manuscript, including in figure 1. The results in figure 1 do reflect a flow-activated increase in K^+ current as this response is partially inhibited by apamin/tram-34 (Mackay et al. eLife 2020). We state that there is “a reduction in inward current” as the entire current range in these experiments is negative of 0 pA.

5.Endothelium-specific deletion of PC-1 increased blood pressure, implying that the proposed role for PC-1 is generally applicable to the resistance arterial network; yet here, only small mesenteric vessels were studied. Given the known heterogeneity in the regulation of vascular tone by sheer stress among different arterial beds, is the identified role of PC-1 observed outside of the mesenteric circulation?

We agree that the blood pressure phenotype in Pkd1 ecKO mice suggest that flowactivates PC-1 in endothelial cells of other vascular beds to induce vasodilation. We have now discussed this concept in the manuscript.

SciScore for 10.1101/2022.02.01.478647: (What is this?)

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

Table 1: Rigor

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

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The PVDF membranes were incubated with primary antibodies against HA-Tag (C29F4) (3724, CST, USA) or GAPDH (30201ES20, Yeasen, China), then incubated with second antibodies against IgG (Yeasen, China)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HA-Tag</div><div>suggested: (Cell Signaling Technology Cat# 3724, RRID:AB_1549585)</div></div><div style="margin-bottom:8px"><div>GAPDH</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then, second antibodies FITC-AffiniPure Goat Anti-Rabbit IgG (33107ES60, Yeasen, China) and Cy3-AffiniPure Goat Anti-Mouse IgG (33208ES60, Yeasen, China)were used to incubate the cells at a 1:1,000 dilution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Rabbit IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-Mouse IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture and treatment: Vero E6 and Raw 264.7 cells were grown in 90% DMEM basal medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C under 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Raw 264.7</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">It was propagated and titrated with Vero E6 cells, and its associated operations were performed in a biosafety level 3 (BSL-3) facility.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Determination of lysosome pH: Vero cells were transfected with Lyso-pHluorin (addgene, 70113) and 2-E plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vector pET28a was used for protein purification; vector pcDNA5 and pcDNA3.1 were used for cell survival assay and cell imaging.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET28a</div><div>suggested: RRID:Addgene_114145)</div></div><div style="margin-bottom:8px"><div>pcDNA5</div><div>suggested: RRID:Addgene_49428)</div></div><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Determination of lysosome pH: Vero cells were transfected with Lyso-pHluorin (addgene, 70113) and 2-E plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>2-E</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data were processed using pClamp 10.2 software (Molecular Devices, US).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pClamp</div><div>suggested: (pClamp, RRID:SCR_011323)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The percentages of differently labeled cells were calculated by FlowJo 7.6.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometric data were collected and quantified using CytoExpert 2.4 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CytoExpert</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These ten structures were employed to construct ten homology models of the mutant envelope protein using Modeller [A. Sali, T.L. Blundell, Comparative protein modelling by satisfaction of spatial restraints, J Mol Biol 234 (1993) 779-815. 10.1006/jmbi.1993.1626.].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Modeller</div><div>suggested: (MODELLER, RRID:SCR_008395)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistics were performed in GraphPad Prism.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

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

---

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

Results from rtransparent:

---

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

---

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

---

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About SciScore

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

</footer>

To find this urn address for your .pdf files and their fingerprints (particularly local/private files not otherwise hosted in the cloud):

1. Visit https://jonudell.info/h/facet/
2. Search for your Hypothes.is username (and perhaps a tag) to find the document name and annotations you made on it.
3. You should be able to click on the document title which will take you to a non-loading web page with an address that looks something like this: urn:x-pdf:1ab23cd45e678fgh9012i34j56k78l90

If others know of alternate/faster methods of finding URIs for local pdf files I'd be happy to hear about them.

If you've annotated local (private) files within your browser using Hypothes.is, you'll need to find the uri path (a rough equivalent to http address for web pages) for your .pdf file with its "fingerprint" (a long unique number).

To do this 1. Visit https://jonudell.info/h/facet/ 2. Search for your Hypothes.is username (and perhaps a tag) to find the document name and annotations you made on it. 3. You should be able to click on the document title which will take you to a non-loading web page with an address that looks something like this: urn:x-pdf:1ab23cd45e678fgh9012i34j56k78l90 4. Copy and paste that address you get into Hypothesidian as the address for an appropriate option like "Retrieve my annotations for a web article or web pdf". 5. Hit enter/return. 6. Hypothesidian should return the appropriate annotations for that document.

If others know of alternate/faster methods of finding URis for local pdf files I'd be happy to hear about them.

Suppose the server were sending rel=canonical in a Link header for non-document resources (e.g. images like this one). Is the Hypothesis bookmarklet/extension thorough enough to deal with this?

SciScore for 10.1101/2022.01.31.478460: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blots were incubated either with anti-His antibody (6x-His Tag Monoclonal Antibody (HIS.H8), Alexa Fluor 488; ThermoFisher Scientific-MA1-21315-A488; 1:5000) or with anti-Strep-tag mouse monoclonal antibody (anti-Strep-tag mouse monoclonal, C23.21; PROGEN-910STR; 1:5000) overnight at 4°C in dilution buffer (TBS-T containing 5% bovine serum albumin (BSA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Strep-tag</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK 293T cells were seeded onto 96-well white plates before 24 h of transfection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For this, fragments BstXI-mNG-Mpro-Nter-auto-NLuc-XhoI and BstXI-mNG-Mpro-Nter-auto-L-NLuc-XhoI were synthesized (Integrated DNA Technologies, IDT; Iowa, USA) and inserted into pIDTSmart (Kan) vectors to generate the plasmid constructs pIDT-mNG-Mpro-Nter-auto-NLuc and pIDT-mNG-Mpro-Nter-auto-L-NLuc, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pIDT-mNG-Mpro-Nter-auto-NLuc</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pIDT-mNG-Mpro-Nter-auto-L-NLuc</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Restriction enzymes BstX-I and XhoI were used to excise the two DNA fragments of interests from entry clones pIDT-mNG-Mpro-Nter-auto-NLuc and pIDT-mNG-Mpro-Nter-auto-L-NLuc and ligated into similarly digested destination plasmid pmNeonGreen-DEVD-NLuc [Addgene: 98287]49 and further confirmed by Sanger sequencing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pmNeonGreen-DEVD-NLuc</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For bacterial expression and purification of the Mpro sensor, the mNG-Mpro-Nter-auto-NLuc plasmid construct was digested with HindIII and XhoI and the mNG-Mpro-Nter-auto-NLuc fragment was subcloned into similarly digested pET-28b(+) plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>mNG-Mpro-Nter-auto-NLuc</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pET-28b(+ )</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Live cell, BRET-based Mpro proteolytic cleavage activity assays: Live cell Mpro proteolytic cleavage activity assays were performed by co-transfecting HEK 293T cells with either the pmNG-Mpro-Nter-auto-NLuc or the pmNG-Mpro-Nter-auto-L-NLuc Mpro sensor plasmid constructs along with either pLVX-EF1alpha-SARS-CoV-2-nsp5-2xStrep-IRES-Puro (Mpro WT) (a gift from Nevan Krogan (Addgene plasmid # 141370; http://n2t.net/addgene:141370 ; RRID:Addgene_141370)85or pLVX-EF1alpha-SARS-CoV-2-nsp5-C145A-2xStrep-IRES-Puro (C145A mutant Mpro) plasmid (a gift from Nevan Krogan (Addgene plasmid # 141371 ; http://n2t.net/addgene:141371 ; RRID:Addgene_141371)85 in 96-well white flat bottom plates (Nunc; 136101).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pmNG-Mpro-Nter-auto-L-NLuc</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_141370)</div></div><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_141371)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For dose-response experiments, the filler plasmid (a pcDNA3.1-based plasmid) is also co-transfected.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-based</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Live cell Mpro proteolytic cleavage inhibitor assay: HEK 293T cells were co-transfected with either pmNG-Mpro-Nter-auto-NLuc or pmNG-Mpro-Nter-auto-L-NLuc plasmid along with either pLVX-EF1alpha-SARS-CoV-2-nsp5-2xStrep-IRES-Puro (Mpro WT) (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pmNG-Mpro-Nter-auto-NLuc</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Live cell, FlipGFP-based Mpro proteolytic assay: For live cell FlipGFP-based Mpro proteolytic activity assays, HEK 293T cells were seeded onto 24-well plates and co-transfected with the FlipGFP sensor plasmid (pcDNA3 FlipGFP(Mpro)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3</div><div>suggested: RRID:Addgene_15475)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mpro N-terminal autocleavage sequence analysis: A total of 1984 sequences for the SARS-CoV-2 pp1a polyprotein available at the NCBI Virus database (https://www.ncbi.nlm.nih.gov/genome/viruses/) were downloaded and aligned using MAFFT server (https://mafft.cbrc.jp/alignment/server/)63, 64.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>https://www.ncbi.nlm.nih.gov/genome/viruses/</div><div>suggested: (NCBI Viral Genomes, RRID:SCR_013789)</div></div><div style="margin-bottom:8px"><div>MAFFT</div><div>suggested: (MAFFT, RRID:SCR_011811)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Models were generated using MODELLER (10.1 release, Mar. 18, 2021)66.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MODELLER</div><div>suggested: (MODELLER, RRID:SCR_008395)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The NAMD output structure was then used as an input for Gaussian accelerated molecular dynamics (GaMD) simulation utilizing the integrated GaMD module in NAMD and its default parameters73, 74 which included 2 ns of conventional molecular dynamics (cMD) equilibration run in GaMD, to collect potential statistics required for calculating the GaMD acceleration parameters, and another 50 ns equilibration run in GaMD after adding the boost potential74, 75, and finally GaMD production runs for 1000 ns.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NAMD</div><div>suggested: (NAMD, RRID:SCR_014894)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Restriction enzymes BstX-I and XhoI were used to excise the two DNA fragments of interests from entry clones pIDT-mNG-Mpro-Nter-auto-NLuc and pIDT-mNG-Mpro-Nter-auto-L-NLuc and ligated into similarly digested destination plasmid pmNeonGreen-DEVD-NLuc [Addgene: 98287]49 and further confirmed by Sanger sequencing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Addgene</div><div>suggested: (Addgene, RRID:SCR_002037)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The ImageJ macro script used for the analysis is provided in the Supporting Text.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data analysis and figure preparation: GraphPad Prism (version 9 for macOS, GraphPad Software, La Jolla California USA; www.graphpad.com), in combination with Microsoft Excel, was used for data analysis and graph preparation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>Microsoft Excel</div><div>suggested: (Microsoft Excel, RRID:SCR_016137)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Figures were assembled using Adobe Illustrator.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Adobe Illustrator</div><div>suggested: (Adobe Illustrator, RRID:SCR_010279)</div></div></td></tr></table>

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

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

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

Results from rtransparent:

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

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

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About SciScore

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

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SciScore for 10.1101/2022.02.01.478657: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: Mice: BALB/c mice (Taconic Biosciences Inc.) 2-3 months age (female) were purchased and housed in the Emory University Division of Animal Resources (DAR) facility and used according to the University IACUC guidelines.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Mice: BALB/c mice (Taconic Biosciences Inc.) 2-3 months age (female) were purchased and housed in the Emory University Division of Animal Resources (DAR) facility and used according to the University IACUC guidelines.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies and proteins: Purified anti-mouse GM-CSF (clone MP1-22E9) and anti-mouse IL-12 (clone C17.8) were from BioXcell and used for affinity chromatography purification of GPI-RBD-GM-CSF fusion protein and GPI-IL-12, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse GM-CSF</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>GPI-IL-12</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-spike RBD antibody (clone MM57) obtained from Sino Biologicals (Cat#40592).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-spike RBD antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-spike RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FITC-conjugated goat secondary antibody against mouse IgG/IgM was purchased from BD Pharmingen (Cat# 555988).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>mouse IgG/IgM</div><div>suggested: (Novus Cat# NBP1-75215, RRID:AB_11005727)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Peroxidase (HRP)-conjugated goat anti-mouse IgG F(ab’)2 specific antibody was from ThermoFisher Scientific/Pierce (Cat#31436).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: (Thermo Fisher Scientific Cat# 31436, RRID:AB_228313)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HRP-conjugated donkey anti-human IgG antibody was obtained from Jackson Immunoresearch (Cat#709-036-098).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HRP-conjugated donkey anti-human IgG antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 709-036-098, RRID:AB_2340497)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression of both the S1 RBD and GM-CSF on the surface of transfected CHO-S cells was confirmed by flow cytometry using fluorophore conjugated antibodies against RBD, Clone MM57 (Sino Biologicals) and GM-CSF, Clone MP1-22E9 (BioLegend).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GM-CSF</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After transfer, the membranes with the proteins were blocked for 1 hr at room temperature with 5 % milk in phosphate buffered saline and 0.2 % Tween 20 (PBS-T) and incubated with a primary antibody (anti-RBD, anti-mouse GM-CSF or anti-mouse IL-12) overnight at 4 °C in PBS-T with on a shaker at low speed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Next day, membranes were washed three times with PBS-T and then incubated with appropriate secondary antibody conjugated with alkaline phosphatase that provides a visual color change upon addition of the chromogenic substrate (mixture of BCIP (5-bromo-4chloro-3-indolyl phosphate-catalog# 34040) and NBT (nitro-blue tetrazolium chloride, catalog# 34035 from Thermo Scientific)).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>5-bromo-4chloro-3-indolyl phosphate-catalog# 34040</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Resulting incorporation was detected by western blot and flow cytometry analysis using anti-RBD mAb, clone MM57 (Sino Biologicals), anti-mouse GM-CSF (clone MP1-22E9, BioLegend), and anti-mouse IL-12 (clone C17.8, Invitrogen) antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IL-12</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Enzyme-linked immunosorbent assay (ELISA): SARS-CoV-2 S protein RBD specific antibodies of different subtypes (IgG, IgG1, IgG2a) were determined in sera by enzyme-linked immunosorbent assay (ELISA) using an approach similar to one previously described using PR8 or WSN as targets (44, 45).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG1, IgG2a</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were washed and diluted secondary antibody (HRP-conjugated) against mouse total IgG or immunoglobulin isotypes (IgG1, IgG2a) were added and incubated for 30 minutes at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>mouse total IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To measure influenza antigen-specific antibody levels in immune sera, inactivated A/PR8 H1N1 virus (200 ng/well) was coated onto ELISA plates, followed by addition of diluted immune sera.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antigen-specific</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IgG isotypes were measured using goat anti-mouse immunoglobulin (Ig) G, IgG1 and IgG2a, and horse-radish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotechnology).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse immunoglobulin (Ig) G, IgG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plaque Reduction Neutralization Test (PRNT): The titers of anti-SARS-CoV-2 neutralizing antibodies were measured in the serum of BALB/c mice using PRNT assay as described previously (41).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 neutralizing</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For mouse sera, CHO-S cells were incubated with diluted serum samples (100-100,000 times diluted in FACS buffer).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO-S</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibody-virus mixture was then added to Vero cells and incubated at 37° C for 1 hour.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mice: BALB/c mice (Taconic Biosciences Inc.) 2-3 months age (female) were purchased and housed in the Emory University Division of Animal Resources (DAR) facility and used according to the University IACUC guidelines.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This construct was cloned into the pCHO 1.0 vector using the AvrII and BstZ17l sites (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCHO 1.0</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Peroxidase (HRP)-conjugated goat anti-mouse IgG F(ab’)2 specific antibody was from ThermoFisher Scientific/Pierce (Cat#31436).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ThermoFisher Scientific/Pierce</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After incubation with secondary antibody, cells were washed with FACS buffer and resuspended in FACS buffer and acquired in a FACSCalibur (BD Biosciences) flow cytometer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FACSCalibur</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data analyzed using FlowJo software (FlowJo LLC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The FRNT-mNG50 titers were interpolated using a 4-parameter nonlinear regression in GraphPad Prism 8.4.3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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

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

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The limitation of the current study is that a comparative analysis of GPI-RBD-GM-CSF and GPI-RBD was not carried out to demonstrate the contribution of GM-CSF as an adjuvant in VLP vaccine. Attempts to make GPI-RBD in CHO cells were not successful. Further, our study did not investigate whether incorporating VLPs with different amounts of GPI-anchored fusion protein results in stronger immune response to SARS-CoV-2. However, the comparison of antibody response induced by purified GPI-RBD-GM-CSF molecule with RBD-His-Tag suggests that GM-CSF stimulated antibody production against RBD. Further, our study focused only on the RBD domain of SARS-CoV-2 S protein but not the full-length S protein which may limit the breadth of protective immune response. In summary, our results demonstrate that influenza VLP-based delivery of SARS-CoV-2 RBD protein in combination with cytokine adjuvants can be used as a platform to develop multivalent vaccines targeting the variant strains of viruses which are currently observed in ongoing SARS-CoV-2 pandemic. Our fusion protein vaccine design also allows for creation of fusion proteins with new variant sequences and quickly purify using anti-GM-CSF mAb affinity chromatography. Further, use of immobilized cytokines as adjuvants will provide a safer way to induce anti-viral immunity with minimal side-effects.

