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    1. 2. Worms A worm is similar to a virus; the difference is that worms spread on their own instead of attaching to a program and infecting it and others. A lot of the time, worms spread over a network, exploiting a vulnerability to jump from machine to machine. As they continue to recursively spread, worms infect machines at a faster rate. This wastes the network's bandwidth at a minimum, while nastier worms can spread ransomware or other problems across an entire business network.

      AAAAA

    2. 10. Exploits and Vulnerabilities While not a form of malware, exploits and vulnerabilities are important terms in online security. Because no programmer or software is perfect, every program, OS, and website has some kind of vulnerability. Malicious actors work to find these flaws so they can exploit them to run malware or similar. advertising function refreshcontentwordcount9(){ if(contentwordcount9Changed == 'false'){ googletag.cmd.push(function(){ googletag.pubads().refresh([contentwordcount9]); googletag.display('div-gpt-ad-1555342976270-7'); }); contentwordcount9Changed = 'true'; }; }; For example, say someone discovered a bug that let you create a new admin account with no password in Windows by following certain steps. Someone could write malware to run these steps on someone's PC, get admin access, and then wreak havoc. The best way to stay safe from these threats is keeping your OS and all software up-to-date. Developers patch these problems as they find them, so staying on the latest version keeps you safe from old and known exploits.

      WWWWW

    3. 8. Rootkit A rootkit (a term which merges the admin "root" account on Unix systems and the "kit" they use) is a type of malware that gains access to restricted parts of a computer and then disguises or otherwise hides itself. Typically, a rootkit gets installed when the attacker has admin (or root) access to a machine. Once the rootkit is installed, it has privileges to do whatever the owner wants on the system. Rootkits abuse this to hide their intrusion—for example, it might cloak its presence from the installed antivirus app. Obviously, a piece of malware having complete control over your system is quite dangerous. A lot of the time, you'll have to completely reinstall the OS to get rid of a rootkit.

      SSSSS

    4. 5. Spyware Spyware is another type of malware that can take several forms. It refers to programs that track your computer usage for some purpose and reports it back to an entity. Most programs—and even operating systems like Windows 10—collect data about your usage and report it back to the developer. They use this to improve their tools with real-world data. Proper spyware is distinguished by the fact that it collects this data without letting the user know. advertising function refreshcontentwordcount5(){ if(contentwordcount5Changed == 'false'){ googletag.cmd.push(function(){ googletag.pubads().refresh([contentwordcount5]); googletag.display('div-gpt-ad-1555342976270-3'); }); contentwordcount5Changed = 'true'; }; }; While spyware often collects your data for advertising purposes, nastier spyware can also collect sensitive information like login credentials. Extreme spyware includes keyloggers, which are programs that record every keystroke you make on your machine.

      DDD

    1. Utterly encapsulating gapless dark ambient experience.

      Now there's a touchstone for the ages

    2. Terrifying in its bleakness, Elegy is a record dedicated to its portrayal of loneliness, telling a story with firm roots in tension contextualised by the title’s disheartening implications.

      A dedicated sponge in the blank countenance of empty space and flat time, I'd say.

    1. 10 Principles of Accessibilities:

      1. Blindness (covers most of accessibility issues through testing)
      2. Images (Alt-Text)
      3. Tag hamburger menus
      4. Don't place important content out of the way
      5. Test for accessibility with real users.
      6. Don't disable zoom in mobile interfaces.
      7. Accessibility is cheaper when done up front.
      8. Be aware of visual bias.
      9. Check mobile accessibility separately.
      10. Embrace all access attitude.
    1. SciScore for 10.1101/2021.01.13.21249429: (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">Informed consent was obtained from the patient, or next of kin if the patient was unable to give consent.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">All experiments were performed in both female and male mice.</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">Lung lysates were immunoprecipitated with a rabbit anti-mouse thrombomodulin antibody (ab230010, Abcam) attached to protein G conjugated Dynabeads (10004D, Thermo Fisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-mouse thrombomodulin</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blots were blocked with 5% BSA for 1 h and incubated overnight with mouse anti-6X His tag antibody (27E8, 2366, Cell Signaling).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-6X</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing and incubating with anti-mouse HRP conjugated secondary antibody (NA931, Sigma), proteins were visualized using ECL plus detection reagents (GERPN2232, Sigma).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-mouse HRP</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>GERPN2232</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blots were stripped with Re-Blot Plus Strong solution (2504, Millipore), blocked, and probed with antithrombomodulin antibody followed by anti-rabbit HRP conjugated secondary antibody (711035-152, Jackson Immuno Research).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>antithrombomodulin</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-rabbit HRP</div> <div>suggested: (Jackson ImmunoResearch Labs Cat# 711-035-152, RRID:AB_10015282)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Total thrombomodulin in lung lysates was done as above and correlated to loading control, anti-Gapdh HRP conjugated antibody (ab9482, Abcam).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-Gapdh HRP</div> <div>suggested: (Abcam Cat# ab9482, RRID:AB_307272)</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">Mice for other experiment came from in house breeding on a C57BL6/J background.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>C57BL6/J</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 density was quantified with ImageJ (NIH).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ImageJ</div> <div>suggested: (ImageJ, RRID:SCR_003070)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical analysis was done in GraphPad Prism 8.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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

      One limitation of our assay is that the measured APC levels were close to the detection limit, hence, possible differences between groups would not be detected. In contrast, a recent study did not find changes in APC in critically ill COVID-19 patients (36). However, reduced levels of APC are found in a majority of patients in sepsis and are associated with increased risk of death (42-45). APC formation may be impaired because of down-regulation or shedding of thrombomodulin induced by inflammatory cytokines (46), and, as we hypothesize herein, by ANGPT2 binding to thrombomodulin. Recent data show that circulating thrombomodulin was elevated in critically ill COVID-19 patients, suggesting shedding of thrombomodulin from the endothelium (15, 36). Furthermore, Goshua et al, reported that soluble thrombomodulin correlates with mortality in critically ill COVID-19 patients (36). In the current study, we noted that injection of ANGPT2 in mice resulted in the loss of thrombomodulin in lung tissue (Fig. 3). Further studies are needed to investigate if this is a direct or indirect effect of ANGPT2. To investigate if ANGPT2 can directly affect coagulation in vivo, we performed several experiments in mice. One simple but highly relevant experiment to evaluate coagulation is tail bleeding time (47). In these experiments, recombinant ANGPT2 and ANGPT1 fragments corresponding to the thrombomodulin and TIE2 binding region were injected before the measurement of tail bleeding time. These ex...


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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 10.1101/2021.01.13.426626: (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 animal studies were approved by the Institutional Animal Ethics committee (IAEC) (RR/IAEC/61-2019, Invivo/GP/084).</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 and Guinea Pig Immunizations Immunizations of BALBc mice (n=5/group, female, 3-4 weeks old, ~16-18 g) and Hartley strain guinea pigs (n=5/group, female, 6-8 weeks old, ~300 g) were performed with freshly adjuvanted (AddaVax™ (vac-adx-10)) protein (1:1 v/v Antigen : AddaVax™ ratio per animal/dose, 20 µg protein in 50 µl PBS (pH 7.4) and 50 µl AddaVax™) (InvivoGen, USA).</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 expression levels were checked using a dot blot analysis with Anti-his tag antibodies conjugated with HRP enzyme.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Anti-his tag</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SPR-binding of hCMP-mRBD analyte to immobilized ACE2-hFc/CR3022 hCMP-mRBD protein kinetic binding studies to ACE2-hFc and CR3022 antibody were performed on a ProteOn XPR36 Protein Interaction Array V.3.1 (Bio-Rad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>CR3022</div> <div>suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Next, three washes were performed (200 µL of PBST/well) and anti- Human IgG secondary antibody (Sigma-Aldrich)</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;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">Stable CHO and HEK293 cell lines expressing hCMP-mRBD were constructed and the corresponding protein was as immunogenic as the protein expressed from transient transfection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>CHO</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>HEK293</div> <div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The adherent Flp-In™-CHO cells were grown in Ham's F-12 Nutrient Mix media (Thermo Fisher Scientific Catalog #: Cat # 11765054) supplemented with 10% FBS, 100 U/ml Penicillin-Streptomycin and 100 µg/ml</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Flp-In™-CHO</div> <div>suggested: RRID:CVCL_U424)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The clone was confirmed by sequencing. vector using HindIII site containing forward primer (5’— Generation of adherent polyclonal Flp-In stable lines T25 flasks (5 ml media) having either adherent Flp-In™-293 or Flp-In™-CHO cells (~80 % confluent) were co-transfected with pOG44 (10 µg) and hCMP-mRBD-HRV-Tg- pcDNA5/FRT/TO (5µg) plasmid DNA using 35 µg of Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher Scientific, Cat # 11668030) in serum free media as per the manufacturers instruction for 4 hr.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Flp-In</div> <div>suggested: RRID:CVCL_U423)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(Thermofisher Scientific Cat# 10687010) for Flp-In™-293 and 750 µg/ ml for Flp-In™-CHO cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Flp-In™-293</div> <div>suggested: RRID:CVCL_U421)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The virus-serum incubated premix samples were serially diluted and plated in duplicates in VeroE6 cell containing 96 well plate (104/well) and cultured for 48/96 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>VeroE6</div> <div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK293T cells were transiently transfected with plasmid DNA pHIV-1 NL4.3Δenv-Luc and Spike-Δ19-D614G by using Profection mammalian transfection kit (Promega Inc) following the instructions in the kit manual.</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">Patient derived convalescent sera (n = 40) were tested for neutralization in both 293T-ACE-2 and Vero/TMPRSS2 cells whereas animal sera were tested only in Vero/TMPRSS2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero/TMPRSS2</div> <div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</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 total binding titer in ELISA measured against whole protein (ie; 42149 (Alhydrogel+CpG), 16048 (AddaVax™)) were 2.4 - 4.5 fold higher compared to guinea pig sera (GMT ID50: Additionally, we performed a dose sparing study involving 5 μg hCMP-mRBD adjuvanted with AddaVax™.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>AddaVax™</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The unbound tagless proteins concentration was determined by absorbance (A280) using NanoDrop™2000c with the theoretical molar extinction coefficient calculated using the ProtParam tool (ExPASy)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ProtParam</div> <div>suggested: (ProtParam Tool, RRID:SCR_018087)</div> </div> <div style="margin-bottom:8px"> <div>ExPASy</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reference-free 2D classification using single-particle analysis The evaluation of micrographs was done with EMAN 2.1 (58).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>EMAN</div> <div>suggested: (EMAN, RRID:SCR_016867)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reference free 2D classification of different projections of particle were calculated using simple_prime2D of SIMPLE 2.1 software (59).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SIMPLE</div> <div>suggested: (SIMPLE, RRID:SCR_009389)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical Analysis The p values for ELISA binding titers, neutralization titers, ACE2 receptor competition titers were analysed with a two-tailed Mann-Whitney test using the GraphPad Prism software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


      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. SciScore for 10.1101/2021.01.13.426436: (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 received free access to food and water and were handled according to the approved protocols of the Animal Committee of Osaka University (Suita, Osaka, Japan) as No.28-021-028, the Ethics Committee for Animal Experiments of the Safety Research Institute for Chemical Compounds Co. Ltd. (Sapporo, Hokkaido, Japan) as No. AN20200617-03 and the Animal Committee of KAC Co. Ltd. (Kusatsu, Shiga, Japan) as No. 20–0508.</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">Animals The rat study was performed using 7-week-old male Crl: CD (SD) rats (Charles River Laboratories Japan Inc., Kanagawa, Japan).</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">Anti-COVID19 spike protein (S1+S2) antibody titer at 2, 4, and 6 weeks after the first injection was measured using enzyme-linked immunosorbent assay (ELISA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Anti-COVID19 spike protein (S1+S2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This meant that the dose-dependent anti-SARS-CoV-2 spike protein (S1+S2) antibody production was induced as described in our previous study using the OVA expression plasmid DNA4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-SARS-CoV-2 spike protein (S1+S2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All wells were washed with PBST (200 l/well) seven times, and incubated with 1:1000 diluted Amersham ECL anti-rat IgG horseradish peroxidaselinked species-specific whole antibody (NA935, GE Healthcare UK Limited, UK) or Amersham ECL anti-mouse IgG horseradish peroxidase-linked species-specific whole antibody (NA931, GE Healthcare UK Limited, UK) in the blocking buffer for 3 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-rat IgG</div> <div>suggested: (GE Healthcare Cat# NA935, RRID:AB_772207)</div> </div> <div style="margin-bottom:8px"> <div>anti-mouse IgG</div> <div>suggested: (GE Healthcare Cat# NA931, RRID:AB_772210)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 binding inhibition assay To analyze the binding inhibition of S1+S2 and ACE2 by neutralizing antibodies in the immunized rat and mouse serum, 96-well plates were coated with human ACE2 recombinant protein (1g/ml, mFc tag; # 83986, Cell Signaling Technology), and then blocked using PBS containing 5% skim milk for 2 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ACE2</div> <div>suggested: (GenWay Biotech Inc. Cat# GWB-243C96, RRID:AB_10280272)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Wells were washed with PBST and then incubated with anti-His antibody conjugated with HRP (GTX21187, GeneTex, Inc., Irvine, CA) for 2 h at room temperature.</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">Sixteen weeks after the first administration, sera were collected from immunized mice and tested for antibody induction against 2019-nCoV spike protein (S1+S2) and the RBD region of the spike protein, as well as for the inhibition of binding to the recombinant hACE2 protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>2019-nCoV spike protein ( S1+S2</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">Pseudoviruses were incubated with a series of dilutions of inactivated rat serum for 1 h at 37 °C and then added to Vero E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero E6</div> <div>suggested: RRID:CVCL_XD71)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The homogenate solution was serially diluted 10 folds in DMEM containing 2% FBS and loaded on VeroE6/TMPRSS2 cells to determine the value.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>VeroE6/TMPRSS2</div> <div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div> </div> </td></tr><tr><td style="min-width:100px;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">The mouse study was performed using 8-10 weeks old C57BL/6NCrSlc mice (C57BL/6N) (Japan SLC,Inc.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>C57BL/6NCrSlc</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>C57BL/6N</div> <div>suggested: RRID:MGI:5651595)</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">ELISA Precisely 1 g/ml of recombinant 2019-nCoV spike protein (S1+S2) (ECD, His tag) (BLPSN0986P, BETA Life Sciences, Fairfield, NJ, USA) was immobilized on a 96-well plate (442404,</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>BETA</div> <div>suggested: (BETA, RRID:SCR_007556)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Percentage inhibition of immunized blood serum at various time points was calculated and plotted using GraphPad Prism software, from which the neutralization titer at ID75 was derived.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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 10.1101/2021.01.13.426537: (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">In additional experiments, the anti-CS antibody was added to trypsin-treated LV(CoV2-S) for 4 hrs at 37°C, followed by incubation with anti-CS sdAb (100ng/ml) for 1 hrs on ice.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-CS</div> <div>suggested: (LifeSpan Cat# LS-C128175-100, RRID:AB_11181559)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The blocked PVDF membrane was probed with the sdAb (having both 6x(HIS)-tag and biotin- tag) and then monoclonal anti-6x(HIS)-tag antibody was probed followed by detection with anti-mouse IgG conjugated with alkaline phosphatase (A3562: 1ml Sigma, USA) was used for developing the blots.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-6x(HIS)-tag</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-mouse IgG</div> <div>suggested: (Sigma-Aldrich Cat# A3562, RRID:AB_258091)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Since the spike construct contains the Flag tag, immunoblotting was performed to detect the presence of spike protein in supernatant collected 24 hours post transduction as well as in cell lysate of 72 hours post transduced Vero E6 cells using anti-flag mouse antibody (F1804-50UG, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-flag</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For detection of sdAb, anti-6x(HIS) antibody was incubated for 1 hr at RT followed by washing and incubation with antimouse antibody conjugated with alkaline phosphatase for 1.5 hrs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-6x(HIS</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>antimouse</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Different concentrations of S protein were incubated for 5 minutes to measure the binding affinity with the immobilised anti-CS and anti-RBD antibodies, and their dissociation kinetics was measured in PBS.</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;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">TG1 cells were then infected with helper phage M13K07 under static conditions at 30°C for 40 mins.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>TG1</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Above mixture was kept at RT for 15 minutes and co-transfected in HEK239T cells (Human Embryonic Kidney).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK239T</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Modified Vero cells (Vero E6) originated from kidney epithelial cells of African green monkey were used for measuring the internalization using concentrated pseudoviruses.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero</div> <div>suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Measuring neutralization of pseudoviruses by sdAbs Different dilutions of anti-CS and anti-RBD sdAbs were used for measuring the internalization of LV(CoV2-S) pseudovirus particles by Vero E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero E6</div> <div>suggested: RRID:CVCL_XD71)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Subsequently, the mix was added to 70-80% confluent Vero-E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero-E6</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Western Blotting To determine the specificity and immune reactivity of the sdAbs with the SARS-CoV2 S protein purified from transfected HEK293T cells or the one displayed by the produced pseudoviruses, the polypeptides in the prepared samples were resolved using SDS-PAGE and after transferring to PVDF membrane were immunoblotted with the sdAb containing 6x(HIS)-tag or their biotinylated versions after blocking of the membrane with 5% skim milk.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK293T</div> <div>suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)</div> </div> </td></tr><tr><td style="min-width:100px;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 available data was analysed using flowJo X software (TreeStar)44.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>flowJo</div> <div>suggested: (FlowJo, RRID:SCR_008520)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis and scaling of all the taken images were done using ImageJ software44</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ImageJ</div> <div>suggested: (ImageJ, RRID:SCR_003070)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical analysis were done using Graphpad prism 8.0.2(263).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Graphpad prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


