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    1. Alors que l’identifiant est unique et isole, le tag permet de regrouper des fiches qui ne peuvent être liées directement, mais qui partagent le(s) même(s) sujet(s).

      Formulation un peu confuse.

      • "isole" sonne bizarre. L'identifiant permet de distinguer de manière non ambigüe.
      • on peut très bien avoir deux fiches avec le même tag et un lien entre elles, ce n'est pas mutuellement exclusif du tout
      • une étiquette n'est pas forcément un mot-clé thématique, ça peut être fonctionnel et lié à des traitements (ex : #mes_publications)
    2. tag

      Je mettrais plutôt étiquette ou mot-clé. On n'a pas tous la même opinion sur la loi Toubon… mais moi je préfère tout traduire.

    1. SciScore for 10.1101/2020.08.02.230839: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The number of red/green color cells in random fields was determined using thresholding and particle analysis in the Fiji modification of ImageJ and expressed as a dead/live cell ratio.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Cell lines were authenticated and tested to ensure the cultures were free of mycoplasma infection.</td></tr></table>

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Abnova ACE2 polyclonal antibody #PAB13444, Santa Cruz Biotechnology ACE2 Antibody (E-11) #sc-390851, Sigma Anti-TMPRSS2 Antibody, clone P5H9A3 #MABF2158, Sigma Anti-IL6 antibody produced in rabbit #SAB1408591, Santa Cruz Biotechnology TMPRSS2 Antibody (H-4) #sc-515727, TMPRSS2 (EMD Millipore #MABF2158), Cell Signaling Technology Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Anti-IL6</div> <div>suggested: (Sigma-Aldrich Cat# SAB1408591, RRID:AB_10742282)</div> </div> <div style="margin-bottom:8px"> <div>#SAB1408591</div> <div>suggested: (Sigma-Aldrich Cat# SAB1408591, RRID:AB_10742282)</div> </div> <div style="margin-bottom:8px"> <div>TMPRSS2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Rabbit mAb #4370, Cell Signaling Technology p44/42 MAPK (Erk1/2) Antibody #9102, Ran (BD Biosciences #610341)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Erk1/2</div> <div>suggested: (Cell Signaling Technology Cat# 9102, RRID:AB_330744)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, Caspase-8 (Cell Signaling #9746), Sigma Monoclonal Anti-β-Actin antibody produced in mouse #A5441.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Anti-β-Actin</div> <div>suggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Invitrogen Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP # 31460 and Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP # 31430 were diluted 1:5000 in 2.5% non-fat milk. qRT-PCR methods and primers Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen)</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# 31460, RRID:AB_228341)</div> </div> <div style="margin-bottom:8px"> <div>anti-Mouse IgG</div> <div>suggested: (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Western blots evaluating cleaved caspase 8 were performed using Cell Signaling antibody (#9746).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>cleaved caspase 8</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 treated H1299, MST0211H, and Calu-3 cells with 5 µM remdesivir, 5 µM VS-6766 or the combination.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>H1299</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines and culture conditions Normal human primary small airway epithelial cells HSAEC, normal human bronchial epithelial cells BEAS-2B, normal human lung fibroblast MRC-5, human NSCLC cells H1975, H1299, Calu3,</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HSAEC</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>BEAS-2B</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>H1975</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Calu-6, human mesothelioma cells MSTO-211H, NSCLC patient-derived cell line, human natural killer cells NK-92, normal human colon epithelial cells CCD 841 CoN and human colorectal cancer cells HT-29, HCT116 were used in this study.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Calu-6</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>HT-29</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>HCT116</div> <div>suggested: NCI-DTP Cat# HCT-116, RRID:CVCL_0291)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MRC-5, Calu-3, Calu-6 and CCD 841 CoN cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>MRC-5</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">NK-92 cells were cultured in Alpha Minimum Essential medium without ribonucleosides and deoxyribonucleosides but with 2 mM Lglutamine and 1.5 g/L sodium bicarbonate supplemented with 0.2 mM inositol; 0.1 mM 2mercaptoethanol; 0.02 mM folic acid; 100 U/ml recombinant IL-2, 12.5% horse serum and 12.5% FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>NK-92</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK293T cells at 75% confluency were co-transfected with the backbone vector pHAGE-fullEF1α-ZsGreen-IRES-Puro(R), plasmids expressing lentiviral proteins Tat, Rev and Gag/Pol, and plasmids expressing D614 or G614 S protein (a gift from Dr. Hyeryun Choe, The Scripps Research Institute, Jupiter, FL).</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">SARS-CoV-2 pseudoviruses (5 x 106) or VSV-G lentivirus (2 x 105) were used to spin-infect (931 g for 2 hours at 30°C) Calu-3 or BEAS-2B cells in a 6-well plate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Calu-3</div> <div>suggested: BCRJ Cat# 0264, RRID:CVCL_0609)</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 number of red/green color cells in random fields was determined using thresholding and particle analysis in the Fiji modification of ImageJ and expressed as a dead/live cell ratio.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Fiji</div> <div>suggested: (Fiji, RRID:SCR_002285)</div> </div> <div style="margin-bottom:8px"> <div>ImageJ</div> <div>suggested: (ImageJ, RRID:SCR_003070)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry analysis of ZsGreen+ cells was carried out 48 h after infection on a BD LSRII flow cytometer and with the FlowJo software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>FlowJo</div> <div>suggested: (FlowJo, RRID:SCR_008520)</div> </div> </td></tr></table>

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


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

      This is a limitation of the work, as is lack of direct evidence that MEKi attenuate SARS-CoV-2 infection of human cells. Our evidence is indirect, and the effects are predicted based on current knowledge of SARS-CoV-2 infectivity factors, and consistent with recent results showing SARS-Cov-2 effects on the kinome including MAPK p38 activation [45]. Our results showing infection of SARS-CoV-2-S pseudovirus of human bronchial epithelial cells or lung cancer cells provide an experimental model system to discover or test therapeutics with potential to block coronavirus infection. The observed effect of MEKi to reduce SARS-CoV-2-S pseudovirus infection of human cells is consistent with our other evidence that MEKi may attenuate coronavirus infectivity factors to inhibit infection. In pursuit of a therapeutic agent that could attenuate cytokine storm while reducing viral infectivity and boosting NK cells activity, we found that VS-6766 decreases G-CSF and other cytokines. These cytokines of interest were increased in COVID-19-(+) patient plasma samples in our study. The combination of remdesivir and VS-6766 was not associated with increased cytokine expression at nontoxic doses of the drugs. The MEKi plus remdesivir drug combinations do not block NK-mediated cell killing and in fact the MEKi stimulate NK killing activity towards target cells. Moreover, the drug combinations do not inhibit TRAIL-mediated killing of target cells. Our results support the idea that MEK inhibitors as a drug class may suppress COVID-19 infectivity factors while allowing (or in some cases boosting) NK-mediated (but not T-cell mediated) killing of target cells and suppressing inflammatory cytokines. The results support the idea that MEKi could be tested in the clinic to suppress early COVID-19 infection and that in combination with an anti-viral such as remdesivir, MEKi may provide a means to lessen the infection spread which may potentiate anti-viral effects. Limitations of this work include a focus on host factors without the presence of actual SARS-CoV-2 infection, although some experiments employed recombinant SPIKE protein fragments. However, we did create a SARS-CoV-2-S pseudovirus bearing D614 or G614 SPIKE protein variants on the envelope of a lentiviral core, demonstrated infection of human airway epithelial cells or lung cancer cells, and showed that MEKi attenuate the viral infectivity. We are conducting additional experiments evaluating the effects of MEKi on pseudovirus infection of other cell lines and also investigating the impact of MEKi on NK cell killing of cells already infected by the pseudovirus. Our understanding of the pathogenesis of COVID-19 has evolved rapidly over the course of the pandemic [24, 46, 47]. Several classes of drugs are being evaluated for the management of COVID-19: antivirals, antibodies, anti-inflammatory agents, targeted immunomodulatory therapies, anticoagulants, and antifibrotics. However, no proven drug for the treatment of COVID19 is currently available [48]. Our inhibitors capable of both inhibiting viral infection and modulating immune responses may synergize to block the disease at multiple stages over its natural history. Our approach with lentiviral vector based pseudovirus has its limitations. For example, how the display of S proteins on a heterologous virus impacts viral entry compared to infectious SARSCoV-2 is not known. Also, the ability of lentivirus to elicit immune response in infected cells is limited. Further studies of SARS-CoV-2 virus cell entry and its inhibition with a replication competent surrogate coronavirus [49, 50] are warranted. It is important to note that the SARS-CoV-2-S pseudovirus approach should allow more laboratories to work on the infectivity problem at biosafety level 2, including drug screening that we are also currently conducting. We realize that the data in this manuscript so far are preliminary but we wish to share them given the seriousness of the current pandemic and the need to perform more research on a larger scale, including in vivo, to promote translational directions, and other collaborative research. Based on the data in this manuscript it may be reasonable to consider further preclinical experiments as well as clinical translation of the MEKi results. Some of the open questions include a more detailed understanding of how the MAPK pathway activates ACE2, more direct evidence for effects of MEKi on actual SARS-CoV-2 infectivity of human cells, and more evidence for their effects on COVID-19 infection spread in preclinical models. In the clinic, it may be reasonable to test MEKi such as VS-6766 or trametinib in COVID-19 infected but less severely ill patients to test the idea that MEKi could keep the infection from getting worse while allowing the body’s NK cells and innate immune mechanisms to more effectively attack virally infected cells prior to severe infection. Consideration could be given to evaluation of MEKi -/+ antiviral agents such as remdesivir given results suggesting potentially favorable drug interactions that may allow suppression of infectivity, suppression of inflammatory cytokines, stimulation of NK cell (but not T-cell) activity, and lack of suppression of TRAIL-mediated cytotoxicity. These effects may help antiviral agents achieve more potent disease suppression to attenuate or block COVID-19 infection that may be of use as a therapeutic approach in patients with early or less severe COVID19 disease. Materials and Methods Human Plasma Samples COVID-19 (+) human plasma samples were received from the Lifespan Brown COVID-19 biobank at Rhode Island Hospital (Providence, Rhode Island). All patient samples were deidentified but with available clinical information as described in the manuscript. The IRB study protocol “Pilot Study Evaluating Cytokine Profiles in COVID-19 Patient Samples” did not meet the definition of human subjects research by either the Brown University or the Rhode Island Hospital IRBs. Normal, healthy, COVID-19 (-) samples were commercially available form Lee BioSolutions (99158-PS-1, Lee BioSolutions, Maryland Heights, Missouri). All samples were thawed and centrifuged to remove cellular debris immediately before the assay was ran. Cytokine Measurements of Culture Supernatants and Plasma Samples A MilliPlex MILLIPLEX® MAP Human Cytokine/Chemokine/Growth Factor Panel A- Immunology Multiplex Assay (HCYTA-60K-13, Millipore Sigma, Burlington, Massachusetts) was run on a Luminex 200 Instrument (LX200-XPON-RUO, Luminex Corporation, Austin, Texas) according to the manufacturer’s instructions. Production of granulocyte colony-stimulating factor (G-CSF), interferon gamma (IFNγ), interleukin 1 alpha (IL-1α), interleukin-1 receptor antagonist (IL-1RA), IL-2, IL-6, IL-7, IL-12, interferon γ-induced protein 10 (IP-10), monocyte chemoattractant protein1 (MCP-1), macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein-1 alpha (MIP-1α), and tumor necrosis factor alpha (TNFα) in the culture supernatant were measured. All samples were run in triplicate. Cell lines and culture conditions Normal human primary small airway epithelial cells HSAEC, normal human bronchial epithelial cells BEAS-2B, normal human lung fibroblast MRC-5, human NSCLC cells H1975, H1299, Calu3, Calu-6, human mesothelioma cells MSTO-211H, NSCLC patient-derived cell line, human natural killer cells NK-92, normal human colon epithelial cells CCD 841 CoN and human colorectal cancer cells HT-29, HCT116 were used in this study. HSAEC was cultured in Airway Epithelial Cell Basal Medium (ATCC® PCS-300-030™) supplemented with Bronchial Epithelial Cell Growth Kit (ATCC® PCS-300-040™). BEAS-2B was cultured in BEGMTM Bronchial Epithelial Cell Growth Medium BulletKitTM (Lonza Catalog No. CC-3170). TALL-104 T cells were from the ATCC (ATCC® CRL-11386™) and were used as we recently described [31]. MRC-5, Calu-3, Calu-6 and CCD 841 CoN cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS). H1975, H1299, MSTO-211H and NSCLC patient-derived cell line were cultured in RPMI-1640 medium supplemented 10% FBS. NK-92 cells were cultured in Alpha Minimum Essential medium without ribonucleosides and deoxyribonucleosides but with 2 mM Lglutamine and 1.5 g/L sodium bicarbonate supplemented with 0.2 mM inositol; 0.1 mM 2mercaptoethanol; 0.02 mM folic acid; 100 U/ml recombinant IL-2, 12.5% horse serum and 12.5% FBS. HCT116 and HT-29 were cultured in McCoy's 5A (modified) medium supplemented 10% FBS. All cell lines were incubated at 37 °C in humidified atmosphere containing 5% CO2. Media containing 1% serum was used for serum starvation experiments. Cell lines were authenticated and tested to ensure the cultures were free of mycoplasma infection. Collection of Culture Supernatants Used in Cytokine Measurements Cells were plated at 3.5 × 104 cells in a 48 well plate (Thermo Fisher Scientific, Waltham, MA) in CM and incubated at 37°C with 5% CO2. At 24 h after plating, almost all the tumor cells were adherent to the bottom of the flask and then the CM was completely replaced. Subsequently, the culture supernatants were collected after another 48 h of incubation, centrifuged to remove cellular debris, and then frozen at –80°C until the measurement of cytokines was performed. Transfections and reporter assays Human ACE2-Luc promoter constructs containing 1119, 252, and 202 base pairs of the ACE2 promoter linked to firefly luciferase were obtained from Addgene. Human tumor cell lines were transfected with each ACE2-promoter luciferase-reporter using lipofectamine 2000 as described in the protocol (Invitrogen, USA). At 24 hours after the transfection, the cells were trypsinized and seeded into 96-well black plates at a density of 3 × 104 cells/well. The next day, cells on the 96well plate were treated with drugs for 24 hours. The luciferase activity was evaluated by bioluminescence imaging in cells using the IVIS imaging system (Xenogen, Alameda, CA). DMSO treatment was used as a negative control in each screened plate. The bioluminescence in each treatment was normalized to DMSO treatment. Cell viability assays Cells were plated at a density of 3 x 103 cells per well of a 96-well plate. Cell viability was assessed using the CellTiter Glo assay (Promega). Cells were mixed with 25 µl of CellTiter-Glo reagents in 100 µl of culture volume, and bioluminescence imaging was measured using the Xenogen IVIS imager. Western blots and antibodies Immunoblotting for proteins was performed using the following antibodies: Cell Signaling Technology ACE2 Antibody #4355, Abnova ACE2 polyclonal antibody #PAB13444, Santa Cruz Biotechnology ACE2 Antibody (E-11) #sc-390851, Sigma Anti-TMPRSS2 Antibody, clone P5H9A3 #MABF2158, Sigma Anti-IL6 antibody produced in rabbit #SAB1408591, Santa Cruz Biotechnology TMPRSS2 Antibody (H-4) #sc-515727, TMPRSS2 (EMD Millipore #MABF2158), Cell Signaling Technology Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370, Cell Signaling Technology p44/42 MAPK (Erk1/2) Antibody #9102, Ran (BD Biosciences #610341), Caspase-8 (Cell Signaling #9746), Sigma Monoclonal Anti-β-Actin antibody produced in mouse #A5441. Primary antibodies are diluted according to their datasheets. Proteins were extracted from cells in denaturing sample buffer and an equal amount of protein lysate was electrophoresed through 4-12% SDS-PAGE (Invitrogen) then transferred to PVDF membranes. The PVDF membrane was blocked with 5% non-fat milk (Sigma) in 1x PBS. The primary antibodies indicated in the figures were incubated with the transferred PVDF in blocking buffer at 4°C overnight. Antibody binding was detected on PVDF with appropriate Pierce HRPconjugated secondary antibodies by the Syngene imaging system. Invitrogen Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP # 31460 and Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP # 31430 were diluted 1:5000 in 2.5% non-fat milk. qRT-PCR methods and primers Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen). Reverse transcription was performed with random primers using the SuperScript II First-Strand Synthesis System (Invitrogen). qRT-PCR reactions used SYBR Green Master Mix with the Real-Time PCR Detection systems (Bio-Rad). Primers for ACE2 were obtained from Origene. Forward Sequence: 5’-TCCATTGGTCTTCTGTCACCCG-3’. Reverse Sequence: 5’-AGACCATCCACCTCCACTTCTC-3’. The ACE2 mRNA gene expression levels were normalized with GAPDH. NK-cell co-culture system and microscopic imaging for data analysis Tumor cells were plated and allowed to grow for 48 hours in their culture media before addition of NK cells. Green fluorescent tumor cells were co-cultured with NK-92 cells [30] at a 1:1 effector target cell ratio (E:T) imaged at various timepoints. Cells were treated with indicated drugs for several hours (drugs added at time of NK cell addition) as indicated in the figures. A total of 1 µM red fluorescent ethidium homodimer (EthD-1) was added at the beginning of drug and NK cell incubation to detect dead cells (Invitrogen, Waltham, MA). For the quantification of dead/live cells, fluorescence microscopy was used to take images at 10x magnification. The number of red/green color cells in random fields was determined using thresholding and particle analysis in the Fiji modification of ImageJ and expressed as a dead/live cell ratio. At least 100 cells were evaluated per sample, with 3 independent replicates. TRAIL cell killing assays Cells were plated at a density of 5 x 105 per well of a 6-well plate. After 16 hours of incubation at 37 degrees Celsius in 5% CO2, cells were treated with 5 µM remdesivir (RDV), VS-6766, or the combination. 24 hours later, cells were treated with TRAIL (50 ng/mL) for an additional 4 hours. Western blots evaluating cleaved caspase 8 were performed using Cell Signaling antibody (#9746). Ran was probed with BD Biosciences antibody (#610341) as a loading control. SARS-CoV-2 pseudovirus production, quantification, and infection. We used a lentiviral packaging system to produce a replication incompetent SARS-CoV-2 pseudovirus. Briefly, HEK293T cells at 75% confluency were co-transfected with the backbone vector pHAGE-fullEF1α-ZsGreen-IRES-Puro(R), plasmids expressing lentiviral proteins Tat, Rev and Gag/Pol, and plasmids expressing D614 or G614 S protein (a gift from Dr. Hyeryun Choe, The Scripps Research Institute, Jupiter, FL). A plasmid expressing VSV-G protein instead of the S protein was used to generate a pantropic control lentivirus. The SARS-CoV-2 S protein gene used in the production of pseudoviruses was codon-optimized and synthesized by Integrated DNA Technologies based on the protein sequence (GenBank YP_009724390). The S protein gene is fused to the FLAG tag at its C-terminus. Pseudovirus containing culture supernatants were collected at 48 h and 72 h post transfection, cleared through 0.45 μm filters, and concentrated using ultra-centrifugation, aliquoted and frozen at -80°C immediately. Lenti-X™ p24 Rapid Titration ELISA Kit (TaKaRa) was used to determine virus titer. Equal number of lentiviral particles were analyzed on an SDS-PAGE gel followed by Western blot to detect FLAG-tagged S protein. SARS-CoV-2 pseudoviruses (5 x 106) or VSV-G lentivirus (2 x 105) were used to spin-infect (931 g for 2 hours at 30°C) Calu-3 or BEAS-2B cells in a 6-well plate. Fluorescence microscopic images were taken 18 h after infection. Flow cytometry analysis of ZsGreen+ cells was carried out 48 h after infection on a BD LSRII flow cytometer and with the FlowJo software. To test the inhibitors, Calu-3 or BEAS-2B cells were pre-treated with the inhibitors for 48 h, spun-infected with pseudovirus followed by another 48 h incubation with the inhibitors. Flow cytometry analysis of live cells that were ZsGreen+ was carried out. Statistical methods The statistical significance of differences between groups was determined using the Student t test. The minimal level of significance was P < 0.05. Following symbols * and ** represent, P < 0.05 and P < 0.01, respectively.


      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 used on pages 28, 28, 36, 37, 37, 37, 54, 54, 54, 55, 59, 59, 59, 59, 59, 59, 60, 60, 60, 60, 60, 61, 61, 61, 61, 61 and 61. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.


      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.

    1. SciScore for 10.1101/2020.08.01.20166553: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consecutive out-patients diagnosed at the same 4 hospitals prior to March 15th and on a convenience sample of later days were approached for consent to collect serum and saliva at 412 weeks post onset of symptoms.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing 4 times, 10 µl of one of the following secondary antibodies (all from Jackson ImmunoResearch) diluted in 1% BLOTTO in PBS-T were added at the indicated concentrations followed by incubation for 2 hr at room temperature: Goat anti-human IgG Fcy – 035-129; 1:12,000 or 0.66 ng per well) or Goat anti-human IgA a chain - HRP (#109-035-127; 1:10,000 or 0.8 ng per well).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-human IgG</div> <div>suggested: (Jackson ImmunoResearch Labs Cat# 109-035-127, RRID:AB_2337587)</div> </div> <div style="margin-bottom:8px"> <div>anti-human IgA</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies used for the standard curves were: Human anti-spike S1 IgG (A02038, GenScript), anti-spike S1 IgM (A02046, GenScript) and Ab01680 anti-spike IgA (Ab01680-16, Absolute Antibody), all used at 0.5 to 10ng per well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-spike S1 IgG</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>A02038</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-spike S1 IgM</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>A02046</div> <div>suggested: (GenWay Biotech Inc. Cat# GWB-A02046, RRID:AB_10276779)</div> </div> <div style="margin-bottom:8px"> <div>anti-spike IgA</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-human Ig antibody (Southern Biotech, 2010-01) diluted 1:1000 in PBS was added to 96-well Nunc MaxiSorp™ plates (ThermoFisher, 44-2404-21).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Anti-human Ig antibody</div> <div>suggested: (SouthernBiotech Cat# 2010-01, RRID:AB_2687525)</div> </div> <div style="margin-bottom:8px"> <div>Anti-human Ig</div> <div>suggested: (SouthernBiotech Cat# 2010-01, RRID:AB_2687525)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HRP-conjugated secondary antibodies against IgA and IgG (goat anti-human IgA- and IgG-HRP, Southern Biotech, IgA: 2053-05, IgG: 2044-05) were added to the appropriate wells at 1:1000 in 2.5% BLOTTO and incubated for 1 hour at 37oC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HRP-conjugated secondary antibodies against IgA and IgG (goat anti-human IgA- and IgG-HRP, Southern Biotech, IgA: 2053-05, IgG: 2044-05)</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>HRP-conjugated secondary antibodies against IgA and IgG</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>goat anti-human IgA-</div> <div>suggested: (InvivoGen Cat# hrp-iga, RRID:AB_11124937)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Horseradish peroxidase (HRP)-conjugated Goat anti human-IgA and anti-IgG secondary antibodies (Southern Biotech, 2053-05 and 2044-05) were added to wells at dilutions of 1:2000 and 1:1000 in 2.5% BLOTTO, respectively, and incubated for 1 hour at 37oC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Goat anti human-IgA</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti human-IgA</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;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 stabilize the spike protein in a trimeric form, the cDNA was cloned in-frame with the human resistin cDNA (aa 23-108) containing a Cterminal FLAG-(His)6 tag (Cricetulus griseus codon bias, GenScript) into a modified cumateinducible pTT241 expression plasmid and transfected in CHO2353 cells (Stuible et al.,</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>CHO2353</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral stock was expanded using Vero E6 as previously described such that stored aliquots of stock contain 2% FBS.</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">Initial experiments were done with Triton X-100 (Sigma-Aldrich) serially diluted and applied to Vero-E6 cells in 96-well flat bottom plates to determine the minimum concentration required to prevent toxicity to cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero-E6</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antigen production – serum assays Spike trimer was expressed as follows: the SARS-CoV-2 spike sequence (aa 1-1208 from Genebank accession number MN908947 with the S1/S2 furin site (residues 682–685) mutated [RRAR->GGAS] and K986P / V987P stabilizing mutations was codon-optimized (Cricetulus griseus codon bias) and synthesized by Genscript.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Genebank</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A fourparameter logistic curve was used to determine the line of best fit for the standard curve, and sample Ig quantities were interpolated accordingly, using Prism Graphpad, Version 8.3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Graphpad</div> <div>suggested: (GraphPad, RRID:SCR_000306)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All analysis was performed in SAS 9.4M6 (SAS Institute, Cary, NC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SAS Institute</div> <div>suggested: (Statistical Analysis System, RRID:SCR_008567)</div> </div> </td></tr></table>

      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 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.


      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.