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

Results from rtransparent:

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

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

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About SciScore

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

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Author Response:

Reviewer #1 (Public Review):

This manuscript is a follow-up of an earlier manuscript using the LRET technology, but extends the study by identifying a new "open" state and using experimental distance constraints to provide molecular models of the different states. All in all, the manuscript is well written, the experiments are described in sufficient details and experiments are done to high quality with the appropriate controls. The data corroborate the partially open state as published early, but extend the study to a second, open state. It is very good to see that the observed states are not only present in the catalytic head but the authors also use the full-length protein and find similar states. However, in the present manuscript, I find the conceptual advance with respect to the mechanism of MR somewhat limited. The authors curiously do not include any DNA in their structural studies, so the observed states are only relevant for the free MR complex, but not the complex "in action" bound to DNA where quite different conformations might occur. As one consequence, the structurally proposed states do not directly correlate with the functional nuclease states that are necessarily bound to DNA. Perhaps as a consequence, in the author's model, Rad50 is merely a gate-keeper for Mre11, but this is not the case as recent structural work shows that Rad50 forms a joint DNA binding surface with Mre11. Likewise, biochemical studies are done with physiologically unclear/less relevant 3' exonuclease activity only, but not with the physiological important 5' endonuclease activity. In my opinion, it is important for a publication in a journal with the scope of eLife and addressed to a broad audience to provide structural analysis in the presence of DNA and validate the structures using the endonuclease activity.

We thank the reviewer for these comments.

Specific recommendations:

1) Instead of using the physiological unclear exo activity, I suggest to use the more relevant endonuclease activity to validate the mutants.

We now include plate- and gel-based endonuclease activity assays, using a variety of DNA substrates, for all of the validation mutants. We have expanded Fig. 3 and included a new Supplemental Fig. S4 to show this data. We have expanded the Results section of the modified manuscript to present and discuss these findings.

2) Since the authors mutated one side of newly identified/proposed salt-bridges, I also suggest to test whether a charge reversal on both sides of the salt bridge rescues the phenoptype. I find this important because MR has quite many conformations, and mutating a single residue might not unambiguously validate the proposed conformation, a rescue by a charge reversed salt bridge is much stronger.

We thank the Reviewer for this suggested experiment, and we tried to do it. Although we were successful in generating each of the charge reversal mutations in full-length Rad50, all of the mutants unfortunately had issues with either expression or purification. For example, the 6x His-tag for several of the new Rad50 mutants was not accessible to the TEV protease for cleavage indicating that the mutated proteins were mis-folded (the His-tag of the WT full-length Rad50 is readily cleaved off by TEV). As such, we did not feel confident using these proteins in subsequent MR activity assays.

3) Since all LRET experiments are done without DNA, the authors do not capture relevant DNA processing states and comparison of structural (w/o DNA) and biochemical data (w/ DNA) is not really justified, in my opinion. Also, they might miss critical conformations. Is there a technical reason for not including DNA in the LRET studies?

We have collected LRET data on ATP-bound MRNBD in the presence of a hairpin DNA or a ssDNA as substrates. We still observe three states in the presence of both DNAs; however, the open conformation appears to be slightly more compact (i.e., closer distance between Rad50NBD protomers) in the presence of ssDNA. As described above, we have added to the Results section of the modified manuscript and included a new figure (Fig. 4) describing these data.

4) If the authors want to claim processive movement coupled to partially open/open state interchanges, they should provide experimental evidence. Where would the energy come from for such a movement, this is not clear from the model?

On the surface, ATP hydrolysis by Rad50 would seem to be the perfect source of energy for the conformational changes that drive the sequential and/or processive nuclease functions of the MR complex. However, the D313K mutant is not as good at ATP hydrolysis as the wild type enzyme (Fig. 3E), and the data in Fig. 3 and Supplemental Fig. S4 clearly demonstrate that D313K is by far the best nuclease. If the free energy for the movement does not come from ATP hydrolysis, where else could it come? Richardson and co-workers measured a release of -5.3 kcal mol-1 (-22.17 kJ mol-1) of free energy for the hydrolysis of a DNA phosphodiester bond (Dickson, K.S. et al. 2000 J. Biol. Chem. 275:15828–15831). Thus, the free energy released from the Mre11 nuclease activity could be the driving force for the conformational changes we propose. We have made this point in the Discussion of the revised manuscript.

5) The SAXS data for the "open" state do not validate the model, in my opinion. Experimental data and model are not inconsistent, but the curve looks to me as if the open state is perhaps much more flexible (i.e. an ensemble) or extended? Please comment.

We agree with the Reviewer on this point. We have updated Fig. 5A (original Fig. 4) to include the two-state fits to the experimental SAXS data. Although the multi-state fit to the apo MR SAXS data is better than any of the single model fits (2 = 1.05 vs. 1.26, respectively), the 2 is still larger than the multi-state fits to the ATP-bound MR SAXS data. Thus, an additional unobserved conformation (perhaps the so-called “extended”) might be present in solution for apo MRNBD. We have added a sentence to the revised manuscript with this point.

To explore the possibility that the previously described “extended” structure might be contributing to the SAXS data, we built a model of the extended conformation of Pf MRNBD based on the Tm MRNBD structure (PDB: 3QG5) and used Rosetta to connect the coiled-coils and add the linker to the Mre11 HLH. When this model was used in the FoXS calculations for the apo SAXS data, the 2 was 4.77 (versus 2 of 1.26 for the “open” model). The MultiFoXS two-state fit gave 90% open + 10% closed (2 of 1.04), whereas the three-state fit gave 65% open + 20% extended + 15% part open (2 of 0.84). Thus, there is some improvement when using the extended model, but since that model is not measurable in our LRET experiments and we are unsure of its validity as we have modeled it for Pf MR, we have chosen to omit it from the analysis.

6) Distance errors for the full complex are much smaller than those for the catalytic module only (Fig. 1d). Does that mean that the full complex is more rigid, please comment?

From looking at the data presented in Fig. 1D, it is logical to suggest that the full-length complex may be more rigid or better defined by the LRET data. However, we note that there are nearly as many distance errors which are similar between MRNBD and MR as there are MR errors less than MRNBD. And although many are not identical, most are of a similar magnitude. Because of this, we do not think the variations in LRET errors are systematic (i.e., related to a more rigid full-length complex).

https://github.com/lmichan/BioDBS

Auto-Discovery

Publishers SHOULD implement auto-discovery of GBFS feeds by linking to the location of the gbfs.json auto-discovery endpoint.

• The location of the auto-discovery file SHOULD be provided in the HTML area of the shared mobility landing page hosted at the URL specified in the url field of the system_information.json file.

• This is referenced via a link tag with the following format:

  • <link rel="gbfs" type="application/json" href="https://www.example.com/data/gbfs.json" />

SciScore for 10.1101/2022.01.20.22269581: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Ethics committee review was not required for this review.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We searched the COVID-19 living evidence database [20], which uses automated workflow processes to: (1) provide daily updates of searches of four electronic databases (Medline, PubMed,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Medline</div><div>suggested: (MEDLINE, RRID:SCR_002185)</div></div><div style="margin-bottom:8px"><div>PubMed</div><div>suggested: (PubMed, RRID:SCR_004846)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, Ovid Embase, bioRxiv and medRxiv), using medical subject headings and free-text keywords for SARS-CoV-2 infection and COVID-19; (2) de-duplicate the records; (3) tag records that are preprints; and (4) allow searches of titles and abstracts using Boolean operators.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>bioRxiv</div><div>suggested: (bioRxiv, RRID:SCR_003933)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two authors independently assessed the risk of bias, using a customised online tool, which saved responses into the REDCap database.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>REDCap</div><div>suggested: (REDCap, RRID:SCR_003445)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We used the metaprop and metabin functions from the meta package (version 4.11-0) [26] and the ggplot2 package (version 3.3.5) in R (version 3.5.1) to display the study findings in forest plots and synthesise their results, where appropriate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ggplot2</div><div>suggested: (ggplot2, RRID:SCR_014601)</div></div></td></tr></table>

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

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

Strengths and weaknesses of the living systematic review methods: A strength of the methodology of this review is the transparent reporting, with openly available data and changes over different versions reported in the protocol. Our inclusion criteria attempted to reduce risks of bias and we developed a new tool to address potential biases in the studies included in this review. In contact investigations, we subtracted index cases from the total number of people with SARS-CoV-2 to avoid underestimation of the proportion asymptomatic [14]. We examined heterogeneity in detail and, as a result of the wide prediction interval, we chose not to report an overall summary estimate [19, 146]. A limitation of the methods for this living systematic review is that this update only includes published studies up to 2 February 2021. Although we made extensive efforts to comply with the planned 3-monthly updates, with weekly searches and a continuous process of screening, data extraction and risk of bias assessment, the pace of publications about SARS-CoV-2 exceeds the capacity of our crowd of reviewers [8, 20]. In reviews of observational epidemiological studies, search terms are broad so the number of studies that needs to be screened is high, but the yield of included studies is low. We are in the process of updating our findings and preliminary analyses show that the main findings do not change when including studies published up to April 2021. The four databases that we searched are no...

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Results from TrialIdentifier: No clinical trial numbers were referenced.

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

---

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

---

Results from rtransparent:

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

---

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

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About SciScore

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

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SciScore for 10.1101/2022.01.26.22269848: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Patients and samples: The study was approved by the Institutional Review Board of Washington University School of Medicine (Approval # 202104138).<br>Consent: Inclusion criteria included males and females over 18 years of age, health care provider-documented PAD syndrome including common variable immunodeficiency (CVID), specific antibody deficiency, or hypogammaglobulinemia, and the ability to give informed consent.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Inclusion criteria included males and females over 18 years of age, health care provider-documented PAD syndrome including common variable immunodeficiency (CVID), specific antibody deficiency, or hypogammaglobulinemia, and the ability to give informed consent.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Exclusion criteria included participation in an investigational study of SARS-CoV-2 vaccines within the past year, history of HIV infection, an active cancer diagnosis, treatment with immunosuppressive medications, history of hematologic malignancy, treatment with anti-CD20 monoclonal antibody, receipt of live-attenuated vaccine within 30 days or any inactivated vaccine within 14 days of SARS-CoV-2 vaccination, blood or blood product donation within 30 days prior to study vaccination, and planned blood donation at any time during or 30 days after the duration of subject study participation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-CD20</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Of the 30 subjects, 9 were diagnosed with a prior SARS-CoV-2 infection with a positive nasal swab RT-PCR test, and one received treatment with an anti-SARS-CoV-2 monoclonal antibody (bamlanivimab) 90 days prior to study enrollment.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Unbound antibodies were washed away, and antigen-bound antibodies were detected by using a PE-coupled detection antibody for each subclass and isotype (IgG1, IgG2, IgG3, IgA1, and IgM; Southern Biotech), and FcγRs were fluorescently labeled with PE before addition to immune complexes (FcγR2a, FcγR2b, FcγR3a, FcγR2b; Duke Protein Production facility).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antigen-bound</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG2, IgG3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgA1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PE median fluorescent intensity (MFI) is reported as a readout for antigen-specific antibody titers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antigen-specific</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were washed and sequentially incubated with an oligoclonal pool of SARS2-2, SARS2-11, SARS2-16, SARS2-31, SARS2-38, SARS2-57, and SARS2-7148,49 anti-spike antibodies and HRP-conjugated goat anti-mouse IgG (Sigma) in PBS supplemented with 0.1% saponin and 0.1% BSA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS2-57</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>SARS2-7148,49</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-spike</div><div>suggested: (Sigma-Aldrich Cat# ZMS1076, RRID:AB_2893440)</div></div><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37°C for 1 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 spike and RBD protein expression: Genes encoding SARS-CoV-2 spike protein (residues 1-1213, GenBank: MN908947.3) and RBD (residues 319-514) were cloned into a pCAGGS mammalian expression vector with a C-terminal hexahistidine tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_127347)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quantification and statistical analysis: Statistical significance was assigned using Prism Version 9 (GraphPad) when P < 0.05.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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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: We detected the following sentences addressing limitations in the study:

One limitation of our study is the heterogeneity in the PAD patient cohort, which included those with CVID, hypogammaglobulinemia, or specific antibody deficiency. Although this was a limitation of the cohort available for study, we did not observe substantive differences between the patient subgroups. Instead, the most significant differences in antibody response to mRNA vaccines were between patients that had or lacked a prior history of SARS-CoV-2 infection. Many of our PAD patients who historically had poor immune responses to bacterial and other protein antigens (e.g., Streptococcus pneumoniae polysaccharides, tetanus toxoid, and diphtheria toxin) as part of their initial immune workup (Table S3) responded to mRNA vaccines. The basis for this difference remains unclear, although it could be due to the unique adjuvant properties of the lipid nanoparticle or in vitro-synthesized mRNA35-37. In comparison, although numbers in our cohort were clearly small (n = 3), we detected little to no antibody response at 35, 60 or 90 days after immunization of COVID-naive PAD patients with the Ad26.COV2.S adenoviral-vectored vaccine (Fig S3). Because PAD is a heterogeneous clinical entity, with many of the genetic defects unknown7-11, certain classes of adjuvants or antigens may overcome specific deficiencies and promote B cell responses, albeit at lower levels than healthy counterparts. Our data suggest that the mRNA platform may have utility for vaccination of PAD patients. That said,...