      Results from Barzooka: We 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 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. Note: your arrow must be an HTMLElement (not an SVGElement). To use an SVG arrow, wrap it in a <div> tag with the data-popper-arrow attribute.
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    1. SciScore for 10.1101/2021.01.09.426032: (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">For use of patient serum ethical approval was granted by the Ethics Committee at the Medical Faculty of LMU Munich (vote 20-225 KB) in accordance with the guidelines of the Declaration of Helsinki.</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 (6 to 10 week-old) were purchased from Charles River Laboratories (Sulzfeld, Germany).</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 blots were blocked in a phosphate buffered saline (PBS) buffer containing 5% Bovine Serum Albumin (BSA) (Sigma- Aldrich, Taufkirchen, Germany) and 0.1% Tween-20 (Sigma-Aldrich, Taufkirchen, Germany) and incubated for 60 min with primary antibody, monoclonal anti-HAtag antibody (1:8000; HA Tag mAb 2-2.2.14, Thermo Fisher Scientific, Planegg, Germany) or COVID-19 patient serum (1:200).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-HAtag</div> <div>suggested: (InvivoGen Cat# ab-hatag, RRID:AB_391833)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Permeabilized cells were probed with a monoclonal antibody against the HA-tag epitope (1:1000; HA Tag mAb 2- 2.2.14, Thermo Fisher Scientific, Planegg, Germany) to detect SARS-2-S protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HA-tag epitope</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Non- permeabilized cells were stained with a mouse monoclonal antibody obtained against the S protein of SARS-CoV-1 (SARS-1-S; 1:200; GeneTex) before fixation with PFA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARS-1-S</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Polyclonal goat anti-mouse secondary antibody (1:1000; Life Technologies, Darmstadt, Germany) was used to visualize S- specific staining by red fluorescence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-mouse</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To test for the presence of neutralizing anti-SARS- CoV-2-S serum antibodies we used surrogate virus neutralization test as described before with slight modifications (25).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-SARS- CoV-2-S serum</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were extensively washed with phosphate- buffered saline/0.05% Tween-20 (PBST), followed by incubation for 1 h at 37 °C with an HRP- conjugated anti-His-tag antibody (1.2 µg/ml; clone HIS 3D5).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-His-tag</div> <div>suggested: (MBL International Cat# D291-3, RRID:AB_10597733)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To remove background effects, the mean percentage of inhibition from non-specific mouse serum (Invitrogen) was deducted from sample values and neutralizing anti-SARS-CoV2-S antibodies titres were determined as serum dilution that still had binding reduction > mean + 2 SD of values from sera of vehicle-treated mice.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-SARS-CoV2-S</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After incubation, we fixed the cells with 4% formaldehyde/phosphate-buffered saline (PBS) and stained the https://www.sinobiological.com) and a secondary peroxidase-labeled goat anti-rabbit IgG (Dako, cells with polyclonal rabbit anti-SARS-CoV antibody (Sino Biological, https://www.agilent.com).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-rabbit IgG</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-SARS-CoV</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">Vero cells (ATCC CCL-81) were maintained in Dulbecco`s Modified Eagle’s Medium (DMEM), 10% FBS and 1% MEM non-essential amino acid solution (Sigma-Aldrich, Taufkirchen, Germany).</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">Human A549 cells (ATCC® CCL-185™) (LGC standards) were maintained in DMEM with high glucose and 10% FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>A549</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Human HeLa cells (ATCC CCL-2) were maintained in Minimum Essential Medium Eagle (MEM) (Sigma-Aldrich, Taufkirchen, Germany), 7% FBS and 1% MEM non-essential amino acid solution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HeLa</div> <div>suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Human HaCat cells (CLS Cell Lines Service GmbH, Eppelheim, Germany) were maintained in DMEM, 10% FBS, 1% MEM non-essential amino acid solution and 1% HEPES solution (Sigma-Aldrich, Taufkirchen, Germany).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HaCat</div> <div>suggested: CLS Cat# 300493/p800_HaCaT, RRID:CVCL_0038)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To obtain vaccine preparations, recombinant MVA–SARS-2-S were amplified on CEF or DF-1 cell monolayers grown in T175 tissue culture flasks, purified by ultracentrifugation through 36% sucrose and reconstituted to high titre stock preparations in Tris-buffered saline pH 7.4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>DF-1</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We then added 50 μL of virus suspension (400 plaque-forming units) to each well and incubated at 37°C for 1 h before placing the mixtures on Vero-E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero-E6</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cytopathic effects (CPE) on VeroE6 cells (ATCC CRL1586) were analyzed 4 days after infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>VeroE6</div> <div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div> </div> </td></tr><tr><td style="min-width:100px;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">Female BALB/c mice (6 to 10 week-old) were purchased from Charles River Laboratories (Sulzfeld, Germany).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>BALB/c</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;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 incubation, we fixed the cells with 4% formaldehyde/phosphate-buffered saline (PBS) and stained the https://www.sinobiological.com) and a secondary peroxidase-labeled goat anti-rabbit IgG (Dako, cells with polyclonal rabbit anti-SARS-CoV antibody (Sino Biological, https://www.agilent.com).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>https://www.sinobiological.com</div> <div>suggested: (Sino Biological, RRID:SCR_003697)</div> </div> <div style="margin-bottom:8px"> <div>https://www.agilent.com</div> <div>suggested: (Agilent Technologies, RRID:SCR_013575)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Next, cells were permeabilized using Intracellular Staining Permeabilization Wash Buffer (Perm Wash buffer; Biolegend; dilution 1:10), and stained intracellularly in 100 µl/well of anti-mouse IFN-γ (clone XMG1.2, 1:200, Biolegend) plus anti-mouse TNF-α (clone MP6-XT22, 1:200, Biolegend) diluted in Perm Wash buffer for 30 min at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Biolegend</div> <div>suggested: (BioLegend, RRID:SCR_001134)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For each antibody, single colour controls were prepared using OneComp eBeads™ Compensation Beads (eBioscience, Thermo Fisher Scientific) and cells for the viability dye Zombie Aqua.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>OneComp</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data was acquired by the MACSQuant VYB Flow Analyser (Miltenyi Biotec) and analyzed using FlowJo (FlowJo LLC, BD Life Sciences, Ashland, OR, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>FlowJo</div> <div>suggested: (FlowJo, RRID:SCR_008520)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were prepared using GraphPad Prism version 5 (GraphPad Software Inc., San Diego CA, USA) and expressed as mean ± standard error of the mean (SEM).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> <div style="margin-bottom:8px"> <div>GraphPad</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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.

  2. Jan 2021
    1. but you'll find that wherever you have an img tag inside a link, the image will have a red border under it.
    1. SciScore for 10.1101/2021.01.10.426120: (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">HexaPro Spike was captured through its C-terminal his-tag over an anti-his antibody surface.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-his</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The binding affinities of full IgG antibodies to SARS-CoV-2 spike protein were determined using surface plasmon resonance (SPR) and a BIAcore T200 instrument (GE Healthcare) at 25°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>SARS-CoV-2 spike protein</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">DMEM supplemented with anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) was added to the infected cells and they were cultured overnight as described above.</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>I1</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Ca distance calculation The Ca distances per residue between superimposed ligand-free and antibody-bound NTD were calculated by Python 3.8.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>antibody-bound NTD</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">They were expressed in Human Embryonic Kidney (HEK) 293 Freestyle cells (Invitrogen) in suspension culture using serumfree media (Invitrogen) and transfected into HEK293 cells using polyethyleneimine (Polysciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>HEK293</div> <div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV-2-spike (kindly provided by Dr. Peihui Wang, Shandong University, China) using FuGENE 6 (Promega).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK293T</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, Vero E6 cells were seeded in a 96-well plate at a concentration of 2 × 104 cells per well.</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">The data was processed and fit to 1:1 single cycle model using the Scrubber 2.0 (BioLogic Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Scrubber</div> <div>suggested: (Scrubber2, RRID:SCR_015745)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting data were fit to a 1:1 binding model using Biacore Evaluation Software and were plotted using Graphpad.</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">The IC50 values were calculated using non-linear regression in GraphPad Prism.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To identify somatic hypermutations, each antibody sequence was aligned to the assigned germline gene using MUSCLE v3.8.31 (Edgar, 2004).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>MUSCLE</div> <div>suggested: (MUSCLE, RRID:SCR_011812)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Symmetry expansion and focused classification in RELION 3.1 (Scheres, 2012) was used for S2P spike complex with 2-51.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>RELION</div> <div>suggested: (RELION, RRID:SCR_016274)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Diffraction data were processed with XDS (Kabsch, 2010) and scaled using AIMLESS (Evans and Murshudov, 2013) from the CCP4 software suite (Winn et al., 2011).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>CCP4</div> <div>suggested: (CCP4, RRID:SCR_007255)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Structure refinement was performed with a 3.65 Å high-resolution cutoff using Phenix refine (Adams et al., 2010) and PDB-redo (Joosten et al., 2014) alternated with manual model building using Coot.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Coot</div> <div>suggested: (Coot, RRID:SCR_014222)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Ca distance calculation The Ca distances per residue between superimposed ligand-free and antibody-bound NTD were calculated by Python 3.8.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Python</div> <div>suggested: (IPython, RRID:SCR_001658)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RMSD calculation The root mean square deviation (RMSD) of antibody epitopes was calculated by PyMOL 2.4 between two superimposed NTDs – ligand-free and antibody-bound structures.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>PyMOL</div> <div>suggested: (PyMOL, RRID:SCR_000305)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequence entropy of betacoronavirus spike Human coronavirus reference amino acid sequences of OC43 (UniProt ID: QHO60594.1) sequence reported in Washington state were aligned using MAFFT software with default parameters (Katoh et al., 2002).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>MAFFT</div> <div>suggested: (MAFFT, RRID:SCR_011811)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cryo-EM data were processed and analyzed using cryoSPARC and Relion.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>cryoSPARC</div> <div>suggested: (cryoSPARC, RRID:SCR_016501)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cryo-EM and crystallographic structural statistics were analyzed using Phenix, Molprobity, EMringer and Chimera.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Phenix</div> <div>suggested: (Phenix, RRID:SCR_014224)</div> </div> <div style="margin-bottom:8px"> <div>Molprobity</div> <div>suggested: (MolProbity, RRID:SCR_014226)</div> </div> </td></tr></table>

      Results from OddPub: Thank you for sharing your data.


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


      Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on page 45. 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. Reviewer #3:

      The authors present a simple model that explains important outstanding controversies in the field of long-range gene regulation. These controversies include the fact that insulation boundaries tend to be weak; that acute inactivation of CTCF or cohesin (that leads to inactivation of insulation boundaries) leads to only minimal gene expression and that in live cells enhancer-promoter contacts appear not correlated with transcriptional bursting. The model involves a futile cycle of tag addition and removal from promoters, stimulation of more tag addition when tag is already present, and stimulation of tag addition by contacts with distal enhancers. The authors show that such a model explains all the above controversies, and indicate that the controversies are not inconsistent with mechanisms where long-range gene activation is driven by physical contacts with distal regulatory elements.

      The authors have explained and explored the properties of the model well. I have only minor comments.

      1) An alternative explanation for TAD-specific enhancer action is that an E-P interaction within a TAD (between two convergent CTCF sites), one that is brought about by extruding cohesin, is not equivalent to an interaction that occurs between two loci on either side of a CTCF site and that can be a random collision that is not mediated by extruding cohesin. In other words, two interactions can be of the same frequency but can be of a very different molecular nature. I agree that this model would not explain the results of the experiment where cohesin is acutely removed.

      2) In the beginning of the introduction the authors introduce TADS. I recommend that the authors present this in a more nuanced way: compartment domains also appear as boxes along the diagonal, an issue that has led some in the chromosome folding field to be confused. This reviewer believes TADS are those domains that strictly depend on cohesin mediated loop extrusion, whereas compartment domains are not. If the authors agree, perhaps they can rewrite this section?

      3) If I understand the model correctly, the nonlinearity arises because of the increased rate of tag addition when tag is already present. The authors then speculate histone modifications can be one such tag. However, there are only so many sites of modification at a promoter. Can the authors analyze how the possible range of tag densities affects performance of the model? Is the range required biologically plausible?

      4) Can the authors do more analysis to explore how rapid changes in gene expression may occur (e.g. upon signaling a gene may go up within minutes)? How much more frequent does the E-P interaction need to be for rapid switch to the active promoter state? Can the authors do an analysis where they change the rates of the futile cycle upon some signal: at what time scale does transcription then change (keeping E-P frequency the same)?