    1. Concreto pero bastante entendible. Aunque quedan muchas dudas, creo que picándole aprenderemos..

    1. Excelente taller, creo que sería muy útil otro para reforzar lo aprendido hoy. Gracis

    1. Curso Hypothesis DGBSDI

      Segunda parte del Taller, excelente herramienta

    1. SciScore for 10.1101/2020.01.31.929042: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 an incubation period of 1 h at 37 °C, the inoculum was removed and cells were washed with PBS before medium supplemented with anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) was added (no antibody was added to cells expressing VSV-G).</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>

            <div style="margin-bottom:8px">
              <div><b>I1</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The following primary antibodies were used: Mouse anti-HA tag (Sigma-Aldrich, H3663, 1:2,500), mouse anti-ß-actin (Sigma-Aldrich, A5441, 1:2,000), mouse anti-VSV matrix protein (Kerafast, EB0011, 1:2,500).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-HA</b></div>
              <div>suggested: (Sigma-Aldrich Cat# H3663, <a href="https://scicrunch.org/resources/Any/search?q=AB_262051">AB_262051</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-ß-actin</b></div>
              <div>suggested: (Leinco Technologies Cat# B517, <a href="https://scicrunch.org/resources/Any/search?q=AB_2828227">AB_2828227</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>A5441</b></div>
              <div>suggested: (Sigma-Aldrich Cat# A5441, <a href="https://scicrunch.org/resources/Any/search?q=AB_476744">AB_476744</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-VSV matrix protein ( Kerafast , EB0011</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As secondary antibody we used a peroxidase-coupled goat anti-mouse antibody (Dianova, 115-035-003, 1:10000)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-mouse</b></div>
              <div>suggested: (Jackson ImmunoResearch Labs Cat# 115-035-003, <a href="https://scicrunch.org/resources/Any/search?q=AB_10015289">AB_10015289</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">S. Gierer et al., The spike protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2, and is targeted by neutralizing antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>TMPRSS2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">J Virol 87, 5502-5511 (2013). H. Kleine-Weber et al., Mutations in the Spike Protein of Middle East Respiratory Syndrome Coronavirus Transmitted in Korea Increase Resistance to Antibody-Mediated Neutralization.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Antibody-Mediated Neutralization .</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In agreement with these findings, directed expression of human and bat ACE2 but not human DPP4, the entry receptor used by MERS-CoV (20), or human APN, the entry receptor used by HCoV-229E (21), allowed 2019-nCoV-S- and SARS-Sdriven entry into otherwise non-susceptible BHK-21 cells (Fig. 2C), indicating that 2019-nCoV-S like SARS-S uses ACE2 for cellular entry.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>BHK-21</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Moreover, ammonium chloride treatment strongly inhibited 2019-nCoV-Sand SARS-S-driven entry into TMPRSS2- 293T cells while inhibition of entry into TMPRSS2+ Caco-2 cells was less efficient, which would be compatible with 2019-nCoV-S priming by TMPRSS2 in Caco-2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>Caco-2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293T (human, kidney), BHK-21 (Syrian hamster, kidney cells), Huh-7 (human, liver), LLC-PK1 (pig, kidney), MRC-5 (human, lung), MyDauLu/47.1 (Daubenton's bat [Myotis daubentonii], lung), NIH/3T3 (Mouse, embryo), RhiLu/1.1 (Halcyon horseshoe bat [Rhinolophus alcyone], lung), Vero (African green monkey, kidney) cells were incubated in Dulbecco’s’ modified Eagle medium (PAN-Biotech).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Huh-7</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>MRC-5</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Calu-3 (human, lung), Caco-2 (human, colon), MDBK (cattle, kidney) and MDCK II (Dog, kidney) cells were incubated in Minimum Essential Medium (ThermoFisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Calu-3</b></div>
              <div>suggested: BCRJ Cat# 0264, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0609">CVCL_0609</a></div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>MDCK</b></div>
              <div>suggested: CLS Cat# 602280/p823_MDCK_(NBL-2), <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0422">CVCL_0422</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A549 (human, lung), BEAS-2B (human, bronchus) and NCI-H1299 (human, lung) cells were incubated in DMEM/F-12 Medium with Nutrient Mix (ThermoFisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>A549</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>BEAS-2B</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>NCI-H1299</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Phylogenetic analysis Phylogenetic analysis (neighbor-joining trees) was performed using the MEGA7.0.26 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>MEGA7.0.26</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For all statistical analyses, the GraphPad Prism 7 software package was used (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GraphPad Prism</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>GraphPad</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
          </td></tr></table>
      

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.01.22.914952: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">Human samples, including oral swabs, anal swabs, blood, and BALF samples were collected by Jinyintan hospital (Wuhan) with the consent from all patients.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">All cell lines were tested free of mycoplasma contamination, applied to species identification and authenticated by microscopic morphologic evaluation.</td></tr></table>

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 culture supernatant was examined for presence of virus by qRT-PCR developed in this study, and cells were examined by immunofluorescent using SARSr-CoV Rp3 NP antibody made in house (1:100).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Rp3 NP</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">An anti-Human IgG-HRP conjugated monoclonal antibody (Kyab Biotech Co., Ltd, Wuhan, China) was used at a dilution of 1:40000.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-Human IgG-HRP</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 expression was detected using mouse anti-S tag monoclonal antibody followed by FITC-labelled goat anti-mouse IgG H&L (Abcam, ab96879)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-S</div> <div>suggested: (Abcam Cat# ab96879, AB_10687475)</div> </div>

            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG</b></div>
              <div>suggested: (Abcam Cat# ab96879, <a href="https://scicrunch.org/resources/Any/search?q=AB_10687475">AB_10687475</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral replication was detected using rabbit antibody against the Rp3 NP protein (made in house, 1:100) followed by cyanin 3-conjugated goat anti-rabbit IgG (1:50, Abcam, ab6939).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Rp3 NP protein</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-rabbit IgG</b></div>
              <div>suggested: (Abcam Cat# ab6939, <a href="https://scicrunch.org/resources/Any/search?q=AB_955021">AB_955021</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We then successfully isolated the virus (named nCoV-2019 BetaCoV/Wuhan/WIV04/2019), in Vero and Huh7 cells using BALF sample from ICU-06 patient.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For b and c cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls. 01 .1 2 1 20 2 0. 01 .0 1 2 20 . . 9. 12 .3 12 30 0.0 20 19 ELISA OD ratio 0.4 01 b OS ELISA OD ratio BALF e Fig. 3 | Virions. a, viral particles in the ultrathin sections under electron microscope at 200 kV, sample from viral infected Vero E6 cells Fig. 4 | Analysis of nCoV-2019 receptor usage.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero E6</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The following cells were used for virus isolation in this study: Vero, Vero E6, and Huh7 that were cultured in DMEM +10% FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Huh7</b></div>
              <div>suggested: CLS Cat# 300156/p7178_HuH7, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0336">CVCL_0336</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HeLa cells transiently expressing ACE2 were prepared by a lipofectamine 3000 system (Thermo Fisher Scientific) in 96-well plate, with mock-transfected cells as controls.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HeLa</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The raw NGS reads were firstly processed by Cutadapt (v1.18) with minimum read length of 30bp.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Cutadapt</b></div>
              <div>suggested: (cutadapt, <a href="https://scicrunch.org/resources/Any/search?q=SCR_011841">SCR_011841</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BWA (v0.7.12-r1039) was utilized to align reads to local database with a filter hits parameter at 0.8 FMM value and minimum alignment score at 30.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>BWA</b></div>
              <div>suggested: (BWA, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010910">SCR_010910</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">NGS reads were assembled into genomes using Geneious (v11.0.3) and MEGAHIT (v1.2.9).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Geneious</b></div>
              <div>suggested: (Geneious, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010519">SCR_010519</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequence alignment and editing were conducted using ClustalW and GeneDoc.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>ClustalW</b></div>
              <div>suggested: (ClustalW, <a href="https://scicrunch.org/resources/Any/search?q=SCR_017277">SCR_017277</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Maximum Likelihood phylogenetic trees based on nucleotide sequences of full-length ORF1b and S genes were constructed using the Jukes-Cantor model with bootstrap values determined by 1000 replicates in the MEGA6 software package.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>MEGA6</b></div>
              <div>suggested: (MEGA Software, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000667">SCR_000667</a>)</div>
            </div>
          </td></tr></table>
      

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  2. Aug 2020
    1. SciScore for 10.1101/2020.07.31.229781: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">Materials and methods Ethics statement All procedures in this study involving animals were reviewed and approved by Minnan Normal University Animal Ethics and Welfare Committee (</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Mice Groups of 8-week-old hACE2 and wild-type male mice (C57BL/6 background) were purchased from the GemPharmatech (Nanjing, China).</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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 and reagents Anti-His-Tag, Anti-p-IKK, Anti-p-JNK, Anti-p-c-jun were purchased from Affinity Biosciences, Inc. (</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Anti-p-IKK</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>Anti-p-JNK</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>Anti-p-c-jun</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-β-actin was purchased from Cell Signal Technology (Beverly, MA, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Anti-β-actin</b></div>
              <div>suggested: (Cell Signaling Technology Cat# 8457, <a href="https://scicrunch.org/resources/Any/search?q=AB_10950489">AB_10950489</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FITC-conjugated sheep and donkey anti-mouse IgG, cy3-conjugated sheep and donkey anti-rabbit IgG and His-Tag antibody were purchased from Affinity Biosciences, Inc. (Cincinnati, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>FITC-conjugated sheep and donkey anti-mouse IgG , cy3-conjugated sheep and donkey anti-rabbit IgG</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG , cy3-conjugated sheep and donkey anti-rabbit IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">With the addition of three secondary antibodies FITC-conjugated sheep anti-mouse IgG (1:300, Affinity, USA), Cy3conjugated sheep anti-rabbit IgG (1:300, Affinity, USA) and DAPI (1:300, Affinity, USA)), cells were re-suspended and incubated in dark at 37 °C for 60 min.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-rabbit IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Confocal Microscopy For immunofluorescence on cell culture, cells mounted on glass slides were permeabilized with PBS containing 0.1% TritonX-100 and 0.1mol/L of glycine for 15min, and blocked with 1% donkey serum albumin in PBS for 30min at room temperature, followed by an incubation with primary antibodies (His-Tag (1:200, affinity, T0009) and ACE2 (1:200, Abcam,ab15348) ) in PBS for overnight at 4°C and detected by FITC-labeled anti-donkey IgG(1:400) or anti-donkey IgG conjugated with Cy3(1:400) at room temperature for 1h.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-donkey IgG(1:400</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-donkey IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibody was incubated for overnight in a humidifified chamber at 4°C, followed by detection using the by FITC-labeled antidonkey IgG (1:400) or anti-donkey IgG conjugated with Cy3 (1:400) at room temperature for 1h.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>antidonkey IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Multiplex antibody panels applied in this study are: His-Tag (1:200, affinity, T0009) and ACE2 (1:200, Abcam, ab15348); after all the antibodies were detected sequentially, the slices were imaged using the confocal laser scanning microscopy platform Leica. Native-PAGE Analysis 6% resolving gel and 5% condensing gel were used in the Native-PAGE analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>His-Tag</b></div>
              <div>suggested: (Affinity Biosciences Cat# T0009, <a href="https://scicrunch.org/resources/Any/search?q=AB_2839416">AB_2839416</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The membrane was blocked in 5% non-fat milk for 1 h at room temperature, incubated with primary antibodies (including anti-ACE2, anti-p-IKK, anti-p-JNK</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-ACE2</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-p-IKK ,</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then the membrane was washed by TBST for 3 times and incubated with secondary antibody (HRP-linked anti-immunoglobulin) for 1h.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-immunoglobulin</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">II Vero E6 Cell model in vitro of β-chitosan against the binding of SARS-CoV-2SRBD with ACE2 The results of cell immunofluorescence assay showed (Figure 2A) that ACE2 (red) and SARS-CoV-2S-RBD (green) presented strong fluorescence intensities after the addition of SARS-CoV-2S-RBD into Vero E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero E6</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_XD71">CVCL_XD71</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow Cytometry When Vero-E6 cells grow to 80%, they were treated according to the experimental design.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero-E6</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody and reagents Anti-His-Tag, Anti-p-IKK, Anti-p-JNK, Anti-p-c-jun were purchased from Affinity Biosciences, Inc. (</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Affinity Biosciences</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Animal experiments For the animal experiments, specific pathogen-free, male transgenic hACE2 mice were obtained from the experimental animal center of GemPharmatech (Nanjing, China)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GemPharmatech</b></div>
              <div>suggested: (GemPharmatech, <a href="https://scicrunch.org/resources/Any/search?q=SCR_017239">SCR_017239</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The stained protein gel was scanned using the Gel Doc 2000 imaging system (Bio-Rad, Hercules, CA, USA) and analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Quantity One</b></div>
              <div>suggested: (Quantity One 1-D Analysis Software, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014280">SCR_014280</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis All data were analyzed with GraphPad Prism 8.0 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GraphPad Prism</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
          </td></tr></table>
      

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.07.29.227785: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant SARS-CoV-2 S protein (S1+S2 ECD, His tag) expressed by insect cells (High Five) via a baculovirus, and S protein (S1, His tag) expressed by human embryonic kidney (HEK293) cells were purchased from Sino Biological (Beijing, China).</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, CVCL_0045</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Model building based on the Cryo-EM structure (PDB: 6VSB) of the SARS-CoV-2 S protein was performed using PyMOL. 3. RESULTS AND DISCUSSION 3.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>PyMOL</div> <div>suggested: (PyMOL, SCR_000305)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After ZIC-HILIC enrichment and PNGase F digestion, intact O-glycopeptides were analyzed using a high-resolution mass spectrometer, and their spectra were characterized using Byonic™ software and validated manually. B. SCE-HCD-MS/MS spectrum of reported representative O-glycopeptide 320 VQPTESIVR328 with deduced GalNAcGal glycan detected on site Thr323 of human spike protein subunit 1. C. SCE-HCD-MS/MS spectrum of this O-glycopeptide with deduced GalNAcGalNeuAc glycan detected on site Ser325. D. SCE-HCD-MS/MS spectrum of this O-glycopeptide with deduced GalNAcGal glycan on site Thr323 and deduced GalNAcGalNeuAc glycan on site Ser325.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Byonic™</div> <div>suggested: (PMI-Byonic, SCR_016735)</div> </div> </td></tr></table>

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.07.29.227595: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">In Vivo hAd5 S-Fusion + N-ETSD Vaccine Immunogenicity Studies Based on the evidence that S-Fusion + N-ETSD resulted in enhanced expression of physiologically-relevant RBD and that N-ETSD successfully translocated to the endosomal / lysosomal compartment, we chose the bivalent hAd5 S-Fusion + N-ETSD vaccine for inoculation of 7-week old female CD-1 mice.</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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Both the T-cell and antibody immune responses to S and N demonstrated a T-helper 1 (Th1) bias.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Th1</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Enhanced HEK 293T cell-surface expression of RBD following transfection with Ad5 SFusion + N-ETSD As shown in Figure 2, anti-RBD-specific antibodies did not detect RBD on the surface of HEK 293T cells transfected with hAd5 S-WT (Fig. 2a) or hAd5 S-WT + N-ETSD (Fig. 2b) constructs, while hAd5 S-Fusion alone was slightly higher (Fig. 2e).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-RBD-specific</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>2a</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As expected, both constructs with RBD, hAd5 RBD-ETSD and RBD-ETSD + N-ETSD, showed high binding of anti-RBD antibody (Fig. 2c and d).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-RBD</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell surface RBD expression with (a) hAd5 S-WT, (b) S-Fusion, and (c) S-Fusion + N-ETSD in HEK 293T cells shows high correlation with (d) expression of S in immunoblots of HEK 293T cell lysates probed using anti-full length (S2) antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-full length (S2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">There was significant production of both anti-S (Fig. 7a) and anti-N (Fig. 7c) antibodies in the sera from CD-1 mice vaccinated with hAd5 S-Fusion + N-ETSD at Day 28 in the study.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-N (Fig. 7c)</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Compared to anti-S antibodies, anti-N antibodies were higher in sera, given the dilution factor for sera was 1:90 for anti-N antibody analysis and 1:30 for anti-S antibody analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-S</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These values were used to calculate that hAd5 S-Fusion + NETSD vaccination generated a geometric mean value of 5.8 µg S-specific IgG and 42 µg N-specific IgG per mL of serum, therefore the relative µg amount of anti-N antibodies is higher than that for anti-S antibodies and reflects the strong contribution of N to anti-SARS-CoV-2 antibody production.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-N</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-SARS-CoV-2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Fig. 7 Anti-spike and anti-nucleocapsid antibody responses in sera from hAd5 S-Fusion + N-ETSD vaccinated mice.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Anti-spike</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Based on absorbance, there was significant production of both (a) anti-S antibodies and (c) anti-nucleocapsid antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-nucleocapsid</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">G4 pool – mice with S-specific antibodies; M1, M2, M3, M4 – mouse ID; +C – convalescent serum; and media – media only negative control.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>M4</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">hAd5 S-Fusion + N-ETSD generates Th1 dominant responses both in humoral and T-cell immunity Antibody Th1 dominance in response to N and S IgG2a, IgG2b, and IgG3 represent Th1 dominance; while IgG1 represents Th2 dominance.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>S IgG2a</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For both anti-S (Fig, 9a) and anti-N (Fig, 9c) antibodies in sera from hAd5 S-Fusion + N-ETSD vaccinated mice, IgG2a and IgG2b isotypes were predominant and significantly higher compared to the hAd5 Null control.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-N (Fig, 9c)</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(a, c) IgG2a and IgG2b isotype anti-spike and anti-nucleocapsid antibodies were significantly increased for hAd5 S-Fusion + NETSD mice compared to hAd5 Null mice.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>IgG2b isotype anti-spike and anti-nucleocapsid</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Th1 predominance was seen again in humoral responses, where the ratio based on ng equivalence of Th1 related antibodies (IgG2a, IgG2b, and IgG3) to Th2 related antibodies (IgG1) for both anti-S and anti-N antibodies is greater than 1 in all mice (Fig. 11b).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Th2 related antibodies (IgG1</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were harvested 1, 2, 3, and 7 days post transfection by gently pipetting cells into medium and labeled with an anti-RBD monoclonal antibody (clone D003 Sino Biological Catalog # 40150-D003) and F(ab’)2-Goat anti-Human IgG-Fc secondary antibody conjugated with Rphycoerythrin (ThermoFisher Catalog # H10104).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-Human IgG-Fc</b></div>
              <div>suggested: (Thermo Fisher Scientific Cat# H10104, <a href="https://scicrunch.org/resources/Any/search?q=AB_2536546">AB_2536546</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To label N, cells were then incubated with an anti-flag monoclonal (Anti-Flag M2 produced in mouse, Sigma cat# F1804) antibody at 1:1000 in phosphate buffered saline with 3% BSA overnight at 4oC, followed by washes in PBS and a 1 hour incubation with a goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 (Life Technologies, Cat# A32727) at 1:500.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-flag</b></div>
              <div>suggested: (Sigma-Aldrich Cat# F1804, <a href="https://scicrunch.org/resources/Any/search?q=AB_262044">AB_262044</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For co-localization studies, cells were also incubated overnight at 4oC with a sheep anti-Lamp1 Alexa Fluor 488conjugated (lysosomal marker) antibody (R&D systems, Cat# IC7985G) at 1:10 or a rabbit antiCD71 (transferrin receptor, endosomal marker) antibody (ThermoFisher Cat# PA5-83022) at 1:200.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-Lamp1</b></div>
              <div>suggested: (LifeSpan Cat# LS-C106557-200, <a href="https://scicrunch.org/resources/Any/search?q=AB_10624612">AB_10624612</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>antiCD71 (transferrin receptor, endosomal marker)</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After removal of the primary antibody, two washes in PBS and three 3 washes in PBS with 3% BSA, cells were incubated with fluor-conjugated secondary antibodies when applicable at 1:500 (Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, Life technologies, A-11034) for 1 hour at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-Rabbit IgG</b></div>
              <div>suggested: (Thermo Fisher Scientific Cat# A-11034, <a href="https://scicrunch.org/resources/Any/search?q=AB_2576217">AB_2576217</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-Spike S2 (SinoBiological Cat #40590-T62) was used as the primary antibody and IRDye® 800CW Goat anti-Rabbit IgG (H + L) (Li-Cor, 925-32211) as the secondary antibody using the Ibind Flex platform.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Anti-Spike S2</b></div>
              <div>suggested: (Imported from the IEDB Cat# S2, <a href="https://scicrunch.org/resources/Any/search?q=AB_2833224">AB_2833224</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were incubated with ACE2-Fc for 20 minutes and, after a washing step, were then labeled with a PE conjugated F(ab’)2-goat anti-human IgG Fc secondary antibody at a 1:100 dilution, incubated for 20 minutes, washed and acquired on flow cytometer.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-human IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Fluorescent-conjugated antibodies against mouse CD8b antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), and TNF-α (clone MP6-XT22, BD) and staining was performed in the presence of unlabeled anti-CD16/CD32 antibody (clone 2.4G2).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>mouse CD8b</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>CD4</b></div>
              <div>suggested: (BioLegend Cat# 391503, <a href="https://scicrunch.org/resources/Any/search?q=AB_2721611">AB_2721611</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>IFN-γ</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>TNF-α</b></div>
              <div>suggested: (Leinco Technologies Cat# T798, <a href="https://scicrunch.org/resources/Any/search?q=AB_2832121">AB_2832121</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-CD16/CD32</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cells (2-4 x 105 cells per well of a 96-well plate) were added to the ELISpot plate containing an immobilized primary antibodies to either IFN-g or IL-4 (BD), and were exposed to various stimuli (e.g. control peptides, target peptide pools/proteins) comprising 2 μg/mL peptide pools or 10 μg/mL protein for 36-40 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>IL-4 (BD)</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ELISA for detection of antibodies For antibody detection in sera from inoculated mice, ELISAs specific for spike and nucleocapsid antibodies, as well as for IgG subtype (IgG1, IgG2a, IgG2b, and IgG3) antibodies were used.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>IgG subtype (IgG1</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>IgG3</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After incubation, the wells were washed with PBST and 100 μL of a 1/5000 dilution of anti-mouse IgG HRP (GE Health Care; Cat # NA9310V), or anti-mouse IgG1 HRP (Sigma; Cat # SAB3701171), or antimouse IgG2a HRP (Sigma; Cat # SAB3701178), or anti-mouse IgG2b HRP (Sigma; catalog# SAB3701185), or anti-mouse IgG3 HRP conjugated antibody (Sigma; Cat # SAB3701192) was added to wells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG1</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>antimouse IgG2a</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG2b</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG3</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Using these values, we were able to calculate that hAd5 S-Fusion + N-ETSD vaccination generated a geometric mean value of 5.8 µg S-specific IgG and 42 µg N-specific IgG per milliliter of serum. cPassTM Neutralizing Antibody Detection The GenScript cPassTM (https://www.genscript.com/cpass-sars-cov-2-neutralization- antibody-detection-Kit.html) for detection of neutralizing antibodies was used according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>antibody-detection-Kit</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell surface RBD expression with (a) hAd5 S-WT, (b) S-Fusion, and (c) S-Fusion + N-ETSD in HEK 293T cells shows high correlation with (d) expression of S in immunoblots of HEK 293T cell lysates probed using anti-full length (S2) antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK 293T</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunocytochemical labeling of hAd5 infected HeLa cells To determine subcellular localization of N after infection or transfection of HeLa cells with hAd5 N-wild type (WT) or hAd5 N-ETSD (each with a flag tag to allow labeling), 48 hours after infection or transfection cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.4% Triton X100, in PBS) for 15 min.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HeLa</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant ACE2-IgG1Fc protein was produced using Maxcyte transfection in CHO-S cells that were cultured for 14 days.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>CHO-S</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For detection of neutralization, 120 µL of the virus/sample mixture was transferred to the Vero E6 cells and incubated for 48 hours before fixation with 4% PFA.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero E6</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were harvested 1, 2, 3, and 7 days post transfection by gently pipetting cells into medium and labeled with an anti-RBD monoclonal antibody (clone D003 Sino Biological Catalog # 40150-D003) and F(ab’)2-Goat anti-Human IgG-Fc secondary antibody conjugated with Rphycoerythrin (ThermoFisher Catalog # H10104).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>ThermoFisher Catalog</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Fluorescent-conjugated antibodies against mouse CD8b antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), and TNF-α (clone MP6-XT22, BD) and staining was performed in the presence of unlabeled anti-CD16/CD32 antibody (clone 2.4G2).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>ThermoFisher</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry was performed using a Beckman-Coulter Cytoflex S flow cytometer and analyzed using Flowjo Software.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Flowjo</b></div>
              <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div>
            </div>
          </td></tr></table>
      

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  3. Jul 2020
    1. SciScore for 10.1101/2020.07.26.222026: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">29 Antibodies and reagents Rabbit anti-DYKDDDDK Tag (D6W5B), rabbit anti-IRF3 (D83B9), rabbit anti-pIRF3 (4D46), rabbit anti-TBK1 (3031S), rabbit anti-pTBK1 (D52C2), and rabbit anti-TRAF3 were from Cell Signaling Technology (</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-DYKDDDDK</div> <div>suggested: (Cell Signaling Technology Cat# 14793, RRID:AB_2572291)</div> </div> <div style="margin-bottom:8px"> <div>anti-IRF3</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-pIRF3 ( 4D46)</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-TBK1</div> <div>suggested: (Cell Signaling Technology Cat# 14590, RRID:AB_2798527)</div> </div> <div style="margin-bottom:8px"> <div>anti-pTBK1</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-TRAF3</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Alexa Fluor 488 goat anti-rabbit IgG secondary antibody, Alexa Fluor 568 goat anti-mouse IgG secondary antibody, Alexa Fluor 488 goat anti-mouse 10 / 55 IgG secondary antibody, and Alexa Fluor 568 goat anti-rabbit IgG secondary antibody were from Thermo Fisher Scientific (USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Alexa Fluor 488 goat anti-rabbit IgG secondary antibody</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-rabbit IgG</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-mouse IgG</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-mouse 10 / 55 IgG</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Coimmunoprecipitation and immunoblotting For coimmunoprecipitation assays, HEK293T cells were collected 24 hours after transfection and lysed in lysis buffer [1.0% (v/v) NP-40, 50 mM Tris-HCl, pH 7.4, 50 mM EDTA, 0.15 M NaCl] supplemented with a protease inhibitor cocktail (Sigma, USA) and a phosphatase inhibitor cocktail (Sigma, USA) as described in our previous publications.30,31 After centrifugation for 10 min at 14,000 g, the supernatants were collected and incubated with the indicated 13 / 55 antibodies, followed by the addition of protein A/G beads (Santa Cruz, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>NP-40</div> <div>suggested: (Covance Cat# MMS-503P-100, RRID:AB_291448)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The expression plasmids of the SARS-CoV-2 M protein and plasmids expressing RIG-I or MDA-5 were cotransfected into HEK293T cell, 24 hours later, MAVS antibodies were used to perform coimmunoprecipitation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>MAVS</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">9 / 55 Materials and methods Cell culture and transfection HEK293, HEK293T, HeLa, and Vero-E6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) with 10% heat-inactivated fetal bovine serum (FBS, Gibco, USA).</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">Constructs and plasmids The RIG-I, RIG-IN, MDA-5, MAVS, TBK1, IKK , IRF3-5D, TRIF, and STING ε genes were cloned into pcDNA6B-Flag, pcDNA6B-Myc, pcDNA6B-V5, pCAG-Flag or pCMV-HA-N expression vectors using standard molecular cloning methods as described in our previous publications.30-32 The IFN- β luciferase reporter plasmid pGL3-IFN- -Luc vector was constructed in our β previous study.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>MDA-5</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Confocal immunofluorescence microscopy Confocal immunofluorescence microscopy studies were performed as described in our previous publications.30,31 Briefly, HeLa cells were grown on 12-well slides one day before transfection with the indicated plasmids.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HeLa</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral plaque assays Viral plaque assays were performed on Vero-E6 cells to measure the titer of VSV-eGFP as described in our previous study.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero-E6</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">32 Briefly, Vero cells were seeded on 24-well plates.</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">VSV-eGFP was used to infect HEK293 cells transfected with empty vector or the SARS-CoV-2 M protein plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK293</div> <div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 removing the solid agarose-medium mix, the cells were stained with 0.05% crystal violet, and the plaques on the monolayer were then counted to calculate the virus titer. 15 / 55 Bioinformatics analysis The transmembrane motifs were predicted with the TMHMM server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>http://www.cbs.dtu.dk/services/TMHMM/</div> <div>suggested: (TMHMM Server, RRID:SCR_014935)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For statistical analysis, two-tailed unpaired Student's t-tests were performed by GraphPad Prism 8.0 and Microsoft Excel.</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>Microsoft Excel</div> <div>suggested: (Microsoft Excel, RRID:SCR_016137)</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 Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).


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


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    2. SciScore for 10.1101/2020.07.26.222026: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">29 Antibodies and reagents Rabbit anti-DYKDDDDK Tag (D6W5B), rabbit anti-IRF3 (D83B9), rabbit anti-pIRF3 (4D46), rabbit anti-TBK1 (3031S), rabbit anti-pTBK1 (D52C2), and rabbit anti-TRAF3 were from Cell Signaling Technology (</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-DYKDDDDK</div> <div>suggested: (Cell Signaling Technology Cat# 14793, RRID:AB_2572291)</div> </div> <div style="margin-bottom:8px"> <div>anti-IRF3</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-pIRF3 ( 4D46)</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-TBK1</div> <div>suggested: (Cell Signaling Technology Cat# 14590, RRID:AB_2798527)</div> </div> <div style="margin-bottom:8px"> <div>anti-pTBK1</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-TRAF3</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Alexa Fluor 488 goat anti-rabbit IgG secondary antibody, Alexa Fluor 568 goat anti-mouse IgG secondary antibody, Alexa Fluor 488 goat anti-mouse 10 / 55 IgG secondary antibody, and Alexa Fluor 568 goat anti-rabbit IgG secondary antibody were from Thermo Fisher Scientific (USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Alexa Fluor 488 goat anti-rabbit IgG secondary antibody</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-mouse IgG</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-mouse 10 / 55 IgG</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-rabbit IgG</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Coimmunoprecipitation and immunoblotting For coimmunoprecipitation assays, HEK293T cells were collected 24 hours after transfection and lysed in lysis buffer [1.0% (v/v) NP-40, 50 mM Tris-HCl, pH 7.4, 50 mM EDTA, 0.15 M NaCl] supplemented with a protease inhibitor cocktail (Sigma, USA) and a phosphatase inhibitor cocktail (Sigma, USA) as described in our previous publications.30,31 After centrifugation for 10 min at 14,000 g, the supernatants were collected and incubated with the indicated 13 / 55 antibodies, followed by the addition of protein A/G beads (Santa Cruz, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>NP-40</div> <div>suggested: (Covance Cat# MMS-503P-100, RRID:AB_291448)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The expression plasmids of the SARS-CoV-2 M protein and plasmids expressing RIG-I or MDA-5 were cotransfected into HEK293T cell, 24 hours later, MAVS antibodies were used to perform coimmunoprecipitation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>MAVS</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">9 / 55 Materials and methods Cell culture and transfection HEK293, HEK293T, HeLa, and Vero-E6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) with 10% heat-inactivated fetal bovine serum (FBS, Gibco, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero-E6</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">32,35 Briefly, approximately 0.5 x 105 HEK293T cells were seeded in 48-well plates and transfected 12 hours later with the luciferase reporter plasmid and the expression vector plasmids of RIG-I, RIG-IN, MDA-5, MAVS, TBK1, IKK , IRF3-5D, TRIF, and STING, alone or together with the plasmid ε expressing the SARS-CoV-2 M protein, as indicated in the experiments.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>MDA-5</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viruses and infection VSV-enhanced green fluorescent protein (eGFP) and SeV were used to infect HeLa, HEK293, or HEK293T cells as described in our previous publications.30-32 Briefly, before infection, prewarmed serum-free DMEM medium at 37°C was used to wash the target cells, after which the virus was diluted to the desired multiplicity of infection (MOI) in serum-free DMEM and incubated with the target cells for 1-2 hours.</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>HEK293</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">32 Briefly, Vero cells were seeded on 24-well plates.</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">In a ll cases,ava lue ofP<0.0 5was conside red tobestatisti callysi gnifican t.16/ 55R esultsT heSAR S-Co V-2Mprot ei ninhibitst ypeIandI IIIFNin ductio nbySeV andpol y(I: C)To exp lorewhethertheSA RS-Co V-2Mpr otein affec tstype IandI III FNpro duct ion,HEK2 93 Tcel ls e xpressingt heSARS-Co V-2M pr otei nwe reinfect edwi thSeVortran sfec tedwith adsRNAmi mic ,pol y(I:C ).</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;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 removing the solid agarose-medium mix, the cells were stained with 0.05% crystal violet, and the plaques on the monolayer were then counted to calculate the virus titer. 15 / 55 Bioinformatics analysis The transmembrane motifs were predicted with the TMHMM server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>http://www.cbs.dtu.dk/services/TMHMM/</div> <div>suggested: (TMHMM Server, RRID:SCR_014935)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For statistical analysis, two-tailed unpaired Student's t-tests were performed by GraphPad Prism 8.0 and Microsoft Excel.</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>Microsoft Excel</div> <div>suggested: (Microsoft Excel, RRID:SCR_016137)</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 Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).