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

---

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About SciScore

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

</footer>

According to Bhagavad Gita Society, Sankhya-yog means "a philosophy established by sage Kapila. Essence of Sankhya yoga is removal of suffering by cultivating discrimination and by releasing the soul (purusha) from its entanglement in matter (Prakriti)".

Sankhya Yoga | Bhagavad Gita Society. bhagavadgitasociety.com/tag/sankhya-yoga/.

Samkhya Yoga." Samkhya or Sankhya means number. Yoga means union. Samkhya yoga means the union of numbers. The numbers are with regard to the number of realities (tattvas) that are present in existence. Samkhya Yoga deals with the union or the combination of a number of hidden realities, which manifest the existential reality. Those who are familiar with the Indian philosophies know that the Samkhya Philosophy is one of the six schools (Darshanas) of Hinduism. It is said to have been founded by Kapila and expounded by Isvara Krishna (6th Century AD) in his work, the Samkhya Karika.

practice tag

SciScore for 10.1101/2022.01.26.477937: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: Crystallization, X-ray diffraction data collection, and processing: Crystallization was carried out by the sitting-drop vapor diffusion method at 20 °C.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The serum dilution or antibody concentration causing a 50% reduction of RLU compared to control (ED50 or IC50, respectively) were reported as the neutralizing antibody titers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ED50</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IC50</div><div>suggested: (GeneTex Cat# GTX21096, RRID:AB_384608)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The vector was transfected into Expi293 cells and incubated at 37°C for 4 days.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentiviral vector, pWPI-ffLuc-P2A-EGFP for luciferase reporter assay and pTRC2puro-ACE2-P2A-TMPRSS2 for the generation of 293T cell line susceptible to SARS-CoV-2 infection was created from pWPI-IRES-Puro-Ak-ACE2-TMPRSS2, a gift from Sonja Best (Addgene, no.154987) by In-Fusion® technology (Takara Bio).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus production and neutralization: Pseudoviruses bearing SARS-Cov2 S-glycoprotein and carrying a firefly luciferase (ffLuc) reporter gene were produced in LentiX-293T cells by transfecting with pWPI-ffLuc-P2A-EGFP, psPAX2, and either of S variant from Wuhan, D614G</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>LentiX-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus titers were measured by infecting 293T/TRCAT cells for 72 hours before measuring luciferase activity (ONE-Glo™ Luciferase Assay System, Promega, Madison, WI)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T/TRCAT</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization assay with authentic SARS-CoV-2 viruses: VeroE6/TMPRSS2 cells (African green monkey kidney-derived cells expressing human TMPRSS2, purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, JCRB1819) were maintained in DMEM containing 10% FBS and 1 mg/mL G418 at 37 °C in 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6/TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PCR fragments were assembled into a linearized pcDNA vector using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA</div><div>suggested: RRID:Addgene_66792)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pcDNA3 (Invitrogen) vectors containing an Ig light chain gene and the pcDNA4 (Invitrogen) vectors containing an Ig heavy chain gene were simultaneously transfected into Expi293 cells using Expi293 Expression System Kit (Thermo Fisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3</div><div>suggested: RRID:Addgene_15475)</div></div><div style="margin-bottom:8px"><div>pcDNA4</div><div>suggested: RRID:Addgene_45907)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A soluble version of the S protein (amino acids 1–1213), including the T4 foldon trimerization domain, a histidine tag, and a strep-tag, was cloned into the mammalian expression vector pCMV.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_16459)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">psPAX2 (Addgene, no.12260) was a gift from Didier Trono. pCDNA3.3_CoV2_B.1.1.7 (Addgene, no.170451) for Alpha-S and pcDNA3.3-SARS2-B.1.617.2 (Addgene, no.172320) for Delta-S proteins, were gifts from David Nemazee25.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>pCDNA3.3_CoV2_B.1.1.7</div><div>suggested: RRID:Addgene_170451)</div></div><div style="margin-bottom:8px"><div>pcDNA3.3-SARS2-B.1.617.2</div><div>suggested: RRID:Addgene_172320)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentiviral vector, pWPI-ffLuc-P2A-EGFP for luciferase reporter assay and pTRC2puro-ACE2-P2A-TMPRSS2 for the generation of 293T cell line susceptible to SARS-CoV-2 infection was created from pWPI-IRES-Puro-Ak-ACE2-TMPRSS2, a gift from Sonja Best (Addgene, no.154987) by In-Fusion® technology (Takara Bio).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pWPI-IRES-Puro-Ak-ACE2-TMPRSS2</div><div>suggested: RRID:Addgene_154987)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pcDNA3.4 expression plasmids encoding SARS-CoV-2 S proteins with human codon optimization and 19 a.a deletion of C-terminus (C-del19) from Wuhan, D614G, and Omicron were generated by assembly of PCR products, annealed oligonucleotides, or artificial synthetic gene fragments (Integrated DNA Technologies, IDT) using In-Fusion® technology.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.4</div><div>suggested: RRID:Addgene_131198)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">lentiviral vector VSV-G-pseudotyped lentivirus carrying ACE2 and TMPRSS2 genes were produced in LentiX-293T cells by transfecting with pTRC2puro-ACE2-P2A-TMPRSS2, psPAX2 (gag-pol), and pMD2G-VSV-G (envelope) using PEI-MAX (Polysciences)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTRC2puro-ACE2-P2A-TMPRSS2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pMD2G-VSV-G</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus production and neutralization: Pseudoviruses bearing SARS-Cov2 S-glycoprotein and carrying a firefly luciferase (ffLuc) reporter gene were produced in LentiX-293T cells by transfecting with pWPI-ffLuc-P2A-EGFP, psPAX2, and either of S variant from Wuhan, D614G</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pWPI-ffLuc-P2A-EGFP</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ED50 or IC50 were calculated using a nonlinear regression curve fit (GraphPad Prism software Inc., La Jolla, CA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Paired-end, 300 bp sequencing was performed using MiSeq (Illumina) with the MiSeq reagent kit v3 (Illumina, MS-102-3003).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MiSeq</div><div>suggested: (A5-miseq, RRID:SCR_012148)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Structure determination and analyses: Phase determinations were carried out by the molecular replacement method using the program Phaser in the PHENIX package32 and the program Molrep33 with the combination between RBD structure (PDB ID:7EAM) and Fab structures (PDB ID:7CHB and 7CHP) as search models.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All figures of structures were generated by the program pymol (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr></table>

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

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About SciScore

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

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Name chosen as a nod to the use of Template:Should be SVG on Wikipedia

SciScore for 10.1101/2022.01.25.22269808: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: These studies were reviewed and approved by the Mount Sinai Hospital Institutional Review Board (IRB-20-03374,IRB-16-00791).<br>Consent: All participants signed written consent forms prior to sample and data collection.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After three washes with PBS-T, 50 μl/well of the pre-diluted secondary anti-human IgG (Fab-specific) horseradish peroxidase antibody (produced in goat; Sigma-Aldrich) diluted 1:3,000 in PBS-T containing 1% milk powder were added and plates were incubated for one hour incubation at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>the pre-diluted secondary anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The biotinylated mAb 1C7C7, a mouse anti-SARS nucleoprotein monoclonal antibody generated at the Center for Therapeutic Antibody Development at the Icahn School of Medicine at Mount Sinai ISMMS (Millipore Sigma), was used for NP staining at a concentration of 1μg/ml in PBS, 1% BSA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were incubated with an anti-SARS-CoV spike primary antibody directly conjugated to Alexafluor-647 (CR3022-AF647) for up to 4 hours at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following this, 50 μL per well of 1X MSD SULFO-TAG Anti-Human IgG detection antibodies were added and incubated for 1 hour.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Human IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, residues 1-1208 of the spike protein (Wuhan-1 strain numbering) were codon optimized with proline substitutions at residues 986 and 987, the furin cleavage site modified to “GSAS”, and a T4 fibritin trimerization motif and a 8X HIS tag were added on the C-terminus and the construct was cloned into pCDNA3.4. 293F cells were transfected with plasmid using PEIMax in FreeStyle 293 Expression Medium (Fisher) and cultured for three days at 32°C, 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293F</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero.E6 cells were seeded in 96-well high binding cell culture plates (Costar) at a density of 20,000 cells/well in complete Dulbecco’s modified Eagle medium (cDMEM) a day before infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero.E6</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibody-virus mixture was then added to Vero.E6-TMPRSS2 cells and incubated at 37°C for 1 hour.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero.E6-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences for the proteins were cloned into a mammalian expression vector, pCAGGS, as previously described and proteins were purified after transient transfections with each respective plasmid (23, 26).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_127347)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, residues 1-1208 of the spike protein (Wuhan-1 strain numbering) were codon optimized with proline substitutions at residues 986 and 987, the furin cleavage site modified to “GSAS”, and a T4 fibritin trimerization motif and a 8X HIS tag were added on the C-terminus and the construct was cloned into pCDNA3.4. 293F cells were transfected with plasmid using PEIMax in FreeStyle 293 Expression Medium (Fisher) and cultured for three days at 32°C, 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA3.4</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Area under the curve (AUC) values were calculated and plotted using Prism 9 software (GraphPad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed using GraphPad Prism 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All the analyses were performed using Prism 7 software (GraphPad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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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: We detected the following sentences addressing limitations in the study:

Our study has several limitations. We include a limited number of samples, and our dataset comes from a comparison of imperfectly matched groups from a clinical trial in Thailand and an observational cohort study in New York City. Strengths of the study include the use of authentic SARS-CoV-2 for neutralization assays and that the findings could be replicated with different methods in a different, blinded, and independent laboratory. In summary, we show that a vaccine candidate which can be produced locally in LMICs at low cost induces neutralizing antibody titers to SARS-CoV-2 comparable to those observed in cohorts having received mRNA-based COVID-19 vaccines. The NDV-HXP-S vaccine candidate induces a strong RBD focused immune response resulting in a high proportion of neutralizing antibodies, which are associated with protection from infection and severe disease (20, 24, 25).

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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">NCT04871737</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Study of a Live rNDV Based Vaccine Against COVID-19</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT05181709</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Live Recombinant Newcastle Disease Virus-vectored COVID-19…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04830800</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Phase 1/2 Safety and Immunogenicity Trial of COVID-19 Vacc…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04764422</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Assess the Safety and Immunogenicity of NDV-HXP-S Vaccine in…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04993209</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Clinical Trial of the COVID-19 Vaccine (Recombinant, Inactiv…</td></tr></table>

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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.

---

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

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About SciScore

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

</footer>

SciScore for 10.1101/2022.01.25.477753: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: The absence of mycoplasma contamination in cells was routinely tested.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blots were probed with the following antibodies: rabbit anti-eIF4E2/4EHP (Proteintech, 12227-1-AP), rabbit anti-DDX6 (Proteintech, 14632-1-AP), rabbit anti-CNOT9 (Proteintech, 22503-1-AP), mouse anti-Flag M2 (Sigma-Aldrich, F1804), rabbit anti-GIGYF2 (Proteintech, 24790-1-AP)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-eIF4E2/4EHP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-DDX6</div><div>suggested: (Proteintech Cat# 14632-1-AP, RRID:AB_2091264)</div></div><div style="margin-bottom:8px"><div>anti-CNOT9</div><div>suggested: (Proteintech Cat# 22503-1-AP, RRID:AB_11232413)</div></div><div style="margin-bottom:8px"><div>anti-Flag</div><div>suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)</div></div><div style="margin-bottom:8px"><div>F1804</div><div>suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)</div></div><div style="margin-bottom:8px"><div>anti-GIGYF2</div><div>suggested: (Proteintech Cat# 24790-1-AP, RRID:AB_2879727)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flp-In T-REx 293 cells (Thermo Fisher Scientific) were grown in similar conditions supplemented with 100μg/ml zeocin and 15μg/ml blasticidin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Flp-In T-REx 293</div><div>suggested: RRID:CVCL_U427)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The full-length cDNA encompassing the coding region of human 4EHP, obtained by RT-PCR using total RNA from HEK293T cells, were cloned into the pCI-Neo vector (Promega) at the XhoI-NotI sites in frame with a sequence encoding a V5 tag inserted at the NheI-XhoI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CRISPR-Cas9-mediated genome editing of HEK293 cells was performed according to Ran et al. (53).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cell line inducibly expressing Flag-NSP2 was generated by co-transfecting pcDNA5-FRT-TO-FH-Nsp2 (Addgene plasmid 157683) and pOG44 (Thermo Fisher Scientific) with a 1:10 ratio.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA5-FRT-TO-FH-Nsp2</div><div>suggested: RRID:Addgene_157683)</div></div><div style="margin-bottom:8px"><div>pOG44</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Truncated versions of NSP2 were generated by replacing the sequence encoding full-length NSP2 by PCR-amplified fragments at the BamHI-XhoI sites of the pcDNA5-FRT-TO-FH vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA5-FRT-TO-FH</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mCherry2-NSP2 expressing plasmids were created by cloning a PCR-amplified NSP2 fragment into the mCherry2-C1 (Addgene plasmid 54563) and mCherry2-N1 vectors (Addgene plasmid 54517) using EcoRI and BamHI restriction enzymes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>mCherry2-NSP2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>mCherry2-C1</div><div>suggested: RRID:Addgene_54563)</div></div><div style="margin-bottom:8px"><div>mCherry2-N1</div><div>suggested: RRID:Addgene_54517)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate the pCI-λNV5-GIGYF2 vector, a fragment containing the GIGYF2 sequence was obtained by PCR from the pcDNA4/TO/GFP-GIGYF2 vector (Addgene plasmid 141189) (34) and inserted into the pCI-λNV5 at the XhoI-NotI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-λNV5-GIGYF2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA4/TO/GFP-GIGYF2</div><div>suggested: RRID:Addgene_141189)</div></div><div style="margin-bottom:8px"><div>pCI-λNV5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A similar strategy was used to generate the V5-tagged fragments of GIGYF2 using primers to isolate the following domains encompassing residues: 1-267; 257-495; 485-752; 742-1,085; 1,075-1,320. pCI-eGFP-GIGYF2 was generated by inserting the eGFP sequence, PCR-amplified from pEGFP-C1 (Clontech), at the NheI-XhoI sites of the pCI-Neo vector and then by adding the fragment encoding GIGYF2 at the XhoI-NotI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-eGFP-GIGYF2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pEGFP-C1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The full-length cDNA encompassing the coding region of human 4EHP, obtained by RT-PCR using total RNA from HEK293T cells, were cloned into the pCI-Neo vector (Promega) at the XhoI-NotI sites in frame with a sequence encoding a V5 tag inserted at the NheI-XhoI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-Neo</div><div>suggested: RRID:Addgene_16574)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate the pCI-λNV5-GW182SD vector, a fragment encoding the residues 1382-1690 was cloned by PCR as a XhoI-NotI fragment from the pFRT/TO/FLAG/HA-DEST TNRC6C vector (Addgene plasmid 19885) (52) and inserted into the pCI-λNV5 at the XhoI-NotI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-λNV5-GW182SD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFRT/TO/FLAG/HA-DEST TNRC6C</div><div>suggested: RRID:Addgene_19885)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For recombinant GST-fused protein expression, full-length 4EHP and GIGYF2742-1,085 coding sequences contained in PCR-amplified BamHI-NotI and XhoI-NotI fragments, respectively, were cloned into a pGEX-6P-1 vector (Amersham) in frame with the GST coding sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEX-6P-1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pET-28b vector (EMD Biosciences) was used to express NSP2 as a recombinant protein fused to an His6 tag at the N-terminus.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-28b</div><div>suggested: RRID:Addgene_47327)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate KO HEK293 cells, we transfected 700,000 cells with the pSpCas9(BB)-2A-Puro plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSpCas9 ( BB)-2A-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the reporter containing the 3’UTR of Ifnb1, 20 ng of psiCHECK2-RLuc-Ifnb1 3’ UTR reporter (31), or empty psiCHECK2 (Promega), was added along with 100 ng of vectors encoding Flag-NSP2 or Flag (empty vector).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psiCHECK2</div><div>suggested: RRID:Addgene_40763)</div></div></td></tr></table>