    1. SciScore for 10.1101/2021.01.07.425806: (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">Five antibodies were included from Brouwer et al.28, four of which (COVA1-12, COVA2-04, COVA2-07, COVA2-15) have low-resolution negative-stain electron-micrograph reconstructions available28.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>COVA2-15</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Yeast scFv display levels were evaluated prior to sorting (data not shown) by incubating the yeast with a 1:1000 dilution of Chicken anti C-MYC Epitope Tag Primary Antibody from Exalpha Biologicals Inc.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti C-MYC</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Catalog No. ACMYC), followed by incubation with a 1:1000 dilution of Goat anti-Chicken IgY (H+L) Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 647 from Thermo Fisher Scientific (Catalog No. A32933</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-Chicken IgY</div> <div>suggested: (Thermo Fisher Scientific Cat# A32933, RRID:AB_2762845)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD antigen binding during selection rounds 3 and 5 was evaluated using a 1:1000 dilution of rabbit anti-His FITC secondary antibody (Bethyl Laboratories).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-His FITC</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After a baseline step in assay buffer, RBD-coated biosensors were dipped in a 300 nM solution of ACE2 or assay buffer for 5 min before being dipped in a 100 nM solution of a given antibody.</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;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 protein expression and purification IgGs, ACE2-huFc-Avi, and coronavirus RBDs were expressed and purified from Expi293F cells cultured in 33% Expi293 Expression / 66% FreeStyle Expression medium (Thermo Fisher Scientific) and grown in baffled polycarbonate shaking flasks (Triforest) at 37 °C and 8% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Expi293F</div> <div>suggested: RRID:CVCL_D615)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 6 x 106 HEK293T cells cultured in D10 medium (DMEM, 10% fetal bovine serum (Gemini Bio-Products), 2 mM L-glutamine, 1% penicillin/streptomycin, and 10 mM HEPES [pH 7.0]) were transfected at ~50-70% confluency using calcium phosphate transfection20 with 5 lentiviral production plasmids:</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">HeLa cells overexpressing ACE219 were plated at 5,000 cells per well in clear-bottom white-walled 96-well plates 1 day prior to infection in D10 medium.</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">Medium was removed from HeLa/ACE2 cells and replaced with virus/inhibitor mixtures which were then incubated for 48 h at 37 °C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HeLa/ACE2</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">Data from neutralization assays were processed using GraphPad Prism 8.4.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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. free text and tag search

      free text and tag search

    1. SciScore for 10.1101/2021.01.04.20248897: (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">Study Approval This study was approved by the Ethics Committees of Centro Hospitalar e Universitário de Coimbra (CHUC-084-20).</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">The investigators were blinded to sample allocation during experiments.</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">Among the negative subjects, the median cohort age was 37.5 years (21-60 years), 9/19 (47%) were male, and 10/19 (53%) were female.</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 a washing step, 100 μL antihuman IgG antibody conjugated with horseradish peroxidase (Sigma-Aldrich) diluted at 1:5000 was added and the plates were incubated at 37 °C for 1 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>100 μL antihuman IgG antibody</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>antihuman IgG</div> <div>suggested: (GeneTex Cat# GTX28798, RRID:AB_374523)</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">Nucleocapsid-His recombinant Protein (YP_009724397.2(335Gly/Ala)) (Met1-Ala419) expressed in Baculovirus-Insect Cells with a polyhistidine tag at the C-terminus (Reference 40588-V08B); Spike protein S1 Subunit (YP_009724390.1) (Val16-Arg685) expressed in HEK293 Cells with a polyhistidine tag at the C-terminus (Reference 40591-V08H); Spike protein Receptor Binding Domain (RBD) (YP_009724390.1) (Arg319-Phe541) expressed in HEK293 Cells with a polyhistidine tag at the C-terminus.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK293</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;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">Negative SARS-CoV-2 group (n=19) included subjects with a negative qRT-PCR performed following risk-contact tracking and monitored by IgG serology (Alinity i, Abbott).</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">Samples were acquired in a LSRFortessaTM flow cytometer (BD Biosciences CA, USA) using the FACSDivaTM software v8.0 (BD Biosciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>FACSDivaTM</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data and Statistical analysis Microsoft Excel v.14.1.0</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Statistical analysis Microsoft Excel</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(Microsoft Corporation, WA, USA) and Prism 7 (GraphPad software, CA, USA) were used for plotting and statistical analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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. das Ziel, die Weltim Positiven zu verändern (vgl. https://www.biblio2030.de/)

      Dieses Ziel scheint mir etwas nichtssagend bzw. zu generisch zu sein ohne weitere Ausführungen. (Ich versuche z.B. jeden Tag einen positiven (und nicht negativen) Beitrag zum (Welt-)Geschehen zu machen und denke das gilt für viele andere auch.)

      Ebenfalls ist mir nicht klar, in welcher Relation dies alles mit der verlinkten Agenda 2030 steht; da sollte m.E. ein expliziter Bezug im Text gemacht werden.

    1. SciScore for 10.1101/2021.01.07.425674: (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 animal work in the current study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) with animal study protocol approval number TFBS2020-019 and Academia Sinica (approval number: 20-10-1526).</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">Immunization and challenge of hamsters The hamsters were randomized from different litters into four groups (n=10 for each group): hamsters were vaccinated intramuscularly with 2 injections of vehicle control (PBS), 1 or 5 µg of S-2P protein adjuvanted with 150 µg CpG 1018 and 75 µg aluminum hydroxide (alum), or adjuvant alone at 3 weeks apart.</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">Briefly, HEK293-hACE2 cells were seeded in 96- well white isoplates and incubated overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK293-hACE2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 10-fold serial dilutions of each sample were added onto Vero E6 cell monolayer in quadruplicate and incubated for 4 days.</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;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">Lung histopathology scoring system Statistical analysis The analysis package in Prism 6.01 (GraphPad) was used for statistical analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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

      There are a few limitations of this study. Firstly, the hamsters were challenged with SARS-CoV-2 at 29 days after the second immunization, a relatively short time that did not allow for the evaluation of the durability of protective antibodies. Secondly, none of the animals died in the pre-test or challenge study within the observation time. Thus, the model is not suitable for the evaluation of severe disease or mortality prevention but, rather, is appropriate for evaluation of the effects of immunization on viral challenge-induced moderate disease. Thirdly, nasal swab was not conducted, thus the study did not evaluate the vaccine’s ability to block viral entry or prevent upper respiratory tract infection. Further studies are needed to evaluate the durability of the protective antibody, the capacity of MVC-COV1901 to prevent severe disease, mortality, or viral entry. Methods Production of S-2P protein ectodomains from ExpiCHO-S cells SARS-CoV-2 (Wuhan-Hu-1 strain, GenBank: MN908947) S-2P proteins containing residues 1–1208 with a C-terminal T4 fibritin trimerization domain, an HRV3C cleavage site, an 8×His-tag and a Twin-Strep-tag were produced in ExpiCHO-S cells (ThermoFisher) as described previously [10]. Pseudovirus-based neutralization assay and IgG ELISA Lentivirus expressing the Wuhan-Hu-1 strain SARS-CoV-2 spike protein was constructed and the neutralization assay performed as previously described [10]. Briefly, HEK293-hACE2 cells were seeded in 96- well white isoplate...


      Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04695652</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Study to Evaluate MVC-COV1901 Vaccine Against COVID-19 in ...</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04487210</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Study to Evaluate the Safety and Immunogenicity of MVC-COV...</td></tr></table>


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


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


      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 10.1101/2021.01.06.20249035: (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">Data and samples were collected only from voluntary, non-remunerated, adult donors as described previously [27], and who provided written informed consent as part of routine donor selection and blood collection procedures, that were approved by the Ethics Advisory Council of Sanquin Blood Supply Foundation.</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">For anti-RBD IgG1 and IgG3, the calibrator consisted of recombinantly expressed IgG1 and IgG3 monoclonal antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-RBD IgG1</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>IgG3</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">The ACE2 was produced in HEK cells with a HAVT20 leader peptide, 10xhis-tag and a BirA-tag as described by Dekkers et al., [ 28].</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">African green monkey (Vero-E6) cells were added in a concentration of 2 × 10 4 cells per well and incubated for 3 days at 35°C in an incubator with 5% carbon dioxide.</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">For the remaining 676 samples collected between March 30 and August 14, the longitudinal changes in IgG antibody levels were analyzed by linear mixed effects modeling in R (v3.6.0) using the LmerTest package (v3.1.2).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>LmerTest</div> <div>suggested: (R package: lmerTest, RRID:SCR_015656)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis were carried out using Graphpad Prism 7.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Graphpad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></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:

      A limitation is the substantial uncertainty in the individual half-life estimates especially at the low end, since the available data will sometimes only cover a fraction of a half-life. Long-term maintenance of antibody production is provided by a pool of long-lived plasma cells and memory B-cells that can last a lifetime [35]. Several time scales may be identified, of which the longest- lasting can have half-lifes in the order of many years, and thus beyond scope of the current study [36]. It will be interesting to continue following the decline of anti-RBD IgG in time and investigate whether the longevity of the antibody response is similar to other coronaviruses, such as SARS-CoV that can still be detected in most individuals 3-years after recovery [37]. The antibody titer required for protection against re-infection in humans is not yet known, and has to be evaluated also in the context of a recall response upon re-infection. Recent studies provide evidence that upon infection, individuals develop SARS-CoV-2 specific memory B and memory T cells that can be detected for up to 240 days, with numbers of IgG memory B cells increasing over time and plateauing after ca. 150 days [20, 38]. The relationship between long-lasting antibody production and the T and B cell memory compartments requires more investigation. We also quantitatively examined the IgG1 and IgG3 subclass response and found that in our study population IgG1 appeared to be by far the dominant IgG subtype. This ...


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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. So I think with Dropbox specifically, and I’d say also at Notion now, we’re really looking at what the customer feedback is telling us to drive towards. So Notion has an amazing, what we call a tagging database, where all the conversations that come in, we tag them and have a very robust way of organizing that customer feedback. And that helps us drive our roadmap. So mixed in with customer feedback and a little bit of intuition, helps us determine what type of problems you should be solving that are going to really make the most impact for us long term.

      This is where I've felt the same way and the value of integrating support into the product as opposed to making the users go outside the product (e.g., support portal).

      Make it easy to get feedback - whether it's an idea or problem the user using our product runs into.

    1. Reviewer #3:

      In the manuscript by Kim et al., show that, beyond its roles of preventing somatic differentiation in the germline of embryos, Zn-finger protein PIE-1 also functions in the adult germline, where it is both SUMOylated as well as interacts with the SUMO conjugating machinery and promotes SUMOylation of protein targets. They identify HDA-1 as a target of PIE-1-induced SUMOylation. Here too, I find the claims interesting, however data is sometimes missing or does not fully support the claims.

      Main concerns:

      1) A key claim of novelty over previously proposed "glue" functions of SUMO is based on the fact that they find that temporally regulated SUMOylation of a very specific residue in a specific protein is affecting protein activity: The observation that "SUMOylation of HDA-1 only appears to regulate its functions in the adult germline" and not in the embryo together with the finding that "other co-factors such as MEP-1 are SUMOylated more broadly, these findings imply that SUMOylation in the context of these chromatin remodeling complexes, does not merely function as a SUMO-glue (Matunis et al., 2006) but rather has specificity depending on which components of the complex are modified and/or when."

      I find this claim poorly supported by the data. In fact, I find that the data supports that multiple SUMOylations contribute to formation of larger complexes: The His-SUMO IP (Fig 2B) brings down far more un-SUMOylated HDA-1 than SUMOylated. This argues for the presence of large complexes with different factors being SUMOylated and many bringing down unmodified HDA-1. The chromatography experiments (Fig 3B-C) also provide hits that are in complex and not direct interactors. Finally, HDA-1 SUMOylation is indicated to regulate MEP-1 interaction with numerous factors (Fig 3D). If all these factors are in one complex, it is hard to imagine how a single SUMO residue would mediate all of these simultaneously. It is quite likely (and not tested) that loss of HDA-1 SUMOylation leads to (partial?) dissociation of a large complex, rather than loss of individual interactions with the SUMO residue of HDA-1. Unlike claimed by the authors, there is no evidence that the "activity" of HDA-1 is regulated by SUMO modification.

      2) Based on loss of MEP-1/HDA-1 interaction upon pie-1 RNAi and smo-1 RNAi (Fig 4B), the authors conclude that "SUMOylation of PIE-1 promotes the interaction of HDA-1 with MEP-1 in the adult germline".

      The evidence that it is PEI-1 SUMOylation that is affecting MEP-1/HDA-1 interaction is fairly weak. In fact, based on Fig 4A, MEP-1 and HDA-1 interact without expression of PIE-1, and in PIE-1 K68R (sumoylation-deficient), although due to poor labeling of the panel it is not clear whether lane 1 and 4 refer to the WT pie-1 locus without tag or lack of pie-1.

      In 4B the HDA-1 band that is present in L4440 but not in pie-1 or smo-1 RNAi is very faint, and in our experience such weak signal is not linear i.e., bands can disappear or appear depending on the exposure. Importantly, according to the data, seemingly unmodified HDA-1 immunoprecipitated with MEP-1 (Fig 4B). This data contradicts the authors' claim that "These findings suggest that in the adult germline only a small fraction of the HDA-1 protein pool, likely only those molecules that are SUMOylated, can be recruited by MEP-1 for the assembly of a functional NURD complex".

      Furthermore, the fact that pie-1 and smo-1 depletion eliminate the interaction between HDA-1/MEP1 doesn't mean that the SUMOylation of pie-1 specifically is required for the interaction: perhaps un-SUMOylated pie1, and SUMOylation of something else, are both necessary for the interaction. The authors show that MEP-1 is also SUMOylated (Fig3C). When IP-ing GFP-MEP-1, they precipitate all its modified forms and associated factors. One alternative possibility for why smo-1 RNAi abolishes MEP-1/HDA-1 interaction is that MEP-1 SUMOylation is needed for interaction with HDA-1 (independently of pie-1). (On a side note, why are the authors not including MEP-1 SUMOylation in the model?)

      3) On page 13 the authors write: "These findings suggest that SUMOylation of PIE-1 on K68 enhances its ability to activate HDA-1 in the adult germline" and "We have shown that PIE-1 is also expressed in the adult germline where it engages the Krüppel-type zinc finger protein MEP-1 and the SUMO-conjugating machinery and functions to promote the SUMOylation and activation of the type 1 HDAC, HDA-1 (Figure 6)". Activation of HDA-1 is misleading and was never tested. If not performing in vitro assays for HDAC activity, the authors at least need to look at whether pie loss (degron) leads to acetylation of genomic HDA-1 targets and whether it affects HDA-1 (and/or MEP-1) recruitment to these sites. This could be done by ChIP-seq of HDA-1 and H3K9ac in WT and pie-1 degron animals.

    1. There is a dimension of personal preference to it. I don't like to expose more than strictly necessary to external consumers, because it makes it harder to track usages. If you find a bind:prop in a consumer, you know prop is used (which you already kind of knew since the prop is part of the "public" API of the component). Done. If you find a bind:this, you now need to track all usages of this this.
    1. SciScore for 10.1101/2020.12.31.424987: (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">METHOD DETAILS Human Sample Collection Informed consent was obtained for all study participants under IRB-AAAS9010 (Hong Kong 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">CPE from the resulting cell incubations were visually scored for each well in a blinded fashion by two independent observers.</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">4-3 is an anti-RBD antibody encoded by IGHV3-66 that does not compete with ACE2, and serves as a heavy chain gene-matched control.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-RBD</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>ACE2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Right panel: close-up views of the Fab:RBD interface for the eight IGHV3-53/3-66 antibodies superimposed on RBD.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>IGHV3-53/3-66</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">( D) Heavy chain genetic elements associated with the IGHV3-53/3- antibody class.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>IGHV3-53/3-</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">DMEM supplemented with anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL- 2700; ATCC) and was added to the inoculated cells.</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>I1</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For staining with the NHS-Biotin S-Trimer Protein probe, cells were mixed with 20nM un-labeled antigen and a monoclonal anti-FLAG-FITC marker (Monoclonal ANTI-FLAG M2-FITC antibody, Sigma-Aldrich, St. Louis, MO, USA) used to measure VL surface expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-FLAG-FITC marker</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>M2-FITC</div> <div>suggested: (Sigma-Aldrich Cat# F4049, RRID:AB_439701)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Samples were then washed 3x with staining buffer, and resuspended in a common mix containing the monoclonal anti-FLAG-FITC marker and a monoclonal anti-His-PE antibody (PE anti-His Tag Antibody, BioLegend, San Diego, CA, USA) to label surface expressed, antigen bound Fab.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-FLAG-FITC</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-His-PE</div> <div>suggested: (Miltenyi Biotec Cat# 130-098-810, RRID:AB_2751027)</div> </div> <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">Enrichment ratios were calculated by comparing sequence prevalence in each sorted libraries to the unsorted, Fab-expressing (VL+) antibody library.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>VL+</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were washed and 50uL of a secondary anti-human kappa light chain detection antibody (A18853, Invitrogen, Carlsbad, CA) was added to each well and incubated at room temperature for 1 hr.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-human kappa light chain</div> <div>suggested: (Thermo Fisher Scientific Cat# A18853, RRID:AB_2535630)</div> </div> <div style="margin-bottom:8px"> <div>A18853</div> <div>suggested: (Thermo Fisher Scientific Cat# A18853, RRID:AB_2535630)</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">Cell line Expi293F cell was purchased from Thermo Fisher Scientific.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Expi293F</div> <div>suggested: RRID:CVCL_D615)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell line Vero C1008 (Vero-E6 cell) was purchased from ATCC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero C1008</div> <div>suggested: ATCC Cat# CRL-1586, RRID:CVCL_0574)</div> </div> <div style="margin-bottom:8px"> <div>Vero-E6</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, expression vectors encoding the ectodomain of the SARS-CoV-2 S protein was transiently transfected into Expi293 cells and then purified five days post transfection using on-column purification methods.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Expi293</div> <div>suggested: RRID:CVCL_D615)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were grown to 80% confluency then used for transfection of pCMV3-SARS- CoV-2-spike (kindly provided by Dr. Peihui Wang, Shandong University, China) using FuGENE (Promega).</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;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">( F) The local resolution of the two final maps are shown generated through cryoSPARC using an FSC cutoff of 0.5; left: sample 1, right: sample 2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>cryoSPARC</div> <div>suggested: (cryoSPARC, RRID:SCR_016501)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, Illumina sequences were quality-filtered to improve read quality, followed by V(D)J gene identification and annotation of CDR3 regions using IgBLAST (Ye et al., 2013).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>IgBLAST</div> <div>suggested: (IgBLAST, RRID:SCR_002873)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, protein structures of IGHV3-53/3-66 antibodies complexed with RBD or spike were selected for analysis, and the buried surface area (BSA) (https://www.ebi.ac.uk/pdbe/pisa/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>https://www.ebi.ac.uk/pdbe/pisa/</div> <div>suggested: (PISA, RRID:SCR_015749)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Automated and manual model building were iteratively performed using real space refinement in Phenix (Adams et al., 2004) and Coot (Emsley and Cowtan, 2004) respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Coot</div> <div>suggested: (Coot, RRID:SCR_014222)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">EMRinger (Barad et al., 2015) and Molprobity (Davis et al., 2004) were used to validate geometry and check structure quality at each iteration step.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Molprobity</div> <div>suggested: (MolProbity, RRID:SCR_014226)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data was processed and fit to 1:1 single cycle model using the Scrubber 2.0 (BioLogic Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Scrubber</div> <div>suggested: (Scrubber2, RRID:SCR_015745)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">QUANTIFICATION AND STATISTICAL ANALYSIS IC50 calculations were reported using GraphPad Prism software (version 8.4.3).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry analysis was carried out using FlowJo software (version 10.4).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>FlowJo</div> <div>suggested: (FlowJo, RRID:SCR_008520)</div> </div> </td></tr></table>

      Results from OddPub: Thank you for sharing your data.