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


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    3. SciScore for 10.1101/2020.07.26.222026: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">29 Antibodies and reagents Rabbit anti-DYKDDDDK Tag (D6W5B), rabbit anti-IRF3 (D83B9), rabbit anti-pIRF3 (4D46), rabbit anti-TBK1 (3031S), rabbit anti-pTBK1 (D52C2), and rabbit anti-TRAF3 were from Cell Signaling Technology (</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-DYKDDDDK</div> <div>suggested: (Cell Signaling Technology Cat# 14793, AB_2572291)</div> </div>

            <div style="margin-bottom:8px">
              <div><b>anti-IRF3</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-pIRF3 ( 4D46)</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-pTBK1</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-TRAF3</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Alexa Fluor 488 goat anti-rabbit IgG secondary antibody, Alexa Fluor 568 goat anti-mouse IgG secondary antibody, Alexa Fluor 488 goat anti-mouse 10 / 55 IgG secondary antibody, and Alexa Fluor 568 goat anti-rabbit IgG secondary antibody were from Thermo Fisher Scientific (USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Alexa Fluor 488 goat anti-rabbit IgG secondary antibody</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-mouse 10 / 55 IgG</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-rabbit IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Coimmunoprecipitation and immunoblotting For coimmunoprecipitation assays, HEK293T cells were collected 24 hours after transfection and lysed in lysis buffer [1.0% (v/v) NP-40, 50 mM Tris-HCl, pH 7.4, 50 mM EDTA, 0.15 M NaCl] supplemented with a protease inhibitor cocktail (Sigma, USA) and a phosphatase inhibitor cocktail (Sigma, USA) as described in our previous publications.30,31 After centrifugation for 10 min at 14,000 g, the supernatants were collected and incubated with the indicated 13 / 55 antibodies, followed by the addition of protein A/G beads (Santa Cruz, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>NP-40</b></div>
              <div>suggested: (Covance Cat# MMS-503P-100, <a href="https://scicrunch.org/resources/Any/search?q=AB_291448">AB_291448</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The expression plasmids of the SARS-CoV-2 M protein and plasmids expressing RIG-I or MDA-5 were cotransfected into HEK293T cell, 24 hours later, MAVS antibodies were used to perform coimmunoprecipitation.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>MAVS</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">When using the TBK1 antibody to perform endogenous coimmunoprecipitation, MAVS was detected in the TBK1 immunoprecipitates (Fig. 5c).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>TBK1</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">12 The binding of the type I or III IFNs to their specific receptors, the type I IFN receptor (IFNAR) and the type III IFN receptor (IFNLR), respectively, triggers the activation of the receptor-associated Janus kinase 1 (JAK1)/tyrosine kinase 2 (TYK2), which stimulates the phosphorylation of STAT1 and STAT2.9,13 JAK2 also participates in type III IFN-induced STAT phosphorylation.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>STAT2.9,13 JAK2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This study reveals a previously undiscovered mechanism of SARS-CoV-2 in evading host antiviral immunity, which may partially explain the clinical features of impaired antiviral immunity in COVID-19 patients and provide insights into the viral pathogenicity and treatment. 9 / 55 Materials and methods Cell culture and transfection HEK293, HEK293T, HeLa, and Vero-E6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) with 10% heat-inactivated fetal bovine serum (FBS, Gibco, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero-E6</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viruses and infection VSV-enhanced green fluorescent protein (eGFP) and SeV were used to infect HeLa, HEK293, or HEK293T cells as described in our previous publications.30-32 Briefly, before infection, prewarmed serum-free DMEM medium at 37°C was used to wash the target cells, after which the virus was diluted to the desired multiplicity of infection (MOI) in serum-free DMEM and incubated with the target cells for 1-2 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK293</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">32 Briefly, Vero cells were seeded on 24-well plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The results indicated that both SeV infection and poly (I:C) transfection strongly stimulated the expression of IFN- , IFN- 1, β λ ISG56, and CXCL10 in the control HEK293T cells (Fig. 1).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK293T</b></div>
              <div>suggested: KCB Cat# KCB 200744YJ, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0063">CVCL_0063</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">When the SARS-CoV-2 M protein was overexpressed, the binding between RIG-I and MAVS was reduced (Fig. 5a, lanes 2 compared to lane 3); however, in the same condition, the interaction between MDA-5 and MAVS was not affected (Fig 5b, lanes 2 compared to lane 3), indicating that the SARS-CoV-2 M protein impedes the complex formation of RIG-I and MAVS but has no effect on the interaction between MDA-5 and MAVS.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>MDA-5</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To address the effect of the SARS-CoV-2 M protein on virus-induced IRF3 phosphorylation, control HeLa cells and HeLa cells expressing the SARS-CoV-2 M protein were infected with SeV.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HeLa</b></div>
              <div>suggested: CLS Cat# 300194/p772_HeLa, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0030">CVCL_0030</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After removing the solid agarose-medium mix, the cells were stained with 0.05% crystal violet, and the plaques on the monolayer were then counted to calculate the virus titer. 15 / 55 Bioinformatics analysis The transmembrane motifs were predicted with the TMHMM server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>http://www.cbs.dtu.dk/services/TMHMM/</b></div>
              <div>suggested: (TMHMM Server, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014935">SCR_014935</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For statistical analysis, two-tailed unpaired Student's t-tests were performed by GraphPad Prism 8.0 and Microsoft Excel.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GraphPad Prism</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>Microsoft Excel</b></div>
              <div>suggested: (Microsoft Excel, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016137">SCR_016137</a>)</div>
            </div>
          </td></tr></table>
      

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.07.29.227249: (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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Subsequently, the antibodies anti-ACE2 (1:100), S-RBD-His-tag (1:50), antiLAMP1 (1:50) and anti-LC3b (1:50) were added and slides were incubated overnight at 4°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-ACE2</div> <div>suggested: (Enzo Life Sciences Cat# ALX-804-722-C100, RRID:AB_11180102)</div> </div> <div style="margin-bottom:8px"> <div>S-RBD-His-tag</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>antiLAMP1</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following blocking, membranes were incubated overnight at 4°C with the primary antibodies anti-ACE2 (1:1000), anti-LAMP1 (1:2000), anti-LC3b (1:1000) and anti-His-tag (1:1000).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-LAMP1</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-LC3b</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-His-tag</div> <div>suggested: (AnaSpec; EGT Group Cat# 29673-1000, RRID:AB_11232932)</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein Extraction and Immunoblot HUVEC cells were rinsed twice with ice-cold PBS and proteins were extracted with M-PER for whole cell lysis, respectively (Thermo Fisher).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HUVEC</div> <div>suggested: JCRB Cat# IFO50271, RRID:CVCL_2959</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HUVECs at 60%–70% confluence were supplemented with hypoglycemic, normal or hyperglycemic media 24 hours before incubation with 10μg of recombinant S-RBD-His-tag overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HUVECs</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">9 weeks old male C57BL/6 were purchased from The Jackson Laboratories, USA.</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;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Band intensity was quantified using ImageJ software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ImageJ</div> <div>suggested: (ImageJ, RRID:SCR_003070)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, mice were anesthetized with sevoflurane inhalation (Abbott) and placed in dorsal recumbency.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Abbott</div> <div>suggested: (Abbott, RRID:SCR_010477)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed, and graphs were prepared with Prism 6.0 (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Prism</div> <div>suggested: (PRISM, RRID:SCR_005375)</div> </div> <div style="margin-bottom:8px"> <div>GraphPad</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The OPLSAA based DoGlycans software was used for building all models35 (S-T1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>DoGlycans</div> <div>suggested: (doGlycans, RRID:SCR_015758)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Molecular interactions were analyzed with LigPlot software 58 and the pdb files required was constructed with fortran based own computer programs and the statistical data results.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>LigPlot</div> <div>suggested: (LigPlot+, RRID:SCR_018249)</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 Barzooka: We did not find any issues relating to the usage of bar graphs.


      Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on pages 22, 23, 23, 24 and 32. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    2. SciScore for 10.1101/2020.07.29.227249: (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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Subsequently, the antibodies anti-ACE2 (1:100), S-RBD-His-tag (1:50), antiLAMP1 (1:50) and anti-LC3b (1:50) were added and slides were incubated overnight at 4°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>S-RBD-His-tag</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>antiLAMP1</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following blocking, membranes were incubated overnight at 4°C with the primary antibodies anti-ACE2 (1:1000), anti-LAMP1 (1:2000), anti-LC3b (1:1000) and anti-His-tag (1:1000).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-ACE2</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-LAMP1</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-LC3b</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-His-tag</b></div>
              <div>suggested: (AnaSpec; EGT Group Cat# 29673-1000, <a href="https://scicrunch.org/resources/Any/search?q=AB_11232932">AB_11232932</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We extend these in silico observations by also demonstrating that entry of the S-RBD can be augmented in vitro by exposure of cultured HUVECs to a hyperglycemic environment that increases ACE2 glycosylation.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HUVECs</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein Extraction and Immunoblot HUVEC cells were rinsed twice with ice-cold PBS and proteins were extracted with M-PER for whole cell lysis, respectively (Thermo Fisher).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HUVEC</b></div>
              <div>suggested: JCRB Cat# IFO50271, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_2959">CVCL_2959</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Malhotra is supported by a COVID-19 Fast Grant (fastgrants.org), NHLBI R01 HL142809, the American Heart Association grant 18TPA34230025, and the Wild Family Foundation. Dr.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>American Heart Association</b></div>
              <div>suggested: (American Heart Association, <a href="https://scicrunch.org/resources/Any/search?q=SCR_007210">SCR_007210</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Preprint at bioRxiv https://doi.org/10.1101/2020.06.20.163097 (2020). 29. Goodman, W. A. et al.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>bioRxiv</b></div>
              <div>suggested: (bioRxiv, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003933">SCR_003933</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Band intensity was quantified using ImageJ software.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>ImageJ</b></div>
              <div>suggested: (ImageJ, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003070">SCR_003070</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, mice were anesthetized with sevoflurane inhalation (Abbott) and placed in dorsal recumbency.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Abbott</b></div>
              <div>suggested: (Abbott, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010477">SCR_010477</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed, and graphs were prepared with Prism 6.0 (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Prism</b></div>
              <div>suggested: (PRISM, <a href="https://scicrunch.org/resources/Any/search?q=SCR_005375">SCR_005375</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>GraphPad</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The OPLSAA based DoGlycans software was used for building all models35 (S-T1).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>DoGlycans</b></div>
              <div>suggested: (doGlycans, <a href="https://scicrunch.org/resources/Any/search?q=SCR_015758">SCR_015758</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To address the structural interactions, we performed molecular dynamics simulations using GROMACS (v.2019.4)46 with the OPLS/AA force field parameters47.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GROMACS</b></div>
              <div>suggested: (GROMACS, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014565">SCR_014565</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The PatchDock algorithm discard all unacceptable complex and results are assorted by geometry shape complementarity score.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>PatchDock</b></div>
              <div>suggested: (PatchDock, <a href="https://scicrunch.org/resources/Any/search?q=SCR_017589">SCR_017589</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Molecular interactions were analyzed with LigPlot software 58 and the pdb files required was constructed with fortran based own computer programs and the statistical data results.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>LigPlot</b></div>
              <div>suggested: (LigPlot+, <a href="https://scicrunch.org/resources/Any/search?q=SCR_018249">SCR_018249</a>)</div>
            </div>
          </td></tr></table>
      

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. Why was the container type inferred? Because we did not specify a height attribute for the amp-img tag. In HTML, reflow can be reduced by always specifying a fixed width and height for elements on a page. In AMP, you need to define the width and height for amp-img elements so that AMP can pre-determine the aspect ratio of the element.
    2. While stylesheets can be reworked relatively easily with AMP by inlining the CSS, the same is not true for JavaScript. The tag 'script' is disallowed except in specific forms. In general, scripts in AMP are only allowed if they follow two major requirements: All JavaScript must be asynchronous (i.e., include the async attribute in the script tag). The JavaScript is for the AMP library and for any AMP components on the page. This effectively rules out the use of all user-generated/third-party JavaScript in AMP except as noted below.
    3. The above errors can be resolved by simply adding the ⚡attribute to the <html> tag like so: <html ⚡ lang="en">
    4. The meta charset information must also be the first child of the <head> tag. The reason this tag must be first is to avoid re-interpreting content that was added before the meta charset tag.

      But what if another tag also specified that it had to be the first child "because ..."? Maybe that hasn't happened yet, but it could and then you'd have to decide which one truly was more important to put first? (Hopefully/probably it wouldn't even matter that much.)

    1. The next step is to link the canonical article to the AMP page. This is achieved by including a <link rel="amphtml"> tag to the <head> section of the canonical article.
  4. learn-eu-central-1-prod-fleet01-xythos.s3.eu-central-1.amazonaws.com learn-eu-central-1-prod-fleet01-xythos.s3.eu-central-1.amazonaws.com
    1. FLAG tag

      This is a fairly hydrophilic sequence ((DYKDDDDK)) that a number of very specific antibodied that have been raised to it. Being so hydrophilic, it tends not to denature proteins to which it is fused (presumably hydrophobic peptides could interfere with proteins' folding - the "molten globule" - as the protein is synthesised from the ribosome..

    1. “How heavenly; how simply heavenly!”

      This reminds me of what this text would look like with a POS tag on it. There are so many adjectives that reapeat themselves. This one is funny becuase it is a repition of words to describe something else.

    1. So why does Qui-Gon keep letting Jar Jar tag along? It’s the same reason he butts heads with the Jedi council: his connection to the living Force. His compassion is greater than the rigid and, frankly, arrogant views of the Jedi.

      Great positive viewpoint about Jar Jar Binks

    1. SciScore for 10.1101/2020.07.24.205583: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">Animals All animal studies were conducted at Oregon Health and Sciences University and approved by the Institutional Animal Care and Use Committee (IACUC, IP00001707).</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Female BALB/c mice (8-12 weeks) were anesthetized using ketamine/xylazine cocktail.</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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">It was observed that anti-V5 antibody was able to immunoprecipitate the hsACE2V5 from cell-free conditioned media (Fig. 4c) while the unbound hsACE2 V5 was detected in the flow-through samples (Fig. 4d).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-V5</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We also conducted a reciprocal co-immunoprecipitation, in which anti-His antibody was used to capture RBDHis (Fig. 4e, f).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-His</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The secondary antibodies used were goat polyclonal anti-rabbit HRP (Jackson ImmunoResearch, 111-035-003) and anti-mouse HRP (115-035-003).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-rabbit HRP</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>anti-mouse HRP</b></div>
              <div>suggested: (Jackson ImmunoResearch Labs Cat# 115-035-003, <a href="https://scicrunch.org/resources/Any/search?q=AB_10015289">AB_10015289</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibodies used for pulldown were mouse monoclonal anti-His tag (sc-8036) or anti-V5 tag (sc-81594) antibody (Santa Cruz Biotechnology).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-His tag</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>sc-81594</b></div>
              <div>suggested: (Santa Cruz Biotechnology Cat# sc-81594, <a href="https://scicrunch.org/resources/Any/search?q=AB_1131162">AB_1131162</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To probe hACE2, anti-ACE2 antibody (Santa Cruz Biotechnology, sc-390851) and anti-mouse HRP were used as the primary and secondary antibodies at 1:200 and 1:2,000, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-ACE2</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>sc-390851</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293T/17 cell line was purchased from ATCC (CRL-11268).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T/17</b></div>
              <div>suggested: ATCC Cat# CRL-11268, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_1926">CVCL_1926</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Calu-3 cells were cultured in MEM supplemented with 10% heatinactivated FBS, 1% penicillin/streptomycin, non-essential amino acids, and sodium pyruvate.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Calu-3</b></div>
              <div>suggested: BCRJ Cat# 0264, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0609">CVCL_0609</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In vitro Fluc mRNA transfection assay For in vitro Fluc mRNA transfection assays, cells were seeded on a white 96 well plate at 4×103 cells/well for 293T and Hep G2 cells or at 104 cells/well for Calu-3, followed by overnight incubation for cell attachment.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>Hep G2</b></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 neutralization assay, 293T-hACE2 cells were seeded into white 96-well plates at 2×104 cells/well and grown for 24 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T-hACE2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr></table>
      

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.07.25.217158: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">After this step, the pellets were placed on ice and resuspended with propidium iodide, which is a viability marker of cells and the cytotoxic activity of antibodies was determined by flow cytometry, after gating on lymphocytes, based on their morphology.</td></tr></table>

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing, bound FcR molecules were revealed with peroxidase conjugated anti-tag antibody (Miltenyi, 120003-811; 1/5000 in 2% bovine serum albumin) and tetramethylbenzidine (TMB) reagent.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-tag</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bound pig IgG were revealed with a secondary anti-mouse-HRP-conjugated antibody (Sigma-Aldrich, L’Isle d’Abeau, France) diluted in washing buffer, at 1:1000, incubated 1h at RT and washed 3 times.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-mouse-HRP-conjugated</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The human Fc tag was then revealed with a specific HRP-conjugated anti-human IgG secondary antibody (diluted in in PBS-Tween-0.05%-1% skimmed milk powder at 1:1000 , incubated 1h at RT and washed 3 times).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-human IgG</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To explore the actual impact of removal of αGal and Neu5Gc epitopes on recognition of swine IgG by pre-existing human natural antibodies, human serums from healthy volunteers were assessed in a binding assay against wild-type or glycohumanized swine IgG, or against rabbit IgG used as a comparator.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>rabbit IgG</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Shen et al 37 reported efficacy after two consecutive transfusions with 200 mL of CP with end point dilution titers greater than 1:40, corresponding to SARSCoV-2–specific antibody (IgG) binding titer greater than 1:1000.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARSCoV-2–specific</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The procedure has been initially developed to circumvent problems related to the occurrence of high titers of anti-αGal and anti-Neu5Gc antibodies following administration of anti-T lymphocytes rabbit immunoglobulins, which is potentially causing allergies 38 , serum sickness “xenosialitis”, a systemic inflammation associated with cardiovascular events 10,11 26 and .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-αGal</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>anti-Neu5Gc</b></div>
              <div>suggested: (BioLegend Cat# 146903, <a href="https://scicrunch.org/resources/Any/search?q=AB_2562884">AB_2562884</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-T</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Although our experiments do not directly address the clinical importance of avoiding anti-αGal/Neu5Gc antibody responses, we provide for the first time data showing that “natural” anti-pig antibodies found in human serum are essentially directed against Neu5Gc and that reactivity against rabbit IgG is higher than against pig IgG (Figure 2).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-αGal/Neu5Gc</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-pig</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>rabbit IgG is higher than against pig IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As enveloped viruses might be sensitive to direct complement-mediated lysis, administration of anti-SARS-CoV-2 pig antibodies might therefore lead to viral neutralization without targeting viral particles to inflammatory cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-SARS-CoV-2 pig</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Owing to the high antibody titers produced by CMAH/GGTA1 KO animals in a short period of time and to the significantly shorter, cheaper and easier pharmaceutical development of polyclonal antibodies as compared to mAbs or mAb cocktails 46 , glyco-humanized anti-SARS-CoV-2 pig immunoglobulins might be among the first treatments specific to COVID-19 reaching the clinic.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-SARS-CoV-2</b></div>
              <div>suggested: (Abcam Cat# ab272854, <a href="https://scicrunch.org/resources/Any/search?q=AB_2847844">AB_2847844</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CPE reduction assay was performed as follows: Vero E6 cells were seeded in 96-well clusters at a density of 5000 cells/well 2 day before infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero E6</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Binding to RFcg The binding of IgG from DKO swine to FcγRI, FcγRIIa and FcγRIIb receptor was tested using an ELISA assay and BIAcore surface plasmon resonance (SPR).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>BIAcore</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">His-Tagged FcRI, FcγRIIa and FcγRIIb receptors (CD64/32a/32b, R&D Systems; 100µL, 2.4 µg/mL in 2% bovine serum albumin) were then added and incubated for 2h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>R&D Systems</b></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 on GraphPad Software (GraphPad Software, San Diego, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GraphPad</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
          </td></tr></table>
      

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.07.21.214759: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">All plasma samples were obtained under protocols approved by Institutional Review Boards at both institutions.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">All cell lines have been tested negative for contamination with mycoplasma.</td></tr></table>

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Abstract Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARS-CoV-2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARSCoV-2 agents.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-SARSCoV-2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Results Selection of SARS-CoV-2 S variants using a replication competent VSV/SARS-CoV-2 chimeric virus To select SARS-CoV-2 S variants that escape neutralization by antibodies, we used a recently described replication-competent chimeric virus based on vesicular stomatitis virus that encodes the SARS-CoV-2 spike (S) protein and green fluorescent protein (rVSV/SARS-CoV2/GFP) (</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>rVSV/SARS-CoV2/GFP</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies that constitute at last part of the neutralizing activity evident in COV-NY plasma appear to recognize an epitope that includes and K444 and V445.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>V445</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody binding and ACE2 binding inhibition assay A conformationally stabilized (6P) version of the SARS-CoV-2 S protein(25), appended at its C-terminus with a trimerization domain, a GGSGGn spacer sequence, NanoLuc luciferase, Strep-tag, HRV 3C protease cleavage site and 8XHis (S-6P-NanoLuc) was expressed and purified from the supernatant of 293T Expi cells.</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">Mutants thereof were also expressed and purifies following substitution of sequences encoding the RBD that originated from the unmodified Sexpression plasmids For antibody binding assays, 20ng, 40ng, or 80ng S-6P-NanoLuc (or mutants thereof) were mixed with 100ng of antibodies, C121, C135, or C144, \ diluted in LI-COR Intercept blocking buffer, in a total volume of 60μl/well in 96-well plate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>C121</div> <div>suggested: (Leinco Technologies Cat# C121, AB_2828361)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Patent applications submitted by Rockefeller University are pending for anti SARS-CoV-2 antibodies (MCN, DR, inventors) and VSV/SARS-CoV-2 chimeric virus (PDB, TH FS and YW, inventors) A 10μg/ml C121, C135, C144 or plasma dilution 10μg/ml C121, C135, C144 or plasma dilution 1x106 IU rVSV/SARS-CoV-2/GFP p1 B p2 No Antibody Sequence, Isolate mutants by limiting dilution Plasma [5x initial] Sequence, Isolate mutants by limiting dilution Sequence Plasma [5x initial] p3 p4 10μg/ml C121 C Figure 1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti SARS-CoV-2</div> <div>suggested: (Abcam Cat# ab273074, AB_2847846)</div> </div>

            <div style="margin-bottom:8px">
              <div><b>C135</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>p3</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Selection of SARS-CoV-2 S mutations that confer antibody resistance. A. Outline of serial passage experiments with replication competent VSV derivatives encoding the SARS-CoV-2 S envelope glycoprotein and a GFP reporter (rVSV/SARS-CoV-2/GFP) in 293T/ACE2(B) cells in the presence of neutralizing antibodies or plasma. B. Representative images of 293T/ACE2(B) cells infected with 1x106 PFU of rVSV/SARS-CoV2/GFP in the presence or absence of 10μg/ml of the monoclonal antibody C121. C.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>SARS-CoV-2 S envelope glycoprotein</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">C144 passaged rVSV/SARS-CoV-2/GFP Populations Mutant purification by limiting dilution rVSV/SARS-CoV-2/GFP (E484K) rVSV/SARS-CoV-2/GFP (Q493R) Figure 3 - supplement 1 Example of plaque purification of individual viral mutants from populations passaged in the presence of antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>rVSV/SARS-CoV-2/GFP</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Amino acids whose substitution confers partial or complete (IC50 >10μg/ml) resistance to each monoclonal antibody in the HIV-pseudotype assays are indicated for C121 (red) C135 (green) and C144 (purple). E. Binding of S-NanoLuc fusion protein in relative light units (RLU) to 293T or 293T/ACE2cl.22 cells after preincubation in the absence or presence of C121, C135 and C144 monoclonal antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>C144</b></div>
              <div>suggested: (Leinco Technologies Cat# C144, <a href="https://scicrunch.org/resources/Any/search?q=AB_2828501">AB_2828501</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines HEK-293T cells and derivatives were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37oC and 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK-293T</b></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 with pHIVNLGagPol, pCCNanoLuc2AEGFP and a WT or mutant SARS-CoV-2 expression plasmid (pSARS-CoV2Δ19) using polyethyleneimine.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Amino acids whose substitution confers partial or complete (IC50 >10μg/ml) resistance to each monoclonal antibody in the HIV-pseudotype assays are indicated for C121 (red) C135 (green) and C144 (purple). E. Binding of S-NanoLuc fusion protein in relative light units (RLU) to 293T or 293T/ACE2cl.22 cells after preincubation in the absence or presence of C121, C135 and C144 monoclonal antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T/ACE2cl.22</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The PCR products were gel-purified and sequenced either using Sanger-sequencing or NGS as previously described (31).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>NGS</b></div>
              <div>suggested: (NGSadmix, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003208">SCR_003208</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For analysis of NGS data, the raw paired-end reads were pre-processed to remove adapter sequences and trim low-quality reads (Phred quality score <20) using BBDuk.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Phred</b></div>
              <div>suggested: (Phred, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001017">SCR_001017</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Information regarding RBD-specific variant frequencies, their corresponding P-values, and read depth were compiled using the Python programming language (version 3.7) running pandas (1.0.5), numpy (1.18.5), and matplotlib (3.2.2).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Python</b></div>
              <div>suggested: (IPython, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001658">SCR_001658</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>matplotlib</b></div>
              <div>suggested: (MatPlotLib, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008624">SCR_008624</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The half maximal inhibitory concentrations for plasma (NT50), and monoclonal antibodies (IC50) was calculated using 4-parameter nonlinear regression curve fit to raw or normalized infectivity data (GraphPad Prism).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GraphPad</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
          </td></tr></table>
      