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

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About SciScore

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

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SciScore for 10.1101/2022.01.25.477789: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: The mouse and hamster studies were carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals under the approval of the Institutional Animal Care and Use Committee of Xiamen University.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Rhesus monkey immunizations: Ten rhesus monkeys were allocated randomly into two groups (three females and two males per group).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Rhesus monkey immunizations: Ten rhesus monkeys were allocated randomly into two groups (three females and two males per group).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">Microscopic evaluation of pathological lung lesions was performed blindly by pathologists following a semiquantitative scoring system with the inclusion of three indicators (49): (i) alveolar septum thickening and consolidation; (ii) hemorrhage, exudation, pulmonary edema, and mucous; and (iii) recruitment and infiltration of inflammatory immune cells.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two weeks after boosting, serum samples were collected for antibody analyses, including measurement of anti-spike IgG, anti-RBD IgG, pseudovirus neutralizing antibody, and authentic neutralizing antibody titers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-spike IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-RBD IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, single-cell suspensions were obtained from mouse spleen (106 cells per well) through grinding in 70 μm cell strainers and were seeded in anti-mouse IFN-γ antibody precoated ELISpot plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IFN-γ</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mixtures (150 μL per well) were added to a monolayer of Vero cells in a 96-well plate and incubated at 37 °C supplying with 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Experimental animals: BALB/c and C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Co.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse immunizations: Six to eight-week-old BALB/c mice (n=6 per group) were immunized with STFK vaccines at 0.01, 0.1, 1, or 10 μg per dose in 150 μL through intramuscular injection following a two-dose schedule at weeks 0 and 3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The tag-free STFK and STFK variants were constructed into a pGS01b vector, modified from the pCGS3 vector containing glutamine synthetase (GS) selection marker (Sigma Aldrich).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGS01b</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCGS3</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were acquired using the SerialEM software on an FEI Tecnai F30 transmission electron microscope (ThermoFisher Scientific) operated at 300 kV and equipped with a Gatan K3 direct detector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SerialEM</div><div>suggested: (SerialEM, RRID:SCR_017293)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing and 3D reconstruction: Drift and beam-induced motion correction were performed with MotionCor2 (38) to produce a micrograph from each movie.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MotionCor2</div><div>suggested: (MotionCor2, RRID:SCR_016499)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We initially fitted the templates into the corresponding final cryo-EM maps using Chimera (43), and further corrected and adjusted them manually by real-space refinement in Coot (44).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting models were then refined with phenix.real_space_refine in PHENIX (45).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The final atomic models were validated with Molprobity (46, 47).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Molprobity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical analyses were conducted in GraphPad Prism 8 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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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.

---

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

---

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About SciScore

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

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SciScore for 10.1101/2022.01.26.477860: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">A pool of DNA constructs containing a randomized 18 bp barcode sequence (N18) was cloned into the NotI and AscI sites upstream of the LexA promoter sequence via restriction digestion, ligation and transformation into chemically competent E. coli.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">15 µg of total cellular protein was resolved by SDS-PAGE, transferred to a PVDF membrane, and probed using an anti-his antibody (ref).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-his</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">W303 cells were transformed with the p416LexA-UbMpro(C145A)-his construct and the resulting yeast cells were grown to exponential phase in SD-ura media at 30°C. 125 nM β-estradiol was added when indicated and cells were grown for an additional eight hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>W303</div><div>suggested: Coriell Cat# GM03889, RRID:CVCL_W303)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Gal4, Gal80 and Pdr5 genes were disrupted to create the following strain: W303 HO::Gal1-GFP-v5-His3; gal4::trp1; gal80::leu2 pdr5::natMX.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gal4 , Gal80</div><div>suggested: RRID:BDSC_51280)</div></div><div style="margin-bottom:8px"><div>HO::Gal1-GFP-v5-His3; gal4::trp1; gal80::leu2 pdr5::natMX</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Construction of WT Ub-Mpro vector (p416LexA_UbMpro(WT)_B112): The Ubiqutin-Mpro gene fusion was constructed using overlapping PCR of the yeast ubiquitin gene and SARS-CoV-2 Mpro gene (Jin, Du et al. 2020) and was inserted into the pRS416 vector after digestion with SpeI and BamHI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRS416</div><div>suggested: RRID:Addgene_158144)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(Addgene plasmid # 58434; http://n2t.net/addgene:58434 ; RRID:Addgene_58434))(Ottoz, Rudolf et al. 2014) and inserted between the SacI and SpeI sites upstream of the ubiquitin-Mpro gene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_58434)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Addgene plasmid # 58437; http://n2t.net/addgene:58437; RRID:Addgene_58437))(Ottoz, Rudolf et al. 2014) and inserted into the KpnI site.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_58437)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Barcode association: To associate barcodes with Mpro variants, we digested the p416-UbMpro(lib)-B112 plasmid upstream of the N18 sequence and downstream of the Mpro sequence with NotI and SalI enzymes (NEB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416-UbMpro(lib)-B112</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The randomized 18 bp barcode sequence (N18) was cloned between the NotI and AscI sites upstream of the LexA promoter sequence in the p416LexA-Ub-Mpro(WT)-B112 vector with the goal of the WT sequence being represented by approximately 100 barcodes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-Ub-Mpro(WT)-B112</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting DBD-MproCS-AD fusion gene was inserted between the EcoRI and SacI sites downstream of the CUP promoter in the integrative bidirectional pDK-ATC plasmid (kindly provided by D. Kaganovich)(Amen and Kaganovich 2017).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDK-ATC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mCherry gene was subsequently cloned into the XhoI/BamHI sites downstream of the TEF promoter in the opposite orientation to create the plasmid pDK-CUP-DBD-MproCS-AD-TEF-mCherry.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDK-CUP-DBD-MproCS-AD-TEF-mCherry</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bulk Split transcription factor (TF) competition experiment: Barcoded WT UbMpro (p416LexA-UbMpro(WT)-N18) plasmid was mixed with the barcoded UbMpro library (p416LexA-UbMpro(lib)-N18) at a ratio of 20-fold WT to the average library variant.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-UbMpro(WT)-N18</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The CyPet gene was amplified by PCR from the pCyPet-His vector (pCyPet-His was a gift from Patrick Daugherty (Addgene plasmid # 14030 ; http://n2t.net/addgene:14030 ; RRID:Addgene_14030)) with a forward primer containing the BamHI site (BamHI_F) and a reverse primer containing the extending MproCS overhang sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_14030)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The YPet gene was amplified by PCR from the pYPet-His vector (pYPet-His was a gift from Patrick Daugherty (Addgene plasmid # 14031 ; http://n2t.net/addgene:14031 ; RRID:Addgene_14031)) with a forward primer containing the extending MproCS overhang sequence and a reverse primer containing the XhoI site (XhoI_R).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_14031)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting CyPet-MproCS-YPet gene was inserted between the BamHI and XhoI sites downstream of the TEF promoter in the integrative bidirectional pDK-ATG plasmid (kindly provided by D. Kaganovich)(Amen and Kaganovich 2017).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDK-ATG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis of Mpro expression and Ubiquitin removal by Western Blot: To facilitate analysis of expression levels of Mpro and examine effective removal of Ubiquitin, a his tag was fused to the C-terminus of Mpro to create the plasmid p416LexA-UbMpro-his6-B112.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-UbMpro-his6-B112</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">W303 cells were transformed with the p416LexA-UbMpro(C145A)-his construct and the resulting yeast cells were grown to exponential phase in SD-ura media at 30°C. 125 nM β-estradiol was added when indicated and cells were grown for an additional eight hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-UbMpro(C145A)-his</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PacBio circular consensus sequences (CCS) were generated from the raw reads using SMRTLink v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SMRTLink</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Illumina sequence reads were filtered for Phred scores > 10 and strict matching of the sequence to the expected template and identifier sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phred</div><div>suggested: (Phred, RRID:SCR_001017)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The figures were generated using Matplotlib (Hunter 2007), PyMOL and GraphPad Prism version 9.3.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Matplotlib</div><div>suggested: (MatPlotLib, RRID:SCR_008624)</div></div><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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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: We detected the following sentences addressing limitations in the study:

However, there are a couple of caveats that should be kept in mind when utilizing these data sets. For example, we do not fully understand how Mpro’s biochemical function relates to viral fitness. Having some Mpro function is essential to the virus, so mutations that destroy Mpro function will form non-functional viruses. Function-fitness relationships tend to be non-linear (Heinrich and Rapoport 1974, Kacser and Fell 1995, Jiang, Mishra et al. 2013) and it may be likely that Mpro function must be decreased by a large amount in order to cause measurable changes in viral replication efficiency. This relationship between Mpro function and SARS-CoV-2 fitness would need to be determined in order to translate our functional scores to fitness scores. Additionally, our TF and FRET screens quantify cleavage at one defined site (Nsp4/5) and it may be important to analyze all sites in order to fully understand the selection pressures acting on Mpro. Another important caveat is that our fitness landscape captures single amino acid changes and therefore does not provide information on the potential interdependence or epistasis between double and higher order mutations. Information regarding epistasis will be important for accurately predicting the impacts of multiple mutations on fitness. Despite these caveats, the similarity in fitness landscapes for the TF and FRET screens with the yeast growth screen suggests that all three capture fundamental and general aspects of Mpro selection. In...

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

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About SciScore

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

</footer>

SciScore for 10.1101/2022.01.26.477856: (What is this?)

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

Table 1: Rigor

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

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After incubation for 1h at room temperature and washing, the ACE-2 mFc Tag was revealed using a peroxidase-conjugated anti-mouse secondary antibody (1:1000 dilution).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 clinical isolates (D614G variant; GenBank accession number MW322968) , Alpha (GenBank accession number MW633280) , Beta (GenBank accession number MW580244) , Gamma (Gene accession number pending), Delta (Gene accession number pending) and Omicron (Gene accession number pending) were isolated from SARS-CoV-2 RT-PCR confirmed patients by inoculating Vero cells with sputum sample or nasopharyngeal swabs in the biosafety level-3 (BSL-3) facility of the Pitié-Salpêtrière University Hospital.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Hundred μl of a Vero E6 cell suspension (3 × 105 cells/ml) were then added to the mixture and incubated at 37°C under an atmosphere containing 5% CO2 until microscopy examination on day 4 to assess cytopathogenic effect (CPE), as previously described (Vanhove et al</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IC50s were analyzed by nonlinear regression using a four-parameter dosage-response variable slope model with the GraphPad Prism 8.0.2 software (GraphPad Software, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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

---

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

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Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr style="background-color:#FF0000"><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT0492830</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Trial number did not resolve on clinicaltrials.gov. Is the number correct?</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NA</td></tr></table>

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

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2022.01.24.477625: (What is this?)

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

Table 1: Rigor

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

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Library Design: A library of antibodies directed against SARS-CoV-2 Spike (S) protein was developed using paired antibody sequences, meaning antibody sequences for which the heavy and light chain are both known, from the Coronavirus Antibody Database, CoV-AbDab57.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CoV-AbDab57</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CrossMAb, antibody, hFc-ACE2, and Lentiviral plasmids were maxi prepped in the same fashion.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>hFc-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two microliters of mouse anti-hexa His antibody (BioLegend) were added to the 10 mL sample and incubated for 1 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-hexa</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral transfections were done in HEK293T cells using calcium phosphate transfection reagent.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resultant 10mL was added to plated HEK cells from which the medium had been removed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization: The target cells used for infection in viral neutralization assays were from a HeLa cell line stably overexpressing the SARS-CoV-2 receptor, ACE2, as well as the protease known to process SARS-CoV-2, TMPRSS2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa</div><div>suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2/TMPRSS2/HeLa cells were plated one day prior to infection at 5,000 cells per well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2/TMPRSS2/HeLa</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Equimolar aliquots of each scFv plasmid were pooled and the resultant pool was amplified using primers which annealed to the hexa-his Tag (reverse primer) or signal peptide (forward primer) and had a 50bp overlap with the pPNL6 vector digested with NheI and BamHI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>scFv</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Yeast were resuspended in electroporation buffer (10 mM Tris Base, 250 mM sucrose, 2mM MgCl2) containing the gel extracted library amplification and digested pPNL6 vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pPNL6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Light chain (LC) and LC-ACE2 fusion proteins: Antibody sequences were cloned into the CMV/R plasmid backbone for expression under a CMV promoter.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CMV/R</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">hCoV spike proteins constructs: Spike proteins from six hCoVs were cloned into a pADD2 vector between the rBeta-globin intron and β-globin poly(A).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pADD2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentivirus plasmids: Plasmids encoding the full-length spike proteins with native signal peptides were cloned into the background of the HDM-SARS2-Spike-delta21 plasmid (Addgene Plasmid #155130).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HDM-SARS2-Spike-delta21</div><div>suggested: RRID:Addgene_155130)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">They are: pHAGE-Luc2- IRS-ZsGreen (NR-52516), HDM-Hgpm2 (NR-52517), pRC-CMV-Rev1b (NR-52519), and HDM- tat1b (NR-52518).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHAGE-Luc2- IRS-ZsGreen</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The EtOH solution was then added to a geneJET plasmid miniprep column and the regular wash steps and elution steps were followed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>geneJET</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Digested FAbs were purified by SP/AKTA using 50 mM NaOAc, pH5 with gradient NaCl elution (using 50 mM NaOAc + 1M NaCl, pH5).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pH5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmids were added to D10 medium in the following ratios: 10 µg pHAGE-Luc2-IRS-ZsGreen, 3.4 µg FL Spike, 2.2 µg HDM-Hgpm2, 2.2 µg HDM-Tat1b, 2.2 µg pRC-CMV-Rev1b in a final volume of 1000 µL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRC-CMV-Rev1b</div><div>suggested: RRID:Addgene_164443)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequence alignment was again produced using MUSCLE31 and sequence identity was calculated using Geneious</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Geneious</div><div>suggested: (Geneious, RRID:SCR_010519)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences used were: 6VXX, UniRef90_U5NJG5, _L7UP8, _A0A7U3W1C7, _K9N5Q8, _A0A2I6PIW5, _A0A3Q8AKM0, _U5WHZ7, _A0A5H2WTJ3, _A0A0U1WJY8, _A0A166ZND9, _A0A678TRJ7, _A0A2R4KP93, _A0A2Z4EVK1, _A0A7R6WCE7, _E0ZN36, _A0A6M3G9R1, _F1DAZ9, _A0A0U1UYX4, _A0A2R3SUW7, _A0A2Z4EVN5, _A0A2Z4EVN2, _U5LMM7, _A0A5Q0TVR4, _E0XIZ3, _A0A023Y9K3, _A0A2R4KP86, _A0A088DJY6, _A0A7G6UAJ9, _S4X276, _A0A4Y6GL90, _A3EXG6, _F1BYL9, _E0ZN60, _A0A0K1Z054, _A0A0U1WHI2, and NCBI accession numbers: YP_009047204.1, QLR06867.1, AAK32191.1, AGZ48828.1, AAT84362.1, QHR63300.2, ABD75513.1, YP_003767.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NCBI</div><div>suggested: (NCBI, RRID:SCR_006472)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences were first aligned using the MUSCLE algorithm, and then two phylogenetic trees were made, both using PhyML 3.3.20180621.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MUSCLE</div><div>suggested: (MUSCLE, RRID:SCR_011812)</div></div><div style="margin-bottom:8px"><div>PhyML</div><div>suggested: (PhyML, RRID:SCR_014629)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences were then analyzed by sequence alignment using SnapGene software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SnapGene</div><div>suggested: (SnapGene, RRID:SCR_015052)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FAb Production from IgGs: 1/10 volume of 1M Tris, pH 8 was added to IgGs at ∼2 mg/mL in PBS. 2 µL of a 1 mg/mL stock of Lys-C (stock stored at -70C) was added for each mg of human IgG1 and digested for 1 hour at 37 °C with moderate rotation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgGs</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Final data analysis was done in Prism.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