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


      Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on pages 51, 51, 51, 52, 52, 52 and 52. 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.

  3. Dec 2020
    1. SciScore for 10.1101/2020.12.30.424801: (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">After the cells were washed three times with PBS, incubated with serum free media with primary antibody anti-ACE2 (1:100</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> </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">For CSNPs-binding, hACE2-overexpressed Hek293 cells were incubated with 10 uM peptide for 1 hour and then treated and incubated with 5 uM SARS-CoV-1 S1 protein-His Tag (AcroBiosystems, S1N-C52H3-100UG, USA) for 24 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Hek293</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;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">For electrostatic surfaces isolation of the proteins, APBS and APBSrun plugins in Pymol and VMD, and for monitoring changes in the secondary structures of the peptides as function of time the sscache.tcl script was used in VMD.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Pymol</div> <div>suggested: (PyMOL, RRID:SCR_000305)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For molecular dynamics simulations GROMACS 2019.6 was used (66).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GROMACS</div> <div>suggested: (GROMACS, RRID:SCR_014565)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For CSNPs-binding, hACE2-overexpressed Hek293 cells were incubated with 10 uM peptide for 1 hour and then treated and incubated with 5 uM SARS-CoV-1 S1 protein-His Tag (AcroBiosystems, S1N-C52H3-100UG, USA) for 24 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>AcroBiosystems</div> <div>suggested: (ACRObiosystems, RRID:SCR_012550)</div> </div> </td></tr></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:

      To overcome this limitation, two research groups have stabilized the α1 helix by increasing the helical bundles and designed peptide biologics that could effectively inhibit SARS-CoV-2 cell entry (44, 45). However, this modification increases the size of these peptides by ~3-4 folds, increasing the cost of synthesis and formulation. We and others have previously used peptide stapling to enhance the target specificity of therapeutic peptides (28, 51). In fact, Fiarlie and his co-workers found that the helical-constrained compounds hold comparatively similar biological potencies in PPI as their parent proteins. They constructed four such compounds and proposed that downsizedconstrained peptides could be of great value in biological PPIs and medicine (52). Unfortunately, we could not synthesize the CSNP1 to compare its potency with CSNP2; however, CSNP3 (a relatively shorter version of CSNP1) was unable to bind SARS-CoV-2 S1 (Figure 5). This notion suggests that in addition to the structural integrity, optimum length and the featured pharmacophores are vital for CSNP to bind RBD. Together, we suggest that structure guided computational medicinal chemistry and click chemistry approaches might be useful in designing CSNPs to enhance tethering to their targets. Collectively, we suggest that CSNP2 and CSNP4 are stable peptide that deter the binding of ACE2 and S1 subunit of SARS-CoV-2 and could be potent antidote for COVID-19. Methods Peptides Designing Rationale and Synthesis The c...


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


      Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on page 28. 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 10.1101/2020.12.29.424779: (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">Mice studies were carried out at Akdeniz University Experimental Animal CareUnit under permission of the Local Ethics Committee for Animal Experiments at Akdeniz University with thesupervision of a veterinarian.</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></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">Serum samples were collected on days -1 (pre-bleed) and 42 (post 2nd vaccination) and assessed for anti-N, antigRBD, anti-dRBD anti-(N+gRBD) and anti-gS1 antibody responses by an IgG ELISA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-N, antigRBD, anti-dRBD anti-(N+gRBD</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-gS1</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After transfer, Western blot membranes were blocked with I-Block (Applied Biosystems, Carlsbad, CA) and recombinant proteins N, RBD, S1 and N+RBD were detected with anti-DYKDDDDK antibody (cat. no. 651503, BioLegend, for N, RBD or N+RBD)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-DYKDDDDK</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, anti-His antibody (for S1) and anti-RBD of S protein of SARC-CoV-2 monoclonal antibody (cat. no. MBS7135930, for RBD) or Human Novel Coronavirus Nucleoprotein (N) (1419aa) monoclonal Antibody (MyBioSource, cat. no. MBS7135930).</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">After washing, wells were incubated with anti-rabbit IgG antibody (Cat. no. MBS440123, MyBioSource, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-rabbit IgG</div> <div>suggested: (MyBioSource Cat# MBS440123, RRID:AB_10571491)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Serum samples were collected on days -1 (pre-bleed) and 42 (post 2nd vaccination) and assessed for anti-N, antiRBD, anti- N+RBD and anti-S1 domain antibody responses by an IgG ELISA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-N , antiRBD ,</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-S1 domain</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After transfer, Western blot membranes were blocked with I-Block (Applied Biosystems, Carlsbad, CA) and recombinant proteins detected with an anti-FLAG (N, S1 and N+S1) or anti-His Antibody (S2), or anti-SARS-COV2 COVID 19 Spike Protein Coronavirus Monoclonal Antibody (MyBioSource, cat. no. MBS2563837).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-SARS-COV2 COVID 19 Spike Protein Coronavirus</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">N protein from N. benthamiana plant was purified using anti-FLAG antibody resin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-FLAG</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Membranes were probed with anti-Flag (B) or anti-N protein monoclonal antibody (C).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-Flag ( B )</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-N protein</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">B: Western blot analysis of gRBD and dRBD using antiFlag antibody. pp Endo H, plant produced and purified Endo H protein with Molecular mass of ~30 kDa19. pp PNGase F, plant produced and purified PNGase F protein with Molecular mass of ~35 kDa19. C: Western blot analysis of gRBD and dRBD using commercial available anti-RBD antibody (MBS2563840, MyBiosource, USA);</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>antiFlag</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Membranes were probed with anti-His tag monoclonal antibody (B) or commercial available anti-RBD antibody (MBS2563840, MyBiosource, USA); M: color prestained protein standard (NEB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-His tag</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-RBD</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">Total protein content was estimated using the BioDrop and then analyzed by SDSPAGE and western blot.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>BioDrop</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The degradation of gRBD, dRBD or gS1 protein bands were calculated and quantified based on SDS- PAGE and WB analysis using highly sensitive Gene Tools software (Syngene Bioimaging, UK) and ImageJ software as described previously21.</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">GraphPad Prism software was used for all statistical analyses.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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. Wo sind die Produktionsstätten im ganzen Land, die Tag und Nacht laufen, um Leben zu retten und Leben zu ermöglichen? Bundeskanzlerin und Gesundheitsminister wussten lange genug, dass es die brauchen wird. Sie könnten auch jetzt noch eingreifen und die finanzielle und gesetzgeberische Macht des Staates, mit der sie Lockdowns verhängt und wirtschaftlich abgefedert haben, zum Aufbau neuer Produktionsanlagen nutzen.

      Chemie- und insbesondere Pharmaanlagen werden nicht in Tagen oder Wochen, sondern eher in Jahren gebaut. Dass wir überhaupt jetzt schon impfstoffe haben, ist nur deshalb möglich, weil Anlagen bereits vor der Zulassung gebaut wurden, damit ein Impfstoff dann möglichst schnell zur Verfügung stellt. Das bedeutet aber auch, dass jede dieser Anlagen ein Risiko darstellt. Es wäre halt betriebswirtschaftlich schwachsinnig, die Anlagen so auszulegen, dass innerhalb des ersten Monats eine Produktion durchläuft, die für die Welt reicht und danach die Anlage abzureißen.

    1. 2.3.1. Mail Objects SMTP transports a mail object. A mail object contains an envelope and content. The SMTP envelope is sent as a series of SMTP protocol units (described in Section 3). It consists of an originator address (to Klensin Standards Track [Page 11] RFC 5321 SMTP October 2008 which error reports should be directed), one or more recipient addresses, and optional protocol extension material

      The SMTP envelope is sent as a series of SMTP protocol units (described in Section 3). It consists of

      • an originator address (to which error reports should be directed),

      MAIL FROM that refers to the originator (a.k.a., reverse path, backward-pointing address) of the request

      • one or more recipient addresses,

      Multiple RCPT TO for each "to:" rfc822 message header in the mail data (see annotation)

      • and optional protocol extension material.

      DATA (see below)


      (See also envelope-vs-mail tags.)

    1. SciScore for 10.1101/2020.12.24.20248821: (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">Materials and methods Human samples and ethical declaration Anonymized samples from blood donors (n=100/sampling week) and pregnant women (n=100/sampling week) were randomly selected from their respective pools by the department of Clinical Microbiology, Karolinska University Hospital.</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">Design: In this cross-sectional prospective study, otherwise-healthy blood donors (n=2,100) and pregnant women (n=2,000) were sampled at random for consecutive weeks (at three intervals) between 14th March and 11th December 2020.</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">Secondary HRP-conjugated anti-human antibodies were diluted in blocking buffer and incubated with samples for 1 hour at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-human</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Secondary antibodies (from Southern Biotech) and dilutions used: goat anti-human IgG (2014- 05) at 1:10,000.</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">Briefly, we used a logistic regression over anti-RBD and -S training data (from n=595 historical blood donor controls and n=138 SARS-CoV-2 PCR+ individuals across the clinical spectrum) to model the relationship between the ELISA measurements and the probability that a sample is antibody-positive.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-RBD</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>antibody-positive</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">The RBD domain (RVQ – QFG) of SARS-CoV-2 was cloned upstream of a Sortase A recognition site (LPETG) and a 6xHIS tag, and expressed in 293F cells as described above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>293F</div> <div>suggested: RRID:CVCL_D615)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In vitro virus neutralisation assay Pseudotyped viruses were generated by the co-transfection of HEK293T cells with plasmids encoding the SARS-CoV-2 spike protein harboring an 18 amino acid truncation of the cytoplasmic tail2; a plasmid encoding firefly luciferase; a lentiviral packaging plasmid (</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">Approximately 15,000 HEK293T-ACE2 cells were then added to each well and the plates incubated at 37°C for hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK293T-ACE2</div> <div>suggested: None</div> </div> </td></tr></table>

      Results from OddPub: Thank you for sharing your data.


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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. We're born feeling. It's simple response to a stimulus. But it takes years of effort and discipline to subjugate our emotions to our reason, to be more than a dog salivating at the sound of a bell, to become worthy of the tag homo sapiens.

      Almost universal education and this is still a large scale problem.

    1. SciScore for 10.1101/2020.12.21.423721: (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 myocardial samples (right ventricle or atrium) were discarded material from surgical repair of congenital heart defects (ethical approval number 15/LO/1064 from the North Somerset and South Bristol Research Ethics 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">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 (ACE2, dilution 1:100; CD147, 1:500, 6x-HIS-tag (Invitrogen MA121315), 1:1000) were incubated for 16 hrs at 4°C. β-Actin was used as a loading control (Sigma, A5441, 1:10000)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ACE2</div> <div>suggested: (LifeSpan Cat# LS-C349-500, RRID:AB_1271970)</div> </div> <div style="margin-bottom:8px"> <div>6x-HIS-tag</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>β-Actin</div> <div>suggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)</div> </div> <div style="margin-bottom:8px"> <div>A5441</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">Where required, cells were incubated with the anti-CD147 neutralizing antibody (20 μg/mL).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-CD147</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PCs were pre-incubated with antiACE2 (20 μg/mL, as described before5) or anti CD147 (20 μg/mL) antibodies for 1 hat 37 °C and then exposed to the S protein (500 ng/mL - 2·9 nM) for 1 h at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>antiACE2</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti CD147</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">Western blot data were analyzed using the ImageJ software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ImageJ</div> <div>suggested: (ImageJ, RRID:SCR_003070)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistics Data were analyzed using Prism version 8.0 and expressed as individual values and as means ± standard error of the mean.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Prism</div> <div>suggested: (PRISM, RRID:SCR_005375)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


      Results from Barzooka: We 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 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 10.1101/2020.12.19.423584: (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">Then, endogenous human NPC1 and HSP90 were purified using immobilized Recombinant Protein G Resin (Generon) and µg of specific antibodies against NPC1 (Abcam, ab108921) or HSP90 (Enzo Life Sciences, ADI-SPA-835) respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HSP90</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>NPC1</div> <div>suggested: (Abcam Cat# ab108921, RRID:AB_11143218)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After that, plates were washed with PBST (PBS 0.1%Tween20) and the binding of NPC1 to SARS-CoV-2 N protein was detected with a rabbit anti-NPC1 antibody (1:2000), revealed with an anti-rabbit-horseradish peroxidase (HRP) (1:2000) using a colorimetric substrate (OPD) and finally, quantified by absorbance at nm in the EnSight multimode plate reader of PerkinElmer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-rabbit-horseradish peroxidase (HRP</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then, the binding of purified NPC1 protein to viral N protein was determined with an anti-NPC1 antibody revealed with an anti-rabbit-HRP.</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> </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">Materials and Methods Cell culture and viruses Human embryonic kidney cells 293T/17 (HEK 293T; ATCC-CRL-11268) were cultured in Dulbecco modified Eagle medium (DMEM) at 37 ºC and 5% CO atmosphere, supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 1X GlutaMAX (Thermo Fisher) and 10% heat-inactivated fetal bovine serum (FBS).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK</div> <div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Huh-7 Lunet C3 cells, a gift from T. Pietschman (Twincore, Germany), were cultured at 37 ºC in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 10mM HEPES, 1X NEAA and 10% of heat-inactivated fetal bovine serum (FBS).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Lunet C3</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression of tagged-N protein and EGFP in HEK 293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK 293T</div> <div>suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cytotoxicity assays Huh-7 cells were seeded in 96-well plates and incubated with DMEM containing each compound at concentrations ranging from 0 to 100 µM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Huh-7</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">To generate the SARS-CoV-2 N with N-terminal EGFP tag (EGFP-N), a codon optimized cDNA sequence for the ORF of SARS-CoV-2 N (NCBI reference sequence number: NC_045512) was cloned into the pEGFP- C1 (by GeneArt-Thermo Fisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GeneArt-Thermo Fisher Scientific</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blots then were developed using enhanced chemiluminescence reagent (Bio-Rad) and detected with ChemiDoc™ XRS Gel Imaging System using Image Lab™ software (Bio- Rad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Image Lab™</div> <div>suggested: (Image Lab Software, RRID:SCR_014210)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of SARS-CoV-2 N protein in the baculovirus system The sequence of the N protein published in the NCBI database was selected (GenBank accession number: 43740575 / NCBI reference sequence number: NC_045512).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>NCBI</div> <div>suggested: (NCBI, RRID:SCR_006472)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In order to determine the percentage of infected cells per condition, 8,000 cells/time point were scored using FACS Canto II flow cytometer (BD Sciences) and analyzed using 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><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis The experimental data was analyzed by one-way ANOVA by Graph Pad Prism software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Graph Pad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