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. A # character always indicates a block opening tag. A / character always indicates a block closing tag. A : character, as in {:else}, indicates a block continuation tag.
    1. SubsecTimeA tag used to record fractions of seconds for the DateTime tag.Tag =37520 (9290.H)Type=ASCIICount=AnyDefault=none
    1. SciScore for 10.1101/2020.07.20.213280: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Red, anti-S antibody; green, anti-β-actin antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-S</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(D) Western blot analysis of pseudovirions bearing SARS-CoV-2 S, SARS-CoV-2 S ΔPRRA, bat raTG13 S, raTG13 S+PRRA and Pangolin GX S. Red, anti-p24 antibody; green, anti-Spike antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-p24</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>anti-Spike</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(A) The expression of ACE2 ortholog plasmids in 293T cells were detected using mouse anti-V5 tag monoclonal antibody targeting the C-terminal V5 tag (in Red).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-V5</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Green, anti-β-actin antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-β-actin</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">By contrast, infection of the human lung epithelial cell line Calu3 as well as the human intestinal cell line Caco-2 by the same pseudoviruses were reduced by 9050%, respectively, compared to pseudoviruses bearing the wild type SARS-CoV-2 S or raTG13 S (Fig. 3B&C).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Calu3</b></div>
              <div>suggested: BCRJ Cat# 0264, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0609">CVCL_0609</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Addition of PRRA to raTG13 Spike further enhanced the cleavage but decreased the relevant pseudovirus infectivity on 293T-hACE2 cells, indicating that addition of PRRA does not enhance raTG13 Spike-driven entry.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T-hACE2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RNA-seq analyses of SARS-CoV-2 infected Calu-3 and Caco-2 cells revealed different protease repertoires with cathepsin A being expressed to a much higher level in Caco-2 (Figure S4).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Caco-2</b></div>
              <div>suggested: CLS Cat# 300137/p1665_CaCo-2, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0025">CVCL_0025</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">It is conceivable that the spike protein is cleaved by different host proteases among 293T, Calu-3 and Caco-2.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Calu-3</b></div>
              <div>suggested: BCRJ Cat# 0264, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0609">CVCL_0609</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Huh7.5.1 was a gift of F. Chisari and maintained in (DMEM+10% FBS) (Zhong et al., 2005).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Huh7.5.1</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_E049">CVCL_E049</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 isolate USA-WA1/2020 was obtained from BEI Resources, NIAID, NIH, and had been passed three times on Vero cells and 1 time on Vero E6 cells prior to acquisition.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero E6</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_XD71">CVCL_XD71</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero cells were plated the day before infection into 96 well plates at 1.5 x 104 cells/well.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">24 h post-transfection, these recipient cells were mixed at a 1:1 ratio with 293T cells expressing Cre and CoV S (donor cells) to initiate cell–cell fusion.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T</b></div>
              <div>suggested: KCB Cat# KCB 200744YJ, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0063">CVCL_0063</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">STAR Methods Resource Availability Lead Contact Further information and requests for reagents may be directed to and will be fulfilled by Lead Contact Tony Wang (Tony.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>STAR</b></div>
              <div>suggested: (STAR, <a href="https://scicrunch.org/resources/Any/search?q=SCR_015899">SCR_015899</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data was analyzed by FlowJo software.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>FlowJo</b></div>
              <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div>
            </div>
          </td></tr></table>
      

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.07.20.213249: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">The cohort comprised of 138 females, 56 males (2 not recorded) with a 198 median age of 37 years (range 1-66y).</td></tr></table>

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 serum was calibrated against research 128 reagents NIBSC 20/130 and NIBSC 20/124 distributed by the National Institute for 129 Standards and Biological Control (NIBSC, Potters Bar, UK, https://www.nibsc.org/) for 130 the purpose of development and evaluation of serological assays for the detection of 131 antibodies against SARS-CoV-2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARS-CoV-2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SULFO-TAG™ Anti-Human IgG 160 Antibody).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Anti-Human IgG</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The S antigen was the most sensitive of the three, with a 273 sensitivity of 96.2% and 97.9% >14 days and >21 days since onset of symptoms 274 respectively. 275 276 Antibody concentration relationship between antigens 277 The concentration of anti-S, RBD and N antibody all correlated significantly with each 278 other (p<0.0001; Figure 4A-C), the strongest association was between S and RBD 279 (r=0.882) (Figure 4A).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-S</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In this study we describe a 308 novel assay that can measure antibody to several SARS-CoV-2 antigens 309 simultaneously as well as evaluating the functional capacity of anti-Spike antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-Spike</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">370 In summary, the MSD multiplexed coronavirus panel assay evaluated in this study is 371 highly reproducible, specific and sensitive for the detection of anti-SARS-CoV-2 372 antibody over 14 days since the onset of COVID-19 symptoms.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-SARS-CoV-2 372</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 172 173 Statistical analysis 174 Statistical analysis was performed using MSD Discovery Workbench and GraphPad 175 Prism version 8.0 (GraphPad, San Diego, CA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad</div> <div>suggested: (GraphPad Prism, SCR_002798)</div> </div> </td></tr></table>

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. Author Response

      Reviewer #1:

      In this manuscript, Cobb and colleagues report on the biochemical and functional characterization of redox active ER proteins in the malaria parasite Plasmodium falciparum. They studied a protein called PfJ2, which contains HSP40 J and Trx domains and is homologous to human ERdj5. Using the TetR-PfDOZI aptamer system to tag PfJ2 and conditionally regulate its expression, they show that PfJ2 is localized in the parasite ER and is essential for parasite growth during the asexual blood stages. Using co-immunoprecipitation combined with mass spectrometry, they identify partner proteins of PfJ2 including other ER proteins such as PDI and BIP. Using a chemical biology approach based on DVSF crosslinker, they document the redox activity of PfJ2 and identify redox substrates of PfJ2, which include PDI8 and PDI11 protein disulfide isomerases. They further functionally characterized PDI8 and PDI11 using the glmS ribozyme for conditional knockdown. These experiments confirm that PDI8 and PDI11 are partners of PfJ2 and show that knockdown of PDI8 impairs parasite blood stage growth. Finally, the authors show that inhibitors of human PDI inhibit parasite growth (at best in the micromolar range) and block the redox activity of PfJ2 and parasite PDI.

      This is an interesting study combining genetic and chemical biology approaches to investigate an understudied compartment of the malaria parasite. The manuscript is clearly written and the work technically sound. In summary, this study illustrates that ER redox proteins in the malaria parasite perform similar functions as in other organisms. The main limitation of this study is that evidence showing that redox ER parasite proteins are druggable is rather weak. PfJ2 is very similar to human ERdj5 in terms of active redox site and function, and the authors used inhibitors that are active on human PDI. It thus remains uncertain whether an antimalarial strategy targeting such conserved pathways is achievable.

      RESPONSE: We thank the reviewer for their appreciation of our work. While PfJ2 shares some similarity to human ERdJ5, we disagree that they are functionally similar. Our data show that, unlike ERdJ5, PfJ2 substrates are primarily other redox chaperones. In terms of the redox active site, our data clearly identifies a pathway that is targeted by a small molecule inhibitor. There is a lot of precedence for targeting conserved pathways as an antimalarial strategy. For example, anti-translational and anti-proteasomal inhibitors are being widely studied for their potency as antimalarials (Baragana et al 2015 Nature; Li et al 2016 Nature; Wong et al 2017 Nat. Microbiol.; Kirkman et al 2018 PNAS; Stokes et al 2019 PLoS Path.), several proteases (with conserved active sites) are well known antimalarial targets (Sleebs et al 2014 PLoS Biol.; Nasamu et al 2017 Science; Pino et al 2017 Science; Favuzza et al 2020 Cell Host Microbe), and effective inhibitors targeting a parasite chaperone has been repurposed for antimalarial drug development (Lu et al 2020 PNAS). We thank the reviewer for recognizing that there is a long road ahead of us to develop a more specific inhibitor for PfJ2, however, that is beyond the scope of this study.

      In addition, a number of specific points should be addressed to improve the quality of the manuscript:

      Although PDI8 and PDI11 gene edition were performed in the PfJ2apt line, the authors did not attempt to knockdown both PfJ2 and PDI8/11 simultaneously (because PfJ2 is essential). Therefore, referring to "double conditional mutants" is misleading.

      RESPONSE: We are open to alternative ways to refer to these mutants. Since we have orthogonal systems for knockdown of two proteins, we refer to these as double conditional mutants.

      The authors should provide details on the parasite lysis conditions used for the co-IP experiments to identify interacting proteins (Table 1) and redox partners (figure 3). In their proteomic analysis, the authors considered proteins with a 5-fold increase in the specific versus control conditions. A more stringent analysis would retain only proteins identified exclusively in the modified J2apt line.

      RESPONSE: We will include this in a new version. We agree that a more stringent analysis would lead to fewer proteins being identified, however, it also runs the risk of missing real interactors. We chose to use a 5-fold cutoff based on previously published work (Boucher et al 2018 PLoS Biol; Florentin et al 2020 PNAS).

      In figure 6, the authors should probe the blots for a control protein that is not co-immunoprecipitated with PfJ2 or PDI8. In Supplementary fig 4, control untreated parasites should be analyzed in parallel to GlcN-treated parasites.

      RESPONSE: We will do this once our labs reopen after the pandemic.

      The partial reduction of protein levels (Fig S4) shows that the glmS system is not very efficient here, which might explain why there is no phenotype in the PDI11 mutant (Fig5B). This questions the conclusion that PDI11 is dispensable.

      RESPONSE: We agree and we state that “These results...suggest that PfPDI11 may be dispensable... conclusions are supported by a genome-wide essentiality screen performed in P. falciparum” (Lines 319-322). We will add more discussion to explain this result.

      Reviewer #2:

      The claim here is of having discovered a druggable cellular process in P. falciparum, one that opens the door to therapeutic intervention in the most deadly form of malaria.

      The study commences with a focus on what appears to be the Pf homologue of a eukaryotic protein disulphide isomerase, known to many as ERdJ5 and referred to here as PfJ2. Its cellular contingents were identified by cross-linking and pull down, it’s (predicted) thiol reactivity explored with agents that react with reduced thiols and it’s functional importance to parasite fitness (in the lab) explored by gene knockout. These experiments provide evidence that PfJ2 and it’s associated Pf PDIs engage in thiol redox chemistry in the ER of the parasite and that integrity of this biochemical process is important to viability of the parasite.

      Lacking all expertise in molecular parasitology, this reader is unable to judge the specific significance of these findings to the field nor indeed the extent to which these are hard-won discoveries.

      RESPONSE: We are gratified to note that the reviewer is cognizant of their limitations and their ability to judge the significance of this work.

      However, from the perspective of the fundamentals of ER redox chemistry the findings represent a modest advance, showing that what is true of yeast and mammals is also true of Apicomplexa. The important mystery related to the juxtaposition of a J-domain and thioredoxin domains in PfJ2, remains.

      The most important claim however is the one with translational potential, namely that one might be able to discover (electrophilic) compounds that, despite the monotony of shared chemical features of thiol chemistry, will nonetheless possess sufficient specificity towards this or that malarial protein to be converted one day to a useful drug. However, in regards to this important point the authors offer very little in the way of evidence how and if this might be achieved.

      RESPONSE: We disagree. The work does not reconfirm the ‘fundamentals of ER redox’ chemistry. There is no work, in any system, that has shown that PfJ2-like proteins act as reductases for PDIs. In fact, as we state in the paper, in other model systems, there is a lot of redundancy built in the ER redox systems and PfJ2-like proteins work with specific clients like SERCA pumps or LDL receptor. Thioredoxin domain proteins in the ER of other eukaryotes have not been shown to work with each other or other chaperones. Furthermore, our data actually does suggest a reason why the J-domain is juxtaposed to thioredoxin domains. It recruits BiP to the mixed disulfides formed by PDIs. This insight would not have been possible in other systems because of the redundant redox mechanisms. In terms of the translational aspect, this work identifies an essential, pathway and a starting point for developing better inhibitors. As the reviewer may be aware, once a starting drug-like molecule has been identified, one has to embark on a medicinal chemistry program to develop more potent inhibitor. However, this is beyond the scope of this manuscript.

      Therefore, the main conclusions to draw from this paper are that ER-localised thiol chemistry is also important in malaria parasites and that, assuming one were able to explore localised context-specific features of thiol reactivity in malarial proteins, it may one day be possible to develop anti-malarial drugs that exploit this as a mechanism of action. The generic nature of these considerations limits the significance of the conclusions one might draw from this paper.

      RESPONSE: We are disappointed that we were unable to satisfy the reviewer’s need for ‘a giant leap for mankind’ insights.

      Reviewer #3:

      This paper describes redox-active proteins in the ER of malaria parasites. The authors start out with PfJ2, a J- and Trx-domain containing protein. They find that it is an essential ER protein that interacts with other chaperone and Trx domain proteins. Using a crosslinker with specificity for redox-active cysteines they identify PfPDI8 and PfPDI11 as redox-partners that together may aid folding of other proteins in the secretory pathway. Finally the authors use inhibitors that act on human PDIs and show that they inhibit parasite growth, albeit at rather high concentrations. This may be fortunate as this suggests different specificities for host and parasite PDIs. However, it also means that from this work it is difficult to judge if the parasite PDIs can be specifically targeted.

      RESPONSE: We thank the reviewer for recognizing the important insights gained from this work. We agree that the specific inhibitor identified is not an ideal antimalarial. There is a lot of precedence in the field for antimalarial inhibitors that target conserved mechanisms such as protein translation (Baragana et al 2015 Nature; Wong et al 2017 Nat. Microbiol.), aspartic proteases (Sleebs et al 2014 PLoS Biol.; Nasamu et al 2017 Science; Pino et al 2017 Science; Favuzza et al 2020 Cell Host Microbe), the proteasome (Li et al 2016 Nature; Kirkman et al 2018 PNAS; Stokes et al 2019 PLoS Path.), the TRiC chaperone complex (Lu et al 2020 PNAS) etc. We are starting a medicinal chemistry program to identify more potent inhibitors of these redox chaperones. However, that is beyond the scope of this paper.

      This is an interesting paper and rightly emphasises that it addresses a much understudied process and organelle in the parasite. The DVSF-crosslinking and the knockdown cell lines are highlights (although the knockdown cell lines were not fully exploited). The paper covers a lot of ground. However, this comes at the cost of depth. The actual function of the studied proteins on folding of other proteins and on the state of the ER was not evaluated and it is also not clear if the human PDI inhibitors indeed target the parasite enzymes. The high concentrations of inhibitors needed to show an effect on DVSF-crosslinking might indicate a secondary effect due to loss of parasite viability. As a result it is not fully clear if the studied proteins are indeed critical for folding of relevant substrates and if this process is druggable. More work is needed to support the main conclusions of the paper.

      RESPONSE: We thank the reviewer for appreciating the diverse toolsets used here to gain important insights into the ER of malaria-causing parasites. Due to the short time-frame of the DVSF-crosslinking experiment (30 mins vs 48h life cycle), we are able to conclude that the effect of the drug is not secondary. A new version will clarify this.

      Major points:

      1) The authors describe conditional knockdown lines and find that PfJ2 and PfPDI8 are essential but these lines are not further exploited for functional studies. Did the knock downs have any effect on proteins they mention as potential substrates (Table 1)? Did it affect the state/morphology of the ER? Did knock down of PfPDI8 remove/shift one of the PfJ2 bands after DVSF-crosslinking, as would be expected? Is there an effect on BiP? A general folding problem in the ER with such a lethal phenotype might have profound effects on the morphology of the organelles receiving protein from the ER. What happens to other cellular markers after knock down of these proteins? Were the knock down cells analysed by EM? Was there an effect on protein export? As it stands the knock down data does not show a role of the complex in the folding of any type of substrate and the function in oxidative folding, as indicated in the title, remains tentative.

      RESPONSE: The morphology of the ER is difficult to address due the fact that in these lifecycle stages the ER is quite condensed. Further, the ER is not clearly identifiable via EM. The knockdown of PDI8 is not complete, therefore, it is not possible to perform the suggested experiment as we will always see the residual PDI8 crosslinked with PfJ2. We are not sure what or if there’s any effect on BiP upon knockdown of PfJ2. BiP does not crosslink with PfJ2 and its expression levels do not change. We are not sure what other effect the reviewer expects on BiP. The co-IP data show that BiP is part of a complex with PfJ2 and PDI8, this complex has not been previously observed in the ER of any organism. Since the parasites die during the trophozoite/early schizont stages, several of these organelles such as Rhoptries, micronemes etc probably do not form. Once the lab reopens after the pandemic, we will test for the presence of these organelles via immunofluorescence microscopy as well as EM. Similar experiments could show an effect on protein export. However, since we didn’t identify any exported proteins to be putative substrates of PfJ2 (despite the expectation that chaperones are sticky and bind everything), and therefore, any effect we observe is likely to be indirect. Given the published data establishing the function of PDIs as oxidative folding chaperones, their high degree of conservation, and in vitro characterization, we conclude that they function in oxidative folding. Furthermore, we show that PfJ2 regulates the function of Plasmodium PDIs as well as recruits BiP to the mixed disulfide complex. BiP is a highly conserved chaperone that has clear function in protein folding. Based on this and the data presented here, we conclude that PfJ2 functions as a regulator of oxidative folding in P. falciparum.

      2) While I like the idea to use established commercial drugs as novel potential antimalarials, those used here are specific for non-infectious human diseases and target the host which is not a desirable property. Considering this, their rather low activity against the parasite can be taken as a positive result. However, the low activity is less convincing to establish the folding pathway in the parasite ER as a drug target. Beside the issue that it is unclear if indeed oxidative folding is the essential function of the PfJ2 complex (see previous point), the data in Fig. 7 does not clearly establish that this function is targeted by the inhibitors used. The effect is only seen at concentrations of 5xIC50. It is possible that this severely reduced viability which could be a non-specific reason for the lack of DVSF-crosslinked products. This needs to be examined in more depth. For instance, is the crosslink still seen after equivalent treatment of cultures with 5xIC50 of other unrelated drugs? Were other, unrelated processes unaffected? What was the effect of exposure to the drug on the ER and parasite morphology? Was the appropriate parasite stage affected? Can it be tested how fast exposure to 5xIC50 of the drug kills the parasites (at least morphologically, but preferably also by more specific means)?

      RESPONSE: We agree that the drugs identified here are not ideal antimalarials but rather they are starting molecules for a larger medicinal chemistry program, that is beyond the scope of this manuscript. While we see significant loss of DVSF crosslinking (for PfJ2) even at the IC50, the relationship between protein activity inhibition and parasite death isn’t always linear. We are testing analogs of 16F16 to identify more potent inhibitors of these proteins. We thank the reviewer for the suggested experiments, and when the pandemic is no long limiting access to the lab, we will perform some of these.

      3) While generally sound, a few experiments would have benefitted from more controls. A reducing sample from the same parasites for Fig. S7 (loaded a couple of lanes away to avoid interference of the reducing agent) would have been nice for comparison to show specificity of the higher molecular weight adducts. Detection of a control protein not expected to co-purify (for instance a cytosolic protein or a membrane-bound protein to control for residual parasite material) would have been appropriate for the co-immunoprecipitations (e.g. Fig. 6A,D, Fig. S9).

      RESPONSE: We show that there are no non-specific bands for PDI11, because when we mutate the cysteines, we do not observe any cross-linking. We will include the control proteins for the co-IPs, they were not included for the sake of clarity.

    2. Reviewer #1:

      In this manuscript, Cobb and colleagues report on the biochemical and functional characterization of redox active ER proteins in the malaria parasite Plasmodium falciparum. They studied a protein called PfJ2, which contains HSP40 J and Trx domains and is homologous to human ERdj5. Using the TetR-PfDOZI aptamer system to tag PfJ2 and conditionally regulate its expression, they show that PfJ2 is localized in the parasite ER and is essential for parasite growth during the asexual blood stages. Using co-immunoprecipitation combined with mass spectrometry, they identify partner proteins of PfJ2 including other ER proteins such as PDI and BIP. Using a chemical biology approach based on DVSF crosslinker, they document the redox activity of PfJ2 and identify redox substrates of PfJ2, which include PDI8 and PDI11 protein disulfide isomerases. They further functionally characterized PDI8 and PDI11 using the glmS ribozyme for conditional knockdown. These experiments confirm that PDI8 and PDI11 are partners of PfJ2 and show that knockdown of PDI8 impairs parasite blood stage growth. Finally, the authors show that inhibitors of human PDI inhibit parasite growth (at best in the micromolar range) and block the redox activity of PfJ2 and parasite PDI.

      This is an interesting study combining genetic and chemical biology approaches to investigate an understudied compartment of the malaria parasite. The manuscript is clearly written and the work technically sound. In summary, this study illustrates that ER redox proteins in the malaria parasite perform similar functions as in other organisms. The main limitation of this study is that evidence showing that redox ER parasite proteins are druggable is rather weak. PfJ2 is very similar to human ERdj5 in terms of active redox site and function, and the authors used inhibitors that are active on human PDI. It thus remains uncertain whether an antimalarial strategy targeting such conserved pathways is achievable.

      In addition, a number of specific points should be addressed to improve the quality of the manuscript:

      Although PDI8 and PDI11 gene edition were performed in the PfJ2apt line, the authors did not attempt to knockdown both PfJ2 and PDI8/11 simultaneously (because PfJ2 is essential). Therefore, referring to "double conditional mutants" is misleading.

      The authors should provide details on the parasite lysis conditions used for the co-IP experiments to identify interacting proteins (Table 1) and redox partners (figure 3). In their proteomic analysis, the authors considered proteins with a 5-fold increase in the specific versus control conditions. A more stringent analysis would retain only proteins identified exclusively in the modified J2apt line.

      In figure 6, the authors should probe the blots for a control protein that is not co-immunoprecipitated with PfJ2 or PDI8.

      In Supplementary fig 4, control untreated parasites should be analyzed in parallel to GlcN-treated parasites.

      The partial reduction of protein levels (Fig S4) shows that the glmS system is not very efficient here, which might explain why there is no phenotype in the PDI11 mutant (Fig5B). This questions the conclusion that PDI11 is dispensable.

    1. SciScore for 10.1101/2020.07.16.20153437: (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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Here, we utilize multiomics single-cell analysis to probe dynamic immune responses in patients with stable or progressive manifestations of COVID-19, and assess the effects of tocilizumab, an antiIL-6 receptor monoclonal antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>antiIL-6 receptor</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The signaling pathways driven by IL-1β, TNF-α, and IL-6 have been implicated in the pathogenesis of COVID-1910 and antibodies against IL-6 receptor have shown early promise9,11-13, including our own experience14; however, large-scale randomized trials are needed to adequately evaluate their efficacy.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>TNF-α</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>antibodies against IL-6</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Here, we employed a single-cell multi-omics approach in order to study the dynamics of the innate and adaptive immune system responses in COVID-19, explore the molecular mechanisms that contribute to the progression of the diseases, and assess the effects of tocilizumab, a humanized anti-IL-6 receptor monoclonal antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-IL-6</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Tocilizumab effects differ across cell types and are associated with the levels of expression of IL6R and IL6ST Eight of ten COVID-19 patients in our study were treated with tocilizumab, an antiIL6 receptor (IL6R) antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>IL6ST</b></div>
              <div>suggested: (MBL International Cat# D023-3, <a href="https://scicrunch.org/resources/Any/search?q=AB_591799">AB_591799</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>antiIL6</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>IL6R</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To better identify cellular multiplets and enable us to superload the cells onto the 10x platform, we used Cell Hashing technique and multiplexed 56 samples in each 10x reaction by using six hashing antibodies61.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>antibodies61</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody-derived tag (ADT) and Hashtag oligonucleotide (HTO) sequencing libraries were generated.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Antibody-derived tag ( ADT )</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following unsupervised clustering, annotation for CITE-seq cells was performed with both gene expression and antibody-derived counts (ADT) by using a manually curated marker gene list (Supp Table ST8).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>antibody-derived counts ( ADT</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Yale Center for Genome Analysis/Keck Biotechnology Resource Laboratory, Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, CT, USA. 14. SJTU-Yale Joint Center for Biostatistics and Data Science, Department of Bioinformatics and Biostatistics, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China. 15</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Genome Analysis/Keck Biotechnology Resource Laboratory</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>Biostatistics</b></div>
              <div>suggested: (BWH Biostatistics Center, <a href="https://scicrunch.org/resources/Any/search?q=SCR_009680">SCR_009680</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">D.A.H. has received research funding from Bristol-Myers Squibb, Novartis, Sanofi, and Genentech.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Genentech</b></div>
              <div>suggested: (Genentech, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003997">SCR_003997</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Automated annotation using SingleR package22</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>SingleR</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Gene set enrichment analysis (GSEA) further demonstrated that dividing T cells in the progressive COVID-19 patients exhibited more terminally exhausted T cell signature and type 1 IFN response signature than those in stable patients (Fig 4J, Supp Table ST9).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Gene set enrichment analysis</b></div>
              <div>suggested: (Gene Set Enrichment Analysis, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003199">SCR_003199</a>)</div>
            </div>
          </td></tr></table>
      

      Data from additional tools added to each annotation on a weekly basis.