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About SciScore

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

</footer>

SciScore for 10.1101/2022.01.24.477037: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The 293 cells were co-transfected with split GFP vectors, and fluorescence intensity was determined by 5 random areas with the same exposure, followed by ImageJ analysis.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies against GSK-3β (610202, BD Transduction Laboratories), pS9-GSK-3β (9323S, Cell Signaling), pY216-GSK-3β (612312, BD Transduction Laboratories), Flag (F-3165, Sigma), Snail (L70G2, Cell Signaling), β-catenin (610154, BD Transduction Laboratories), and Tubulin (LF-PA0146, AbFrontier) were obtained from the commercial vendors</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pS9-GSK-3β ( 9323S , Cell Signaling) , pY216-GSK-3β ( 612312</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Snail ( L70G2 , Cell Signaling) ,</div><div>suggested: (Cell Signaling Technology Cat# 3895, RRID:AB_2191759)</div></div><div style="margin-bottom:8px"><div>β-catenin</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Tubulin ( LF-PA0146 , AbFrontier )</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Phosphorylation status of N protein was determined by anti-flag antibody and mobility shift on a Phos-tag gel (Wako) as described previously (38).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-flag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were then washed three times with PBS containing 0.1% Tween 20 followed by incubation with anti-mouse-Alexa Fluor-488 (for green) or anti-rabbit-Alexa Fluor-594 (for red) secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse-Alexa</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit-Alexa</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 293 cells were co-transfected with split GFP vectors, and fluorescence intensity was determined by 5 random areas with the same exposure, followed by ImageJ analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293</div><div>suggested: NCI-DTP Cat# NCI-293TT, RRID:CVCL_1D85)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The expression vector pGBW-m4134490 (plasmid number 152580) having codon optimized N of SARS-CoV, pGBW-m4134909 (plasmid number 151901) having N of human coronavirus 229E,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGBW-m4134490</div><div>suggested: RRID:Addgene_152580)</div></div><div style="margin-bottom:8px"><div>pGBW-m4134909</div><div>suggested: RRID:Addgene_151901)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pGBW-m4134899 (plasmid number 151902) having N of human coronavirus OC43 and pGBW-m4134901 (plasmid number 151922) having N of human coronavirus HKU1 229E were obtained from Addgene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGBW-m4134899</div><div>suggested: RRID:Addgene_151902)</div></div><div style="margin-bottom:8px"><div>pGBW-m4134901</div><div>suggested: RRID:Addgene_151922)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Those N expression vectors were subcloned into pcDNA3.1 with C-terminal flag or EGFP tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies against GSK-3β (610202, BD Transduction Laboratories), pS9-GSK-3β (9323S, Cell Signaling), pY216-GSK-3β (612312, BD Transduction Laboratories), Flag (F-3165, Sigma), Snail (L70G2, Cell Signaling), β-catenin (610154, BD Transduction Laboratories), and Tubulin (LF-PA0146, AbFrontier) were obtained from the commercial vendors</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pS9-GSK-3β</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">TCF/LEF and E-cadherin reporter assay: For TCF/LEF and E-cadherin reporter assay, the cells were transfected with 50 ng of the Super-Top or E-cad(−108)-Luc reporter vector (30,39) and 1 ng of pSV40-Renilla expression vector in combination with N or N-mutant as indicated.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>E-cad(−108)-Luc reporter</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pSV40-Renilla</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 293 cells were co-transfected with split GFP vectors, and fluorescence intensity was determined by 5 random areas with the same exposure, followed by ImageJ analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">: Amino acid sequences of N for SARS-CoV-2 (P0DTC9), SARS-CoV (P59595), HKU1 (Q0ZME3), HCoV-OC43 (P33469), HCoV-229E (PP15130), HCoV-NL63 (Q6Q1R8) were obtained from UniProt (https://www.uniprot.org/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>UniProt</div><div>suggested: (UniProtKB, RRID:SCR_004426)</div></div><div style="margin-bottom:8px"><div>https://www.uniprot.org/</div><div>suggested: (Universal Protein Resource, RRID:SCR_002380)</div></div></td></tr></table>

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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.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2022.01.21.477288: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The study protocol was approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00010350)</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two hours after infection, the cells were washed 5 times with DMEM and grown in DMEM supplemented with 10% FBS and 1% PenStrep along with an anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-VSV-G</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK-293T cells and HEK-293T cells stably expressing the human ACE2 receptor (HEK-ACE2)38 were grown in DMEM supplemented with 10% FBS and 1% PenStrep at 37°C and 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero cells stably expressing the human protease TMPRSS2 (Vero-TMPRSS2) were grown in DMEM supplemented with 10% FBS, 1% PenStrep, and 8 µg/mL puromycin at 37°C and 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant protein expression and purification: To produce the SARS-CoV-2 spike ectodomain containing the E406W mutation, 125 mL of Expi293 cells were grown to density of 2.5 × 106 cells per mL and transfected with 125 µg of DNA using PEI MAX diluted in Opti-MEM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization assays with vaccine-elicited sera and monoclonal antibodies: For neutralization assays using vaccine-elicited sera, HEK-ACE2 cells were seeded in 96-well poly-D-lysine coated plates at a density of 30,000 cells per well and grown overnight until they reached approximately 80% confluency.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralizations were conducted as described above with one modification: prior to the addition of the virus-antibody mixture, Vero-TMPRSS2 cells were washed 3 times with DMEM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The spike ectodomain was codon optimized, stabilized with the hexapro mutations39 and mutation of the furin cleavage site (682RRAR685 to 682GSAS685), and inserted into the pCDNA3.1 vector containing a C-terminal foldon followed by an avi tag and an octa-histidine tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The construct encoding the E406W RBD was generated by performing around-the-horn mutagenesis using a pCMVR vector encoding the wildtype SARS-CoV-2 RBD containing an N-terminal mu-phosphatase signal peptide and a C-terminal avi tag and octa-histidine tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMVR</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reference-free 2D classification was performed using cryoSPARC to select for well-defined particle images42.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These selected particles were then used for 3D classification with 50 iterations (angular sampling 7.5° for 25 iterations followed by 1.8° with local search for 25 iterations) using Relion and a previously reported closed model for the SARS-CoV-2 spike ectodomain (PBD: 6VXX) as the initial model without imposing any symmetry.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Relion</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Luminescence readings from the neutralization assays were normalized and analyzed using GraphPad Prism 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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

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At this point in the article the information that the audience has been met with the introduction that the black liquid being referenced can be used for faces and feet, overhyping jargon, and $110 price tag for what is essentially dirt.

What kind of proof is available about not only the improved brain function, heart health, and toxin eliminator, but also that it can simply work for both all-ages humans and animals?

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

Learn more at Review Commons

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Reply to the reviewers

Manuscript number: RC-2021-01129

Corresponding author(s): Koji Kikuchi

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Reviewer #1

Evidence, reproducibility and clarity (Required):

In this manuscript, Kikuchi et al describe the characterization of MAP7D2 and MAP7D1, two MAP7 family members in mouse with specific expression patterns. Focusing mostly on MAP7D2, they assess its expression pattern across the body and find that it is mostly expressed in certain neuronal subsets. They then characterize the MT-related properties of MAP7D2 based on previous knowledge of other MAP7 family members. They show that MAP7D2 binds MTs (via the N-terminus), determine the binding affinity, and show that it can stimulate MT polymerization (or stabilization) both in vitro and in vivo. Using a specific antibody, they localize MAP7D2 to centrosomes, midbody and neurites in N1-E115 cells. Functionally, they show that loss of MAP7D1/2 mildly affects microtubule stability as judged by acetyl-tubulin staining, and properties of these cells that rely on cytoskeletal elements such as cell migration and neurite growth. Interestingly, there might be a feedback loop regulating MAP7D1/2 expression, as knockdown of MAP7D1 upregulates MAP7D2.

Overall, the experiments and conclusions are very solid and convincing, such that I would not ask for further experiments. This is in part because the experiments are largely based on previous characterizations of other MAP7 family members, which are largely confirmed. The presentation of the data is also very clear.

Significance (Required):

I see the value of the study in the fact that it provides solid and specific research tools for MAP7D1/2 which could be very useful for the microtubule/neuronal cytoskeleton community.

Response: We thank the reviewer very much for appreciating the content of our manuscript.

\*Referees cross-commenting***

Reviewers 2 and 3 criticize that the evidence for an effect of MAP7D1/2 on MT dynamics is weak. I would agree in that ac-tub stainings and in vitro experiments are rather indirect. The experiments suggested by reviewer 2 should clarify this (esp. nocodazole should be easy). I also agree that an experiment addressing the potential involvement of kinesin-1 would help, the involvement of which seems to have been omitted by the authors. A kinesin-binding deficient mutant would add another MAP7D1/2 tool and increase the value for the community.

Response: As for the reviewer’s suggestions listed above, please refer to our responses to the comments of Reviewer #2.

Reviewer #2

Evidence, reproducibility and clarity (Required):

In this study, the authors investigate 2 members from the MAP7 family Map7D2 and Map7D1. They first address the tissue distribution of Map7D2, by northern blotting using a variety of rat tissues. To complement their analysis, they also raised an antibody to look at the protein distribution. From their studies, they concluded that Map7D2 is abundantly expressed in the brain and testis. The authors went on to perform a series of functional assays. First, they biochemically demonstrated that rat Map7D2 directly binds to MTs by MT co-sedimentation assay. The MT binding domain was mapped to the N-terminal half. They performed MT turbidity assay to demonstrate enhanced MT polymerisation in the presence of Map7D2, suggesting that this Map stabilises MTs. The authors went on to characterise in detail the subcellular localisation of Map7D2 which was predominantly present in the centrosome and partially localised to MTs including within neurites from N1-E115 cells. Kikuchi et al. further revealed the overlap in expression between Map7D2 and another family member, Map7D1. The authors continued these studies by a series of functional studies in N1-E115 cells where they performed single or combined knock-downs of Map7D2 and Map7D1 and studied the levels of acetylated and detyrosinated tubulins and the effect of the knock-downs on migration and neurite extension. The main conclusion from this work was that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms which are important in controlling cell motility and neurite outgrowth. Map7D2 is proposed to stabilise MTs by direct binding whereas Map7D1 does it indirectly by affecting acetylation.

Major comments:

The main conclusion from this work that Map7D2 and Map7D1 facilitate MT stabilization and that this is necessary for correct migration and neurite extension has not been convincingly demonstrated. In my opinion, a more detailed study of MT properties to demonstrate a role in MT stabilisation would greatly benefit the work, eg. experiments using MT destabilising agents such as nocodazole. In addition, a series of experiments aiming to study MT dynamics would help to understand the function of these MT regulators. The authors proposed an elevation in microtubule dynamics to explain the increase in migration and neurite extension but no experimental proof was provided.

Response: According to the reviewer’s suggestion, we plan to assess the role of MT stabilization in greater detail by analyzing the sensitivity to the MT-destabilizing agent, nocodazole.

To study MT dynamics, methods such as analyzing the velocity and direction of an EB1-GFP comet are commonly used. We have previously analyzed the roles of Map7 and Map7D1 in MT dynamics using HeLa cells stably expressing EB1-GFP (Kikuchi et al., EMBO Rep., 2018). However, no such tools have been developed for analyzing MT dynamics in N1-E115 cells, which were used in this study. In addition, it is difficult to analyze MT dynamics by transient expression of EB1-GFP because of the low plasmid transfection efficiency. Therefore, we instead plan to assess the effect on MT dynamics by measuring the EB1 comet length by immunofluorescence, referring to Fig. 7D in EMBO J. 32:1293–1306, 2013.

Moreover, considering the possibility that the Map7D2 dynamics are altered when MT stability is changed, e.g., before and after differentiation induction, we analyzed the Map7D2 dynamics at the centrosome by fluorescence recovery after photobleaching (FRAP) using N1-E115 cells stably expressing EGFP-rMap7D2. We found that the dynamics were altered between the proliferative and differentiated states (see the figure below). Compared to the proliferative state, the recovery rate of EGFP-Map7D2 was reduced (lower left panel), and the immobile fraction of Map7D2 was increased in the differentiated state (lower right panel). As these data suggest that the increase in immobile Map7D2 may enhance MT stabilization, we will present them in a new figure in our manuscript along with the results of the above two experiments.