      Results from Barzooka: We 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 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 10.1101/2020.12.18.423418: (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">The membrane was incubated with polyclonal rabbit anti-human SP-D primary antibody (1:1000; MRC Immunochemistry Unit, Oxford) for 1h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-human SP-D</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Next day, the wells were washed and then incubated with anti-sheep IgG-HRP antibodies or anti-His antibodies (Genetex, GTX628914, 0.5 µg/ml) (1:2000) for 2 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-sheep IgG-HRP</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the detection of RBD binding, the wells were further incubated with anti-mouse IgG antibody (Abcam, ab6728, 0.5 µg/ml) (1:2000) for 2 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-mouse IgG</div> <div>suggested: (Abcam Cat# ab6728, RRID:AB_955440)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, both ACE2transfected and non-transfected HEK293T cells (1x105 cells) were incubated with ACE2 antibody [N1N2], N-term (GeneTex, GTX101395) (1:250) for 1 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GTX101395</div> <div>suggested: (GeneTex Cat# GTX101395, RRID:AB_1240451)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following PBS washes, the cells were probed with goat anti-Rabbit IgG (H+L) CrossAdsorbed Secondary Antibody linked to Alexa Fluor 647 (Thermo Fisher Scientific) (0.6 l/100 l per tube) for 1 h at room temperature in dark.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-Rabbit IgG</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For binding experiments using rfhSP-D, SARS-CoV-2 S1 protein containing a C-terminal His-tag (Acro; S1N-C52H3) (5 g/ml) was tagged with anti-His antibody (Genetex; GT395) (1:100) at 4℃ for 1h and followed by pre-incubation with a series of two-fold dilutions of rfhSP-D (10 µg/ml) or mock (cells only) at 4℃ for 1h.</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> <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">After washing, coverslips were blocked with 2% w/v BSA for 1h and incubated with ACE2 antibody [SN0754] (1:250) (GeneTex, GTX01160), followed by Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (1:500) (Thermo Fisher Scientific) for 1 h at room temperature in dark.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ACE2</div> <div>suggested: (Thermo Fisher Scientific Cat# MA5-32307, RRID:AB_2809589)</div> </div> <div style="margin-bottom:8px"> <div>GTX01160</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Purified pseudotyped particles and cell lysate harvested at 48 and 72 h were analyzed via western blotting, and the expression level of SARS-CoV-2 spike protein was determined using anti-S1 monoclonal antibody (data not shown).</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;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">Cell culture and treatments Human embryonic kidney (HEK) 293T or HEK293T cells overexpressing ACE2 receptor (HEK293T-ACE2) were cultured in complete Gibco Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% v/v fetal bovine serum (FBS), 100 U/ml penicillin (Sigma-Aldrich) and 100 µg/ml streptomycin (Sigma-Aldrich), and left to grow at 37°C in the presence of 5% v/v CO2 for approximately 48 h before passaging.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK</div> <div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div> </div> <div style="margin-bottom:8px"> <div>293T</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T-ACE2 cells (1x105 cells) were incubated in DMEM incomplete medium with the mixture of SARSCoV-2 S1 protein, anti-His antibodies and rfhSP-D at 37℃ for 2 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK293T-ACE2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293TACE2 cells (HEK293T cells overexpressing ACE2 receptor) (0.5x105 cells) were preincubated with rfhSP-D (0, 5, 10 and 20 µg/ml) for 24 h and then washed twice with PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK293TACE2</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>HEK293T</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;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">Statistical Analysis GraphPad Prism 6.0 software was used to generate all the graphs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


      Results from Barzooka: We 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 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. Reviewer #1:

      My general assessment of this work is that it is full of good ideas and presents a novel and general approach to examine lipid remodeling in cells and perhaps subsequent transport of lipids, mainly to mitochondria, but it lacks the scientific rigor necessary to be fully confident that their conclusions firmly support their claims. Often, insufficient information about the methods are provided and the manuscript is hard to follow critically.

      More specific comments:

      1) I am surprised that acyl-CoAs are transported into cells. I don't know of any precedent for this. Usually fatty acids are imported into cells and then converted to acyl-CoAs as part of the mechanism of import. Could it be that the acyl--CoAs are hydrolysed before uptake only to be reformed inside the cells? I would suggest feeding the NBD-palmitate plus the lysolipids to the cells as a control to see whether this is the case.

      2) In fig 1 as an example they choose a region to blow up. As one can see there is a large variation, even in the blowups of mitochondrial labeling and if one looks at the originals the variation is confirmed. How have they chosen these areas? Furthermore, in figure 1 there is quite a bit of label with MLCL outside of the mitochondria, in particular in regions that they did not choose to blow up. What are these structures? Remodeling of MLCL is thought to take place in mitochondria.

      3) They speak of transport of lipids from ER to mitochondria, but in fact the demonstration of this is very weak from what they show in the time course in supp fig 1. I am also disturbed by the difference in patterns of the NBD-PA patterns in a and b. They should be the same, but there are problems, maybe focus? I would say anyway that there is no clear evidence that the NBD PA first appears in the ER then goes to mitos. It could be synthesized in both compartments from their data.

      4) The product characterization by TLC is insufficient. There are no standards, no characterization. Would they have seen the free NBD-palm by their methods?

      5) When they use mutants and find less "transport" the mitochondrial signal as seen by mitotracker is always more diffuse. This indicates to me that there is another problem.

      6) In fig 3 the fluorescent pictures do not correspond to what is seen in the quantification. There is more yellow in e than in h.

      7) How did they add cholesterol at 50 or 100 micromolar? It is soluble at less than 1 micromolar in aqueous solution. The cholesterol experiments are puzzling. From what we know about StAR protein it recognizes cholesterol not esters. There is no precedent for cholesterol ester transport into mitochondria. Can they rule out that the esters are transported to the surface of the mitochondria and the NBD-Palm cleaved off and transported into the mitochondria?

      8) The MAG and DAG experiments are overinterpreted. It could just be a kinetic problem since the MAG gets converted to DAG before TAG

      9) They compare to externally added NBD lipids, but we don't know which ones they used. Are they using short chain NBD phospholipids. I could not find this in their manuscript. If they do not have the same NBD-palm in the sn-2 position then the comparison is meaningless.

      10) The excitation and emission spectra of their probes are sometimes overlapping. How did they deal with this? Are they sure that they are not seeing FRET?

    1. There's a bug in Hypothesis (at least the sidebar client) such that it's possible to post annotations with comments to the the public, but if you want to highlight something and make it similarly public, then it's not possible...

      I'm using this tag as a workaround. The annotation comment should be a Markdown-style quote (i.e. set off by an ASCII right-pointing angle bracket / less-than sign).

    1. SciScore for 10.1101/2020.12.13.420406: (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">For quantification of cell-cell fusion, three fields were randomly selected in each well to count the fused and unfused cells.</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">Antigens and antibodies The RBD domain of SARS-CoV-2 S protein (His-tag) were synthesized by Prof. Xuefei Cai at Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Chongqing Medical University.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Antigens</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>His-tag</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The wells were incubated with mouse anti-RBD monoclonal antibody (1:1000 dilution) for 1 hour at 37°C, and then washed with PBST five times and incubated with Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Abmart, Shanghai, China) for 1 hour at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-RBD</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-mouse</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">293T-ACE2 cells were pretreated with 20 μM of each compound for 1 h and then, a SARS-CoV-2 S-G614 pseudovirus (3.8 × 104 copies) mixture containing 20 μM of each compound was added to the cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>293T-ACE2</div> <div>suggested: RRID:CVCL_YZ65)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines and cell culture HEK 293T, A549, and Calu3 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK 293T</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>A549</div> <div>suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)</div> </div> <div style="margin-bottom:8px"> <div>Calu3</div> <div>suggested: KCLB Cat# 30055, RRID:CVCL_0609)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293T cells were The copies of the pseudovirus were expressed as numbers of viral RNA genomes per mL of viral stock solution and determined using RT-qPCR with primers and a probe that targeting LTR.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>293T</div> <div>suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In short, HEK 293T-ACE2 cells were assigned to 96-well plates (4x104 cells/well) and cultured for hours at 37 °C with 5 μM compounds.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HEK 293T-ACE2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cytopathic effect (CPE) assay and quantification of SARS-CoV-2 infection Vero E6 cells were dispensed into 96-well plate (4.0x104 cells/well), pre-treated with medium containing compounds or DMSO for 1 hour at 37 ºC.</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">Statistical analyses Data were analyzed using GraphPad Prism version 6.0 software and were presented as means ± SD.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


      Results from Barzooka: We 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 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. Rabbit polyclonal anti-VSV-G tag

      DOI: 10.1016/j.celrep.2020.108490

      Resource: (Abcam Cat# ab1874, RRID:AB_302646)

      Curator: @Naa003

      SciCrunch record: RRID:AB_302646

      Curator comments: Rabbit Anti-VSV-G tag Polyclonal Antibody, Unconjugated Abcam Cat# ab1874


      What is this?

    1. S tag

      DOI: 10.3390/v12121391

      Resource: (MBL International Cat# PM021, RRID:AB_592663)

      Curator: @Naa003

      SciCrunch record: RRID:AB_592663

      Curator comments: S Polyclonal Antibody MBL International Cat# PM021


      What is this?

    1. Tg(C3-1-TAg)cJeg/JegJ

      DOI: 10.1016/j.devcel.2020.10.004

      Resource: (IMSR Cat# JAX_013591,RRID:IMSR_JAX:013591)

      Curator: @Naa003

      SciCrunch record: RRID:IMSR_JAX:013591

      Curator comments: FVB-Tg(C3-1-TAg)cJeg/JegJ Mus musculus IMSR Cat# JAX:013591


      What is this?

    1. In fact, even <svelte:slot /> feels a bit confusing because it introduces a new kind of slot, where the concept is already a bit crowded (there the <slot /> in the parent component, and the target slot="name" for the slot content).

      tag?: crowded (how do we disambiguate, make it not ambiguous?)

    1. Some devs prefer Svelte’s minimal approach that defers problems to userland, encouraging more innovation, choice, and fragmentation, and other devs prefer a more fully integrated toolkit with a well-supported happy path.

      tag?: what scope of provided features / recommended happy path is needed?

    2. It’s worth mentioning that Svelte limits its scope to being only a UI component framework. Like React, it provides the view layer, but it has more batteries included with its component-scoped CSS and extensible stores for state management. Others like Angular and Vue provide a more all-in-one solution with official routers, opinionated state management, CLIs, and more. Sapper is Svelte’s official app framework that adds routing, server-side rendering, code splitting, and some other essential app features, but it has no opinions about state management and beyond. Some devs prefer Svelte’s minimal approach that defers problems to userland, encouraging more innovation, choice, and fragmentation, and other devs prefer a more fully integrated toolkit with a well-supported happy path.

      tag?: what scope of provided features / recommended happy path is needed?

    3. With the caveat that hero worship can be gross, distorting, and unhelpful to everyone involved, Svelte author Rich Harris (@rich_harris on Twitter) is one of my favorite open source developers. In the JS community he’s well-known among tool authors for spreading interesting ideas. He’s the creator of many open source projects including Rollup, the bundler of choice for many libraries including React and Vue.
    4. Svelte is its own language, not plain HTML+CSS+JS

      its own _

    5. The compiler architecture moves complexity from the runtime and source code to buildtime and tools. Behind Svelte’s simple APIs sits a beefy compiler. Frontend web development has become very tool heavy in the webapp era, so in practice this adds little cost beyond what developers like myself already pay, but increased build complexity is important to acknowledge.

      tool-heavy dependence on build tools / heavy/complex build-time

    1. A preferred medium is the price tag: in New Orleans, where he currently lives, he once ran a lunch cart that asked white patrons to pay more than double what he charged people of color, reflecting the city’s racial income disparities. In Nashville, he hosted a series of dinners where hot chicken was free for the neighborhood’s black residents, while white diners were asked to pledge a hundred dollars for one piece, a thousand dollars for four, and the deed to a property for a whole bird plus sides.

      I absolutely love this concept that he did

    1. First, I'll tag each idea with the book I got it from

      给每个想法以 书名 为标签

      我也是这么想的:)

    1. SciScore for 10.1101/2020.12.07.414706: (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">Intravenous viral administration in vivo The animal protocol was approved by the University of South Carolina IACUC 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">Male wild type C57BL/6J mice (5-6 weeks old) were intravenously administered 100 µl of Spp or VSVG lentivirus (8x108 of viral particles) via tail venous or retro-orbital injection.</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">AntiSpike S1 subunit, anti-low density lipoprotein receptor (LDLR), anti-mannose receptor C-type 1 (MRC1), anti-CD68 and anti-human immunodeficiency viruses (HIV)-1 p24 antibodies were obtained from Novus Biologicals (Littleton, CO, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>AntiSpike S1 subunit, anti-low density lipoprotein receptor (LDLR), anti-mannose receptor C-type 1</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>MRC1</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-CD68</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-human immunodeficiency</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>HIV)-1 p24</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The anti-His tag antibodies were obtained from Proteintech (Rosemont, IL, USA) and Thermo Fisher (Waltham, MA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>The anti-His tag antibodies</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-His tag</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-Spike S1 subunit, anti-HIV-1 p24 antibody, or anti-His antibodies were used to detect the expression of viral proteins and followed by HRP-labeled secondary antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Anti-Spike S1 subunit,</div> <div>suggested: (Active Motif Cat# 91345, RRID:AB_2847847)</div> </div> <div style="margin-bottom:8px"> <div>anti-HIV-1</div> <div>suggested: None</div> </div> <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">RAW264.7 cell culture, viral uptake and electroporation Macrophage-like RAW264.7 (RAW) cells (ATCC® TIB-71™) were cultured in DMEM (10% FBS) medium.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>RAW264.7</div> <div>suggested: ATCC Cat# TIB-71, RRID:CVCL_0493)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In a parallel experiment, RAW cells (5x106) were electroporated with pcDNA3.1, EGFP-N2 or pcDNA-Spike plasmids (10 µg) using the following parameters: 2 mm gap cuvette, 250 ul sample volume and 120V (BTX Harvard Bioscience, Inc., Holliston, MA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>RAW</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">Spp or VSV-G lentivirus (8x108 particles) was intravenously injected into C57BL/6J mice.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>C57BL/6J</div> <div>suggested: RRID:IMSR_JAX:000664)</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">Images were acquired using ImageXpress Pico System (Molecular Device, San Jose, CA, USA) or confocal microscopy system (Carl Zeiss AG, Oberkochen, Germany).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ImageXpress Pico System</div> <div>suggested: None</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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. streptavidin-HRP

      DOI: 10.1007/s11120-020-00806-y

      Resource: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)

      Curator: @Naa003

      SciCrunch record: RRID:AB_2556564

      Curator comments: V5 Tag Monoclonal Antibody Thermo Fisher Scientific Cat# R960-25


      What is this?

    1. RRID:AB_2798161

      DOI: 10.1111/bph.15060

      Resource: (Cell Signaling Technology Cat# 13246, RRID:AB_2798161)

      Curator: @Naa003

      SciCrunch record: RRID:AB_2798161

      Curator comments: T7-Tag (D9E1X) XP® Rabbit mAb antibody Cell Signaling Technology Cat# 13246


      What is this?

    1. Hi Collin, nice project! So these results would be useful for reliable sources to write articles in a way that will increase shares. Do you think social media could also benefit from these results in order to find and tag misleading articles perhaps?