      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.07.09.195263: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Verified by a pseudovirus-based neutralization assay, we found that nearly two-thirds of the VH3-53/VH3-66 antibodies displayed neutralizing abilities against SARS-CoV-2 (Figure 1A).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARS-CoV-2</div> <div>suggested: (Sino Biological Cat# 40143-R019, AB_2827973)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Crystal structures of VH3-53/VH3-66 antibodies in complex with RBD To characterize the molecular interactions between the VH3-53/VH3-66 NAbs and RBD, we determined the crystal structures of RBD in complex with the antigen-binding fragment (Fabs) of BD-236, BD-604, and BD-629 at 2.4 Å, 3.2 Å, and 2.7 Å, respectively (Table S2).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>antigen-binding fragment</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>BD-604</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Furthermore, they bind to the RBD in similar fashions as B38 (Figure 2D), a VH3-53 antibody (Wu et al., 2020); and CB6 (Figure 2E), a VH3-66 antibody (Shi et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>VH3-53</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>CB6</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We subsequently determined the crystal structures of three tripartite complexes consisting of the Fabs of these antibodies and RBD: BD- 236/RBD/BD-368-2, BD-604/RBD/BD-368-2, and BD-629/RBD/BD-368-2.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>BD-604/RBD/BD-368-2</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>BD-629/RBD/BD-368-2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In fact, BD368-2 may further potentiate the activity of the VH3-53/66 antibodies, since it can induce rapid structural changes of the S trimer, which may lead to the exposure of the RBDs that were originally in the “down” state.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>VH3-53/66</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recently, scientists at Regeneron have described such a pair of antibodies, REGN10987 and REGN10933, and showed that their cocktail indeed prevented the generation of escaping mutants using a pseudovirus system (Baum et al., 2020; Hansen et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>REGN10933</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The VH3-53/VH3-66 antibodies engage the “up” RBDs to prevent their interaction with ACE2.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>VH3-53/VH3-66</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>ACE2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Certainly, the SARS-CoV-2 S protein is flexible in nature and exists in multiple conformations, and the presence of some of these antibodies can change the conformation landscape and trigger conformational changes, as shown for BD-368-2 (Figure 6A) and CR3022 (Huo et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>CR3022</b></div>
              <div>suggested: (Imported from the IEDB Cat# CR3022, <a href="https://scicrunch.org/resources/Any/search?q=AB_2848080">AB_2848080</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-His-tag antibodies were loaded to a SA sensor chip by NHS to capture the His-tag labeled S trimer.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Anti-His-tag</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>His-tag labeled S trimer.</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibodies were in vitro expressed using HEK293 cells, and the binding specificities were quantity by ELISA against the Spike protein and the RBD protein, as described previously.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK293</b></div>
              <div>suggested: CLS Cat# 300192/p777_HEK293, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0045">CVCL_0045</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Measurement of antibody neutralization potency The pseudovirus neutralization assays were performed using Huh-7 cell lines, as described previously (Cao et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Huh-7</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BD368-2 IgG was expressed by transient transfection in HEK293F cells and purified from the conditioned media using a Protein A column (GE Life Sciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK293F</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_6642">CVCL_6642</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">STAR Methods In Vitro expression of the antibodies and ELISA quantification All antibody sequences in this manuscript were generated in the previous study (Cao et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>STAR</b></div>
              <div>suggested: (STAR, <a href="https://scicrunch.org/resources/Any/search?q=SCR_015899">SCR_015899</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IC50 and IC80 were determined by a four-parameter logistic regression using GraphPad Prism 8.0 (GraphPad Software Inc.).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GraphPad</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All structures were solved by the molecular replacement method using the Phaser program (McCoy et al., 2007) in Phenix (Liebschner et al., 2019).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Phaser</b></div>
              <div>suggested: (Phaser, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014219">SCR_014219</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>Phenix</b></div>
              <div>suggested: (Phenix, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014224">SCR_014224</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The structural models were then manually adjusted in Coot (Emsley et al., 2010) and refined using Phenix.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Coot</b></div>
              <div>suggested: (Coot, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014222">SCR_014222</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Movies were recorded on a K2 Summit direct electron detector (Gatan) using the SerialEM software (Mastronarde, 2005), in the super-resolution mode at a nominal magnification of 130,000, with an exposure rate of 7.1875 e-/Å2 per second.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>SerialEM</b></div>
              <div>suggested: (SerialEM, <a href="https://scicrunch.org/resources/Any/search?q=SCR_017293">SCR_017293</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Raw movie frames were aligned and averaged into motion-corrected summed images with a pixel size of 1.055 Å by MotionCor2 (Zheng et al., 2017).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>MotionCor2</b></div>
              <div>suggested: (MotionCor2, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016499">SCR_016499</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Figures were prepared using Pymol (Schrödinger) and UCSF Chimera.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Pymol</b></div>
              <div>suggested: (PyMOL, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000305">SCR_000305</a>)</div>
            </div>
          </td></tr></table>
      


      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 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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.07.05.187344: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">COVID19, SARS-CoV-2 spike glycoprotein, N-glycopeptide, structure specific LC-MS/MS glycoprotein analysis, O-glycosylation Materials and Methods Materials Recombinant SARS-CoV-2 spike (R683A, R685A, His-tag) protein expressed in HEK 293 cell line was obtained from Acrobiosystems (Newark, DE, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK 293</div> <div>suggested: CLS Cat# 300192/p777_HEK293, CVCL_0045</div> </div> </td></tr></table>


      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 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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.07.10.197814: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All combos displayed decreased levels of ACE2 and antibody binding compared to S-2P control (Fig 2D, Fig. S6).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ACE2</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>S6</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For CR3022 the heavy and light chain were cloned into a single IgG1 expression vector to express a fully human IgG1 antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>human IgG1</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The acceptor beads were conjugated to an anti-His antibody (Cat#: AL128M, Perkin Elmer), which binds to the His-tag of the construct.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-His</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Acceptor HEK293 cells were transfected in 6-well plates (Corning) with ACE2, TMPRSS2 and the PEP86 subunit, or just the PEP86 subunit (‘Mock’) as negative control.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK293</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cryo-EM image processing Collected movies were imported into RELION-3.1-beta (Zivanov, Nakane et al. 2018) and subjected to beam induced drift correction using MotionCor2 (Zheng, Palovcak et al. 2017) and CTF estimation by CTFFIND-4.1.18 (Rohou and Grigorieff 2015).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>MotionCor2</b></div>
              <div>suggested: (MotionCor2, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016499">SCR_016499</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PHENIX-1.18.261 (Liebschner, Afonine et al. 2019), Coot (Emsley, Lohkamp et al. 2010) and the Namdinator webserver (Kidmose</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Coot</b></div>
              <div>suggested: (Coot, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014222">SCR_014222</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">When purified proteins were analyzed using SEC-MALS, µmMALS detectors were inline and data was analyzed using Astra 7.3 software package.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Astra</b></div>
              <div>suggested: (ASTRA, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016255">SCR_016255</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Binding of SAD-S35, ACE2 and CR3022 to purified combined S trimer variants measured with BioLayer Interferometry, showing the initial slope at the start of binding.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>BioLayer</b></div>
              <div>suggested: (Harvard Medical School Center for Macromolecular Interactions Core Facility, <a href="https://scicrunch.org/resources/Any/search?q=SCR_018270">SCR_018270</a>)</div>
            </div>
          </td></tr></table>
      


      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 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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. Imagine that instead of a dropdown containing the search results, you want a tag-like list of search results that always display:
    1. SciScore for 10.1101/2020.04.20.052126: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">Serum samples were collected from recovered patients at 3-6 months after discharge, with the patients’ written consent and the approval of the ethics review committee (23, 24).</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Apparently, each of mouse antisera had high cross-reactivity with the SARS-CoV-2 S and S2 proteins, but the cross-reactive antibodies specific for the SARS-CoV-2 S1 and RBD were hardly detected except the anti-S1 response in mouse M3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-S1</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Interestingly, both RBD proteins elicited antibodies highly reactive with both the SARS-CoV and SARS-CoV-2 antigens (Fig.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARS-CoV-2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Rabbit antibodies induced by SZ16-RBD but not GD03 can block RBD binding to 293T/ACE2 cells To validate the observed cross-reactive neutralization and explore the underlying mechanism, we purified anti-RBD antibodies from the rabbit antisera above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>antisera above.</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Next, we investigated whether the rabbit anti-RBD antibodies block RBD binding to 293T/ACE2 cells by flow cytometry.</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">Surprisingly, the antibodies purified from SZ16-RBD-immunized rabbits (R-1 and R-2) potently blocked the binding of both the RBD proteins, whereas the antibodies from GD03-RBD-immunized rabbits (R-3 and R-4) had no such blocking functionality except a high concentration of the rabbit R-3 antibody on the SARS-CoV RBD binding (Fig. 6).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>R-4</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>R-3</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For example, CR3022, a neutralizing antibody isolated from a convalescent SARS patient, cross-reacted with the RBD of SARS-CoV-2 but did not neutralize the virus (31, 32).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>CR3022</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Meaningfully, this work found that the RBD proteins derived from different SARS-CoV strains can elicit antibodies with unique functionalities: while the RBD from a palm civet SARS-CoV (SZ16) induced potent antibodies capable of blocking the RBD-receptor binding, the antibodies elicited by the RBD derived from a human strain (GD03) had no such effect despite their neutralizing activities.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>SZ16</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>GD03</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(A) Binding titers of purified rabbit anti-RBD antibodies to SARS-CoV (RBD) and SARS-CoV-2 (S, RBD, and S2) antigens were determined by ELISA.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>S2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A diluted horseradish peroxidase (HRP)-conjugated goat anti-human, mouse or rabbit IgG antibody was added for 1 hour at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-human,</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>rabbit IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washed with PBS two times, cells were incubated with a 1:500 dilution of Alexa Fluor® 488-labeled rabbit anti-His tag antibody (Cell Signaling Technology, Danvers, MA) for 30 min at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-His tag antibody (Cell Signaling Technology,</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Differently, it was reported that plasma from mice infected or immunized by SARS-CoV failed to neutralize SARS-CoV-2 infection in Vero E6 cells (28), and mouse antisera raised against the SARS-CoV RBD were even unable to bind to the S protein of SARS-CoV-2 (9).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero E6</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_XD71">CVCL_XD71</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">S1 to S3 Materials and Methods Recombinant S proteins Two RBD-Fc fusion proteins, which contain the RBD sequence of Himalayan palm civet SARS-CoV strain SZ16 (GenBank: AY304488.1) or the RBD sequence of human SARS-CoV strain GD03T0013 (GenBank: AY525636.1, denoted GD03) linked to the Fc domain of human IgG1, were expressed in transfected 293T cells and purified with protein A-Sepharose 4 Fast Flow in our laboratory as previously described (15).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T</b></div>
              <div>suggested: KCB Cat# KCB 200744YJ, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0063">CVCL_0063</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pseudovirus particles were prepared by co-transfecting HEK293T cells with a backbone plasmid (pNL4-3.luc.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK293T</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Binding activity of RBD proteins to 293T/ACE2 cells determined by flow cytometry.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T/ACE2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Like SARS-CoV and many other CoVs, SARS-CoV-2 utilizes its surface spike (S) glycoprotein to gain entry into host cells (4-6).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>SARS-CoV-2</b></div>
              <div>suggested: (Sino Biological Cat# 40143-R019, <a href="https://scicrunch.org/resources/Any/search?q=AB_2827973">AB_2827973</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">bioRxiv preprint doi: https://doi.org/10.1101/2020.03.15.993097. 29. S. Jiang, C. Hillyer, L. Du, Neutralizing Antibodies against SARS-CoV-2 and Other Human Coronaviruses.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>bioRxiv</b></div>
              <div>suggested: (bioRxiv, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003933">SCR_003933</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis Statistical analyses were carried out using GraphPad Prism 7 Software.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GraphPad Prism</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
          </td></tr></table>
      


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

      Results from 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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.04.02.021469: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Monoclonal antibody against FLAG tag ( ANTI-FLAG M2 ) and β-Actin were purchased from Sigma ( Cat.No. F1804 and A2228 , respectively)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>FLAG</div> <div>suggested: (Sigma-Aldrich Cat# F1804, AB_262044)</div> </div>

            <div style="margin-bottom:8px">
              <div><b>β-Actin</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>A2228</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Monoclonal antibody against human IFITM1 ( Cat.No. 60047-1) , rabbit polyclonal antibody against human IFITM3 ( Cat.No. 11714-1-AP) , which also efficiently recognizes IFITM2 and weakly cross-reacts with IFITM1 , were purchased from Proteintech Group , Inc .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>IFITM1</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>human IFITM3</b></div>
              <div>suggested: (Proteintech Cat# 11714-1-AP, <a href="https://scicrunch.org/resources/Any/search?q=AB_2295684">AB_2295684</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse monoclonal antibody against HCoV-OC43 nucleocapsid ( NP ) protein was purchased from Millipore ( Cat.No. MAB9012)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HCoV-OC43 nucleocapsid ( NP ) protein</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>HCoV-OC43 nucleocapsid ( NP</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After permeabilization with 0.1 % Triton X-100 , the cells were stained with a monoclonal antibody ( 541-8F ) recognizing HCoV-OC43 NP protein .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HCoV-OC43 NP protein .</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The bound antibodies were visualized by using Alexa Fluor 488-labeled ( green ) goat anti-mouse IgG or Alexa Fluor 555-labeled ( red ) goat anti-mouse IgG , Cell nuclei were counterstained with DAPI .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-OC43.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK 293</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoVOC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>A549</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infected C3A cultures also produced approximately 20-fold more progeny viruses than HepG2 cultures did ( Fig . 1D) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HepG2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In order to identify host cellular proteins that may enhance HCoV-OC43 infection of C3A cells or suppress the virus entry into HepG2 cells , we first compared the expression of several cellular genes with known activity to restrict or enhance virus entry into target cells .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>C3A</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_T437">CVCL_T437</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">While the expression of ADAP2 and GILT did not inhibit HCoVOC43pp infection ( Fig . 4B) , expression of LY6E in Flp-In TREx 293 cells efficiently suppressed the infection of lentiviral particles pseudotyped with the envelope glycoproteins of all the human CoVs including SARS-CoV-2 , except for SARS-CoV ( Fig . 4C and 4D) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Flp-In TREx 293</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_U427">CVCL_U427</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Particularly , enhancement of viral infection in Huh7.5 cells that do not have basal levels of IFITM protein expression indicates that LY6E enhancement of RNA viral infection is most likely not through modulating the function of IFITM proteins ( 42) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Huh7.5</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_7927">CVCL_7927</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Third , studying the effects of LY6E on HIV-1 infection of CD4 low-expressing cells , such as Jurkat T cells and primary monocyte-derived macrophages , revealed that HIV-1 entry was inhibited by LY6E ( 46) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Jurkat</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">GP2-293 and Lenti-X 293T cell Lines were purchased from Clontech and cultured in DMEM supplemented with 10 % FBS and 1 mM Sodium pyruvate ( Invitrogen)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HCoV-OC43 ( strain VR1558 ) were purchased from ATCC and amplified in HCT-8 cells according to the instruction from ATCC .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HCT-8</b></div>
              <div>suggested: KCLB Cat# 10244, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_2478">CVCL_2478</a></div>
            </div>
          </td></tr></table>
      


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

      Results from 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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.03.29.013490: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 primary anti-spike rabbit monoclonal antibody (40150-R007, Sino Biological, 1:100) and a goat anti-rabbit secondary antibody (Alexa Fluor 488, Invitrogen, Thermo Fisher Scientific) were employed to stain 2019-nCoV S1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-spike</div> <div>suggested: (Sino Biological Cat# 40150-R007, AB_2827979)</div> </div>

            <div style="margin-bottom:8px">
              <div><b>anti-rabbit</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>S1</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A primary anti-His-tag mouse monoclonal antibody (AF5060, Beyotime, 1:1000) and a goat anti-mouse secondary antibody (A0216, Beyotime, 1:2000) were employed to detect S1.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-His-tag</b></div>
              <div>suggested: (AnaSpec; EGT Group Cat# 29673-1000, <a href="https://scicrunch.org/resources/Any/search?q=AB_11232932">AB_11232932</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-mouse</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">β-actin determined by a mouse monoclonal antibody (AA128, Beyotime, 1:1000) was used as a reference.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>AA128</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The purified S1 expressed by human HEK293 cells intensively bound ACE2 with an affinity of 2.68 nM (Figure 1D).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK293</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">However , S1 pretreated with HD5 was still efficient to contact Caco-2 , as revealed by either immunoblotting or immunofluorescence.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Caco-2</b></div>
              <div>suggested: CLS Cat# 300137/p1665_CaCo-2, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0025">CVCL_0025</a></div>
            </div>
          </td></tr></table>
      


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

      Results from 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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.03.26.010165: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">Immunizations and handling of the 349 llama were performed according to directive 2010/63/EU of the European parliament for the 350 protection of animals used for scientific purposes and approved by the Ethical Committee for 351 Animal Experiments of the Vrije Universiteit Brussel ( permit No. 13-601-1) .</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">384 385 Periplasmic ELISA screen to select MERS- and SARS-CoV directed VHHs 386 After panning , 45 individual colonies of phage infected bacteria isolated after the first panning 387 round on MERS-CoV S-2P or SARS-CoV-1 S-2P protein and 45 individual colonies isolated after 388 the second panning round on MERS-CoV S-2P or SARS-CoV-1 S-2P protein were randomly 389 selected for further analysis by ELISA for the presence of MERS-CoV and SARS-CoV-1 specific 390 VHHs , respectively .</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 epitope of MERS VHH-55 overlaps with the 285 epitopes of several of these MERS-CoV RBD-directed antibodies including C2 , MCA1 , m336 , 286 JC57-14 ,</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>C2</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>MCA1</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">378 Phages displaying SARS-CoV-1 directed VHHs were enriched after 2 rounds of 379 biopanning on 20 μg of SARS-CoV-1 S-2P protein captured with an anti-foldon antibody 380 ( generously provided by Dr. Vicente Mas ) in one well of a microtiter plate ( type II , F96 Maxisorp , 381 Nuc) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-foldon</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Binding was detected by 478 incubating the plates sequentially with either mouse anti-Histidine Tag antibody ( MCA1396 , Abd 479 Serotec ) followed horseradish peroxidase ( HRP)-linked anti-mouse IgG ( 1/2000 , NXA931 , GE 480 Healthcare ) or Streptavidin-HRP ( 554066 , BD Biosciences ) or by an HRP-linked rabbit anti- 481 camelid VHH monoclonal antibody ( A01861-200 , GenScript) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-Histidine Tag</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>MCA1396</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG</b></div>
              <div>suggested: (GE Healthcare Cat# NXA931, <a href="https://scicrunch.org/resources/Any/search?q=AB_772209">AB_772209</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG ( 1/2000 , NXA931 , GE 480 Healthcare )</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>Streptavidin-HRP</b></div>
              <div>suggested: (Cell Signaling Technology Cat# 3999, <a href="https://scicrunch.org/resources/Any/search?q=AB_10830897">AB_10830897</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Binding of the VHH-72-Fc and VHH-72-Fc 624 (S) to cells was determined by an AF633 conjugated goat anti-human IgG antibody and binding 625 to SARS-CoV-1 or SARS-CoV-2 S was calculated as the mean AF633 fluorescence intensity 626 (MFI) of GFP expressing cells (GFP+) divided by the MFI of GFP negative cells (GFP-).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-human IgG</b></div>
              <div>suggested: (R and D Systems Cat# AF633, <a href="https://scicrunch.org/resources/Any/search?q=AB_355491">AB_355491</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>SARS-CoV-1</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>GFP-</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These bivalent SARS VHH-72 constructs were 257 able to bind to both prefusion SARS-CoV-1 S and SARS-CoV-2 RBD-SD1 as demonstrated by 258 ELISA and by a dose-dependent reduction in the binding of SARS-CoV-2 RBD-SD1 to Vero E6 259 cells ( Figure 6C-D and S. Figure 6B-C) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero E6 259</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Supernatants of HEK 293S cells transiently transfected with 262 VHH-72-Fc exhibited neutralizing activity against both SARS-CoV-1 and SARS-CoV-2 S VSV 263 pseudoviruses in the same assay which showed no such cross-reactive neutralization for 264 monovalent SARS VHH-72 ( Figure 6E-F) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK 293S</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_A784">CVCL_A784</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly , pseudoviruses expressing spike genes for MERS-CoV England1 490 ( GenBank ID: AFY13307 ) and SARS-CoV-1 Urbani ( GenBank ID: AAP13441.1 ) were 491 produced by co-transfection of plasmids encoding a luciferase reporter , lentivirus backbone , and 492 spike genes in 293T cells ( Wang et al. , 2015) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T</b></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 VHHs were mixed with 493 pseudoviruses , incubated for 30 min at room temperature , and then added to previously-plated 494 Huh7.5 cells .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Huh7.5</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_7927">CVCL_7927</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the VSV pseudotype neutralization 501 experiments , the pseudoviruses were incubated for 30 min at 37 °C with different dilutions of 502 purified VHHs or with dilution series of culture supernatant of 293S cells that had been 503 transfected with plasmids coding for SARS VHH-72 fused to human IgG1 Fc ( VHH-72-Fc ) or 504 with GFP-binding protein ( GBP: a VHH specific for GFP) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293S</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_A784">CVCL_A784</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Supernatant of 612 non-transfected and VHH-72-Fc transfected HEK293-S cells was applied in a three-fold dilution 613 series in kinetics buffer.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK293-S</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_A784">CVCL_A784</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK 622 293S culture media (1/20 diluted in PBS + 0.5%BSA) of VHH-72-Fc and VHH-72-Fc (S) 623 transformants were incubated with transfected cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK 622</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">632 Vero E6 cells grown at sub-confluency were detached by cell dissociation buffer (Sigma) and 633 trypsin treatment.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Vero E6</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_XD71">CVCL_XD71</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Curve fitting was performed using nonlinear 485 regression ( Graphpad 7.0) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Graphpad</b></div>
              <div>suggested: (GraphPad, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000306">SCR_000306</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting molecular 594 replacement solutions were iteratively rebuilt and refined using Coot, ISOLDE and Phenix 595 (Adams et al., 2002; Croll, 2018; Emsley and Cowtan, 2004).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Coot</b></div>
              <div>suggested: (Coot, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014222">SCR_014222</a>)</div>
            </div>
          </td></tr></table>
      


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

      Results from 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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.03.26.010694: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">In the DMSO group, a typical morphology of stem cells with high nucleus/cytoplasm ratio and close cell membrane contacts was observed, while iPSC-nCoVN after a 7-day induction started to exhibit endothelial cell morphological features and lower nucleus/cytoplasm ratio.</td></tr></table>

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These primary antibodies were [ target , dilution , species , company , product number]: Troponin T Cardiac Isoform , 1:100 , mouse , Thermo Fisher , MA5-12960; alpha-smooth muscle actin , 1:100 , mouse , Bioss , bsm-33187M; S100A4 , 1:100 , rabbit , Bioss , bs-3759R; SSEA4 , 1:250 , mouse , Invitrogen , 14-8843-80;</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>MA5-12960; alpha-smooth muscle actin</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>SSEA4</b></div>
              <div>suggested: (Thermo Fisher Scientific Cat# 14-8843-80, <a href="https://scicrunch.org/resources/Any/search?q=AB_657847">AB_657847</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were washed three times with PBS containing 0.1% Triton X-100, then incubated with the Alexa Fluor 488 goat anti-mouse or Alexa Fluor 555 goat anti-rabbit IgG secondary antibodies at 37°C for 1 hour.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-mouse</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-rabbit IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The nCoVN protein was detected by using an anti-6× His Tag antibody in cells from the Dox group (Supplementary Figure 1).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-6×</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibodies against fibroblast markers vimentin, alpha-smooth muscle actin (α-SMA) and S100A4 were used to verify the cell type of these fibroblast-like cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>antibodies against fibroblast markers vimentin, alpha-smooth muscle actin</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>α-SMA</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>S100A4</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Comparing with the transcriptome of the pluripotent stem cells and fibroblast in the ENCODE project (Consortium, 2012; Davis et al., 2018), iPSC-GFP samples were clustered with H7 and GM23338, which were the embryonic stem cells (ESC) and iPSC, respectively; while iPSC-nCoVN samples were clustered with multiple kinds of fibroblast (Figure 4G).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GM23338</b></div>
              <div>suggested: Coriell Cat# GM23338, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_F182">CVCL_F182</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data were analyzed and plotted using GraphPad Prism 6 . 2.5 Immunofluorescence Staining Cells were fixed with 4 % paraformaldehyde at room temperature for 20 minutes and washed three times with 1× PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GraphPad Prism</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Images were obtained by using the DMi6000 B inverted ′ μ microscope (Leica) or the FV1000 confocal laser scanning microscope (Olympus), then were analyzed by using ImageJ software.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>ImageJ</b></div>
              <div>suggested: (ImageJ, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003070">SCR_003070</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We employed Kallisto (v0.46.0) (Bray et al., 2016) to determine the read count for each transcript and quantified transcript abundance as transcripts per kilobase per million reads mapped (TPM), using gene annotation in the GENCODE database (v32, GRCh38) (Frankish et al., 2019).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Kallisto</b></div>
              <div>suggested: (kallisto, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016582">SCR_016582</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>GENCODE</b></div>
              <div>suggested: (GENCODE, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014966">SCR_014966</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">DESeq2 (v1.26.0) (Love et al., 2014) was used to identify differentially expressed genes (DEGs) (false discovery rate (FDR) <0.05 and abs(log2FoldChange) >3).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>DESeq2</b></div>
              <div>suggested: (DESeq, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000154">SCR_000154</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pathway analysis was performed by ToppGene Suite (Chen et al., 2009).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>ToppGene</b></div>
              <div>suggested: ( ToppGene Suite , <a href="https://scicrunch.org/resources/Any/search?q=SCR_005726">SCR_005726</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Next, we used RNA-seq data from the ENCODE project to evaluate the cell type of iPSC-nCoVN.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>ENCODE</b></div>
              <div>suggested: (Encode, <a href="https://scicrunch.org/resources/Any/search?q=SCR_015482">SCR_015482</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(A) Expression values of ACE2 and GAPDH derived from the Gene Expression Omnibus database.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Gene Expression Omnibus</b></div>
              <div>suggested: (Gene Expression Omnibus (GEO), <a href="https://scicrunch.org/resources/Any/search?q=SCR_005012">SCR_005012</a>)</div>
            </div>
          </td></tr></table>
      


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

      Results from 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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.03.20.20039495: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">Patients and samples The Institutional Ethics Review Committee of Foshan Fourth Hospital , Foshan , China approved this study , and written informed consent was obtained from each patient .</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Indepth analysis showed that the level of S1 IgG positively correlate to age and the level of LDH (lactate dehydrogenase), especially for women, while the level of S1 IgG negatively correlate to Ly% (Lymphocyte percentage).</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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Besides protein N and S1, significant antibody responses to ORF9b and NSP5 were also identified.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>S1</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The anti-6xHis antibody results showed that most of the proteins were nicely immobilized , and the microarray quality if fairly good.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-6xHis</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Since the Fc tag could be recognized by fluorescence labeled anti-human IgG antibody , the ACE2-Fc generated high signals for all the tests , which could serve as control for the anti-human IgG antibody , though the initial reason to include ACE2 on the microarray is for applications other than serum profiling .</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">The purified proteins were analyzed by SDS-PAGE followed by Western blotting using an anti-His antibody ( Merck millipore , USA ) and Coomassie brilliant blue staining .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-His</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein microarray fabrication The proteins , along with negative ( BSA ) and positive controls ( anti-Human IgG and IgM antibody) , were printed in quadruplicate on PATH substrate slide ( Grace Bio-Labs , Oregon , USA ) to generate identical arrays in a 2 x 7 subarray format using Super Marathon printer ( Arrayjet , UK)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>IgM</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The arrays were washed with PBST and bound autoantibodies were detected by incubating with Cy3-conjugated goat anti-human IgG and Alexa Fluor 647-conjugated donkey anti-human IgM ( Jackson ImmunoResearch , PA , USA) , the antibodies were diluted 1: 1,000 in PBST , and incubated at room temperature for 1 h .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-human IgM</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Significant antibody responses were identified for ORF9b and NSP5.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>NSP5</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">It is known that there are 23 N-glycosylation sites on S protein, which is heavily glycosylated, and the glycosylation may play critical roles in antibody- antigen recognition5,41.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>antigen recognition5,41</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 NSP5 is also highly homologous to SARS NSP5 (96% identity, 98% similarity).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>COVID-19 NSP5</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pearson correlation coefficient between two proteins or indicators and the corresponding p value was calculated by SPSS software under the default parameters.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SPSS</div> <div>suggested: (SPSS, SCR_002865)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cluster analysis was performed by pheatmap package in R30.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>pheatmap</div> <div>suggested: (pheatmap, SCR_016418)</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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.03.25.006569: (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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 report name, abbreviation, NCBI code and bp size for each virus.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>NCBI</div> <div>suggested: (NCBI, SCR_006472)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To evaluate scatter of dots from theoretical Wright’s curve, we calculate the module of distance and we drawn box plot calculated with an in-house Python script. 4.7.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Python</div> <div>suggested: (IPython, SCR_001658)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In this case, the three stop codons (TAA, TAG, or TGA) and the three codons for isoleucine (ATT, ATC, and ATA) were excluded in calculation of GC3, and two single codons for methionine (ATG) and tryptophan (TGG) were excluded in all three (GC1, GC2, GC3) (Sueoka 1988).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ATC</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The protein sequences were aligned using Biopython.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Biopython</div> <div>suggested: (Biopython, SCR_007173)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting multiple sequence alignment was used to build a phylogenetic tree by employing a maximum-likelihood (ML) method implemented in the software package MEGA version 10. 1 [15].</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>MEGA</div> <div>suggested: (Mega BLAST, SCR_011920)</div> </div> </td></tr></table>