It has been previously demonstrated that loss of MAP7D2 leads to a decrease in axonal cargo entry to axons resulting in defects in axon development and neuronal migration. The C-terminus is necessary for this function as it mediates interaction with Kinesin-1 (Pan et al., 2019). Such mechanisms could also explain the defects in migration and neurite growth that the authors observed. This possibility has not been considered but instead, the subtle changes in total α-tubulin led to suggest MT stabilisation as a key function without proof of causation. Could the authors provide some further experimental evidence to demonstrate that stability is the main contributor to the phenotypes observed? Eg. by rescuing migration and neurite phenotypes with a variant of MAP7D2 which cannot bind kinesin1.

Response: The reviewer states “Such mechanisms could also explain the defects in migration and neurite growth that the authors observed;” however, our results showed that loss of Map7D2 elevated the rates of both cell motility and neurite outgrowth (original Fig. 5). In contrast, it has been reported in several papers that when Kinesin-1 function is impaired, both cell motility and neurite outgrowth are reduced (Curr. Biol., 23: 1018–1023, 2013; Mol. Cell. Biol., 39: e00109–19, 2019; etc.). Therefore, it is likely that the phenotypes we observed are independent of the functions associated with Kinesin-1 in N1-E115 cells. It is indeed possible that the experiment suggested by the reviewer may reveal relationships between Map7D2 and kinesin-1 in terms of cell motility and neurite outgrowth, however, it is difficult to conduct such an experiment because transient expression of Map7D2 induces MT bundling, as shown in original Fig. 2F. Based on the above, we plan to add a discussion of the relationship between Map7D2 and Kinesin-1.

A key conclusion proposed by the authors is that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms. Such different roles in MT stabilisation are important in controlling cell motility and neurite outgrowth. In my opinion, their data does not fully support this statement and the findings using MT readouts do not match the defects in migration and neurite growth. Loss of Map7D2 leads to a very subtle phenotype on α-tubulin, while Map7D1 decreases both α-tubulin and acetylated tubulin, but Map7D1 seems to have a milder or similar effect on migration and neurite growth than Map7D2. Furthermore, it would be expected that the combined loss of function would lead to a stronger phenotype in cell migration when compared to the single loss of functions due to their distinct roles on MT stability, however, this seems not to be the case.

Response: The fact that no stronger phenotype was observed may be because, besides Map7D2 and Map7D1, other molecules are involved in MT stabilization. Another possible explanation is that the increases in both cell motility and neurite outgrowth caused by decreased MT stabilization are offset by Kinesin-1 dysfunction. We plan to add a discussion of the above two possibilities.

Minor comments:

1) In the first result section, the author refers to Fig. S3 to suggest the expression of MAP7D2 in the cerebral cortex, however, there are no transcripts in the cerebral cortex according to the figure. Similarly, the immunofluorescence analysis done by the authors shows marginal expression of MAP7D2 in the cerebral cortex.

Response: According to the reviewer’s comment, we have changed the order of the data shown in Fig. 1C, top panels. The data from the olfactory bulb, cerebellum, and hippocampus, in which Map7D2 expression was detected in the database, were arranged in the top three rows, and the data from the cerebral cortex, in which Map7D2 expression was not detected in the database, were moved to the bottom row as a negative control. In addition, we have revised the relevant part of the Results section as follows: “Based on RNA-seq CAGE, RNA-Seq, and SILAC database analysis (Expression Atlas, https://www.ebi.ac.uk/gxa/home/), Map7D2 expression was detected in the cerebellum, hippocampus, and olfactory bulb, and not in the cerebral cortex (Fig. S3). We further confirmed Map7D2 expression in the above four brain tissue regions of postnatal day 0 mice by immunofluorescence. Among these regions, Map7D2 was the most highly expressed in the Map2-negative area of the olfactory bulb, i.e., the glomerular layer (Fig. 1C). Weak signals were detected in the cerebellum, and marginal signals were observed in the hippocampus and cerebral cortex (Fig. 1C).” (page 5, lines 4–11)

2) The authors use γ-Tubulin as a housekeeping gene in Fig. 3D, since Map7D2 is enriched in centrosomes this may not be the most appropriate choice.

Response: γ-Tubulin is abundant in both the cytosol and the nuclear compartments of cells (Sig. Transduct. Target Ther. 3: 24, 2018). As it has been used for similar purposes in several other studies (Cancer Res., 61: 7713–7718, 2001; J. Biol. Chem., 291: 23112–23125, 2016; etc.), we considered it acceptable for use as a loading control for immunoblotting.

3) According to the authors, knockdown of Map7D2 leads to a decrease in the intensity of α-tubulin and Map7D1 (Fig. 4C and D). This data doesn't agree with the previous statement made by the authors where they show that Map7D2 knockdown or knockout did not affect Map7D1 expression by Western Blot Analysis (Fig. S2C and S5B)

Response: The immunoblotting results indicate that the total amount of Map7D1 in the cells is not affected by loss of Map7D2. In contrast, the immunofluorescence results indicate that the amount (distribution) of Map7D1 localized around the centrosome is decreased by loss of Map7D2, presumably due to a reduction in the number of MT structures that can serve as scaffolds for Map7D1. We plan to add this interpretation in the Results section.

4) Line 6 page 7 "Endogenous Map7D2 expression is suppressed in N1-E115 cells stably expressing EGFP-rMap7D2 and was restored by specific knock-down of EGFP-rMap7D2 using gfp siRNA (Fig. 3D)". No quantifications and stats are shown. Also, endogenous Map7D2 after knock-down of EGFP-rMap7D2 is not comparable to the control.

Response: According to the reviewer’s suggestion, we have quantified the amount of endogenous Map7D2 or EGFP-rMap7D2, normalized it to the amount of γ-tubulin, and calculated relative values to endogenous Map7D2 in the parental control. The amount of endogenous Map7D2 was decreased to 53% in N1-E115 cells stably expressing EGFP-rMap7D2, suggesting that EGFP-rMap7D2 expression suppressed endogenous Map7D2 expression. In this cell line, the total amount of Map7D2 (EGFP-rMap7D2 + endogenous Map7D2) was increased, however, when EGFP-rMap7D2 was depleted using sigfp in this cell line, endogenous Map7D2 was expressed to the same level as EGFP-rMap7D2 before knock-down. Together with the finding that Map7d1 knock-down increased the amount of Map7D2, these findings indicate that the amount of Map7D2 in the cells is regulated in response to the amount of Map7D1 and exogenous Map7D2. We have added this interpretation in the Results section. (page 7, lines 8–15)

In addition, we have changed the legend of the original Fig. 3D to clarify the quantification method, as follows: “(D) Generation of N1-E115 cells stably expressing EGFP-rMap7D2. To check the expression level of EGFP-rMap7D2, lysates derived from the indicated cells were probed with anti-GFP (top panel) and anti-Map7D2 (middle panel) antibodies. The blot was reprobed for γ-tubulin as a loading control (bottom panel). The amount of endogenous Map7D2 or EGFP-rMap7D2 was normalized to the amount of γ-tubulin, and the value relative to endogenous Map7D2 in the parental control was calculated.” (page 22, lines 18–20)

5) Line 8 page 7 "These results suggest that the expression of Map7D2 was influenced by changes in that of Map7D1" This statement seems in the wrong place, after the Map7D2 and EGFP-rMap7D2 experiment. Instead for clarity, it would be better placed after line 5 where the authors explain the effect of Map7D1 knock-down on the levels of Map7D2.

Response: According to the reviewer’s suggestion, we have rephrased the relevant sentence as “Interestingly, Map7d1 knock-down upregulated Map7D2 expression, as confirmed with three different siRNAs (Fig. S2C), suggesting that Map7D2 expression is affected by changes in Map7D1 expression, not by off-target effects of a particular siRNA.” (page 7, lines 7, 8)

6) Line 8 page 8 "Although the physiological role of the C-terminal region of Map7D2 is currently unknown..." This statement seems not adequate as there are several studies reporting the role of the C-terminal region of Map7D2 in Kinesin1- mediated transport. The authors mention such studies in the discussion.

Response: According to the reviewer’s suggestion, we plan to add a discussion of the relationship between Map7D2 and kinesin-1.

7) Line 6 page 9 " Further, the knock-down of either resulted in a comparable reduction of MT intensity (Fig. 4C and D) ..." This is not visible and/or justified by the images provided and would benefit from some sort of quantification at other regions such as neurites.

Response: Considering the cell motility, quantification of α-tubulin/Ace-tubulin/Map7D1/Map7D2 intensities in neurites is not appropriate. Instead, we have added arrowheads indicating α-tubulin/Ace-tubulin/Map7D1/Map7D2 in Fig. 4C, for better understanding.

8) In Fig. 2B, a band corresponding to his6-rMAP7D2 of molecular weight >97 kDa co-sedimented with the microtubules. However, the cloned rMAP7D2 had a molecular weight of 84.82 kDa and the addition of 6XHis-Tag would add another 2-3 kDa, therefore, the final protein band observed should be less than 90 kDa. It would be beneficial if the authors could specify the molecular weight of the purified protein after the addition of the V5-his tag and/or if there was addition of amino acids due to cloning strategy.

Response: In Fig. 2B, we used full-length GST-tagged rMap7D2, like in Fig. 2E and D; therefore, we have corrected His6-rMap7D2 as GST-rMap7D2. We apologize for the mistake.

9) In Fig. 2C, there is misalignment of the western blot with the panel or text underneath.

Response: We thank the reviewer for pointing this out; we have corrected the misalignment of the CBB staining in Fig. 2C.

10) In Fig. 3C the inset from the first panel seems to correspond to a different focal plane than the main image.

Response: We have revised the relevant part of the figure legend as follows: “In C, images of differentiated cells were captured by z-sectioning, because the focal planes of the centrosome and neurites are different. Each inset shows an enlarged image of the region indicated with a white box at each focal plane. Arrowheads indicate the centrosomal localization of Map7D2.”

11) In Fig. 4A, the cell type is not specified and is referred as "indicated cells", also the material and methods section seems to omit the specific cells used.

Response: We have added “in N1-E115 cells treated with each siRNA” in the legend of Fig. 4A.

12) Fig. S6 is not mentioned in the results.

Response: We apologize for having referred to Fig. S6 only in the Discussion section in the original manuscript. We plan to describe the findings shown in the original Fig. S6 to the Results section and renumber the figures accordingly.

Significance (Required):

MTs play essential roles in practically every cellular process. Their precise regulation is therefore crucial for cellular function and viability. MAPs are specialised proteins that interact with MTs and regulate their behaviour in different manners. Understanding their precise function in different cellular contexts is of utmost importance for many biological and biomedical fields.

MAPs are well known for their ability to promote MT polymerization, bundling and stabilisation in vitro (Bodakuntla et al., 2019). Several members of the Map7 family have been shown to regulate microtubule stability. For instance, MAP7 can prevent nocodazole-induced MT depolymerization and maintain stable microtubules at branch points in DRG neurons (Tymanskyj & Ma, 2019). Ensconsin, the Drosophila Map, is required for MT growth in mitotic neuroblasts by regulating the mean rate of MT polymerization (Gallaud et al., 2014). However, this family of Maps seems to have diverse functions encompassing a variety of mechanisms, as exemplified by a series of studies demonstrating the involvement of MAP7 family proteins in the recruitment and activation of kinesin1 (Hooikaas et al., 2019; Pan et al., 2019) and in microtubule remodelling and Wnt5a signalling (Kikuchi et al., 2018). Further understanding of this family of Maps and how its members differ in their function is important and will help to advance the field.

Response: We appreciate the reviewer’s comments. We believe that our revision plan will greatly improve the quality of our manuscript.

Reviewer #3

Evidence, reproducibility and clarity (Required):

Summary:

Microtubule Associated Proteins (MAPs) are important regulators of microtubule dynamics, microtubule organization and vesicular transport by modulating motor protein recruitment and processivity. In the current manuscript the authors have characterized 2 members of the MAP7 protein family, MAP7D1 and MAP7D2. The authors characterized MAP7D2 expression pattern in the brain and its microtubule binding properties in vitro and in cells. In cells both proteins localize to the centrosome and to microtubules and upon depletion centrosome localized microtubules seem reduced, and cell migration and neurite outgrowth are increased. Surprisingly, they find that microtube acetylation (a common marker for stable microtubules) is reduced upon MAP7D1 depletion but not MAP7D2 depletion. Based on this finding the authors conclude that these proteins have a distinct mechanism in stabilizing MTs to affect cell migration and neurite outgrowth; MAP7D2 stabilizes by binding to MTs, whereas MAP7D1 stabilizes MTs by acetylation.

Main comments:

- Both MAP7 proteins show strong localization to the centrosome and to a lesser degree to MTs. Knockdown of either protein leads to reduced MTs around the centrosome, which lead the authors to conclude the MAP7s are stabilizing the MTs. However, the effect could just as well be an indirect effect due to a function of these MAPs at the centrosome. To address this authors could e.g. quantify microtubule properties in postmitotic cells. In addition, antibody specificity should be tested using knockdown of knockout cells, as this centrosome localization was not observed in Hela cells (Hooikaas, 2019; Kikuchi, 2018). Maybe this localization is specific to rat MAP7s or to the cell line used.

Response: We think that this comment partly overlaps with the comments raised by Reviewer #2. We plan to assess the role of MT stabilization in greater detail by analyzing the sensitivity to the MT-destabilizing agent, nocodazole, and the effect on MT dynamics by measuring the EB1 comet length by immunofluorescence.

Regarding the reviewer’s concern about antibody specificity, we had carefully confirmed the antibody specificity, as shown in Fig. S2 of the original manuscript. Subsequently, Map7D2 localization was confirmed in N1-E115 cells stably expressing EGFP-rMap7D2, as shown in Fig. 3D, E of the original manuscript. In addition, we are currently conducting analyses using Map7d1-egfp knock-in mice, which confirmed that Map7D1 localizes around the centrosome in cortical neurons, as shown below (we would like to disclose these unpublished data to the reviewers only). Therefore, it is thought that the localization pattern of Map7D2 and Map7D1 differs depending on the cell type and cell line. We plan to add this interpretation to the Results section.

- Centrosome nucleated microtubules are typically highly dynamic and little modified. Therefore is the Ac-tub staining at the centrosome really MTs? I cannot identify MTs in the fluorescent images in 4C. Maybe authors could consider ac-tub/alpha-tub ratio in non centrosomal region (e.g. neurites). Moreover, as both Acetylation and detyrosination are associated with long-lived/stable MTs, it is surprising that only acetylated tubulin goes down on WB. Does this suggest that long-lived MTs are still present to normal level? If so, can one still argue that the loss of acetylation is the cause of the lower MT levels? This should at least be discussed.

Response: As for the reviewer’s statement “Centrosome nucleated microtubules are typically highly dynamic and little modified. Therefore is the Ac-tub staining at the centrosome really MTs?”, it has been previously reported that tubulin acetylation is observed around the centrosome in some cell lines (J. Neurosci., 30: 7215–7226, 2010; PLoS One, 13: e0190717, 2018; etc.). N1-E115 is one of the cell lines in which tubulin acetylation is observed around the centrosome.