    1. some common metadata

      添加阅读的起始时间和结束时间,一方面是表明是否读完,另一方面则是 measure。

      添加 推荐人 也是个好想法,增加更多的 connection,无论是书还是人都有更多的 context

      但是需要构建一个 tag system,不光是个不同的 source,甚至还可以通用于非 Roam Research 之外,比如 raindrop

    1. SciScore for 10.1101/2020.12.03.409318: (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 the voluntary donors were informed about the project and gave their consent.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr"><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">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">Antibody fragments were selected against SARS-CoV-2 Antibodies were selected by panning in microtiter plates against SARS-CoV-2 S1 subunit using S1-S2 in the first panning round, RBD in the second panning round and S1-S2 in the third panning round.</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">The V-Gene combinations for kappa 8/49 (Supplementary Data 3) and lambda (Supplementary Data 4) antibodies were analyzed .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>lambda ( Supplementary Data 4 )</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This is in accordance with Cao et al. ( VH3-66 antibodies against RBD were also selected from the naive HAL9/10 antibody gene libraries made long before the SARS-CoV2 outbreak (Kügler et al., 1998) N/A STE90-C11 This paper N/A mouse anti-His epitope tag Dianova Cat#DIA-900-200 mounse anti-penta His (APC conjugated) Qiagen Cat#3466 goat anti-hIgG(Fc)-AP Jackson ImmunoResearch Cat#AB_2337599 goat anti-mIgG-AP Dianova Cat#115-055-071 mouse α-CD138 (FITC conjugated) BioLegend Cat#352304 E. coli XL1 Blue MRF’ Stratagene Cat#200230 E. coli TG1 Lucigen</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-His</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-penta</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-hIgG(Fc)-AP</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-mIgG-AP</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>α-CD138</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All antigens, antibodies and scFv-Fc were run on Superdex 200 Increase 10/300GL (Cytiva) on Äkta or HPLC (Techlab) on an AdvanceBio SEC 300Å 2.7 µm, 7.8x300 mm (Agilent) for quality control.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>antigens ,</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the plasma B-cell sorted library, plasma B-cells were doublestained with mouse α-CD19 APC-conjugated (MHCD1905, Thermo Fisher Scientific, Schwerte, Germany) and mouse α-CD138 (FITC conjugated) antibody (DL-101, BioLegend, San Diego, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>α-CD19 APC-conjugated ( MHCD1905</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>APC-conjugated</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>DL-101</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ScFv-Fc and IgG production Unique scFv sequences isolated by antibody-phage display were subcloned into pCSE2.7hIgG1-Fc-XP using NcoI/NotI (New England Biolabs, Frankfurt, Germany) for mammalian production in Expi293F cells as scFv-Fc (Wenzel et al., 2020a).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ScFv-Fc</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">S1-His and the corresponding mutants were detected with mousen anti-penta His (Qiagen) and goat anti-mFc APC-conjugated antibody (Dianova).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>S1-His</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-mFc APC-conjugated antibody ( Dianova) .</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">Experimental Model: Cell lines Expi293F 18/49 Scientific VeroE6 ATCC ATCC</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>VeroE6</div> <div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div> </div> </td></tr><tr><td style="min-width:100px;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">MN908947 Art ACE2 Thermo Fisher/Gene GenBank:</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Thermo Fisher/Gene</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(2019) OrginLab N/A GraphPad Prism 6.1 GraphPad www.graphpad.com VBASE MRC Centre for Protein Engineering www2.mrclmb.cam.ac.uk/vbase/ VBASE2 (Mollova et al., 2010) www.vbase2.org DataGraph (4.5.1) DataGraph www.visualdatatools.com</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PyMol Schrödinger LLC www.pymol.org/2/ PISA European Bioinformatics Institute http://www.ebi.ac.uk/ pdbe/prot_int/pistart</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>http://www.ebi.ac.uk/</div> <div>suggested: (European Bioinformatics Institute, RRID:SCR_004727)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell Sorter BD N/A Octet qKe</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Cell Sorter</div> <div>suggested: (CHB Cell Sorter Core, RRID:SCR_009706)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Monoclonal binders were sequenced and analyzed using VBASE2 (www.vbase2.org) (Mollova et al., 2010) and possible glycosylation positions in the CDRS were analyzed according to Lu et al (Lu et al., 2019).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>VBASE2</div> <div>suggested: (VBASE2, RRID:SCR_007082)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The IC50 was calculated using the equation f(x)=Amin+(Amax-Amin)/(1+(x0/x)^h)^s and parameters from OriginPro (2019).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>OriginPro</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">EC50 were 28/49 calculated with by GraphPad Prism Version 6.1, fitting to a four-parameter logistic curve.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The model was further improved by manual rebuilding in Coot (Emsley et al., 2010) and computational refinement with phenix. refine (Afonine et al., 2012) including placement of water, TLS refinement and riding hydrogens in the final steps of the procedure.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Coot</div> <div>suggested: (Coot, RRID:SCR_014222)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Depictions of the model were generated with PyMol molecular graphics system (Schrödinger LLC; version 2.3.2).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>PyMol</div> <div>suggested: (PyMOL, RRID:SCR_000305)</div> </div> </td></tr></table>

      Results from OddPub: Thank you for sharing your data.


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


      Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on page 54. 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 10.1101/2020.12.01.407361: (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">A549-ACE2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>A549-ACE2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Serial 1/10 dilutions of virus are made and plated into 4 replicate wells containing fresh cell monolayers of Vero 76 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero 76</div> <div>suggested: JCRB Cat# IFO50410, RRID:CVCL_0603)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lysis of cell monolayer was performed 24 h (for monocytes) or 48 h (for Huh-7 and Calu-3 cells) post infection and culture supernatant was harvested 48 h post infection and virus was titrated by plaque-forming units (PFU) assays in Vero E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Calu-3</div> <div>suggested: None</div> </div> <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">Supernatants were harvested after 24 h, serially ten-fold diluted, and virus titer was determined by TCID50 assay on Huh-7 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Huh-7</div> <div>suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero CCL81 cells were cultivated at 5% CO₂ and 37°C using Dulbecco’s Modified Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero CCL81</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For this experiment, Vero cells were seeded at a density of 10 cells/ well in a 96 well plate prior incubation with a serial dilution of compounds of interest and controls for 72 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Vero</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A codonoptimized gene encoding for SARS-CoV-2 (331 to 528 amino acids, QIS60558.1) was expressed in Expi293 cells (Thermo Fisher Scientific) with human serum albumin secretion signal sequence and fusion tags (6xHistidine tag, Halo tag, and TwinStrep tag) as described before (Premkumar et al., 2020)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Expi293</div> <div>suggested: RRID:CVCL_D615)</div> </div> </td></tr><tr><td style="min-width:100px;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 dye content in each set of wells is converted to a percentage of dye present in untreated control wells using a Microsoft Excel computer-based spreadsheet and normalized based on the virus control.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <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: We detected the following sentences addressing limitations in the study:

      Despite this limitation, valuable information about the virus infection and replication can be learned from studies using these cell lines. A previous study (Chu et al., 2020) assessed 25 cell lines derived from different tissues or organs and host species and reported that cytopathic effects were only seen in VeroE6 and FRhK4 cells after SARS-CoV-2 inoculation for up to 120 hpi. These findings are important for optimization of antiviral assays based on cell protection assessment, because cell lines without obvious cytopathic effects might lead to overestimation of cell viability and drug efficacy (Chu et al., 2020). For efficient SARSCoV-2 research, a cell line, such as Vero cells, that can easily replicate and isolate the virus is essential, but they have been shown not to produce interferon (IFN) when infected with Newcastle disease virus, rubella virus, and other viruses (Desmyter et al., 1968). Under previously described experimental conditions, productive SARS-CoV-2 replication in A549 cells was erratic (Sacramento et al., 2020) which can be overcome by preparing A549 cells overexpressing ACE2 (as used in the current study). The differences in responses in different cell lines could be accounted for by the basic biochemistry, for example hepatic cells, like Huh-7, are equipped with enzymes to synthesize nucleotides, carbohydrates and lipids (Nwosu et al., 2018). Hence it is not surprising that the highest potency of remdesivir against SARS-CoV-2 is found in Huh-7 cells,...


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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 10.1101/2020.12.01.406934: (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">Methods Donors & Ethics Donors were recruited in accordance with the laws of Switzerland and under ethics approval BASEC2016-01260 of the Cantonal Ethics Commission of Zurich.</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">Animals were weighed prior to the start of the study and randomly distributed in the different cohorts.</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 experiments (hamster) A total of 56 Golden Syrian hamsters, 36 male and 20 female, weighing between 80g and 130g were used in the study.</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 titration of anti-SARS-CoV2-Spike protein antibody MTX-COVAB, the IgG1 isotype control 24C03 or anti-CD20 antibody Rituximab was added to cells and incubated overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-SARS-CoV2-Spike protein</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>the IgG1</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-CD20</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Binding of antibodies was detected using a fluorescence labeled anti human IgG.</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">Bound ACE2 was then detected using a fluorescence labeled anti His Tag antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti His Tag antibody.</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Negative control/isotype control antibody (mAb24C3) is a human-derived antibody specific for tetanus toxoid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>mAb24C3</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">Neutralization of SARS-CoPsV-2 HEK293T cells stably expressing full length huACE2 (aa1-805) were seeded and left to adhere.</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">Neutralization of wild type SARS-CoV-2 At day 0, Vero E6 cells were seeded with a concentration of 1*105 cells/well in 300 µl medium.</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">As control cells nontransfected HEK293T and Raji B cells were used.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Raji B</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies were expressed in HEK293 cells after gene synthesis of the variable regions and linkage to the same constant region derived from human IgG1 (Trastuzumab) as COVAB.</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">Table indicates half maximal inhibitory concentration (IC50) for each of the antibodies and for the REGN combi (REGN10933 and REGN10987 in a mixture of equal concentrations) IC50 values were determined using the “log [Inhibitor] vs. normalized response -- Variable slope” fitting model of GraphPad Prism 8.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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. Reviewer #3:

      In this manuscript, Naetar et al. investigate the role of LAP2α binding to A-type lamins in the nucleoplasm. LAP2α was already thought to be important for maintaining the nucleoplasmic pool of soluble A-type lamins, because knockout of LAP2α has previously been shown to reduce nucleoplasmic signal from an antibody that recognizes the lamin-A/C amino terminus. However, by directly tagging A-type lamins with fluorescent proteins and by using an alternative antibody to stain them, Naetar et al. find that the presence of LAP2α does not appreciably affect the pool of soluble lamins in the nucleoplasm. Instead, they find that LAP2α affects the assembly state of soluble lamins within the nucleoplasm, preventing formation of higher order A-type lamin structures that impede the mobility of telomeres within the nucleus.

      There is a lot to like about this paper. I admire the author's mechanistic approach to studying lamin assembly state. The complementary cell biology/microscopy approaches paired with the biochemical approaches in figure 5 lead to an overall convincing story. And finally, I appreciate the efforts the authors made to "show their work," including their genome editing quality control measures.

      Major comments:

      1) Although I appreciate the transparency of the authors in demonstrating their workflow and quality control measures (see above), some of the terminology makes the manuscript difficult to read. At times it feels more like reading a lab notebook than reading a manuscript. For example, The manuscript would be easier to understand if cell lines were given descriptive names (eg: LAP2α KO, or mEos3.2-lmna instead of "WT#21") rather than continuing to refer to them by the small guide RNA that was used to generate them. A second example: it is nice to show biological replicate data as in figure 1, but it took me a while to figure out that the second and third columns in panels A and B were biological replicates; I spent some time trying to determine which experimental condition was different. Perhaps one biological replicate could be displayed in the main text and the second could be moved to the supplement, especially considering that it appears that only one of the clones was used for the quantifications shown in the bottom panels.

      2) Why was the choice made to disrupt LAP2α at the beginning of exon 4? How large are exons 1 and 2, which are not shown in the schematic in the supplemental figures? What percentage of the LAP2α peptide primary sequence is affected by a frameshift mutation at the start of exon 4? Why was this approach preferable to introducing a frameshift mutation closer to the 5' end of the gene? I am concerned that the "LAP2α KO" cells used in the experiments may have some partially functional truncated LAP2α protein.

      3) On page 16, the authors describe a set of experiments that are meant to demonstrate that their failure to see a difference in nucleoplasmic A-type lamins in LAP2α mutants is not due to the fluorescent protein tag used, however, instead of looking at untagged lamins, they elect to look at a cell line that has all lmna alleles tagged. Wouldn't it be better to use the LAP2α KO cells from figure 1 and stain with both the 3A6 antibody and the N18 antibody to determine whether untagged lamins behave the same way as tagged lamins? Perhaps this experiment could be added along with the current data, as it would be nice to compare directly between a cell line with all lmna alleles tagged and a cell line with no lmna alleles tagged.

      This experiment would also give the authors a chance to compare morphology and overall fitness of cells with all untagged lmna with cells with all tagged lmna, to determine whether the tagged proteins are fully functional. Even if the tagged protein is fully functional, it would be appropriate to add a brief discussion of the possibility that fluorescent tags do perturb lamin-A/C function. After all, many lamin mutations do not cause obvious phenotypes in tissue culture cells, but defects can still emerge during development and aging in the context of an animal.

      4) The authors build a convincing case that binding to A-type lamins by LAP2α influences their ability to assemble. But how do cells leverage this relationship for biological functions? Do cells tune the amount of fully soluble vs. partially assembled A-type lamins in the nucleoplasm in order to control nuclear structure or function in response to certain stimuli? Have the A-type lamins in the nucleoplasm been found to be in a different assembly state in different cell types? As the study is currently written, it presents an interesting molecular mechanism but no biological mechanism.

    1. Remora Communiqué

      The Remora Communiqué

      Issued by No Spectator Left, December 2020

      1

      I heard the voice

      Of the Remora speak –

      Slowly, all in silence,

      To wake me from my sleep.

      2

      I heard the voice

      Of its silence say,

      ‘A Plague Ship has been

      Stopped today.’

      3

      ‘Did you even know

      You were at sea?

      Did you ever stop

      To think of me?’

      4

      ‘Know you’d left

      The world behind,

      Or what on Earth

      You hoped to find?’

      5

      ‘Have you heard the whales

      Now have to yell?

      You think they’re singing –

      You can’t tell!’

      6

      ‘It was the droning on & on

      Of your Dread-Nought Destroyer

      That made me sound my calm alarm

      In the ear of your Employer.’

      7

      ‘The Strain & Refrain

      From onboard seemed familiar,

      An updated version of

      “Long Live Caligula!”’

      8

      ‘I stopped his progress, ah

      The hutzpah of karma!

      Rome outweighed

      By the scales of Remora... ’

      9

      ‘Mark Antony

      I scuppered too,

      Underthrown before

      He knew…’

      10

      ‘But today, you thought,

      What need to worry?

      What voodoo-glue can now undo

      Your ship’s world-beating hurry!’

      11

      ‘So I downsized, to fill the role

      I was unborn to play:

      Remember, as the Show Goes On,

      You recast me this way!’

      12

      ‘You even gave new me a name

      (With hollow ring, it’s true):

      Corona-Virus, The Sick Crown,

      Sitting right with you…’

      13

      ‘If you should miss this hint now –

      Heaven knows, I tried! –

      The next ring at the doorbell?

      No more Mr. Nice Guy!’

      14

      ‘For tho’ the story of l’il ole me

      Is soon & simply told

      (N.B., I’m only as little

      As you made the world),

      15

      Perchance in the Grand Scheme

      There’s ‘small’ & then there’s small,

      And your friend the atom

      May do for us all!’

      16

      ‘Fat Man’s little boy

      For purpose trained fit:

      The crack that splits open

      The hull of the ship!’

      17

      ‘Yes, that’s the thing (you’ll see too late),

      It All cracks from inside:

      Nothing in the world left ‘out’

      Now you’ve grown worldwide.’

      18

      ‘So while we’ve a moment –

      And if not now, when? –

      Pray, pay me best attention:

      We may not meet again.’

      19

      ‘And it’s hard to imagine

      But sadly safe to say, you

      May yet remember me

      Fondly one day!’

      20

      ‘For it’s not just the overlooked

      Pit of the Bomb, the

      Abyss that’s grown tired from

      Yawning so long,’

      21

      ‘There’s now – just in case! –

      As the Atomic Clock ticks,

      A new kid on the Doomsday Block,

      A spare Apocalypse!’

      22

      ‘And with two caps melting

      The Dunce is warming to his task,

      Facing down his Mother,

      Preparing Her Death-Mask.’

      23

      ‘But what does Her life matter

      (& who’ll be left to grieve?),

      The Old Girl in the Chokehold

      Croaking “I Can’t Breathe!”’

      24

      ‘O you wring your hands & ring your bells

      While skies & forests fall,

      But “capitalism will adapt!” no doubt:

      It has to, after all!’

      25

      ‘The trusty greenwashed reset button,

      Point missed without fail –

      “Sustainable development”…

      Of the Fairy Tale!’