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


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.03.26.20042184: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">Serum samples for assay The protocols were approved by the institutional ethical committee of Beijing You An Hospital , Capital Medical University</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">As shown in Fig . 2B , on day 16 , both monkeys developed very strong immune reactivity against S1-His , with titers at 1:200 for the male monkey and 1:800 for the female one.</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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Here we report the development and validation of a COVID19/SARS-CoV-2 S1 serology ELISA kit for the detection of total anti-virus antibody (IgG+IgM) titers in sera from either the general population or patients suspected to be infected.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-virus</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For indirect ELISA, CHO-expressed recombinant full length SARS-CoV-2-S1 protein with 6*His tag was used as the coating antigen to capture the SARS-CoV-2-S1 antibodies specifically.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARS-CoV-2-S1</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">COVID-19 , Serological assay for SARS-CoV2 antibodies .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARS-CoV2</div> <div>suggested: (Abcam Cat# ab273074, AB_2847846)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In the initial test , 2 of the 15 samples from Beijing’s group were tested antibody-positive ( Table 1) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>antibody-positive ( Table 1</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In another group of study , shown in Fig . 4D , 23 out of 24 clinic samples were tested positive for SARS-CoV-2 antibodies .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARS-CoV-2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">More works will be carried out to exam the levels of IgG and IgM o those positive samples respectively by simply changing the goat-anti-human IgG ( H+L ) secondary antibodies , which will detect both IgG and IgM , to goat-anti-human IgG Fc specific secondary antibodies and mouse anti-human µ Chain-specific mAb .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>IgG</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>IgM</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK 293F cells and CHO cells and culture media were provided by Zhenge Biotech . , Shanghai , China .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK 293F</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_6642">CVCL_6642</a></div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>CHO</b></div>
              <div>suggested: CLS Cat# 603479/p746_CHO, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0213">CVCL_0213</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Using our patented technology , the transient expression levels of S1-His and S1-Fc in either CHO cells or 293F cells reached 30-72mg/L ( Table S1) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293F</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein expression and purification The full length SRARS-CoV-2 S1 gene ( GenBank: QIC53204.1 ) with C terminal were synthesized by GENEWIZ , China , and inserted into mammalian cell expression vectors with either 6*His tag or fused with human IgG Fc .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GENEWIZ</b></div>
              <div>suggested: (GENEWIZ, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003177">SCR_003177</a>)</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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.03.15.991844: (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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">100μl of HRP anti-His Tag Antibody ( BioLegend , USA ) (</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-His</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These two constructs were transfected into HEK293 cells using polyethyleneimine , respectively .</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, CVCL_0045</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">However, polymorphism and divergence analysis by DnaSP6 (version 6.12.03) (15) showed that the RBD sequences were as diverse as the other regions of the S protein (Fig. 2).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>DnaSP6</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Molecular dynamics simulation was performed using GROMACS 2019 with the following options and parameters: explicit solvent model , system temperature 37°C , OPLS/AA all-atoms force field , LINCS restraints .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GROMACS</div> <div>suggested: (GROMACS, SCR_014565)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Funding statement: This work was supported by grants from the National Key Research and Development Program of China ( 2017YFA0505001/2018YFC0910200/2018YFE0204503) , the National Natural Science Foundation of China ( 81730061) , the Guangdong Key Research and Development Program ( 2019B020226001) , the Natural Science Foundation of Guangdong Province ( 2018B030312010) , and the Guangzhou Healthcare Collaborative Innovation Major Project ( 201803040004 and 201803040007) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Guangzhou Healthcare</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Isolation and Characterization of 2019-nCoV-like Coronavirus from Malayan Pangolins . bioRxiv 2020.02.17.951335 . 9 . Hoffmann M , Kleine-Weber H , Schroeder S , Krüger N , Herrler T , Erichsen S , Schiergens TS , Herrler G , Wu N-H , Nitsche A , Müller MA , Drosten C , Pöhlmann S. 2020 .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>bioRxiv</div> <div>suggested: (bioRxiv, SCR_003933)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">DnaSP 6: DNA sequence polymorphism analysis of large data sets .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>DnaSP</div> <div>suggested: (DnaSP, SCR_003067)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>MAFFT</div> <div>suggested: (MAFFT, SCR_011811)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The electrostatic surface charges of the ACE2 are calculated using Pymol , with the charge unit KbT/ec , as noted in the Pymol manual.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Pymol</div> <div>suggested: (PyMOL, SCR_000305)</div> </div> </td></tr></table>


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

      Results from 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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.03.01.971499: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">The study was approved by the Bioethical Committee of the Medical University of Silesia in Katowice , Poland ( approval no: KNW/0022/KB1/17/10 dated 16.02.2010) .</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Due to the lack of KLK13 specific antibodies , we verified its presence based on RT-PCR ( Fig . 4A) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>KLK13</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A mouse monoclonal anti-TMPRSS2 antibody ( clone P5H9-A3; 1:500 dilution; Sigma-Aldrich , Poland) , followed by incubation with a horseradish peroxidase-labeled anti-mouse IgG ( 65 ng/ml; Dako , Denmark ) diluted in 5 % skimmed milk / TBS-Tween ( 0.1 % ) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-TMPRSS2</div> <div>suggested: (Santa Cruz Biotechnology Cat# sc-101847, AB_2205599)</div> </div>

            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A horseradish peroxidase-labeled anti-His tag antibody ( 1:25000 dilution; Sigma-Aldrich , Poland ) diluted in 5 % skimmed milk / TBS-Tween ( 0.1 % ) was used to detect the His-tagged HmuY proteins .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-His tag antibody</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>anti-His tag</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Subsequently , we transduced RD_ctrl , RD_KLK13 and RD_TMPRSS2 cells with HIV particles pseudotyped with HCoV-HKU1 S glycoprotein ( S-HKU1) , control VSV G protein ( VSV-G ) or lacking the fusion protein ( ΔEnv) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>RD_TMPRSS2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This may be one of the factors limiting the HCoV-HKU1 replication in RD_KLK13 cells , as only minimal replication is observable.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>RD_KLK13</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RD cells grown in 90 % confluency were infected with HCoV-HKU1 ( 108 RNA copies per ml ) in Dulbecco’s MEM ( Thermo Fisher Scientific , Poland</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>RD</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentivirus production and transduction 293T cells were seeded on 10 cm2 dishes , cultured for 24 h at 37°C with 5 % CO2 and transfected with psPAX , pMD2G and third transfer plasmid ( pWPI/KLK13 , pLKO.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr></table>
      


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

      Results from 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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.02.16.951723: (What is this?)

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

      Table 1: Rigor

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

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Due to the relatively low homology of the spike RBDs between SARS-CoV-2 and SARS-CoV , it is of significant interest to investigate whether SARS-CoV neutralizing antibodies possess cross-reactivity to SARS-CoV-2 .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARS-CoV</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two SARS-CoV S-protein rabbit polyclonal antibodies ( Table 1 ) and four monoclonal antibodies ( Table 2 ) were analyzed for cross-reactivity to SARS-CoV-2 S1 protein and cross-neutralizing activities to SARS-CoV-2 PSV .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>S-protein</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Polyclonal nAbs characteristics Antibody ( Polyclonal ) Pseudovirus Neutralization S1 Protein Binding IC50 ( μg/mL ) EC50 ( ng/mL ) SARS-CoV-2 SARS-CoV SARS-CoV-2 SARS-CoV RP01 137 1.1 130 21.5 T52 50.4 0.08 113.6 7.9</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>EC50</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Since SARS-CoV-2 and SARS-CoV are both coronaviruses with over 70 % sequence homology and share the same human receptor ACE2 , analyzing SARS-CoV’s antibodies’ cross-reactivity to SARS-CoV-2 may provide useful information on whether neutralizing epitopes were conserved on the two coronaviruses .</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">Hence it is possible but maybe challenging to identify antibodies with potent neutralizing activities to both SARS-CoV and SARS-CoV-2 , and ideally to their mutant virus strains as well .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARS-CoV-2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, transfection reagent Sinofection ( Cat: STF02) , mammalian expression plasmids of full length S or RBD protein of SARS-CoV-2 ( Cat: VG40589-UT , Wuhan/IVDC-HB-01/2019 ) and SARS-CoV ( Cat: VG40150-G-N , CUHK-W1) , ACE2 ( Cat: HG10108-UT) , polyclonal antibodies against SARS-CoV RP01 ( Cat: 40150-RP01 ) and T52 ( Cat: 40150-T52 ) were purchased from Sino Biological.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>RP01</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>T52</b></div>
              <div>suggested: (Rockland Cat# 600-401-W94, <a href="https://scicrunch.org/resources/Any/search?q=AB_2614544">AB_2614544</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV neutralizing antibodies were generated from mice ( M103 , M127 ) or rabbits ( R314 , R301 , R325 , R302 , R258 , R348 ) immunized with recombinant S1 protein of SARS-CoV .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>R302</b></div>
              <div>suggested: (BioPorto Diagnostics Cat# MON R-3-02, <a href="https://scicrunch.org/resources/Any/search?q=AB_1072801">AB_1072801</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing away the unbound proteins , cells were incubated withPE labelled anti-his-tag antibody for 20 min and went through flow cytometer for detection of cellular binding .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-his-tag</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then 100μL/well of the antibody-PSV mixture was added onto the 293T/ACE2 cell wells and incubated for 37℃ , 5 % CO2 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>antibody-PSV</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines The human embryonic kidney 293T cell line ( Cat:CRL-11268 ) used for pseudovirus ( PSV ) packaging were purchased from ATCC .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T</b></div>
              <div>suggested: ATCC Cat# CRL-11268, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_1926">CVCL_1926</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Both 293T and 293T-ACE2 cells were grown in Dulbecco’s modified Eagle’s medium ( DMEM ) containing 10 % ( v/v ) FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T-ACE2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then 100μL/well of the antibody-PSV mixture was added onto the 293T/ACE2 cell wells and incubated for 37℃ , 5 % CO2 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293T/ACE2</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flowjo and Graphpad softwares were used for data analysis .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Flowjo</b></div>
              <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>Graphpad</b></div>
              <div>suggested: (GraphPad, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000306">SCR_000306</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Software PyMol was used for preparing structural figures39 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>PyMol</b></div>
              <div>suggested: (PyMOL, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000305">SCR_000305</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Alignment of 111 SARS-CoV RBD sequence , used for the generation of sequence conservation , was collected by BLAST via NCBI website34 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>BLAST</b></div>
              <div>suggested: (BLASTX, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001653">SCR_001653</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>NCBI</b></div>
              <div>suggested: (NCBI, <a href="https://scicrunch.org/resources/Any/search?q=SCR_006472">SCR_006472</a>)</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).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

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

      Learn more at Review Commons


      Reply to the reviewers

      We would like to thank Reviewer #1 and #2 for the evaluation of our research and comments to our manuscript. Their comments are highly appreciated and addressed as described below.

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

      **Summary:**

      *Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).*

      Here Ha et al. has further developed their Pumilio RNA tagging methodology for the isolation of UV-crosslinked proteins that are suggested to associate with Xist RNA in mouse embryonic stem cells (mESCs). Within this study the authors claim to have found the Lupus antigen RNA binding protein (La) as a novel Xist interacting partner that influences the efficacy of X-chromosome inactivation (XCI). The authors use a number of different techniques such as qPCR, fluorescent imaging, ATAC-SEQ and SHAPE to show aberration of XCI upon La shRNA knockdown. However, this study has significant flaws in the efficient isolation and validation of Xist associated proteins using their FLAG-out methodology. Furthermore, later experiments predominantly focus on cell death/survival assays, which is somewhat troubling given the essential roles La plays in processes such as cell differentiation and proliferation, ribosome biogenesis, transcriptional control and tRNA maturation. I feel the authors need to robustly address the potential effects La knockdown may be having on their mESCs.

      Reviewer #1 did not fully understand the basic designs of the experimental systems (FLAG-out and iXist), and completely rejected these experimental systems. Reviewer #1 also ignored the majority of the functional analysis on the candidate protein, Ssb. These issues cannot be addressed by additional experiments

      **Major comments:**

      *-Are the key conclusions convincing?*

      My major concern is in their Xist RNA purification.

      First of all, I couldn't find any data on proving the enrichment of Xist RNA itself in their Pumilio pull-down experiment. It would have been useful to show Xist RNA enrichment before benzonase step. Secondly, it is hard to imagine the protocol would successfully isolated Xist RNA-protein complexes from the cell. An earlier report by Clemson et al., (J Cell Biol., 1996) has shown that majority of Xist RNA is still stuck in the nucleus after nuclear matrix prep protocol using detergent, which is not so different from the authors' protocol. Moreover, the authors used UV crosslink, which would have made even harder to purify Xist RNA without sonication. Thirdly, as the tag is located on 5' of Xist RNA, it is rather surprising to see that Spen is not detected in their pulldown. Spen is one of the main functional interactors with Xist, robustly detected by several previous reports. Similarly, other high-affinity binders of Xist such as hnRNP-K and Ciz1 were also lacking from this screen. Finally, the peptides found associated with FLAG-out Xist are extremely low in comparison with other data using glutaraldehyde or formaldehyde crosslinking. For example, HnRNP-M found in Chu et al 2015 has 1120 peptide counts in differentiated cells. The authors here use HnRNP-M as a baseline for specific interactions and show a total of 6 peptide counts in Xist expressing cells and 5 in i-Empty cells (Supplementary excel sheet 1). Similarly, the La protein of interest in this study has 8 counts in i-FLAG-Xist and 6 counts in i-Empty. I struggle to see how this result indicate specific Xist binding. Worryingly this is the starting rationale for the rest of their experiments, it is hard to therefore accept the rest of their conclusions either.

      We have detected Xist RNA after Pumilio pull-down, and added the data in the revised manuscript (Figure S1). The enrichment of Xist RNA by Pumilio pull-down is about 75-fold, comparable to the enrichment reported by Minajigi et al.

      Two out of three previous studies used similar protocols to prep cell lysates for co-IP, including UV cross-linking and detergent (McHugh et al. 2015 and Minajigi et al. 2015). The major difference between their protocols and ours is the co-IP step. They used antisense oligos to pull-down Xist RNA-protein complex, while we take advantage of the specific interaction between PUF and PBS to pull-down Xist RNA-protein complex. With the data in Figure S1, we are confident that our strategy is successful in isolating Xist RNA

      For systematic identification of Xist binding proteins, each method has its own strength and weakness. As we described in the introduction, only 4 proteins were commonly identified by all three studies to systematically identify Xist binding proteins. There is no doubt that our method also missed some authentic Xist binding proteins (false negative) and identified some false positive candidates. Thus, we have to be careful in balancing between the false negative and false positive calls. The reason that we applied the ranking gain to identify Xist binding protein candidates, is to minimize the false negative rate. Meanwhile, we compared our Xist binding protein candidate list with previous identified Xist-binding proteins to enhance the confidence in our candidate lists.

      Regardless the strength and weakness of our method, Ssb is also an Xist-binding protein identified by another study (Chu et al. 2015). More importantly, we have provided experimental validation to confirm Ssb’s involvement in XCI and extensive functional analysis to reveal the protein’s mechanistic role in XCI.

      The other key conclusion the authors make is from the use of numerous cell death/survival assays for both male and female cell lines. This is extremely troubling in the context of assessing their target protein La. La is involved in multiple RNA maturation events of rRNAs, tRNAs and other polIII transcripts. Furthermore, La has been implicated in binding to the mRNA for Cyclin D1 in both human cells and mouse fibroblasts (NIH/3T3 - male) which show a significant effect on cell proliferation upon siRNA knockdown https://www.nature.com/articles/onc2010425. This, along with the observation that La knock-out blastocysts fail to develop any mice or ES cell lines (male or female) show the effect observed in the authors results is most likely not X-linked cell death https://mcb.asm.org/content/mcb/26/4/1445.full.pdf. The authors need to show that their shRNA KD isn't affecting the proliferation and general fitness of their mESC lines.

      The cell death/survival assay was specially designed for analyzing the defect of XCI. The cell death of iXist ESCs upon adding Dox is due to the induction of Xist, which consequently initiates the silencing of the only X chromosome in male cells. Knockdown of genes involved in XCI compromises XCI, thus allowing cell survival. Given the diverse functions of Ssb in cell differentiation and proliferation, ribosome biogenesis, transcriptional control and tRNA maturation, one would expect slow growth and/or cell death of Ssb knockdown cells. Indeed, the result is consistent with our expectation (Figure 2C, without Dox). Nevertheless, more Ssb knockdown cells survive in the presence of Dox, compared with control cells (Figure 2C-E, with Dox), suggesting that Ssb plays an important role in XCI.

      *- Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?*

      As discussed above, I feel the authors have not clearly demonstrated Xist specific protein enrichment and haven't proven X-linked cell death. Due to the lack of necessary control experiments as discussed below, I feel the notion that La is involved directly in XCI as an RNA chaperone is currently preliminary/speculative.

      The FLAG-out experiment just provided an initial point for the study. We have demonstrated the interaction between Xist and Ssb by RIP. And, Ssb knockdown antagonizes the lethal effect of induced XCI in male cells, allowing more cell to survive. This is contradictory to the diverse house-keeping functions of Ssb, which should lead to slow proliferation or cell death. Therefore, the data here (Figure 2C-E) should suggest a role of Ssb in XCI. In addition, we showed that knockdown of Ssb compromises the silencing of X-linked genes (Figure 2F, 2G, and 3E), the compaction of X chromosome (Figure 3D), Xist cloud formation (Figure 4), epigenetic modifications on Xi (Figure 5), Xist RNA folding (Figure 6F-I), and Xist RNA stability (Figure 7C and D). All these data indicate that Ssb is involved in XCI by regulating Xist RNA folding.

      *- Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.*

      I would suggest them to show RT-qPCR results of Xist RNA enrichment from the sample after flagIP before benzonase treatment.

      We have the data, and added it to Figure S1.

      Also, it would have been more convincing if their negative control construct (i-Empty) would contain 25 copies of PBSb RNA at least.

      This is a good alternative design of the negative control. Using i-Empty expressing 25 copies of PBSb RNA will allow us subtract the background causing by proteins binding to PBSb RNA. Yet, as discussed above, regardless how we improve the experimental setting, we cannot completely avoid the issue of false positive and false negative. Our goal of the FLAG-out experiment is to generate a list of Xist binding protein candidates, and their binding to Xist and their functions in XCI should be validated by additional experiments. With our current experimental setting, a list of Xist binding protein candidates has been generated, and we have validated the role of Ssb in XCI with subsequent experiments.

      In Fig1b, the total amount of proteins loaded on the gel is not equivalent between two lanes. The gel should show equivalent amounts of proteins on the gel. It looks like if the negative control sample had been loaded at the same amount as the one with Xist, the band pattern wouldn't be distinguishable between the two samples. Furthermore, as these samples were used in the following mass spectrometry screen it may suggest that the minimal increase in peptide counts observed in the iXist FLAG-out were due to an increased amount of sample being loaded? No controls are conducted to account for this.

      IP samples of i-Empty and i-FLAG-Xist were loaded in the gel in Figure 1b. It is expected that IP sample of i-FLAG-Xist should pull down more proteins than IP samples of i-Empty. The FLAG-PUFb bands (the strongest band in each lane) are about the same amount in two samples, indicating roughly equal amount of loading. After normalization of gel loading according to the FLAG-PUFb bands, the upper part of the i-FLAG-Xist lane showed some unique bands.

      For mass spectrometry analysis, the loading of two samples are independent, therefore, to compare the absolute amount of each protein between the two samples does not always provide valuable information. Yet, the relative amount of different proteins within one sample is not affected by the loading amount, thus, more informative. Therefore, we used the ranking information to estimate the relative amount of different proteins in each sample and used the ranking gain to further identify protein candidates.

      The authors quantify cell death in figures 2C - E. It seems clear that shSsb 1 and 2 have an effect on cell count even in the absence of Dox. The rescue effect seen upon Dox addition is minimal when compared to Empty + Dox 2D. The authors ∆A-iXist line with and without Ssb KD/Dox would be an informative control on whether the increase in cell survival that they see is X-linked.

      As the reviewer pointed out earlier, Ssb plays multiple roles in cellular processes. Inevitably, KD of Ssb leads to slow growth and/or cell death with or without Dox. Thus, it is less meaningful to compare the surviving cell counts in Figure 2D. Rather, the survival rate (Figure 2E) reflects the rescuing effect more precisely. Shown in Figure 2E, both shSsb 1 and 2 increase the survival rate significantly, compared with Empty control.

      Moreover, the data in Figure 3B and C demonstrated that Ssb KD compromises the survival of female differentiating cells, but not the survival of male differentiating cells, also indicating a role of Ssb in XCI. With these experiments, it should be sufficient to conclude that Ssb KD affects X-linked cell death/survival in both iXist male ESCs and WT female differentiating cells

      The qPCR results used to validate silencing defects show minor changes in expression and also don't show significant silencing of X-linked genes sufficient for cell death. Could this be because only ~ 50 - 60% of Male iXist cells seem to be expressing in the movies and that this will have an effect on the observed qPCR results? Furthermore, it seems counterintuitive that expression in the Empty male cells increases in 48h compared to 14h. Is this due to cell death and positive selection of cells less able to silence their X-chromosome? How would these data look in the female XX line? How would the data look in a ∆A-iXist line in the presence and absence of shSsb/Dox?

      First, high-quality live-cell imaging can only be carried out for 2 hours with 2-min time interval. The movies are meant to show the onset of Xist RNA signals. Therefore, they were taken one hour after Dox treatment (figure legend of Figure 4B-D). After overnight Dox treatment, Xist clouds can be seen in majority of cells.

      Second, in Fig. 2F-G, we did not include uninduced iXist male ESCs. Therefore, it is impossible to judge whether induction of Xist in this male ESC line results in Xist-dependent silencing at 14 and 48 hr. However, in our previous publication (Li et al., JMB, 2018, 430: 2734-2746), it has been shown that Gpc4, Hprt, Mecp2, G418, and TomatoRed are silenced (4- to 16-fold reduction) at 24 and 48 hours after Dox induction.

      Third, the qRT-PCR results in 14 h and in 48 h are not normalized to the same internal control. Thus, they are not directly comparable.

      Confusingly, the male line in Fig 3C shows a drop in live cell count at day 6 of differentiation? Surely given their previous results in Fig 2 the Ssb KD should increase cell viability with +Dox? Ssb KD seems to have an adverse effect on ES cells during extended differentiation protocols. In Figure S1 the authors show ~ 8 - 10% survival of male lines during differentiation. Could the recombination of the Xist sequence around the loxP sites enable the cells to outcompete the dead cells? How would iEmpty and ∆A-iXist cells compare here? Have the differentiated cells been tested for their expression of Xist? Additionally, how are there similar live cell counts for male vs female lines when ~90% of male cells die during differentiation? Were more cells plated at day 4? If so, this would bias the competition of male cell survival and therefore make the male line an inappropriate control.

      Given the essential role of La during development a control is needed to prove that this death is X-linked in the female 3F1 line. For example, an XO cell line retaining the Cast allele and shSsb expression could show the amount of death caused from shSsb alone independent of X-linked cell death.

      The reviewer completely misunderstood the experiment. The severe cell death specifically observed in female differentiating ESCs is a strong evidence showing Ssb is involved in XCI (Figure 3).

      The male ESCs in Figure 3C is a WT ESC line without the inducible Xist transgene, in which no XCI occurs upon differentiation. It is completely different from iXist male ESCs with Dox, in which forced Xist induction leads to XCI. Thus, the diverse functions of Ssb might contribute to the slight decrease in live cell count of wild type male cells at day 6 of differentiation.

      Figure S2 shows the differentiation of iXist male ESCs with or without Dox. As explained above, forced Xist induction silences the only X chromosome in male cells, resulting in cell death. In addition, XCI occurs more efficiently in differentiation condition (Figure S2) than in pluripotent status (Figure 2C)

      During differentiation, female ESCs silence one X chromosome, and the other X chromosome remains active. KD of Ssb compromises XCI, and two X chromosomes in some female differentiating cells maintain active, leading to cell death. The reviewer is correct that we need a control to rule out that the essential role of Ssb during development affects cell survival and death. An XO cell line can be used as a control. Similarly, a male cell line (XY) is also a good control. We already included a male cell line as a control in Figure 3B and 3C.

      If I understood correctly, the RNA FISH used dsDNA probes ("Sx9") against 40 kb of the X-inactivation centre (Xic). Surely Tsix or other Xic transcripts will also be visible? Can the authors use their RNA FISH to determine the XX or XO status of their cells? In Figure S5 a number of cells appear to show a single pinpoint of transcription. This could either be low levels of Xist transcripts or Xic transcription from an XO line in which the 129 chromosome is missing. It would be best to solely quantify cells which have two x chromosomes and if a significant amount of X chromosomes have been kicked out, this should be discussed and controlled for.

      This is a valid concern, but this concern can be adequately addressed with the available data in the manuscript.

      First, if the female Ssb KD cell line is an “XO” cell line, in which the X129 allele is “kicked out”, the RNA allelotyping results should show an absolute “silencing” of the X129 allele. However, in complete contrast to this notion, RNA allelotyping detected “more” RNA transcripts from X129, showing the chromosome-wide XCI defects (Figure 3D).

      Second, overexpression of Ssb in Ssb KD female cells restores the Xist clouds and the polycomb marks (Figure S8), suggesting that the Ssb KD female cells are XX, but not XO.

      Third, the severe cell death specifically occurred in female Ssb KD lines is also against the “XO” argument (Figure 3B&C).

      In Fig6, the authors generated a number of Ssb constructs for a rescue assay. However, these results complicate the matter and raise more questions than they address. It seems odd that the ∆RRM1 does not rescue based on comparison with their putative negative control, ∆NLS. However, the ∆RRM1 + 2 and ∆LAM do rescue the phenotype better than the full length Ssb? This makes no logical sense and highlights the inherent variation in cell viability these generated cell lines seem to show.

      Following on from this, figure S7 quantifies the GFP tag mRNA levels, depicting all ∆RRM mutants with expression below ~30%? How can ∆RRM1 or 2 be rescuing in this scenario? Have these lines been tested for their XX or XO status? The loss of an X chromosome would lead to a rescue of the cell death phenotype, which is a process known to occur in XX lines that have been cultured for extended periods of time. Could it also be that the cell lines derived are more or less sensitive to exogenous shRNA expression? Also, further validation is needed to assess the efficiency of KD in these lines as theoretically most of these constructs will be targeted by shRNA? What is the endogenous Ssb expression level in these lines? Where in the mRNA sequence are the shRNAs targeted to? Does this make sense on the relative expression levels of ∆RRM1/2 for example? Further testing of GFP expression could also be assessed by quantitative western blot of GFP or even visualised in their RNA FISH/IF samples (Figure S8), currently neither are shown. In addition, some kind of information of stability of each Ssb protein constructs has not been demonstrated.

      Our shRNA targets the LAM domain, so the expression of ∆LAM is not affected by the shRNA. The reviewer is correct that the detected GFP expression levels of ∆RRM1 and ∆RRM2 are too low to be conclusive. We have removed the data point of ∆RRM1 and ∆RRM2. Meanwhile, it is clear that ∆RRM1&2 has a better rescuing effect than ∆NLS, when ∆RRM1&2 and ∆NLS are expressed at similar levels. Ssb is a well known RNA chaperone/RNA helicase. Identifying Ssb is an Xist-binding protein already suggests the functional role of Ssb in XCI. The data of the plasmid rescue experiments further suggests that Ssb is involved in XCI as a RNA chaperone/RNA helicase.

      As for the Western blot and GFP fluorescence (IF), we have tried both. Neither of them detected GFP signal, reflecting the low expression level of these GFP fusion proteins. As the reviewers pointed out that the shSsb is not targeting the 5’ or 3’-UTR region, therefore, interfering the exogenous Ssb as well. This might be a reason for the low expression of these GFP fusion proteins.

      For the data shown in Figure 7A and B the authors quantify the % of cells with Xist signal. The authors have already shown a defect in Xist visualisation in Ssb KD. Surely it is plausible to assume a faster loss of Xist signal below background in weaker expressing cells. A more appropriate quantification would be the % loss of Xist signal per cell over time.

      With Figure 7C and D, the samples have been treated with actinomycin D which globally affects the transcription of cells even the PolIII associated genes Ssb is needed to mature. This treatment could have an added effect on cell mortality and function. Data confirming that actinomycin D doesn't affect the cells disproportionately is needed. The difference in half-life could be attributed to such a treatment.

      We agree with the reviewer that monitoring Xist signal loss per cell would be a better way to analyze the data. However, in Xist signal loss experiment, snapshot images were taken at four time points (1h, 2h, 3h and 4h). This is not a time-lapse imaging. High-quality time-lapse imaging can only be done within a 2-hour time period with 2-min time interval. Therefore, cell-tracking cannot be done in this experiment. In addition, even though Ssb KD slows down the formation of Xist cloud within the early phase (3 hours) of Xist induction (Figure 4), prolonged (overnight) Xist induction leads to Xist cloud formation in a significant fraction of Ssb KD cells, and the Xist cloud signals are about the same in WT and Ssb KD cells (Figure 7A, 0 h). Similarly, qRT-PCR also revealed that Xist RNA are at the same level in WT and Ssb KD cells (Figure 7C, 0 h). These data argue against that a faster loss of Xist signal in Ssb KD cells is due to weaker initial Xist signal.