It is not surprising that “only acetylated tubulin goes down on WB,” as it has been previously reported that acetylated and detyrosinated tubulins are sometimes not synchronous (J. Neurosci., 23: 10662–10671, 2003; J. Neurosci., 30: 7215–7226, 2010; J. Cell Sci., 132: jcs225805, 2019., etc.). For instance, Montagnac et al. (Nature, 502: 567–570, 2013) showed that defects in the α-tubulin acetyltransferase αTAT1-clathrin-dependent endocytosis axis reduce only tubulin acetylation, resulting in a shift from directional to random cell migration. Although the details of the molecular function of Map7D1 are beyond the main purpose of this study, we plan to add a discussion of the reduced tubulin acetylation by Map7d1 knock-down based on the above.

- MAP7D1 and MAP7D2 depletion leads to subtle defect in cell migration and neurite outgrowth, which the author suggest is caused by reduced MT stability. However, MAP7 proteins have well characterized functions in kinesin-1 transport, and thus the phenotypes may well be caused by defects in kinesin-1 transport. Ideally the authors would do rescue experiments with FL or just the MT binding N-termini to separate these functions. Moreover this is needed to substantiate the claim of the authors that MAP7D1 effect on MT stability is not mediated by direct binding.

Response: As this comment largely overlaps with the comments raised by Reviewer #2, please refer to our responses to the comments of Reviewer #2.

- The authors do not refer well to published work. Several papers have published very similar work (especially to Fig1+2) and it would help the reader much if this would be discussed/compared along the results section and not briefly mention these in the results section. In addition, authors overstate the novelty of their results e.g. page 3: these proteins are not "functionally uncharacterized" nor are their expression patter and biochemical properties analyzed for the first time in this manuscript; page 8 "Although the physiological role of the C-terminal region of Map7D2 is currently unknow, ..." There is a clear function for the C-terminus for the recruitment/activation of kinesin-1.

Response: According to the reviewer’s suggestion, we plan to add a comparison with data on the Map7 family members presented in previous papers in the Results section and rephrase the relevant part regarding the physiological role of the C-terminal region of Map7D2.

Minor comments

- P6 Map7D3 also binds with its N-terminus to MTs, like other MAP7s (Yadav et al)

Response: According to the reviewer’s comment, we have revised this as “Map7D3 binds through a conserved region on not only the N-terminal side, but also the C-terminal side (Sun, 2011; Yadav et al., 2014).” (page 6, lines 4, 5)

- P7 "As Map7D2 has the potential to functionally compensate for Map7D1 loss" where is this based on?

Response: For clarity, we have rephrased this as “As Ma7D2 expression was upregulated upon suppression of Map7D1 expression, Map7D2 has the potential to functionally compensate for Map7D1 loss.” (page 7, line 17, 18)

- Fig2F quality of black-white images is low potentially due to conversion issues

Response: We thank the reviewer for pointing out these conversion issues, and we have made the necessary corrections.

Significance (Required):

At this stage the conceptual advance is limited. Part of the findings are not novel. The finding that MAP7s depletion have a different effect on MTs acetylation may be interesting to cytoskeleton researchers, although the potential mechanism has not been addressed experimentally or textually.

However, their conclusion that this leads to reduced MTs and then to cellar migration and neurite formation defects is not sufficiently supported by experimental evidence.

Response: We appreciate the reviewer’s comments. We believe that our revision plan will greatly improve the quality of our manuscript.

\*Referees cross-commenting***

I completely agree with reviewer #2: At this stage the paper's conclusions are not sufficiently supported by the data. Important will be to further characterize the effect om the MTs (do they really have a different effect) and to look at the possible involvement of the motor recruitment. Maybe that a 3 to 6 months revision time would have been more accurate.

Response: Please refer to our responses to the comments of Reviewer #2.

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

Learn more at Review Commons

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Referee #2

Evidence, reproducibility and clarity

In this study, the authors investigate 2 members from the MAP7 family Map7D2 and Map7D1. They first address the tissue distribution of Map7D2, by northern blotting using a variety of rat tissues. To complement their analysis, they also raised an antibody to look at the protein distribution. From their studies, they concluded that Map7D2 is abundantly expressed in the brain and testis. The authors went on to perform a series of functional assays . First, they biochemically demonstrated that rat Map7D2 directly binds to MTs by MT co-sedimentation assay. The MT binding domain was mapped to the N-terminal half. They performed MT turbidity assay to demonstrate enhanced MT polymerisation in the presence of Map7D2, suggesting that this Map stabilises MTs. The authors went on to characterise in detail the subcellular localisation of Map7D2 which was predominantly present in the centrosome and partially localised to MTs including within neurites from N1-E115 cells. Kikuchi et al. further revealed the overlap in expression between Map7D2 and another family member, Map7D1. The authors continued these studies by a series of functional studies in N1-E115 cells where they performed single or combined knock-downs of Map7D2 and Map7D1 and studied the levels of acetylated and detyrosinated tubulins and the effect of the knock-downs on migration and neurite extension. The main conclusion from this work was that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms which are important in controlling cell motility and neurite outgrowth. Map7D2 is proposed to stabilise MTs by direct binding whereas Map7D1 does it indirectly by affecting acetylation.

Major comments:

The main conclusion from this work that Map7D2 and Map7D1 facilitate MT stabilization and that this is necessary for correct migration and neurite extension has not been convincingly demonstrated. In my opinion, a more detailed study of MT properties to demonstrate a role in MT stabilisation would greatly benefit the work, eg. experiments using MT destabilising agents such as nocodazole. In addition, a series of experiments aiming to study MT dynamics would help to understand the function of these MT regulators. The authors proposed an elevation in microtubule dynamics to explain the increase in migration and neurite extension but no experimental proof was provided.

It has been previously demonstrated that loss of MAP7D2 leads to a decrease in axonal cargo entry to axons resulting in defects in axon development and neuronal migration. The C-terminus is necessary for this function as it mediates interaction with Kinesin-1 (Pan et al., 2019). Such mechanisms could also explain the defects in migration and neurite growth that the authors observed. This possibility has not been considered but instead, the subtle changes in total -tubulin led to suggest MT stabilisation as a key function without proof of causation. Could the authors provide some further experimental evidence to demonstrate that stability is the main contributor to the phenotypes observed? Eg. by rescuing migration and neurite phenotypes with a variant of MAP7D2 which cannot bind kinesin1.

A key conclusion proposed by the authors is that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms. Such different roles in MT stabilisation are important in controlling cell motility and neurite outgrowth. In my opinion, their data does not fully support this statement and the findings using MT readouts do not match the defects in migration and neurite growth. Loss of Map7D2 leads to a very subtle phenotype on -tubulin, while Map7D1 decreases both -tubulin and acetylated tubulin, but Map7D1 seems to have a milder or similar effect on migration and neurite growth than Map7D2. Furthermore, it would be expected that the combined loss of function would lead to a stronger phenotype in cell migration when compared to the single loss of functions due to their distinct roles on MT stability, however, this seems not to be the case.

Minor comments:

1. In the first result section, the author refers to Fig. S3 to suggest the expression of MAP7D2 in the cerebral cortex, however, there are no transcripts in the cerebral cortex according to the figure. Similarly, the immunofluorescence analysis done by the authors shows marginal expression of MAP7D2 in the cerebral cortex.
2. The authors use -Tubulin as a housekeeping gene in Fig. 3D, since Map7D2 is enriched in centrosomes this may not be the most appropriate choice.
3. According to the authors, knockdown of Map7D2 leads to a decrease in the intensity of -tubulin and Map7D1 (Fig. 4C and D). This data doesn't agree with the previous statement made by the authors where they show that Map7D2 knockdown or knockout did not affect Map7D1 expression by Western Blot Analysis (Fig. S2C and S5B)
4. Line 6 page 7 "Endogenous Map7D2 expression is suppressed in N1-E115 cells stably expressing EGFP-rMap7D2 and was restored by specific knock-down of EGFP-rMap7D2 using gfp siRNA (Fig. 3D)". No quantifications and stats are shown. Also, endogenous Map7D2 after knock-down of EGFP-rMap7D2 is not comparable to the control.
5. Line 8 page 7 "These results suggest that the expression of Map7D2 was influenced by changes in that of Map7D1" This statement seems in the wrong place, after the Map7D2 and EGFP-rMap7D2 experiment. Instead for clarity, it would be better placed after line 5 where the authors explain the effect of Map7D1 knock-down on the levels of Map7D2.
6. Line 8 page 8 "Although the physiological role of the C-terminal region of Map7D2 is currently unknown..." This statement seems not adequate as there are several studies reporting the role of the C-terminal region of Map7D2 in Kinesin1- mediated transport. The authors mention such studies in the discussion.
7. Line 6 page 9 " Further, the knock-down of either resulted in a comparable reduction of MT intensity (Fig. 4C and D) ..." This is not visible and/or justified by the images provided and would benefit from some sort of quantification at other regions such as neurites.
8. In Fig. 2B, a band corresponding to his6-rMAP7D2 of molecular weight >97 kDa co-sedimented with the microtubules. However, the cloned rMAP7D2 had a molecular weight of 84.82 kDa and the addition of 6XHis-Tag would add another 2-3 kDa, therefore, the final protein band observed should be less than 90 kDa. It would be beneficial if the authors could specify the molecular weight of the purified protein after the addition of the V5-his tag and/or if there was addition of amino acids due to cloning strategy.
9. In Fig. 2C, there is misalignment of the western blot with the panel or text underneath.
10. In Fig. 3C the inset from the first panel seems to correspond to a different focal plane than the main image.
11. In Fig. 4A, the cell type is not specified and is referred as "indicated cells", also the material and methods section seems to omit the specific cells used.
12. Fig. S6 is not mentioned in the results.

Significance

MTs play essential roles in practically every cellular process. Their precise regulation is therefore crucial for cellular function and viability. MAPs are specialised proteins that interact with MTs and regulate their behaviour in different manners. Understanding their precise function in different cellular contexts is of utmost importance for many biological and biomedical fields.

MAPs are well known for their ability to promote MT polymerization, bundling and stabilisation in vitro (Bodakuntla et al., 2019). Several members of the Map7 family have been shown to regulate microtubule stability. For instance, MAP7 can prevent nocodazole-induced MT depolymerization and maintain stable microtubules at branch points in DRG neurons (Tymanskyj & Ma, 2019). Ensconsin, the Drosophila Map, is required for MT growth in mitotic neuroblasts by regulating the mean rate of MT polymerization (Gallaud et al., 2014). However, this family of Maps seems to have diverse functions encompassing a variety of mechanisms, as exemplified by a series of studies demonstrating the involvement of MAP7 family proteins in the recruitment and activation of kinesin1 (Hooikaas et al., 2019; Pan et al., 2019) and in microtubule remodelling and Wnt5a signalling (Kikuchi et al., 2018). Further understanding of this family of Maps and how its members differ in their function is important and will help to advance the field.

Author Response:

Reviewer #2 (Public Review):

This manuscript by Barton and colleagues explores the roles of the conserved Eco1 transacetylase in modulating cohesin function in meiosis in budding yeast. Numerous studies in mitotically dividing cells have shown that the Eco1 family of transacetylases acetylate the Smc3 subunit of cohesin and that this acetylation renders cohesin on chromosomes resistant to removal by the Wapl (Wpl1 in budding yeast) family of proteins. Cohesins play critical roles in both sister chromatid cohesion and chromatin organization (through the formation of intrachromosomal loops). How cohesins are regulated by Eco1 in meiosis to accommodate meiotic chromosome structures such as the synaptonemal complex, chromatin domains around centromeres, repair of programmed meiotic double strand DNA breaks in prophase, and sequential removal of cohesins - first at arms in meiosis I and centromeres at meiosis II - is largely unexplored. Thus, this manuscript is exploring important new areas.

The authors show that Eco1 persists thru prophase I (longer than it does in vegetative cell cycles), that it is not necessary for cohesin loading at centromeres but is needed to counteract Wpl1 to protect centromeric cohesion, that it is critical for the establishment of chromatin loops on meiotic chromosome arms and that it is critical for protection of the arm cohesin from removal by Wpl1. The authors also provide evidence that, in meiosis, Wpl1 exhibits underappreciated functions in cohesin loading or cohesion establishment in addition to its recognized role in cohesin removal.

The experiments demonstrate that Eco1 is necessary for sharp cohesin boundaries that flank the centromeres and suggest this might be a replication-independent function of Eco1 (the boundaries form in clb5, clb6 cells with no DNA replication phase) but it is unclear if the detectable, but diminished, boundaries in clb5,clb6 cells were formed in the replication-free meiosis or presist from the S-phase associated loading and cohesion establishment from the preceding mitotic cycle.

Entry into meiosis occurs from G1 when there is no cohesin on the chromosomes and boundaries are not present, therefore this would only be a concern if there were persistent mitotic cells in G2 (i.e. after DNA replication). Our flow cytometry shows that the cells used in the experiment were unreplicated, so even if mitotic cells were present, they would not have been through S phase.

Nevertheless, we addressed this point by analysis of pre-S phase meiotic cells (ime1/ime4 block) and by anchoring away Eco1 in unreplicated cells.

Immunofluorescence imaging assays are used to observe the behavior of sister chromatids in meiosis I and meiosis II as a function of Eco1 activity. In wild-type cells sister chromatids co-orient in meiosis I and move to the same pole of the spindle. In mammalian cells and fission yeast this co-orientation requires cohesin while studies in budding yeast have suggested the co-orientation is cohesin-independent. Here, the authors show that when Eco1 is depleted, the sisters often move to opposite poles at meiosis I, and suggest that cohesin (and Eco1) is indeed required for sister co-orientation. An alternate possibility is that the sisters have lost their association in meiotic prophase (due to cohesin failures) before attaching to microtubles and segregating randomly - often to opposite poles.

We agree with this point, but would argue that the “alternative possibility” (which our data support) still leads to the conclusion that cohesin and Eco1 are required for sister co-orientation. A prior study (Monje-Casas et al., 2007) had suggested that monopolin could link sister kinetochores even without cohesin. We now show that this is not the case, which we believe to be an important conclusion.

Our results indicate that establishment of monoorientation requires the cohesin that is localized at centromeres. WPL1 deletion in eco1-aa rescues centromeric cohesion (Figure 2F, Figure 8E), but not chromosome arm cohesion (Figure 2H) or sister chromatid segregation in meiosis II (Figure 8F), indicating that pericentromeric cohesion must still be defective.

For clarity, please note that the relevant data is not immunofluorescence, but live cell imaging (now shown in Figure 8) so these conclusions are based on observation of single chromosomes in individual live cells from prophase I until anaphase II.

In summary the authors show that Eco1 has distinct roles on chromosome arms and centromeres and probably in both replication-linked and replication-independent events, acts to modulate cohesin location and function in meiosis.

Reviewer #3 (Public Review):

This paper investigates the meiotic roles of two regulators of cohesin, the cohesin destabilizer Wpl1 and the cohesin acetyltransferase Eco1. The authors provide evidence that Eco1 antagonizes Wpl1 to allow stabilization of centromeric cohesin, which is important to establish meiotic chromosome segregation patterns. In addition, Eco1 regulates the stable anchoring of cohesin at boundaries to promote defined chromosome loop formation in meiotic prophase.