      26

      ‘And to “listen to the science”

      Isn’t all you need to do:

      If you want to really heal thyself,

      Listen to my silence too!’

      27

      ‘It really is a killer,

      The racket y’all make:

      What kind of f** bully

      Wants to make his Mother Quake?’

      28

      ‘It is what it is,

      Boys will be boys,

      In their noisome

      Kingdom of Noise?’

      29

      ‘Well, until my little finger

      Touched the spinning top,

      Ripped you from the driver’s seat

      Of the Roaring Chariot.’

      30

      ‘But I cannot now take the helm

      Lay in a course that’s true,

      Back to safely grounded land –

      That’s up to all the Crew.’

      31

      ‘For in this emergency,

      All hands on the (burning) deck:

      Check your destiny’s manifest, there

      Are no passengers left!’

      32

      ‘It’s time to call a midnight strike,

      Make love to Mutiny –

      Go overboard, throw overboard

      This plaguey, illthy Bounty!’

      33

      ‘What exactly should you do? You

      Crave a detailed scheme?

      I’m not a power-point, you know,

      Just your own fever-dream!’

      34

      I started when the silence stopped,

      So badly missed its voice:

      Left all alone, onboard to make

      The choice that is no choice –

      35

      To put away so many

      Very foolish things,

      While we can still remember

      What being human means,

      36

      Remember that the question

      ‘To be or not to be?’

      Isn’t just a question

      Of or for humanity,

      37

      Though it wouldn’t be an issue

      Without the threats we pose,

      The constant hammering it takes

      To crucify Life’s Rose,

      38

      To pulverize the Earth that is

      Our only common wealth,

      To tame and tag, gas & gag

      The good wild life of health.

      39

      I cried, ‘my God, I have to rush,

      Right now alert the crew;

      Not those who know they slave & serve –

      The rest, without a clue,

      40

      Who buckle up,

      Enjoy the ride,

      Let those “in the

      Know” decide

      41

      Their fate: “Awake!,” I’d cry,

      “Discern!, deride

      The course laid in

      For Omnicide!”’

      42

      But my voice would

      Not be the Dream’s,

      And I must wake

      To what It means –

      43

      So first things first,

      Some silence, pray:

      High Time to issue

      The Remora Communiqué…

    1. In Python, every object has a unique identification tag

      在Python中,每个对象都有一个唯一的标识标签

      每个object 在堆内存里都有一个id 空间

  4. Nov 2020
    1. Author Response

      Summary: A major tenet of plant pathogen effector biology has been that effectors from very different pathogens converge on a small number of host targets with central roles in plant immunity. The current work reports that effectors from two very different pathogens, an insect and an oomycete, interact with the same plant protein, SIZ1, previously shown to have a role in plant immunity. Unfortunately, apart from some technical concerns regarding the strength of the data that the effectors and SIZ1 interact in plants, a major limitation of the work is that it is not demonstrated that the effectors alter SIZ1 activity in a meaningful way, nor that SIZ1 is specifically required for action of the effects.

      We thank the editor and reviewers for their time to review our manuscript and their helpful and constructive comments. The reviews have helped us focus our attention on additional experiments to test the hypothesis that effectors Mp64 (from an aphid) and CRN83-152 (from an oomycete) indeed alter SIZ1 activity or function. We have revised our manuscript and added the following data:

      1) Mp64, but not CRN83-152, stabilizes SIZ1 in planta. (Figure 1 in the revised manuscript).

      2) AtSIZ1 ectopic expression in Nicotiana benthamiana triggers cell death from 3-4 days after agroinfiltration. Interestingly CRN83-152_6D10 (a mutant of CRN83-152 that has no cell death activity), but not Mp64, enhances the cell death triggered by AtSIZ1 (Figure 2 in the revised manuscript).

      For 1) we have added the following panel to Figure 1 as well as three biological replicates of the stabilisation assays in the Supplementary data (Fig S3):

      Figure 1 panel C. Stabilisation of SIZ1 by Mp64. Western blot analyses of protein extracts from agroinfiltrated leaves expressing combinations of GFP-GUS, GFP Mp64 and GFP-CRN83_152_6D10 with AtSIZ1-myc or NbSIZ1-myc. Protein size markers are indicated in kD, and equal protein amounts upon transfer is shown upon ponceau staining (PS) of membranes. Blot is representative of three biological replicates , which are all shown in supplementary Fig. S3. The selected panels shown here are cropped from Rep 1 in supplementary Fig. S3.

      For 2) we have added the folllowing new figure (Fig. 2 in the revised manuscript):

      Fig. 2. SIZ1-triggered cell death in N. benthamiana is enhanced by CRN83_152_6D10 but not Mp64. (A) Scoring overview of infiltration sites for SIZ1 triggered cell death. Infiltration site were scored for no symptoms (score 0), chlorosis with localized cell death (score 1), less than 50% of the site showing visible cell death (score 2), more than 50% of the site showing cell death (score 3). (B) Bar graph showing the proportions of infiltration sites showing different levels of cell death upon expression of AtSIZ1, NbSIZ1 (both with a C-terminal RFP tag) and an RFP control. Graph represents data from a combination of 3 biological replicates of 11-12 infiltration sites per experiment (n=35). (C) Bar graph showing the proportions of infiltration sites showing different levels of cell death upon expression of SIZ1 (with C-terminal RFP tag) either alone or in combination with aphid effector Mp64 or Phytophthora capsica effector CRN83_152_6D10 (both effectors with GFP tag), or a GFP control. Graph represent data from a combination of 3 biological replicates of 11-12 infiltration sites per experiment (n=35).

      Our new data provide further evidence that SIZ1 function is affected by effectors Mp64 (aphid) and CRN83-152 (oomycete), and that SIZ1 likely is a vital virulence target. Our latest results also provide further support for distinct effector activities towards SIZ1 and its variants in other species. SIZ1 is a key immune regulator to biotic stresses (aphids, oomycetes, bacteria and nematodes), on which distinct virulence strategies seem to converge. The mechanism(s) underlying the stabilisation of SIZ1 by Mp64 is yet unclear. However, we hypothesize that increased stability of SIZ1, which functions as an E3 SUMO ligase, leads to increased SUMOylation activity towards its substrates. We surmise that SIZ1 complex formation with other key regulators of plant immunity may underpin these changes. Whether the cell death, triggered by AtSIZ1 upon transient expression in Nicotiana benthamiana, is linked to E3 SUMO ligase activity remains to be investigated. Expression of AtSIZ1 in a plant species other than Arabidopsis may lead to mistargeting of substrates, and subsequent activation of cell death. Dissecting the mechanistic basis of SIZ1 targeting by distinct pathogens and pests will be an important next step in addressing these hypotheses towards understanding plant immunity.

      Reviewer #1:

      In this manuscript, the authors suggest that SIZ1, an E3 SUMO ligase, is the target of both an aphid effector (Mp64 form M. persicae) and an oomycete effector (CRN83_152 from Phytophthora capsica), based on interaction between SIZ1 and the two effectors in yeast, co-IP from plant cells and colocalization in the nucleus of plant cells. To support their proposal, the authors investigate the effects of SIZ1 inactivation on resistance to aphids and oomycetes in Arabidopsis and N. benthamiana. Surprisingly, resistance is enhanced, which would suggest that the two effectors increase SIZ1 activity.

      Unfortunately, not only do we not learn how the effectors might alter SIZ1 activity, there is also no formal demonstration that the effects of the effectors are mediated by SIZ1, such as investigating the effects of Mp64 overexpression in a siz1 mutant. We note, however, that even this experiment might not be entirely conclusive, since SIZ1 is known to regulate many processes, including immunity. Specifically, siz1 mutants present autoimmune phenotype, and general activation of immunity might be sufficient to attenuate the enhanced aphid susceptibility seen in Mp64 overexpressers.

      To demonstrate unambiguously that SIZ1 is a bona fide target of Mp64 and CRN83_152 would require assays that demonstrate either enhanced SIZ1 accumulation or altered SIZ1 activity in the presence of Mp64 and CRN83_152.

      The enhanced resistance upon knock-down/out of SIZ1 suggests pathogen and pest susceptibility requires SIZ1. We hypothesize that the effectors either enhance SIZ1 activity or that the effectors alter SIZ1 specificity towards substrates rather than enzyme activity itself. To investigate how effectors coopt SIZ1 function would require a comprehensive set of approaches and will be part of our future work. While we agree that this aspect requires further investigation, we think the proposed experiments go beyond the scope of this study.

      After receiving reviewer comments, including on the quality of Figure 1, which shows western blots of co-immunoprecipitation experiments, we re-analyzed independent replicates of effector-SIZ1 coexpression/ co-immunoprecipitation experiments. The reviewer rightly pointed out that in the presence of Mp64, SIZ1 protein levels increase when compared to samples in which either the vector control or CRN83-152_6D10 are co-infiltrated. Through carefully designed experiments, we can now affirm that Mp64 co-expression leads to increased SIZ1 protein levels (Figure 1C and Supplementary Figure S3, revised manuscript). Our results offer both an explanation of different SIZ1 levels in the input samples (original submission, Figure 1A/B) as well as tantalizing new clues to the nature of distinct effector activities.

      Besides, we were able to confirm a previous preliminary finding not included in the original submission that ectopic expression of AtSIZ1 in Nicotiana benthamiana triggers cell death (3/4 days after infiltration) and that CRN83-152_6D10 (which itself does not trigger cell death) enhances this phenotype.

      We have considered overexpression of Mp64 in the siz1 mutant, but share the view that the outcome of such experiments will be far from conclusive.

      In summary, we have added new data that further support that SIZ1 is a bonafide target of Mp64 and CRN83-152 (i.e. increased accumulation of SIZ1 in the presence of Mp64, and enhanced SIZ cell death activation in the presence of CRN83-152_6D10).

      Reviewer #2:

      The study provides evidence that an aphid effector Mp64 and a Phytophthora capsici effector CRN83_152 can both interact with the SIZ1 E3 SUMO-ligase. The authors further show that overexpression of Mp64 in Arabidopsis can enhance susceptibility to aphids and that a loss-of-function mutation in Arabidopsis SIZ1 or silencing of SIZ1 in N. benthamiana plants lead to increased resistance to aphids and P. capsici. On siz1 plants the aphids show altered feeding patterns on phloem, suggestive of increased phloem resistance. While the finding is potentially interesting, the experiments are preliminary and the main conclusions are not supported by the data.

      Specific comments:

      The suggestion that SIZ1 is a virulence target is an overstatement. Preferable would be knockouts of effector genes in the aphid or oomycete, but even with transgenic overexpression approaches, there are no direct data that the biological function of the effectors requires SIZ1. For example, is SIZ1 required for the enhanced susceptibility to aphid infestation seen when Mp64 is overexpressed? Or does overexpression of SIZ1 enhance Mp64-mediated susceptibility?

      What do the effectors do to SIZ1? Do they alter SUMO-ligase activity? Or are perhaps the effectors SUMOylated by SIZ1, changing effector activity?

      We agree that having effector gene knock-outs in aphids and oomycetes would be ideal for dissecting effector mediated targeting of SIZ1. Unfortunately, there is no gene knock-out system established in Myzus persicae (our aphid of interest), and CAS9 mediated knock-out of genes in Phytophthora capsici has not been successful in our lab as yet, despite published reports. Moreover, repeated attempts to silence Mp64, other effector and non-effector coding genes, in aphids (both in planta and in vitro) have not been successful thus far, in our hands. As detailed in our response to Reviewer 1, we considered the use of transgenic approaches not appropriate as data interpretation would become muddied by the strong immunity phenotype seen in the siz1-2 mutant.

      As stated before, we hypothesize that the effectors either enhance SIZ1 activity or alter SIZ1 substrate specificity. Mp64-induced accumulation of SIZ1 could form the basis of an increase in overall SIZ1 activity. This hypothesis, however, requires testing. The same applies to the enhanced SIZ1 cell death activation in the presence of CRN83-152_6D10.

      Whilst our new data support our hypothesis that effectors Mp64 and CRN83-152 affect SIZ1 function, how exactly these effectors trigger susceptibility, requires significant work. Given the substantial effort needed and the research questions involved, we argue that findings emanating from such experiments warrant standalone publication.

      While stable transgenic Mp64 overexpressing lines in Arabidopsis showed increased susceptibility to aphids, transient overexpression of Mp64 in N. benthamiana plants did not affect P. capsici susceptibility. The authors conclude that while the aphid and P. capsici effectors both target SIZ1, their activities are distinct. However, not only is it difficult to compare transient expression experiments in N. benthamiana with stable transgenic Arabidopsis plants, but without knowing whether Mp64 has the same effects on SIZ1 in both systems, to claim a difference in activities remains speculative.

      We agree that we cannot compare effector activities between different plant species. We carefully considered every statement regarding results obtained on SIZ1 in Arabidopsis and Nicotiana benthamiana. We can, however, compare activities of the two effectors when expressed side by side in the same plant species. In our original submission, we show that expression of CRN83 152 but not Mp64 in Nicotiana benthamiana enhances susceptibility to Phytophthora capsici. In our revised manuscript, we present new data showing distinct effector activities towards SIZ1 with regards to 1) enhanced SIZ1 stability and 2) enhanced SIZ1 triggered cell death. These findings raise questions as to how enhanced SIZ1 stability and cell death activation is relevant to immunity. We aim to address these critical questions by addressing the mechanistic basis of effector-SIZ1 interactions.

      The authors emphasize that the increased resistance to aphids and P. capsici in siz1 mutants or SIZ1 silenced plants are independent of SA. This seems to contradict the evidence from the NahG experiments. In Fig. 5B, the effects of siz1 are suppressed by NahG, indicating that the resistance seen in siz1 plants is completely dependent on SA. In Fig 5A, the effects of siz1 are not completely suppressed by NahG, but greatly attenuated. It has been shown before that SIZ1 acts only partly through SNC1, and the results from the double mutant analyses might simply indicate redundancy, also for the combinations with eds1 and pad4 mutants.

      We emphasized that siz1-2 increased resistance to aphids is independent of SA, which is supported by our data (Figure 5A). Still, we did not conclude that the same applies to increased resistance to Phytophthora capsici (Figure 5B). In contrast, the siz1-2 enhanced resistance to P. capsici appears entirely dependent on SA levels, with the level of infection on the siz1-2/NahG mutants even slightly higher than on the NahG line and Col-0 plants. We exercise caution in the interpretation of this data given the significant impact SA signalling appears to have on Phytophthora capsici infection.

      The reviewer commented on the potential for functional redundancy in the siz1-2 double mutants. Unfortunately, we are unsure what redundancy s/he is referring to. SNC1, EDS1, and PAD4 all are components required for immunity, and their removal from the immune signalling network (using the mutations in the lines we used here) impairs immunity to various plant pathogens. The siz1-2 snc1-11, siz1-2 eds1-2, and siz1-2 pad4-1 double mutants have similar levels of susceptibility to the bacterial pathogen Pseudomonas syringae when compared to the corresponding snc1-11, eds1-2 and pad4-1 controls (at 22oC). These previous observations indicate that siz1 enhanced resistance is dependent on these signalling components (Hammoudi et al., 2018, Plos Genetics).

      In contrast to this, we observed a strong siz1 enhanced resistance phenotype in the absence of snc1- 11, eds1 2 and pad4-1. Notably, the siz1-2 snc1-11 mutant does not appear immuno-compromised when compared to siz1-2 in fecundity assays, indicating that the siz1-2 phenotype is independent of SNC1. In our view, these data suggest that signalling components/pathways other than those mediated by SNC1, EDS1, and PAD4 are involved. We consider this to be an exciting finding as our data points to an as of yet unknown SIZ1-dependent signalling pathway that governs immunity to aphids.

      How do NahG or Mp64 overexpression affect aphid phloem ingestion? Is it the opposite of the behavior on siz1 mutants?

      We have not performed further EPG experiments on additional transgenic lines used in the aphid assay. These experiments are quite challenging and time consuming. Moreover, accommodating an experimental set-up that allows us to compare multiple lines at the same time is not straightforward. Considering that NahG did not affect aphid performance (Figure 5A), we do not expect to see an effect on phloem ingestion.