      Actinomycin D was added at the last 11 hours of the experiment. During this period, no obvious adverse effects on cells were observed.

      In summarising the authors claim that La binds Xist to facilitate folding and appropriate spreading of Xist along the X-chromosome. No direct interaction has been shown, CLIP-seq data would resolve this, however I do understand this is a challenging technique. The authors have instead opted for RIP followed by qPCR (Figure S2). However, this process has a greater potential for non-specific recovery of RNAs via indirect binding. Furthermore, qPCR may also amplify the relative abundance of the RNA detected. As multiple nucleolar proteins came down in the mass spec screen and FLAG-Ssb is being over expressed, it is plausible to assume some transient Xist interactions may arise from nucleolar association at which La will be in high abundance. Positive and negative nuclear RNA controls (e.g. 7SK and U1 snRNA respectively) could be used so to determine the amount of non-specific Protein-RNA interactions in their RIP pull downs. Cytoplasmic actin is not an appropriate control as it is cytosolic.

      We have to clarify one point that the mass spec screen analyzed samples pulled down by FLAG-PUFb, but not FLAG-Ssb.

      We did not intend to distinguish whether Ssb directly binds Xist or is just associated with Xist. RIP followed by qPCR is sufficient to prove the association between Ssb and Xist RNA.

      We can include nuclear RNA as controls, if the reviewer regards RIP as a valid method to show protein and RNA association

      Other than this the authors may want to probe (via IF) for the presence of La accumulation on the X? Many other know factors such as Ciz1, hnrnpK and PRC1/2 complexes show clear accumulation on the X. If I understand correctly, there are many La antibodies on the market and endogenous levels on the X could be assessed. These antibodies may be useful in IP's and pull downs also.

      Many XCI factors play extensive roles in the cell and are not clearly enriched on Xi, including Spen (Moindrot et al. 2015). We have tried the immunostaining and did not detect Ssb’s enrichment on Xi. Ssb shows a general distribution in the nucleus without a clear enrichment on Xi (data not shown).

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

      The experiments suggested above are centrally focussed on the cell lines that are currently in the authors possession with maybe exceptions with the ∆A-iXist-shSsb line suggested. However, this should be reasonably quick to obtain given their previous work for this paper. Most experiments suggested will focus on the validation of karyotype, Xist expression, rescue construct expression, further RNA FISH classification and repeating more appropriate positive and negative controls for a number of experiments. In theory this can be obtained relatively simply and quickly from current resources. But with the sheer volume of further experiments that are required here, this may take a significant amount of time.

      One vital improvement needed is the replication of mass spec data and the validation of Xist specific recovery and protein enrichment. As it stands this manuscript seems to not have any replicates of the FLAG-out methodology and mass spec data. This is troubling given the poor recovery and specificity of the protein samples obtained. Repeating these experiments would be costly in time and also financially. As it stands, I feel this is essential to conclusively validate their target of interest.

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

      The data is presented relatively well, however, it would be beneficial if deailed methods were in the main text and not in a supplementary file. Similarly, more information about the process of differentiation and how cell death/survival was quantified and validated is needed.

      The reviewer rejected the basic design of the experimental system and ignored the majority of the functional analysis data. No additional experiment can address these issues

      We can include more information in the main text, regarding Ssb. However, there is limited space for the main text, various depending on the journals. Meanwhile, the current citation on Ssb is adequate to emphasize that Ssb is a versatile RNA binding protein involved in a variety of fundamental RNA processing events in the cell.

      *- Are the experiments adequately replicated and statistical analysis adequate?*

      In the most part yes, however there seems to be no replicates of the FLAG-out mass spec screen which is worrying given the minimal specificity observed in the current data.

      As we mentioned above, the FLAG-out experiment only serves as a starting point to generate a list of Xist binding protein candidates. Rather than repeating the FLAG-out experiment, we compared the result of FLAG-out to previously published lists of Xist binding protein candidates. More importantly, additional experiments are carried out to validate the Xist binding proteins identified by FLAG-out.

      **Minor comments:**

      *- Specific experimental issues that are easily addressable.*

      Unfortunately, the majority of experimental issues need to be addressed with more robust data which are highlighted above. However, some image analysis, quantification and classification can be amended relatively easily. For example, the live-cell imaging data should be quantified as loss of signal as discussed and RNA FISH should be used to classify XX positive cells and the XO cells can be discarded from analysis.

      We have addressed these issue in the previous sections of this rebuttal.

      *- Are prior studies referenced appropriately?*

      Most papers regarding Xist pull down and biology are discussed and referenced appropriately. However, the role in which La plays during development and its aberrant affects upon KD are seemingly downplayed. I would like to see more discussion of potential defects that could be caused due to globally altering cellular RNA folding.

      We have tried to cite key references about Ssb in development and RNA folding. Due to length limitation, we cannot cite all references in the topic. If necessary, we could discuss the possibility of indirect effect of Ssb KD on XCI through globally altering cellular RNA folding.

      *- Are the text and figures clear and accurate?*

      For the most part, lots of the figures are clear and accurate. Apart from these exceptions.

      1.The Y-axis of Figure 2D is confusing. What does 0.3 as a "sum of area" equate to? 30% of the area was ES cells? This doesn't look to be the case from Fig 2C. Also, how does the intensity of the signal compare? The area may not be a good quantification due to ES cells growing in colonies.

      We have revised the Y-axis labelling of Figure 2D to “sum of area cm2”. Thus, “0.3” means that the area of ESCs is 0.3 cm2. ALPP is highly expressed on ES cell surface. ALPP stain usually produce saturated stains on ES cell colonies. Thoroughly stained ES cell colonies, big and small, show similar signal intensity levels. To analyze the “total signal intensity” will be not much different from “sum of area”.

      2.In the Movies S1-7 there are boxes around certain cells and marked with "Figure 5a - c". This seems to be incorrect as figure 5 is currently the IF staining of polycomb marks. I assume this is in relation to Figure 4b-d?

      We have corrected the labelling mistakes.

      3.Similarly, in Movies S1-7, the intensities of Xist foci seem by eye to be similar. In the paper it is claimed that the Xist clouds that do form are lower in intensity. Are the Movies depicting the same range of pixel intensities? If not, this should be amended. Similarly, figure 7 seems to show relatively equivalent RNA signal at 0 h?

      All the images were collected using a fixed standard of the microscope and camera setting, and these movies depict the same range of pixel intensities. Movies S1-S3 are WT control, and Movies S4-S7 are Ssb KD cells. The Xist cloud signals are weaker in Movie S4-S7 (also quantified in Figure 4E). For the Xist cloud signal, not only the intensity, but also the area of Xist cloud, have to be taken into account.

      The 0 h in Figure 7 is after overnight Dox treatment, and different from the time point in Movies S1-7 (maximum 3 hour Dox treatment, figure legend of Figure 4B-D). The discrepancy can be explained by that knockdown of Ssb only slows down the formation of Xist clouds. After overnight forced expression, the Xist RNA still shows an accumulation in the cells. Figure 7 shows the forced accumulation of Xist RNA after prolonged Dox treatment disappears faster after Dox withdraw.

      4.In figure 4A the data is from female XX cells, this should be highlighted to limit confusion with the male iXist data shown below in 4B-E. It would also be helpful to have the male/female icons (as in figure 3B), for each figure that has images of cells. Currently Figure 4, 5, 7, S5 and S8 are lacking these icons.

      We have revised the labelling on Figure 3, 4, 5, 7 S6 and S9 (S5 and S8 before revision).

      5.No explanation of the Flag-Ssb expression is given for figure S2. Furthermore, is it really necessary to express Flag-Ssb? There are reasonably good antibodies out there for Ssb as this was how it was originally found in Systemic Lupus patients. Also, no data showing the amount of Ssb being overexpressed is shown. This may have big implication to the validity of the RIP-qPCR analysis.

      We could perform qRT-PCR to quantify the overexpression level of Flag-Ssb. If required, we could use Ssb antibody to do Western blot to show the amount of Flag-Ssb protein.

      *- Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

      Most of the data is presented reasonably well, but the robustness of the data somewhat retracts from their conclusions. I feel the certainty of their conclusion regarding Xist specific La binding and RNA chaperone activity is still presumptive and should be rewritten unless more robust data can confirm Xist interaction. I would also suggest deciding on the nomenclature for the protein of interest and use either La or Ssb, the continued use of both through the figures and text can get a little confusing to the reader.

      In the current literatures, Ssb seems to be commonly used as a gene name and La is used as a protein name. We have revised the manuscript to use one name “Ssb” to describe both the gene and the protein.

      Reviewer #1 (Significance (Required)):

      *- Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.*

      It was a good trial to use PBSb-PUFb system to purify Xist RNA binding proteins, compared to previous reports had used anti-sense oligo purification using complementary sequence to Xist RNA sequences. But currently the purification still needs further validation and repeats to confirm its use. A potential complementary technique could be to isolate Xist directly by using biotinylated probes against the PBSb sequence.

      The authors further claim the identification of a novel Xist RNA chaperone (La/Ssb) which they say facilitates XCI progression. This would be a novel finding in the field; however, the data is currently not robust enough to support this

      *- Place the work in the context of the existing literature (provide references, where appropriate).*

      This work has focused on the development of a milder methodology for purifying Xist RNA during XCI. Others have published similar methodologies predominantly focusing on purifying Xist RNA directly with biotinylated probes (McHugh et al. 2015; Minaji et al. 2015; and Chu et al. 2015). Although this method boasts a milder purification method, it seems to be low yielding in Xist specific proteins. Others have shown a more robust identification of bona fide Xist binding proteins which are currently missing in this manuscript. A recent preprint from the Plath lab has identified new factors involved in XCI during differentiation and their tethering/rescue experiments are far more convincing than the ones shown in this manuscript https://www.biorxiv.org/content/10.1101/2020.03.09.979369v1. The candidate protein Ha et al. have identified has multiple roles in developing cells and has shown to be important during mouse development. However, Ha et al do not robustly show that the knockdown of Ssb causes X-linked cell mortality. Alternatively, as would be presumed from Ssb's essential role in many housekeeping short non-coding RNAs, the cell death seems more ubiquitous upon shRNA KD. Therefore, the link the authors are making here are relatively weak.

      Ssb KD rescues cell death caused by forced induction of Xist in male ESCs. In addition, Ssb KD leads to cell death in differentiating female ESCs, while it has a negligible effect on cell death in differentiating male ESCs. These data clearly demonstrated X-linked cell survival/mortality by Ssb KD.

      Plath lab’s work is different from ours. In their manuscript, the authors report the observation of a protein condensation which is assembled by Xist but sustains in absence of Xist. TDP-43 (a.k.a. Tardbp) happens to be one protein factor involved in the protein condensation and also one candidate protein selected for further validation in our study. In our study, Tardbp KD did not rescue cell death caused by induced XCI in male cells. Thus, Tardbp is not further studied. In the manuscript, we have discussed the possibility that low efficiency of knockdown and redundancy might contribute to the failure in validation of Tardbp

      *- State what audience might be interested in and influenced by the reported findings.*

      The audience may be interested in the novel technique and the finding of a novel Xist binding protein.

      *- Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.*

      RNA biochemistry and developmental biology

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

      **Summary:**

      This manuscript describes a novel "FLAG-out" system, where the authors sought to identify Xist RNA binding proteins. The authors focused on a specific protein found in their screen and also identified in several other screens for Xist RNA binding proteins, Ssb/La, and further characterize the role of this protein in XCI. This manuscript describes the loss of Ssb/La and suggest that it predominately impacts the canonical 'cloud' formation of Xist RNA on the X chromosome during XCI initiation. Further, they determine that loss of Ssb/La decreases Xist RNA half-life and alters folding of Xist RNA transcripts. Based on their findings, the authors propose that Ssb/La functions to directly bind and fold Xist RNA transcripts in a manner that stabilizes Xist RNA, allowing for proper 'cloud' formation and successful initiation of XCI.

      **Major comments:**

      The authors made an interesting findings that the SLE-relevant autoantigen Ssb/La stabilizes Xist RNA transcripts, and there is some evidence that this occurs by binding and maintaining proper folding of Xist RNA. Despite these intriguing observations, there are many parts of the manuscript that need to be addressed in order to support the authors main conclusions.

      The most troubling aspect of this manuscript is the persistent use of an artificial XCI system in male cells to draw strong conclusions about the function of Ssb in XCI. This issue is prevalent throughout the manuscript, and I question why the authors chose to perform most of their experiments in male cells when the same experiments can be (and have previously been by other groups) performed in female cells. Using male ESCs and then making conclusions for XCI, which is a female-specific process, is a major concern.

      In addition to iXist male ESC line, many experiments, such as cell death/survival (Figure 3B, C), allelotype (Figure 3E), Xist could formation (Figure 4A), H3K27me3 and H2AK119ub IF (Figure 5), were performed in female ESC. We chose to do SHAPE and Xist RNA stability assays in iXist male ESC line, because the onset of XCI is much more synchronized in this system. Moreover, in female cells, Xa causes additional layers of complication/noise in the ATAC-sequencing which may not be fully cleared up by data analysis. On the other hand, inducible Xist expression in male ESCs can be used as an experimental system to recapitulate the silencing step of XCI (Ha et al. 2018; Wutz et al. 2002).

      • Out of the 138 identified binding proteins, the authors chose to only validate three: Mybbp1a, Tardbp, and Ssb/La. The logic for choosing these candidates is weak, and the authors are only able to validate 1 out of 3 of these proteins.

      In theory, all candidate proteins in the list are possibly involved in XCI. There is no method which can help to make accurate prediction. We did not follow a clear-cut logic in selecting candidates for validation, but we do consider the candidate gene’s knockout phenotype, “early embryonic lethality”, as a phenotype consistent with a critical role of the candidate gene in XCI. Meanwhile, in the manuscript, we have discussed why we chose the three proteins for validation as the following:

      “……From the candidate proteins, we shortlisted three proteins for individual validation. Myb-binding protein 1A (Mybbp1a, Q7TPV4) and TAR DNA-binding protein 43 (Tardbp, Q921F2) were selected because they are known transcription repressors (11, 12). The Lupus autoantigen La (P32067, encoding-gene name: Ssb) was selected because systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a strikingly high female to male ratios of 9:1 (13). Moreover, its autoimmune antigen La is a ubiquitous and versatile RNA-binding protein and a known RNA chaperone (14). All the three selected candidates have also been identified as Xist-binding proteins in previous studies (2, 4). Moreover, the knockout of these three genes all lead to early embryonic death. Tardbp knockout causes embryonic lethality at the blastocyst implantation stage (15). Mybbp1a and Ssb knockout affect blastocyst formation (16, 17). Early embryonic lethality is a mutant phenotype consistent with a critical role of the mutated gene in XCI (1)** ……”

      We used cell death/survival assay to further validate the role of Xist binding protein candidates in XCI. This is a stringent assay. It requires not only that Xist binding protein candidates bind to Xist, but also that the candidates have to be functionally important in XCI.

      Indeed, it has been demonstrated by Plath lab (the BioRxix manuscript mentioned by reviewer 1) that Tardbp (also named TDP-43), together with other RBPs, bind to the E repeat of Xist to form a condensate and create an Xi-domain. Yet, Tardbp KD did not rescue cell death caused by forced XCI in male cells in our studies. Thus, only 1 out of 3 of these candidates is validated and further studied. In the manuscript, we also discussed that low efficiency of knockdown and redundancy might contribute to the failure in validation of Tardbp and Mybbp1a.

      • Use of the cell death assay is not strong enough to "confirm that La is involved in induced XCI" as stated by the authors. This is a huge overstatement.

      Given the diverse functions of Ssb in cell differentiation and proliferation, ribosome biogenesis, transcriptional control and tRNA maturation, one would expect less surviving Ssb knockdown cells. In contrast, more Ssb knockdown cells survives in the presence of Dox, suggesting that Ssb plays an important role in XCI. Considering the reviewer’s comment, we revised the sentence to “further suggest that Ssb is involved in induced XCI”.

      While the authors observed differences in X-linked gene expression after Ssb KD, they did not examine expression of these genes in after KD of either Mybbp1a or Tardbp. Are the changes observed in these genes specific to Ssb KD? Or could there still be alterations of X-linked gene expression in the non-validated KDs? This experiment should be performed and included in the manuscript, either within Fig 2 or in the supplemental. As well, inclusion of a well characterized positive control, for example Hnrnpu, as comparison to Ssb should be included.

      Mybbp1a and Tardbp were not validated by the cell death assay. Thus, compared with Ssb, Mybbp1a and Tardbp are less important for XCI functionally. We only focused on Ssb in the subsequent studies. Mybbp1a and Tardbp KD could be additional negative controls. Yet, we have used empty vector as a negative control. We do not need so many controls.

      As mentioned, Tardbp indeed binds to Xist RNA. It is very likely that Tardbp KD might alter some X-linked gene expression. This rules out Tardbp KD as a good negative control.

      If we do not see any effect of Ssb KD on X-linked gene expression, a positive control is absolutely required. However, we have detected that Ssb KD compromises the silencing of several X-linked gene. A positive control might not be essential.

      • The authors perform RIP to validate the interaction of Ssb with Xist, but this is performed in male ES cells with induced Xist RNA and with FLAG-tagged Ssb. Aside from these cells being male, in this system Xist RNA expression is much higher than would be found endogenously. RIP should have been done in female differentiated ESCs if there is in fact a role for XCI.

      • The authors need to include more details in the methods section to explain how the FLAG-Ssb is expressed in these cells, and why the authors chose to use a tagged contrast over endogenous Ssb. Due to these issues the result from this experiment is essentially meaningless and is not convincing of Ssb interaction with Xist RNA. There is no reason RIP cannot be performed in female cells, and the authors should repeat this experiment in the relevant experimental condition. As well, if a validated Ssb antibody exists the authors should perform RIP using the endogenous protein.

      If required, we could try to perform RIP and/or CLIP using Ssb antibody in female cells.

      The authors state in Fig 3A-C that the results of the cell death and differentiation experiments "...support a functional role of La in XCI". The authors state earlier that Ssb is a ubiquitous protein that is embryonic lethal (in both female and males). Based on this, the cell death results shown do not support a functional role of La in XCI as the Ssb KD could be having an indirect affect due to its other developmental functions. This manuscript lacks a direct functional link between Ssb and XCI; more data is necessary.

      Given the diverse functions of Ssb in cell differentiation and proliferation, ribosome biogenesis, transcriptional control and tRNA maturation, one would expect less surviving Ssb knockdown cells. In contrast, more Ssb knockdown cells survives in the presence of Dox, suggesting that Ssb plays an important role in XCI.

      For the data in Fig 3A-C, Ssb KD causes the death of female differentiating cells, but not male differentiating cells. Therefore, it rules out that the death of female cells is due to the general function of Ssb. Rather, the specific role of Ssb in XCI contributes to the female specific cell death.

      In Fig 3D, the authors perform ATAC-seq in inducible male ES cells. The authors claim that the extremely slight reduction in chromatin compaction of the Ssb KD compared to control iXist "directly connect La to the heterochromatinization of Xi, supporting a functional role of La in XCI". This is also an overstatement based on the minimal, and possibly indirect, change in compaction. The positive control i-detaA-Xist sample has significantly less compaction (and thus significantly higher compaction defect) than the Ssb KD again disputing the claim stated above. It is unclear why performing ATAC-seq is even necessary, as Ssb isn't stated to have a function in regulating chromatin architecture. In addition, why the authors performed ATAC-seq in the artificial male XCI system and not in the F1 female cells, and the N of the experiment is unclear. If the authors want to include the ATAC-seq in further revisions it should be repeated n=3 in the female system.

      The male induced XCI system provides a more synchronized onset of XCI. More importantly, in the male induced XCI system, only one X chromosome exists, avoiding the interference from the active X chromosome in female cells. If ATAC-seq was performed in female cells, only loci with SNPs can be distinguished. The sequencing reads from Xa will create additional layers of complication/noise which may not be cleared up fully by data analysis

      “i-delat-Xist” is a positive control to show the experimental system works. It is not justified to compare the chromatin accessibility of the mutant, which is only a Ssb “knockdown” mutant, and the control “i-delat-Xist”, in which the Repeat A is “deleted”. We admit that ATAC-Seq results did not reveal a drastic difference in chromatin accessibility between the wild type sample and the mutant sample. However, as what we discussed in the manuscript, clear difference can still be seen at the 14 h time point. This is shown clearly by the heatmap (Fig. 3E) and the sequencing coverage profile (Fig. S4A).

      • In Fig 6, the authors state in their methods that "The shRNA construct, which worked efficiently against Ssb, was not designed against the 3' UTR of the RNA. Therefore, the shRNA is against some of the rescue plasmid constructs. Nonetheless, transfecting the Ssb knockdown cells with the rescue plasmids should compensate the effect of Ssb knockdown and serve as a rescue assay to study the functional domains of La.". This is troubling and seems like a major experimental issue; the specific rescue constructs that may be impacted by this issue are not stated and should be explicitly mentioned. This becomes more confusing when examining the data from rescue experiments.

      We pointed out this issue in the original manuscript. We agree that the experiment was not perfectly designed. In the revision, we added in the information on the shRNA target site. Our shRNA targets the LAM domain, so the expression of ∆LAM is not affected by the shRNA. We agree that the detected GFP expression levels of ∆RRM1 and ∆RRM2 are too low to be conclusive. In the revision, we have removed the data point of ∆RRM1 and ∆RRM2. Meanwhile, it is clear that ∆RRM1&2 has a better rescuing effect than ∆NLS, when ∆RRM1&2 and ∆NLS are expressed at similar levels. Ssb is a well-known RNA chaperone/RNA helicase. Identifying Ssb is an Xist-binding protein already suggests the functional role of Ssb in XCI. The data of the plasmid rescue experiments further suggests that Ssb is involved in XCI as a RNA chaperone/RNA helicase.

      If it is necessary, we could redo this experiments using a shSsb targeting 3’-UTR or expressing GFP-Ssb immune to shSsb.

      In Figure S7, the expression of the rescue constructs deltaRRM1 and deltaRRM2 is extremely low, yet the authors observe a rescue of the cloud phenotype (fig 6D) from those constructs that reaches almost the level of full length Ssb. This is confusing, and the authors need to address this by performing a western blot to show the protein levels of these rescue constructs and discuss further how such a low level of expression can show a rescue phenotype. The results would also be stronger if the authors examined H3K27me3 and H2AK119ub1 enrichment since they observed decreased overlap of these marks with Xist RNA after Ssb KD. Finally, the authors state that "...all three RNA-binding domains are required for the functionality of La in XCI..." however I have trouble coming to this conclusion based on the above issues. As well, if the authors want to support direct function, they should repeat the RIP experiments with these rescues constructs to show that the domains capable of rescue can still bind to Xist RNA.

      Reviewer 1 raised similar concerns. In Figure 6C, the live cell counts of ∆RRM1 and ∆NLS are about the same. It might be due to the low expression level of ∆RRM1 (Figure S7). It is clear that ∆RRM1&2 has a better rescuing effect than ∆NLS, when ∆RRM1&2 and ∆NLS are expressed as similar levels. To make the data more straight forward, we removed the data point of ∆RRM1 and ∆RRM2, because of their low expression levels.

      As for the Western blot and GFP fluorescence (IF), we have tried both. Neither of them detected GFP signal, reflecting the low expression level of these GFP fusion proteins. The shSsb is not targeting the 5’ or 3’-UTR region, therefore interfering the exogenous Ssb as well. This might be a reason for the low expression of these GFP fusion proteins. If it is necessary, we could redo this experiments using a shSsb targeting 3’–UTR or expressing GFP-Ssb immune to shSsb.

      We deleted the sentence "all three RNA-binding domains are required for the functionality of La in XCI".

      **Minor comments:**

      The authors may want to consider better highlighting the strengths of their "FLAG-out" system. As written, is it difficult to tell how this system sets them apart from the previously published studies referenced in the text, especially as some of these studies used similar crosslinking conditions and cell types. Additionally, the logic and questions the authors pose in the introduction as to why they performed this project are too general and not very strong. For example, the authors mention how might protein machinery may assemble on Xist RNA, and how might Xist RNA may spread on the X chromosome. However neither of these topics are actually addressed in their experiments or discussion. These are interesting questions, but the authors should either discuss them further within the context of their results or take these questions out. It would also be helpful if the authors could better label Figure 4, as it is unclear in the figure itself that Fig 4A is in reference to female cells, but remaining panels are in male cells.

      The inducible XCI in male cells is a valid system to recapitulate the silencing step of XCI. It also provides unique advantages in many experiments, such as ATAC-seq. Meanwhile, we did perform extensive functional analysis on the endogenous XCI process using female cells. However, we do realize that presenting the data of induced XCI in male cells together with the data from female cells is confusing to many readers. We have revised the labelling on Figure 3, 4, 5, 7 S6 and S9 (S5 and S8 before revision).

      To understand “how the protein machinery is assembled by Xist” and “how Xist spreads along its host chromosome territory” are not specifically the initial aims of this study. We removed the sentences from the introduction section. However, we believe Ssb may provide clues for the future studies to fully address these questions, and we did provide the following thoughts in the discussion section:

      “……Secondly, as Ssb is able to utilize ATP to unwind RNA-RNA and RNA-DNA duplex, it may play a more active role in controlling the structural dynamics of Xist in living cells (14, 23). These structural dynamics may be important for recruiting proteins onto the RNA and spreading of the RNA along its host chromosome territory……”

      Reviewer #2 (Significance (Required)):

      I am not convinced the this manuscript, as written, has sufficient novelty. Ssb/La has been previously identified to be an Xist RNA binding protein with older/different approaches. However, there are some interesting observations in this manuscript. Major revisions are necessary.

      We agree with the reviewer that identification of Ssb as an Xist RNA binding protein is not novel. The novelty of our discovery lies in: 1) we developed a new method for isolating lincRNA associated proteins; 2) we confirmed that Ssb is an important player involved in XCI; 3) we showed that Ssb regulates the folding of Xist RNA, consequently the stability of Xist and the formation of Xist cloud.

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      Referee #1

      Evidence, reproducibility and clarity

      Summary:

      Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

      Here Ha et al. has further developed their Pumilio RNA tagging methodology for the isolation of UV-crosslinked proteins that are suggested to associate with Xist RNA in mouse embryonic stem cells (mESCs). Within this study the authors claim to have found the Lupus antigen RNA binding protein (La) as a novel Xist interacting partner that influences the efficacy of X-chromosome inactivation (XCI). The authors use a number of different techniques such as qPCR, fluorescent imaging, ATAC-SEQ and SHAPE to show aberration of XCI upon La shRNA knockdown. However, this study has significant flaws in the efficient isolation and validation of Xist associated proteins using their FLAG-out methodology. Furthermore, later experiments predominantly focus on cell death/survival assays, which is somewhat troubling given the essential roles La plays in processes such as cell differentiation and proliferation, ribosome biogenesis, transcriptional control and tRNA maturation. I feel the authors need to robustly address the potential effects La knockdown may be having on their mESCs.

      Major comments:

      -Are the key conclusions convincing?