The study uses a combination of calibrated ChIP-seq analysis, and chromosome conformation capture techniques to convincingly show that loop formation is altered in wpl1 depletion and eco1 depletion mutants. Well-established cytological techniques are used to demonstrate different effects on chromosome cohesion along arms and at centromeres, and to show that Eco1 is important for establishing the meiotic segregation pattern. The paper is well written and the data largely support the conclusions. As such, this paper is expected to be of substantial interest to the field.

One notable weakness is the poor definition of the eco1 anchor-away allele (eco1-aa), on which much of the eco1 phenotypic analysis is based. The presented data indicate that addition of the FRB-GFP tag alone causes most of the phenotypes, regardless of nuclear depletion. It is well possible that the tag creates a meiosis-specific loss-of-function allele, although it is surprising that the tag does not have mitotic defects even though Eco1 presumably has the same substrate (the cohesin subunit Smc3) in both situations. Encouragingly, some of the phenotypes could be confirmed using a non-acetylatable smc3 mutant. However, the tag may also create neomorphic effects that may contribute to the Wpl1-independent effects and the apparent stronger defects of the eco1-aa allele compared to the non-acetylatable smc3 mutant.

Available evidence suggests that eco1-aa is a loss of function allele.

Reviewer #3 (Public Review):

This paper investigates the meiotic roles of two regulators of cohesin, the cohesin destabilizer Wpl1 and the cohesin acetyltransferase Eco1. The authors provide evidence that Eco1 antagonizes Wpl1 to allow stabilization of centromeric cohesin, which is important to establish meiotic chromosome segregation patterns. In addition, Eco1 regulates the stable anchoring of cohesin at boundaries to promote defined chromosome loop formation in meiotic prophase.

The study uses a combination of calibrated ChIP-seq analysis, and chromosome conformation capture techniques to convincingly show that loop formation is altered in wpl1 depletion and eco1 depletion mutants. Well-established cytological techniques are used to demonstrate different effects on chromosome cohesion along arms and at centromeres, and to show that Eco1 is important for establishing the meiotic segregation pattern. The paper is well written and the data largely support the conclusions. As such, this paper is expected to be of substantial interest to the field.

One notable weakness is the poor definition of the eco1 anchor-away allele (eco1-aa), on which much of the eco1 phenotypic analysis is based. The presented data indicate that addition of the FRB-GFP tag alone causes most of the phenotypes, regardless of nuclear depletion. It is well possible that the tag creates a meiosis-specific loss-of-function allele, although it is surprising that the tag does not have mitotic defects even though Eco1 presumably has the same substrate (the cohesin subunit Smc3) in both situations. Encouragingly, some of the phenotypes could be confirmed using a non-acetylatable smc3 mutant. However, the tag may also create neomorphic effects that may contribute to the Wpl1-independent effects and the apparent stronger defects of the eco1-aa allele compared to the non-acetylatable smc3 mutant.

SciScore for 10.1101/2022.01.20.477107: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: EXPERIMENTAL MODEL AND SUBJECT DETAILS: Blood, PBMCs and HLA typing: Whole blood from COVID-19 convalescent donors, healthy donors, or vaccine recipients was collected under protocol approved by the UMass Chan Medical School Institutional Review Board of the University of Massachusetts and informed consent was obtained from all subjects.<br>Consent: EXPERIMENTAL MODEL AND SUBJECT DETAILS: Blood, PBMCs and HLA typing: Whole blood from COVID-19 convalescent donors, healthy donors, or vaccine recipients was collected under protocol approved by the UMass Chan Medical School Institutional Review Board of the University of Massachusetts and informed consent was obtained from all subjects.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For blocking of antigen-stimulation assays, in-house produced antibodies to HLA-DR (LB3.1), HLA-DQ (SPVL-3), HLA-DP (B7/21), or HLA-ABC (w6/32), were added at a final concentration of 10 μg/mL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HLA-DR</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HLA-DP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HLA-ABC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were collected, washed, and stained using a standard protocol which included: staining for dead cells with Live/Dead Fixable Aqua Dead Cell Stain Kit™ (Life Technologies, Thermo Fisher Scientific, Waltham, MA); blocking of Fc receptors with human Ig (Sigma-Aldrich, St. Louis, MO); staining with the mix of DP4-PE and APC tetramers (final concentration of 2-4 μg/mL each) at 37°C for 2 hours; surface staining antibodies CD3-APC-H7, CD4-PerCP-Cy5.5, CD8-APC-R700, CD14-BV510, CD19-BV510, CD56-BV510 were added for the last 20 minutes of incubation, followed by washes and resuspension in buffer for data acquisition.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD19-BV510</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD56-BV510</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Partially-match HLA cell lines: EBV-transformed LG2 cell line (10984, IPD-IMGT/HLA), 9068 cell line (BM9, IHWG), and mouse DP4-transfected MN605 cell line (M12C3-DPA1*0103/DPB1*0401; (Williams et al., 2018); kindly provided by Dr. S. Kent, UMMS), were maintained in RPMI 1640 medium supplemented with L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and 10% FBS at 37°C/5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MN605</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 6 hours incubation, cells were collected, washed, and stained using a standard protocol, which included: staining for dead cells with Live/Dead Fixable Aqua Dead Cell Stain Kit™ (Life Technologies, Thermo Fisher Scientific, Waltham, MA); blocking of Fc receptors with human Ig (Sigma-Aldrich, St. Louis, MO); surface staining with mouse anti-human CD3-APC-H7 (SK7), CD4-PerCPCy5.5 (RPA-T4), CD8-APC-R700 (RPA-T8), CD14-BV510 (MϕP9), CD19-BV510 (SJ25C1), CD56-BV510 (NCAM16.2); fixation and permeabilization using BD Cytofix/Cytoperm™; and intracellular staining with mouse anti-human IFN-γ-V450 (B27), TNF-α-PE-Cy7 (MAb11), IL-2-BV650 (5344.111), (all from BD Biosciences, San Jose, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD Cytofix/Cytoperm™</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were acquired using a BD LRSII flow cytometer equipped with BD FACSDiva software (BD Biosciences, San Jose, CA) and analyzed using FlowJo v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD FACSDiva</div><div>suggested: (BD FACSDiva Software, RRID:SCR_001456)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Polyfunctional analysis was performed in FlowJo, defining Boolean combinatorial gates for all the markers in the CD3+/CD4+/CD8-population.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These results were visualized in SPICE software v6.0 (Roederer et al., 2011). t-SNE analysis was done in concatenated samples (control, SARS-CoV-2, peptide 163 and peptide 164) from 3 donors using the available plugin in FlowJo.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPICE</div><div>suggested: (SPICE, RRID:SCR_016603)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All primers sequences shown in STAR Methods.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>STAR</div><div>suggested: (STAR, RRID:SCR_004463)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These files were processed using MIGEC v1.2.9 pipeline: Checkout-batch for de-multiplexing and UMI tag extraction, Histogram for MIG (molecular identifier groups) statistics, and Assemble-batch to perform UMI-guided assembly (Shugay et al., 2014); followed by MiXCR v3.0.13: analyze amplicon pipeline, to align, assemble and export clonotypes (Bolotin et al., 2015).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MIGEC</div><div>suggested: (migec, RRID:SCR_016337)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HLA-peptide binding prediction was performed with NetMHCIIpan v4.0 server (Reynisson et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NetMHCIIpan</div><div>suggested: None</div></div></td></tr></table>

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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: We detected the following sentences addressing limitations in the study:

There are several limitations of our study. We mapped the specificity of the cross-reactive response by following IFN-γ-secreting cells, but non-IFN-γ-secreting populations could also contribute to the response. In expanded T cell lines we observed higher frequencies of T cells staining with DP4-163/164 tetramer than responding to the same peptide in IFN-γ ELISPot essays, indicating that some T cells can recognize the epitope but not secrete IFN-γ. We observed the cross-reactive T cell response to involve mostly CD4+ T cells. This might be due to in vitro culture conditions that favor CD4+ over CD8+ T cell populations, or an intrinsic bias of cross-reactive T cells because of the different patterns of pMHC-TCR interaction for MHC-I and MHC-II proteins. We studied a relatively small group of 27 individuals exposed to SARS-CoV-2 antigens by infection or vaccination, mostly over 40 years of age. Younger individuals with more frequent previous exposures to HCoVs might show a different pattern of response. Our initial screen for immunodominant epitopes involved only three donors, all of whom recognized 163/164, but other immudominant cross-reactive epitopes might have escaped our attention, including those recognized by other MHC proteins. For all of the donors, previous HCoV infection was inferred but not observed, and we did not attempt to determine which donors were exposed previously to which of the HCoVs. In conclusion, we identified a pan-coronavirus epitope that dominates t...

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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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 32 and 36. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

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test

SciScore for 10.1101/2022.01.20.477056: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Ethical statement: The study was approved by the ethical review board in Gothenburg (Dnr: 2021-02252).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Levels of human anti-SARS-CoV-2 IgG antibodies in convalescent serum samples: Serum samples from SARS-CoV-2 convalescent individuals (n=24) were obtained from the department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-SARS-CoV-2 antibody reactivity assay: The antibody reactivity towards glycosidase treated proteins were assessed using an enzyme-linked immunosorbent assay (ELISA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The comparison between anti-RBD IgG levels and antibody reactivity towards recombinant RBD was done with Pearson correlation coefficient, assuming normal distribution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lec3.2.8.1 cells (a mutated CHO cell line kindly received from Prof. P Stanley (Chen & Stanley, 2003)) were cultured under the same conditions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The HEK293 derivate HEK293F cell line (Cat nr R79007, Thermo Fisher Scientific) were cultured in Freestyle 293 medium.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293F</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression of recombinant S protein constructs: The receptor-binding domain of the SARS-CoV-2 spike protein (amino acids 319-541) was produced in three cell lines using an expression vector obtained through BEI Resources, NIAID, NIH, which is vector pCAGGS containing the SARS-CoV-2, Wuhan-Hu-1 spike glycoprotein gene RBD with C-terminal Hexa-Histidine tag (NR-52309) (Table EV1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_127347)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Glycan database search and data processing: The acquired data were analyzed using Proteome Discoverer version 2.4 (Thermo Fisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Proteome Discoverer</div><div>suggested: (Proteome Discoverer, RRID:SCR_014477)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 IgG II Quant assay is a chemiluminescent microparticle immunoassay (CMIA) used for quantitative determination of IgG antibodies to SARS-CoV-2 in human serum and plasma on the ARCHITECT System (Abbott Laboratories, Chicago, IL).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Abbott Laboratories</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical analyses were performed using the Graphpad Prism software version 9.3.1</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(GraphPad Software Inc, San Diego, CA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

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

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

SciScore for 10.1101/2022.01.19.476940: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: To generate HEK-293T cells that transiently expressed wild type and mutated spike protein (ATCC; mycoplasma-free low passage stock), the cells were transfected with Spike protein expressing pLP plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol and stained for their spike protein expression 72 hours after the transfection as described in Staining and Flow cytometry Analysis.</td></tr></table>

Table 2: Resources

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

---

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

---

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

---

Results from TrialIdentifier: No clinical trial numbers were referenced.

---

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

---

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 37, 38, 39, 41 and 42. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

Results from rtransparent:

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

---

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

---

<footer>

About SciScore

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

</footer>

In other words, adding a svelte:error tag wouldn't help much.

annotation meta: may need new tag: client/server cooperation?

annotation meta: may need new tag: demonstrating....

Reminds me of that really long series of things on a contamination theory of obesity.

SciScore for 10.1101/2022.01.14.22268980: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Whole blood samples: The 60 samples used for figures 6, S1, S2 and S3 were routine care residues from patients of the Toulouse hospital, where all patients give, by default, their consent for any biological material left over to be used for research purposes after all the clinical tests requested by doctors have been duly completed.<br>Field Sample Permit: Those were anonymized within 24 hours of collection, transferred from the hospital to the research lab, and kept at room temperature until being used for HAT assays within 24 hours (i.e. less than 48 hours after blood samples were collected).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Authentication: Ethical statement: RBCs from O-blood donors were obtained from the Toulouse branch of the Etablissement Français du Sang (EFS), with whom the project was validated under agreement n° 21PLER2020-025.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Polyclonal anti-human Igs secondary antibodies, all conjugated to Alexa-488, were from Jackson laboratories, and purchased from Ozyme (France)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human Igs secondary</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti RBD monoclonal antibodies: CR3022 (ter Meulen et al. 2006) and EY6A (Zhou et al. 2020) were obtained using antibody-expression plasmids, as previously described (Townsend et al. 2021).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti RBD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>antibody-expression</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The activity of those aliquots of working stocks was then evaluated at regular intervals by performing titrations of the IH4-RBD reagent against either the CR3022 monoclonal antibody diluted to a final concentration of 100 ng/ml, or various immune sera diluted to give similar endpoint to those obtained with CR3022.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CR3022</div><div>suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For titration experiments, we prepared stocks containing RBCs and the appropriate amount of the reagent to be kept constant (either the IH4-RBD reagent at 1 µg/ml or IH4 alone at 0.5 µg/ml for plasma titrations, or the appropriate antibody dilution for IH4-RBD titrations).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IH4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">One drop (i.e. ca. 30 µl) of either the pan-specific anti-Ig-GAM secondary fluorescent antibodies, as well as anti-IgG, -IgA or -IgM, all diluted 1/200 in PFN was added to each of the four wells for each sample, and the cells resuspended by gentle shaking of the plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Ig-GAM</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">One drop (i.e. ca. 30 µl) of anti-human secondary antibody conjugated to alexa-488, diluted 1/200 in PFN was added to each of the wells, and the cells resuspended by gentle shaking of the plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human secondary</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>alexa-488</div><div>suggested: (Bioss Cat# bsm-4579M-Alexa488, RRID:AB_11071160)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Covid-19 sera 186, 197 and 203 were obtained from the virology department of the Toulouse hospital; plasmas 79 and 206 were from whole blood samples used in our previous paper describing the Jurkat-S&R-flow test (Maurel Ribes et al. 2021) IH4-RBD (Wuhan and Delta) and IH4 alone were produced by transient transfection of HEK-293T cells, and purified from the supernatant by HIS-tag affinity purification, as previously described (Townsend et al. 2021)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Jurkat-S&R-flow: Briefly, Jurkat-S and Jurkat-R cell lines, obtained and grown as previously described (Maurel Ribes et al. 2021), were resuspended in their own tissue culture medium at a concentration of 2.2 106 cells/ mL before pooling equal volumes of the two.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat-R</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The values used as results are those for specific staining, i.e. the difference between the GMFI (geometric mean fluorescent index) measured on the Jurkat cells expressing the SARS-CoV-2 spike protein and the control Jurkat cells expressing the mCherry fluorescent protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat</div><div>suggested: KCB Cat# KCB 94018YJ, RRID:CVCL_0065)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Post-acquisition analysis of all the samples was performed using the Flowjo software (version 10.7.1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Flowjo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr></table>

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

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