    1. Image-based memes involve, primarily, an image created by somebody. Sometimes the meme creator is also the image creator, but often, when involving movie stills or images of celebrities, the image’s copyright is owned by someone else. American copyright law gives creators the exclusive rights of reproduction, modification, distribution, performance, and display. The viral spread of a meme infringes on theses protections as the original image is modified and then displayed, distributed and reproduced when posted and reposted.

      Memes are basically just ways of making fun of certain pictures, a lot of the time, they happen to be real people caught at a weird or funny moment and the catchy tag you put on the picture is what makes it funny.

    1. None

      I am surprised to see no honorable mention here, because a "book log" sounds a lot like a reference manager. The best free/open-source one I know of is Zotero: https://www.zotero.org

      From your list of desired features above, it can do:

      • tagging of items (automatically when collecting items with the browser button, manually, or a mix of both automatic tags and your own tags)
      • notes as attachments to an item
      • bookmarks as an URL attached to an item (and actually, most item types collected with the browser button have the URL saved by default)
      • making items and their annotations public on your profile on zotero.org
      • shareable format: you can export in many formats, from simple printout kind of formats (HTML) to formats fully re-importable into another instance of Zotero
      • query: not sure it has all the capabilities of a relational database, but you can search based on any piece of metadata found in your items, you can build arbitrarily complex search queries, you can save searches (they will materialize in the interface as "dynamic folders" containing the search results automatically as new items added to your library match the query)

      For dealing with prioritization, you would have to come up with your own system. The workflow described here uses the tag system for this (with custom tags to mark status "to read", "read", etc.): https://incenp.org/notes/2019/managing-academic-literature.html

    2. Allow me to tag books instead of placing them into static lists (think clusters or tag clouds).

      Have your tried Roam Research?

    1. Mouse monoclonal anti-V5

      DOI: 10.1016/j.cub.2020.10.061

      Resource: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)

      Curator: @Naa003

      SciCrunch record: RRID:AB_2556564

      Curator comments: V5 Tag Monoclonal Antibody Thermo Fisher Scientific Cat# R960-25


      What is this?

    1. We’re now 100% powered by renewable and sustainable energy which is great in further minimizing our impact on the planet. Plausible Analytics script weights less than 1 KB which is more than 45 times smaller than the recommended Google Analytics Global Site Tag implementation.

      After speaking to the folks at Plausible they pointed me to this page on the digital ocean community forums:

      https://www.digitalocean.com/community/questions/what-kind-of-electricity-do-you-run-on

      And this one here:

      https://www.interxion.com/why-interxion/sustainability

      The TLDR version is that the servers they are using are run by Digital Ocean, who lease from Interxion, who source the power for the datacentre from renewables.

      Interxion themselves are owned by Digital Realty, who do release figures, but not at a granularity to confirm.

      Once there is info from Interxion, it's possible to confirm this.

    1. 500 iPad-Koffer mit insgesamt 8000 Geräten

      Ich bin ein großer iPad-Fan und nutze meines jeden Tag für Handschriftliches.

      Dieser Aktion ist bestimmt eine gründliche Evaluierung der Optionen vorausgegangen und es ist toll, dass unsere Schulen jetzt besser ausgestattet werden, gar keine Frage.

      Trotzdem nagt die Erkenntnis an mir, dass ein iPad doch in erster Linie ein Konsum- und Kommunikationsgerät und weniger ein Kreativwerkzeug ist. Ich frage mich deshalb, ob die iPads nicht zumindest um günstige Laptops mit Tastatur ergänzt werden sollten.

      Ich schreibe diesen Kommentar übrigens gerade auf einem RaspberryPi für 100€. Davon bekommt man so etwa vier Stück für den Preis eines iPads. Und unglaublich viel mehr Möglichkeiten.

    1. SciScore for 10.1101/2020.11.24.390039: (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">siRNA-mediated knockdown and SP cell preparation HeLa cells (human epithelial cervix carcinoma cells, ATCC, female) were cultured in DMEM supplemented with 10% (v/v) FBS and maintained in a 5% CO2 humidified incubator at 37°C.</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">Following transfer to a PVDF membrane in transfer buffer (0.06 M Tris, 0.60 M glycine, 20% MeOH) at mA for 2.5 h, PVDF membranes were incubated in 1X Casein blocking buffer (10X stock from Sigma-Aldrich, B6429) made up in TBS, incubated with appropriate primary antibodies (1:500 or 1:1000 dilution) and processed for fluorescence-based detection as described by LI-COR Biosciences using appropriate secondary antibodies (IRDye 680RD Donkey anti-Goat, IRDye 680RD Donkey anti-Rabbit, IRDye 800CW Donkey anti-Mouse) at 1:10,000 dilution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>B6429</div> <div>suggested: (LifeSpan Cat# LS-B6429-50, RRID:AB_11134145)</div> </div> <div style="margin-bottom:8px"> <div>anti-Goat</div> <div>suggested: (LI-COR Biosciences Cat# 926-68074, RRID:AB_10956736)</div> </div> <div style="margin-bottom:8px"> <div>anti-Rabbit</div> <div>suggested: None</div> </div> <div style="margin-bottom:8px"> <div>anti-Mouse</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;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">ORF6-OPG2, ORF8-OPG2, M-OPG2 and S-OPG2 were generated by inserting the respective cDNAs in frame between NheI and AflII sites of a pcDNA5/FRT/V5-His vector (Invitrogen) containing a C-terminal OPG2 tag (MNGTEGPNFYVPFSNKTG).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>S-OPG2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Microsomal translation reactions (20 μL) were performed for 30 min at 30°C whereas those using SP HeLa cells were performed on a 1.5X scale (30 μL translation reactions) for 1 h at 30°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>HeLa</div> <div>suggested: None</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


      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 10.1101/2020.11.21.392753: (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">VeroE6 and A549 cells were obtained from American Type Culture Collection (ATCC) and tested at regular intervals for mycoplasma contamination.</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 three times washing with PBS, bound primary antibody was detected with a secondary antibody (anti-mouse IgG), conjugated to horseradish peroxidase.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>anti-mouse IgG</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;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">VeroE6 and A549 cells were obtained from American Type Culture Collection (ATCC) and tested at regular intervals for mycoplasma contamination.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>VeroE6</div> <div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Generation and cultivation of A549 cells stably expressing ACE2 (A549+ACE2) was described recently41.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>A549</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A549+ACE2 cells stably expressing the TMPRSS2 protease were generated by lentiviral transduction.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>A549+ACE2</div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentivirus stocks were produced by transfection of HEK293T cells with a pWPI plasmid encoding for TMPRSS2 and the pCMV-Gag-Pol and pMD2-VSV-G packaging plasmids (kind gifts from D. Trono, Geneva).</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">A549+ACE2+/-TMPRSS2 were seeded at a density of 1.5 x 104 cells per well of a flat bottom 96-well plate (Corning).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>A549+ACE2+/-TMPRSS2</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">For cloning of PLpro with an N-terminal 6xHis-SUMO1 tag, synthetic fragments of the Nsp3 coding sequence derived from the original Wuhan strain were purchased from BioCat.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>BioCat</div> <div>suggested: (BioCAT, RRID:SCR_001440)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All data were analyzed using GraphPad Prism (v8.4)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>GraphPad Prism</div> <div>suggested: (GraphPad Prism, RRID:SCR_002798)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Densitometric analysis of protein bands on gels was performed using ImageJ (v1.52p)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>ImageJ</div> <div>suggested: (ImageJ, RRID:SCR_003070)</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The LC-MS systems used was either a Thermo Fisher Finnigan Surveyor Plus equipped with an Agilent Zorbax 300SB-C18 column (3.5 μm, 3.0 x 150 mm) coupled to a Finnigan LTQ mass spectrometer or an Agilent 1100 Series HPLC equipped with a Luna 4251-E0 C18 column (3 μm, 4.6 x 150 mm) coupled to a PE SCIEX API 150EX mass spectrometer (wavelengths monitored = 220, 254 and 646 nm).</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div>Thermo Fisher Finnigan Surveyor Plus</div> <div>suggested: None</div> </div> </td></tr></table>

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


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


      Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04535167</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">First-In-Human Study To Evaluate Safety, Tolerability, And P...</td></tr></table>


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


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


      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. Anthony Tattersal

      I suggest to add a link to his biography or any other link that tells the reader who he is

    1. Benardou, Agiatis, Panos Constantopoulos, Costis Dallas, et Dimitris Gavrilis. 2010. « Understanding the information requirements of arts and humanities scholarship ». International Journal of Digital Curation 5 (1):18‑33. « British Museum Collection ». 2015. https://old.datahub.io/dataset/british-museum-collection. Brown, Susan. 2011. « Don’t Mind the Gap: Evolving Digital Modes of Scholarly Production across the Digital-Humanities Divide ». In Retooling the humanities: The culture of research in Canadian universities, édité par Daniel Coleman et Smaro Kamboureli, 203‑31. Edmonton: University of Alberta Press. http://hdl.handle.net/10402/era.25382. Brown, Susan, et John Simpson. 2013. « The curious identity of Michael Field and its implications for humanities research with the semantic web ». In 2013 IEEE International Conference on Big Data, 77‑85. IEEE. http://ieeexplore.ieee.org/xpls/abs_all.jsp?arnumber=6691674&tag=1. Bulger, M, E Meyer, G De la Flor, M Terras, S Wyatt, M Jirotka, K Eccles, et others. 2011. « Reinventing research? Information practices in the humanities ». Information Practices in the Humanities (March 2011). A Research Information Network Report. Crane, Gregory. 2006. « What do you do with a million books? » D-Lib magazine 12 (3). Corporation for National Research Initiatives. « DBpedia ». 2015. https://wiki.dbpedia.org/. « Digital Environmental Humanities ». 2015. https://dig-eh.org/. « Dublin Core Metada Initiative ». 2015. https://www.dublincore.org/. Egerton, Frank N. 2013. « History of ecological sciences, part 47: Ernst Haeckel’s ecology ». The Bulletin of the Ecological Society of America 94 (3). JSTOR:222‑44. « eMOP: Early Modern OCR Project ». 2015. https://emop.tamu.edu/. Europeana. 2014. « Linked Open Data ». Europeana Pro. https://pro.europeana.eu/page/linked-open-data. Fons, Ted. 2014. « Transforming bibliographic records into linked open data (LOD) ». Panel presentation at the Coalition for Networked Information Fall 2014. https://www.cni.org/topics/information-access-retrieval/exposing-library-collections-on-the-web-challenges-and-lessons-learned. Godby, Jean, Karen Smith-Yoshimura, Bruce Washburn, Kalan Knudson Davis, Karen Detling, Christine Fernsebner Eslao, Steven Folsom, et al. 2019. « Creating Library Linked Data with Wikibase: Lessons Learned from Project Passage ». OCLC Research Report. https://www.oclc.org/content/dam/research/publications/2019/oclcresearch-creating-library-linked-data-with-wikibase-project-passage.pdf. Hegde, Medha. 2012. « Ecotones: the transitional zones ». Biotech Articles, nᵒ 12. http://www.biotecharticles.com/Biology-Article/Ecotones-The-Transitional-Zones-2191.html. Hendler, Jim, et others. 2011. « Why the Semantic Web will never work ». In 7th Extended Semantic Web Conference (ESWC 2011), Crete, Greece. http://videolectures.net/eswc2011_hendler_work/. Internet Philosophy Ontology (InPhO) Project. s. d. « The InPhO Project ». Consulté le 19 juin 2020. https://www.inphoproject.org/. Jaeger, Paul T, Jimmy Lin, Justin M Grimes, et Shannon N Simmons. 2009. « Where is the cloud? Geography, economics, environment, and jurisdiction in cloud computing ». First Monday 14 (5). Klein, Max. 2012. « VIAFbot Debriefing ». OCLC Research. https://hangingtogether.org/?p=2306. Krafft, Dean, et Tom Cramer. 2014. « Video: Linked Data For Libraries (LD4L) Project Update ». Coalition for Networked Information. https://www.cni.org/news/video-linked-data-for-libraries-ld4l-project-update. Lam, Dominic. 2014. « Big Data Challenges in Social Sciences & Humanities Research ». Datanami. https://www.datanami.com/2014/09/08/big-data-challenges-social-sciences-humanities-research/. « Linked Data for Libraries (LD4L) ». 2014. https://wiki.lyrasis.org/pages/viewpage.action?pageId=41354028. LODE: Linked Open Data Enhancer. s. d. « Gihub Linkedhumanities/lode ». Consulté le 19 juin 2020. https://github.com/linkedhumanities/lode. McCarty, William. 2005. Humanities Computing. Palgrave Macmillan UK. Nardi, Bonnie, et Vicki O’Day. 1999. « Information Ecologies: Using Technology with Heart-Chapter Four ». First Monday 4 (5). Valauskas, Edward J. http://firstmonday.org/ojs/index.php/fm/article/view/672/582. OCLC Research. 2014. « Scholars’ Contributions to VIAF ». https://www.oclc.org/research/areas/data-science/viaf-scholars.html. « Open Annotation Data Model ». 2013. http://www.openannotation.org/spec/core/. Pan-Canadian Documentary Heritage Network. s. d. « Linked Open Data (LOD) Visualization “Proof-of-Concept.” ». Canadiana. Consulté le 13 septembre 2015. http://www.canadiana.ca/sites/pub.canadiana.ca/files/PCDHN\%20Proof-of-concept\_Final-Report-ENG\_0.pdf. Price, Gary. 2012. « Video: “Out of the Trenches: A Linked Open Data Project” From the Pan-Canadian Documentary Heritage Network ». LJ infoDOCKET. https://www.infodocket.com/2012/10/25/video-out-of-the-trenches-a-linked-open-data-project-from-pan-canadian-documentary-heritage-network/. Risser, Paul G. 1990. « The ecological importance of land-water ecotones ». In The ecology and management of aquatic-terrestrial ecotones, édité par H Décamps et Naiman R J, 7‑21. Paris: UNESCO. « Schema.org ». 2015. https://schema.org/. Searle, John R. 1995. The construction of social reality. New York: Simon; Schuster. Simpson, John Edward, Susan Brown, et Lisa Goddard. 2013. « A Humanist Perspective on Building Ontologies in Theory and Practice. » In Digital Humanities Conference Abstracts 2013, édité par University of Nebraska, 403‑5. Lincoln. http://dh2013.unl.edu/abstracts/ab-413.html. Smith-Yoshimura, Karen, David Michelson, et Beth Mardutho. 2013. « Irreconcilable differences? Name authority control & humanities scholarship ». OCLC Research. http://hangingtogether.org/?p=2621. « The Muninn Project ». 2015. http://blog.muninn-project.org/. The Stanford Natural Language Processing Group. s. d. « Software > Stanford Named Entity Recognizer (NER) ». Consulté le 19 juin 2020. https://nlp.stanford.edu/software/CRF-NER.html. Uddin, Mueen, et Azizah Abdul Rahman. 2011. « Techniques to implement in green data centres to achieve energy efficiency and reduce global warming effects ». International Journal of Global Warming 3 (4). Inderscience Publishers:372‑89. « VIAF ». 2015. https://viaf.org/. « VIVO Open Research Networking Community Group ». 2015. https://www.w3.org/community/vivo/. Warren, Robert. 2012. « Creating specialized ontologies using Wikipedia: The Muninn experience ». Proceedings of Wikipedia Academy: Research and Free Knowledge (WPAC2012). Berlin. https://wikipedia-academy.wikimedia.de/w/images.wikipedia-academy-2012/0/0f/21_Paper_Robert_Warren.pdf. Widmer, Rolf, Heidi Oswald-Krapf, Deepali Sinha-Khetriwal, Max Schnellmann, et Heinz Böni. 2005. « Global perspectives on e-waste ». Environmental impact assessment review 25 (5). Elsevier:436‑58. « WorldCat Entities ». 2015. OCLC Developer Network. https://www.oclc.org/developer/develop/linked-data/worldcat-entities.en.html. Wuppleman, William. 2012. « Out of the trenches: A linked open data project ». Canadiana. https://www.canadiana.ca.

      Pour la bibliographie issue d'internet, il faut uniformiser dans un sens où dans l'autre : certains sites portent la mention "Consulté le", d'autres non.

    1. 5 Best Trumpet Cases And Gig Bags in 2020 Any good horn player knows how easily you can easlily damages a trumpet, be it seated at home or on the m