      My major concern is in their Xist RNA purification. First of all, I couldn't find any data on proving the enrichment of Xist RNA itself in their Pumilio pull-down experiment. It would have been useful to show Xist RNA enrichment before benzonase step. Secondly, it is hard to imagine the protocol would successfully isolated Xist RNA-protein complexes from the cell. An earlier report by Clemson et al., (J Cell Biol., 1996) has shown that majority of Xist RNA is still stuck in the nucleus after nuclear matrix prep protocol using detergent, which is not so different from the authors' protocol. Moreover, the authors used UV crosslink, which would have made even harder to purify Xist RNA without sonication. Thirdly, as the tag is located on 5' of Xist RNA, it is rather surprising to see that Spen is not detected in their pulldown. Spen is one of the main functional interactors with Xist, robustly detected by several previous reports. Similarly, other high-affinity binders of Xist such as hnRNP-K and Ciz1 were also lacking from this screen. Finally, the peptides found associated with FLAG-out Xist are extremely low in comparison with other data using glutaraldehyde or formaldehyde crosslinking. For example, HnRNP-M found in Chu et al 2015 has 1120 peptide counts in differentiated cells. The authors here use HnRNP-M as a baseline for specific interactions and show a total of 6 peptide counts in Xist expressing cells and 5 in i-Empty cells (Supplementary excel sheet 1). Similarly, the La protein of interest in this study has 8 counts in i-FLAG-Xist and 6 counts in i-Empty. I struggle to see how this result indicate specific Xist binding. Worryingly this is the starting rationale for the rest of their experiments, it is hard to therefore accept the rest of their conclusions either.

      The other key conclusion the authors make is from the use of numerous cell death/survival assays for both male and female cell lines. This is extremely troubling in the context of assessing their target protein La. La is involved in multiple RNA maturation events of rRNAs, tRNAs and other polIII transcripts. Furthermore, La has been implicated in binding to the mRNA for Cyclin D1 in both human cells and mouse fibroblasts (NIH/3T3 - male) which show a significant effect on cell proliferation upon siRNA knockdown https://www.nature.com/articles/onc2010425. This, along with the observation that La knock-out blastocysts fail to develop any mice or ES cell lines (male or female) show the effect observed in the authors results is most likely not X-linked cell death https://mcb.asm.org/content/mcb/26/4/1445.full.pdf. The authors need to show that their shRNA KD isn't affecting the proliferation and general fitness of their mESC lines.

      - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

      As discussed above, I feel the authors have not clearly demonstrated Xist specific protein enrichment and haven't proven X-linked cell death. Due to the lack of necessary control experiments as discussed below, I feel the notion that La is involved directly in XCI as an RNA chaperone is currently preliminary/speculative.

      - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

      I would suggest them to show RT-qPCR results of Xist RNA enrichment from the sample after flagIP before benzonase treatment.

      Also, it would have been more convincing if their negative control construct (i-Empty) would contain 25 copies of PBSb RNA at least.

      In Fig1b, the total amount of proteins loaded on the gel is not equivalent between two lanes. The gel should show equivalent amounts of proteins on the gel. It looks like if the negative control sample had been loaded at the same amount as the one with Xist, the band pattern wouldn't be distinguishable between the two samples. Furthermore, as these samples were used in the following mass spectrometry screen it may suggest that the minimal increase in peptide counts observed in the iXist FLAG-out were due to an increased amount of sample being loaded? No controls are conducted to account for this.

      The authors quantify cell death in figures 2C - E. It seems clear that shSsb 1 and 2 have an effect on cell count even in the absence of Dox. The rescue effect seen upon Dox addition is minimal when compared to Empty + Dox 2D. The authors ∆A-iXist line with and without Ssb KD/Dox would be an informative control on whether the increase in cell survival that they see is X-linked.

      The qPCR results used to validate silencing defects show minor changes in expression and also don't show significant silencing of X-linked genes sufficient for cell death. Could this be because only ~ 50 - 60% of Male iXist cells seem to be expressing in the movies and that this will have an effect on the observed qPCR results? Furthermore, it seems counterintuitive that expression in the Empty male cells increases in 48h compared to 14h. Is this due to cell death and positive selection of cells less able to silence their X-chromosome? How would these data look in the female XX line? How would the data look in a ∆A-iXist line in the presence and absence of shSsb/Dox?

      Confusingly, the male line in Fig 3C shows a drop in live cell count at day 6 of differentiation? Surely given their previous results in Fig 2 the Ssb KD should increase cell viability with +Dox? Ssb KD seems to have an adverse effect on ES cells during extended differentiation protocols. In Figure S1 the authors show ~ 8 - 10% survival of male lines during differentiation. Could the recombination of the Xist sequence around the loxP sites enable the cells to outcompete the dead cells? How would iEmpty and ∆A-iXist cells compare here? Have the differentiated cells been tested for their expression of Xist? Additionally, how are there similar live cell counts for male vs female lines when ~90% of male cells die during differentiation? Were more cells plated at day 4? If so, this would bias the competition of male cell survival and therefore make the male line an inappropriate control. Given the essential role of La during development a control is needed to prove that this death is X-linked in the female 3F1 line. For example, an XO cell line retaining the Cast allele and shSsb expression could show the amount of death caused from shSsb alone independent of X-linked cell death.

      If I understood correctly, the RNA FISH used dsDNA probes ("Sx9") against 40 kb of the X-inactivation centre (Xic). Surely Tsix or other Xic transcripts will also be visible? Can the authors use their RNA FISH to determine the XX or XO status of their cells? In Figure S5 a number of cells appear to show a single pinpoint of transcription. This could either be low levels of Xist transcripts or Xic transcription from an XO line in which the 129 chromosome is missing. It would be best to solely quantify cells which have two x chromosomes and if a significant amount of X chromosomes have been kicked out, this should be discussed and controlled for.

      In Fig6, the authors generated a number of Ssb constructs for a rescue assay. However, these results complicate the matter and raise more questions than they address. It seems odd that the ∆RRM1 does not rescue based on comparison with their putative negative control, ∆NLS. However, the ∆RRM1 + 2 and ∆LAM do rescue the phenotype better than the full length Ssb? This makes no logical sense and highlights the inherent variation in cell viability these generated cell lines seem to show. Following on from this, figure S7 quantifies the GFP tag mRNA levels, depicting all ∆RRM mutants with expression below ~30%? How can ∆RRM1 or 2 be rescuing in this scenario? Have these lines been tested for their XX or XO status? The loss of an X chromosome would lead to a rescue of the cell death phenotype, which is a process known to occur in XX lines that have been cultured for extended periods of time. Could it also be that the cell lines derived are more or less sensitive to exogenous shRNA expression? Also, further validation is needed to assess the efficiency of KD in these lines as theoretically most of these constructs will be targeted by shRNA? What is the endogenous Ssb expression level in these lines? Where in the mRNA sequence are the shRNAs targeted to? Does this make sense on the relative expression levels of ∆RRM1/2 for example? Further testing of GFP expression could also be assessed by quantitative western blot of GFP or even visualised in their RNA FISH/IF samples (Figure S8), currently neither are shown. In addition, some kind of information of stability of each Ssb protein constructs has not been demonstrated.

      For the data shown in Figure 7A and B the authors quantify the % of cells with Xist signal. The authors have already shown a defect in Xist visualisation in Ssb KD. Surely it is plausible to assume a faster loss of Xist signal below background in weaker expressing cells. A more appropriate quantification would be the % loss of Xist signal per cell over time.

      With Figure 7C and D, the samples have been treated with actinomycin D which globally affects the transcription of cells even the PolIII associated genes Ssb is needed to mature. This treatment could have an added effect on cell mortality and function. Data confirming that actinomycin D doesn't affect the cells disproportionately is needed. The difference in half-life could be attributed to such a treatment.

      In summarising the authors claim that La binds Xist to facilitate folding and appropriate spreading of Xist along the X-chromosome. No direct interaction has been shown, CLIP-seq data would resolve this, however I do understand this is a challenging technique. The authors have instead opted for RIP followed by qPCR (Figure S2). However, this process has a greater potential for non-specific recovery of RNAs via indirect binding. Furthermore, qPCR may also amplify the relative abundance of the RNA detected. As multiple nucleolar proteins came down in the mass spec screen and FLAG-Ssb is being over expressed, it is plausible to assume some transient Xist interactions may arise from nucleolar association at which La will be in high abundance. Positive and negative nuclear RNA controls (e.g. 7SK and U1 snRNA respectively) could be used so to determine the amount of non-specific Protein-RNA interactions in their RIP pull downs. Cytoplasmic actin is not an appropriate control as it is cytosolic.

      Other than this the authors may want to probe (via IF) for the presence of La accumulation on the X? Many other know factors such as Ciz1, hnrnpK and PRC1/2 complexes show clear accumulation on the X. If I understand correctly, there are many La antibodies on the market and endogenous levels on the X could be assessed. These antibodies may be useful in IP's and pull downs also.

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

      The experiments suggested above are centrally focussed on the cell lines that are currently in the authors possession with maybe exceptions with the ∆A-iXist-shSsb line suggested. However, this should be reasonably quick to obtain given their previous work for this paper. Most experiments suggested will focus on the validation of karyotype, Xist expression, rescue construct expression, further RNA FISH classification and repeating more appropriate positive and negative controls for a number of experiments. In theory this can be obtained relatively simply and quickly from current resources. But with the sheer volume of further experiments that are required here, this may take a significant amount of time. One vital improvement needed is the replication of mass spec data and the validation of Xist specific recovery and protein enrichment. As it stands this manuscript seems to not have any replicates of the FLAG-out methodology and mass spec data. This is troubling given the poor recovery and specificity of the protein samples obtained. Repeating these experiments would be costly in time and also financially. As it stands, I feel this is essential to conclusively validate their target of interest.

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

      The data is presented relatively well, however, it would be beneficial if deailed methods were in the main text and not in a supplementary file. Similarly, more information about the process of differentiation and how cell death/survival was quantified and validated is needed.

      - Are the experiments adequately replicated and statistical analysis adequate?

      In the most part yes, however there seems to be no replicates of the FLAG-out mass spec screen which is worrying given the minimal specificity observed in the current data.

      Minor comments:

      - Specific experimental issues that are easily addressable.

      Unfortunately, the majority of experimental issues need to be addressed with more robust data which are highlighted above. However, some image analysis, quantification and classification can be amended relatively easily. For example, the live-cell imaging data should be quantified as loss of signal as discussed and RNA FISH should be used to classify XX positive cells and the XO cells can be discarded from analysis.

      - Are prior studies referenced appropriately?

      Most papers regarding Xist pull down and biology are discussed and referenced appropriately. However, the role in which La plays during development and its aberrant affects upon KD are seemingly downplayed. I would like to see more discussion of potential defects that could be caused due to globally altering cellular RNA folding.

      - Are the text and figures clear and accurate?

      For the most part, lots of the figures are clear and accurate. Apart from these exceptions.

      1.The Y-axis of Figure 2D is confusing. What does 0.3 as a "sum of area" equate to? 30% of the area was ES cells? This doesn't look to be the case from Fig 2C. Also, how does the intensity of the signal compare? The area may not be a good quantification due to ES cells growing in colonies.

      2.In the Movies S1-7 there are boxes around certain cells and marked with "Figure 5a - c". This seems to be incorrect as figure 5 is currently the IF staining of polycomb marks. I assume this is in relation to Figure 4b-d?

      3.Similarly, in Movies S1-7, the intensities of Xist foci seem by eye to be similar. In the paper it is claimed that the Xist clouds that do form are lower in intensity. Are the Movies depicting the same range of pixel intensities? If not, this should be amended. Similarly, figure 7 seems to show relatively equivalent RNA signal at 0 h?

      4.In figure 4A the data is from female XX cells, this should be highlighted to limit confusion with the male iXist data shown below in 4B-E. It would also be helpful to have the male/female icons (as in figure 3B), for each figure that has images of cells. Currently Figure 4, 5, 7, S5 and S8 are lacking these icons.

      5.No explanation of the Flag-Ssb expression is given for figure S2. Furthermore, is it really necessary to express Flag-Ssb? There are reasonably good antibodies out there for Ssb as this was how it was originally found in Systemic Lupus patients. Also, no data showing the amount of Ssb being overexpressed is shown. This may have big implication to the validity of the RIP-qPCR analysis.

      - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

      Most of the data is presented reasonably well, but the robustness of the data somewhat retracts from their conclusions. I feel the certainty of their conclusion regarding Xist specific La binding and RNA chaperone activity is still presumptive and should be rewritten unless more robust data can confirm Xist interaction. I would also suggest deciding on the nomenclature for the protein of interest and use either La or Ssb, the continued use of both through the figures and text can get a little confusing to the reader.

      Significance

      - Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

      It was a good trial to use PBSb-PUFb system to purify Xist RNA binding proteins, compared to previous reports had used anti-sense oligo purification using complementary sequence to Xist RNA sequences. But currently the purification still needs further validation and repeats to confirm its use. A potential complementary technique could be to isolate Xist directly by using biotinylated probes against the PBSb sequence. The authors further claim the identification of a novel Xist RNA chaperone (La/Ssb) which they say facilitates XCI progression. This would be a novel finding in the field; however, the data is currently not robust enough to support this.

      - Place the work in the context of the existing literature (provide references, where appropriate).

      This work has focused on the development of a milder methodology for purifying Xist RNA during XCI. Others have published similar methodologies predominantly focusing on purifying Xist RNA directly with biotinylated probes (McHugh et al. Minaji et al and Chu et al.). Although this method boasts a milder purification method, it seems to be low yielding in Xist specific proteins. Others have shown a more robust identification of bona fide Xist binding proteins which are currently missing in this manuscript. A recent preprint from the Plath lab has identified new factors involved in XCI during differentiation and their tethering/rescue experiments are far more convincing than the ones shown in this manuscript https://www.biorxiv.org/content/10.1101/2020.03.09.979369v1. The candidate protein Ha et al have identified has multiple roles in developing cells and has shown to be important during mouse development. However, Ha et al do not robustly show that the knockdown of Ssb causes X-linked cell mortality. Alternatively, as would be presumed from Ssb's essential role in many housekeeping short non-coding RNAs, the cell death seems more ubiquitous upon shRNA KD. Therefore, the link the authors are making here are relatively weak.

      - State what audience might be interested in and influenced by the reported findings.

      The audience may be interested in the novel technique and the finding of a novel Xist binding protein.

      - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

      RNA biochemistry and developmental biology

    1. One way around this is simply linking to each SVG with an <img> tag, instead of embedding the actual SVG in the DOM. This way, the virtual DOM only needs to track one node per image, instead of hundreds for each SVG. Inline SVG [above] vs linked SVG. But in doing so we’ve crippled our ability to manipulate our SVGs. No longer can we add stroke, move shapes, remove nodes or change fill. In short, if you want :hover to change the fill color, you’re back in the stone age.
    1. You know the trade-off. Use the img tag to display an SVG, and you get clean markup — at the cost of styling the SVG using its properties like fill, stroke, SVG filters and more.
    1. This commit does not belong to any branch on this repository.

      How would I download this commit/changeset with a git client then?? Or is it simply the case that if someone ever deletes the source branch for a merge request and "orphans" those commits, that there is now no longer a way to download it via the usual git fetch methods and the only way now to view these commits is via the web interface?

      Idea: Create a permanent tag for every version of every pull request that gets pushed up. (Which maybe the already do internally to prevent it from being GC'd?)

      https://github.com/ruby/ruby/pull/1758

      Ana06 deleted the Ana06:array-diff branch on Apr 30, 2019

    1. SciScore for 10.1101/2020.06.29.175844: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">Ethics statement All animal experiments are performed according to Swedish Animal Welfare Act SFS 1988:534 and were approved by the Animal Ethics Committee of Malmö/Lund, Sweden.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

      Table 2: Resources

      <table><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 against the His-tag ( 1:2000 , Invitrogen ) were followed by secondary HRP conjugated antibody ( 1:2000 , Dako , Denmark) , for detection of S protein .</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;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">NF-κB; nuclear factor-kappa B , SARS-CoV-2 Spike protein; S protein , TLR4; Toll-like receptor 4 Results SARS-CoV-2 S protein sequence and endotoxin content 2019-nCoV full-length His-tagged S protein ( R683A , R685A) , composed of the S sequence Val 16 - Pro 1213 was produced in HEK293 cells and 1 µg was analyzed on SDS-PAGE followed by staining with Coomassie brilliant blue ( Fig .</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, CVCL_0045</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In order to mimic those LPS levels , we therefore determined the response of the THP-1 cells to doses ranging from 0.25 ng/ml to 1 ng/ml LPS , with or without the presence of 5 nM of S protein .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>THP-1</div> <div>suggested: CLS Cat# 300356/p804_THP-1, CVCL_0006</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td 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 image was obtained using a Gel Doc Imager ( Bio-Rad Laboratories ,</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Bio-Rad Laboratories</div> <div>suggested: (Bio-Rad Laboratories, SCR_008426)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Evaluation of the spectra was performed with Xcalibur v 2.0.7 . software ( from Thermo Fisher Scientific , San José , CA) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Xcalibur</div> <div>suggested: (Thermo Xcalibur, SCR_014593)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis, as indicated in each figure legend, were performed using GraphPad Prism software v8.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, SCR_002798)</div> </div> </td></tr></table>


      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 OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. SciScore for 10.1101/2020.06.30.177097: (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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Interestingly , the dominant population of the SARSCoV-2 S trimer is in the ground prefusion state with inactivated FP and all the RBD domains buried , which may result in “conformational masking” preventing antibody binding and neutralization , similar to that described for HIV-1 envelope ( Env ) ( Kwong et al. , 2002; Munro et al. , 2014) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HIV-1 envelope ( Env )</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-RBD polyclonal antibody and monoclonal antibody ( MAb ) 1A10 were prepared by immunizing BALB/c mice with recombinant SARS-CoV-2 RBD fused with a C-terminal mouse IgGFc tag ( Sino Biological Inc , Beijing , China ) using previously described protocols ( Qu et al. , 2020) .</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">After washing , the corresponding secondary antibodies , horseradish peroxidase ( HRP)conjugated anti-human IgG1 ( Abcam , USA ) or HRP-conjugated anti-mouse IgG ( Sigma , USA) , were added 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>anti-human IgG1</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>anti-mouse IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Key words: SARS-CoV-2 S glycoprotein, SARS-CoV-2-S-ACE2 complex, cryo-EM, conformational dynamic, conformational masking Results An uncharacterized tightly closed state of SARS-CoV-2 S trimer Prefusion stabilized ectodomain trimer of SARS-CoV-2 S glycoprotein was produced from HEK293F cells using the strategy also adopted in other studies (Fig.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK293F</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_6642">CVCL_6642</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Acknowledgements We are grateful to the staffs of the NCPSS Electron Microscopy facility , Database and Computing facility , and Protein Expression and Purification facility for instrument support and technical assistance. Y . C . was supported by grants from the CAS Pilot Strategic Science and Technology Projects B ( XDB37040103) , the National Basic Research Program of China ( 2017YFA0503503) , the NSFC ( 31670754 and 31872714) , the CAS Major Science and Technology Infrastructure Open Research Projects , and the CAS-Shanghai Science Research Center ( CAS-SSRC-YH-2015-01 , DSS-WXJZ-2018-0002) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>National Basic Research Program</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data deposition Cryo-EM maps have been deposited in the Electron Microscopy Data Bank , https://www.ebi.ac.uk/pdbe/emdb/ ( accession nos .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>https://www.ebi.ac.uk/pdbe/emdb/</b></div>
              <div>suggested: (Electron Microscopy Data Bank at PDBe (MSD-EBI), <a href="https://scicrunch.org/resources/Any/search?q=SCR_006506">SCR_006506</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bio-layer interferometry ( BLI ) assay Before BLI experiments , SARS-CoV-2 S trimer protein was biotinylated using the EZLink™ Sulfo-NHS-LC-LC-Biotin kit ( Thermo Fisher ) and then purified using Zeba™ spin desalting column ( Thermo Fisher) , according to manufacturer 's protocols .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Zeba™</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 2D classification , we selected good averages with 13,047 particles for initial model building , which were performed in RELION 3.0 ( Zivanov et al. , 2018) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>RELION</b></div>
              <div>suggested: (RELION, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016274">SCR_016274</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The movies were recorded on a K2 Summit direct electron detector ( Gatan ) operated in the super-resolution mode ( yielding a pixel size of 1.02 Å after 2 times binning) , under low-dose condition in an automatic manner using SerialEM ( Mastronarde , 2005) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>SerialEM</b></div>
              <div>suggested: (SerialEM, <a href="https://scicrunch.org/resources/Any/search?q=SCR_017293">SCR_017293</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All images were aligned and summed using MotionCor2 software ( Zheng et al. , 2017) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>MotionCor2</b></div>
              <div>suggested: (MotionCor2, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016499">SCR_016499</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The S trimer portion without the up RBD was rather stable , could be locally refined to 3.3 Å using local refinement in CryoSparc with non-uniform refinement option chosen .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>CryoSparc</b></div>
              <div>suggested: (cryoSPARC, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016501">SCR_016501</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the missing loop regions in S1 subunit , we either built the homology model based on SARS-CoV S structure ( PDB: 6CRW ) ( Kirchdoerfer et al. , 2018 ) through SWISS-MODEL webserver ( Waterhouse et al. , 2018) , or built the loop manually according to the density in COOT ( Emsley and Cowtan , 2004) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>COOT</b></div>
              <div>suggested: (Coot, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014222">SCR_014222</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the FP region , we first built the homology model by Modeller tool within Chimera by using MERS-CoV S structure ( PDB: 6NB3 ) as template ( Pettersen et al. ,</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Modeller</b></div>
              <div>suggested: (MODELLER, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008395">SCR_008395</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We then refined the combined model against our 3.8 Å resolution SARS-CoV-2 S-ACE2 map using Rosetta and Phenix .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Phenix</b></div>
              <div>suggested: (Phenix, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014224">SCR_014224</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">( G ) Protomer interaction interface analysis of S-ACE2 by PISA .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>PISA</b></div>
              <div>suggested: (PISA, <a href="https://scicrunch.org/resources/Any/search?q=SCR_015749">SCR_015749</a>)</div>
            </div>
          </td></tr></table>
      


      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 OddPub: Thank you for sharing your data.


      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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

    1. Malcolm X

      Hello all! Please As you read, please record three (3) annotations. Here are few ways to use annotations:

      1. Record a question that this reading sparked in your mind (add the tag “raised a question”)
      2. Leave a simple question mark (?) in the margins next to a passage or sentence that you found confusing (no tag needed)
      3. Share the dictionary definition of an unfamiliar word (I recommend Oxford English Dictionary online) or your research on an unknown allusion (add the tag “gloss”)
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      6. Respond to another participant's question or comment. Start a conversation!
    1. SciScore for 10.1101/2020.07.07.20148106: (What is this?)

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

      Table 1: Rigor

      <table><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Institutional Review Board Statement</td><td style="min-width:100px;border-bottom:1px solid lightgray">Written informed consent obtained from all participants in this study and was approved by the following IRBs: 1 ) IRB# SUNY:269846 .</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">In this regard , it is interesting to note that a bruton tyrosine kinase ( BTK ) inhibitor , that targets Fc-receptor signaling in macrophages , is being tested in a randomized clinical trial 32 .</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Subdividing the subjects by sex did not reveal any statistical difference in IgG levels at any of the disease stages , although hospitalized females in the non-ICU setting had significantly lower antibody levels than ICU/deceased patients , whereas the difference in males was not significant ( Fig . 2f) .</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><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</td></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n=87) and were absent in the negative controls.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>CoV-2 Spike protein or Nucleocapsid protein specific IgG</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Other isotype antibodies (IgA, IgG1-4) were also detected.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>IgA, IgG1-4</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CoV-2 infection2-6 To predict protection against CoV-2, it is critical to understand the quantity, quality and duration of the antibody responses during different stages of COVID-19 and in the convalescent period.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>CoV-2</div> <div>suggested: (Abcam Cat# ab272504, AB_2847845)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In this assay, we immobilized biotinylated CoV-2 Spike protein receptor binding domain (RBD) or the Nucleoprotein (N) on streptavidin beads, to detect specific antibodies from patient plasma (Fig.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>CoV-2 Spike protein receptor binding domain (RBD)</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Different antibody isotypes were measured using anti-Ig (IgG, IgA, IgM) specific secondary antibodies conjugated to a fluorescent tag (Fig. 1a).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-Ig ( IgG</div> <div>suggested: None</div> </div>

            <div style="margin-bottom:8px">
              <div><b>IgA , IgM</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Using either anti-SRBD antibody or soluble ACE2-Fc, we show very high sensitivity in detecting Spike protein binding, down to picogram ranges (Fig. 1b).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-SRBD</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Furthermore, Nucleocapsid protein-specific IgG levels and S-RBD specific IgA positively correlated with S-RBD IgG antibodies (Supplementary Fig. 1b, c).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>S-RBD IgG</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Notably, IgG1 subclass antibody levels were comparable to total IgG levels whereas the other subtypes were relatively lower (Fig. 2b).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>IgG1</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To evaluate membrane expression of Spike protein, cells were stained with recombinant soluble ACE2-Fc fusion protein followed by a secondary staining with an anti-Fc antibody (Fig 3a).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-Fc</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 overexpression of ACE2-IRES-GFP or ACE2mKO2 was confirmed by staining with CoV-2 Spike-protein fused with mouse Fc (mFc) and antimFc secondary antibody (Supplementary Fig. 2a, b).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>antimFc</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Of note, there was a significant negative correlation between the number of days and the IgG or IgA to S-RBD, anti-nucleocapsid IgG or the NT50 values ( !" = -0.67) (Fig. 6d), suggesting a potential decline in antibody titers over time.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-nucleocapsid IgG</b></div>
              <div>suggested: (Imported from the IEDB Cat# 3E9, <a href="https://scicrunch.org/resources/Any/search?q=AB_2848062">AB_2848062</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization of the virus by antibodies (NAbs) is one of the goals to achieve protection against CoV-218.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>CoV-218</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">However, another study showed IgA antibodies, but not IgG, increased in severe patients28.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>IgA</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Although it will be important to follow the same individual subject convalescent plasma over time to better assess this finding, our data point towards a relatively short-lived antibody response to COVID-19.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>COVID-19</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">WT and ACE2 over-expressing HEK-293T were also stained with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Mouse IgG2a</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-S-RBD antibody and ACE2-Fc was tested both at 5 µg/mL starting concentration and in additional 5-fold serial dilutions.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Anti-S-RBD</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotype virus neutralization assay Three-fold serially diluted monoclonal antibodies including anti-SARS-CoV-2 Neutralizing human IgG1 Antibody from Acro Biosystems, NAb#3 (Fig 4D), Genscript clone ID 6D11F2, NAb#2 (Fig 4D) and Genscript clone ID 10G6H5, NAb#1 (Fig 4D)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>anti-SARS-CoV-2</b></div>
              <div>suggested: (Abcam Cat# ab272854, <a href="https://scicrunch.org/resources/Any/search?q=AB_2847844">AB_2847844</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>human IgG1</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Percent infection obtained was normalized samples derived from cells infected with CoV-2 or SARS pseudotyped virus in the absence of plasma, ACE2-Fc or monoclonal antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>ACE2-Fc</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In addition, we also developed SARS Spike protein pseudotyped lentivirus, which similarly infected 293-ACE2 cells at almost 100% efficiency at higher virus supernatant volumes (Fig. 3f)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>293-ACE2</b></div>
              <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_DR94">CVCL_DR94</a></div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK-293T cells (ATCC; mycoplasma-free low passage stock) were transfected with the expression plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol as previously described33.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>HEK-293T</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Generating human ACE2 over-expressing cells Wildtype ACE2 sequence was obtained from Ensembl Gene Browser (Transcript ID: ENST00000252519.8) and codon optimized with SnapGene by removing restriction enzyme recognition sites necessary for subsequent molecular cloning steps, preserving the amino acid sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>Ensembl Gene Browser</b></div>
              <div>suggested: None</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>SnapGene</b></div>
              <div>suggested: (SnapGene, <a href="https://scicrunch.org/resources/Any/search?q=SCR_015052">SCR_015052</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry data were analyzed using FlowJo (BD biosciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>FlowJo</b></div>
              <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>GraphPad Prism</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
      
            <div style="margin-bottom:8px">
              <div><b>GraphPad</b></div>
              <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
            </div>
          </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Author contributions M.D., L.K. and D.U. conceived, designed the experiments. M.D., L.K., L.P., M.Y. and R.H. carried out the experiments. B.T.L. designed the clinical research study on UConn Healthcare workers and M.K. recruited participants and executed clinical protocols. R.G. and O.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
            <div style="margin-bottom:8px">
              <div><b>UConn Healthcare</b></div>
              <div>suggested: None</div>
            </div>
          </td></tr></table>
      

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