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Referee #2

Evidence, reproducibility and clarity

This paper focusses on the mechanisms underlying chromosome biorientation in mitosis, an essential process that warrants equal chromosome segregation to the dividing cells. Correction of improper kinetochore-microtubule attachments relies on two conserved protein kinases, Aurora B and Mps1, that detach kinetochores that are not under tension in order to provide them with a second opportunity to establish bipolar connections. In vivo, Aurora B and Mps1 have intertwined functions and share some common targets. For this reason, despite the large body of literature on the subject, their precise roles in chromosome biorientation have been difficult to tease apart.

The authors take advantage of an in vitro reconstitution assay that they previously published (Akyioshi et al., 2010) to identify the critical target(s) of Mps1 in weakening kinetochore-microtubule connections. The assay uses kinetochore particles purified from budding yeast cells that bear Mps1 but are notably deprived of Aurora B. Upon addition of ATP to activate the co-purified kinases (e.g. Mps1), kinetochores are added to coverslip-anchored microtubules to which they attach laterally. Through a laser trap, kinetochores are brought to the microtubule plus-end and pulled with increasing force until the kinetochore detaches, which allows measurements of the average rupture forces that reflect the strength of the attachments. The approach is straightforward and potentially very powerful, first because it provides a simplified experimental set-up in comparison to the cellular context, and second because it directly measures the impact of protein phosphorylation on the strength of attachments.

The authors convincingly show that Mps1-dependent phosphorylation of the N-terminal part of Ndc80 significantly weakens the strength of kinetochore-microtubule attachments in vitro, while phosphorylation of other known Mps1 targets, such as Spc105, does not seem to have an effect. Eight phosphorylation sites in Ndc80, which were previously identified as Mps1-dependent phosphorylation sites (Kemmler et al., 2009), are shown to be critical to destabilise kinetochore-microtubule attachments in the in vitro reconstitution assays. The authors also present evidence for a moderate involvement of Ndc80 phosphorylation by Mps1 in correcting improper attachments in vivo, suggesting that additional mechanisms are physiologically relevant for error correction.

The experiments are mostly well designed, the data are solid and support the main conclusions. However, to my opinion additional experiments could be performed, as outlined below, to strengthen the physiological relevance of the main findings and corroborate some of the conclusions.

Major points:

1. Given the partially overlapping function of Mps1 and Ipl1 (Aurora B) in error correction, the ndc80-8A mutant should display synthetic growth and chromosome mis-segregation defects with ipl1 temperature-sensitive alleles. Conversely, the ndc80-8D mutant should suppress the lethality at high temperatures of mps1-3 mutant cells, which were recently shown to be defective in chromosome biorientation (Benzi et al., 2020). Finally, chromosome mono-orientation could become apparent in ndc80-8A cells upon a transient treatment with microtubule-depolymerising drugs, which should amplify the cellular need for error correction.
2. The authors show that Mps1-dependent phosphorylation of Ndc80 is not involved in the spindle assembly checkpoint, a conclusion that contradicts a previous report (Kemmler et al., 2009). They also find, in contrast with the same report, that the lethal phenotype of the ndc80-14D phospho-mimetic mutant cannot be rescued by disabling the spindle checkpoint. In my opinion, Kemmler et al. convincingly showed, through a number of different experimental approaches, that ndc80-14D cells die because of spindle checkpoint hyperactivation. Not only deletion of checkpoint genes was shown to rescue the lethality, but re-introduction of a wild type copy of the deleted checkpoint gene reinstated lethality. Thus, the explanation invoked here that spontaneous suppressing mutations could underlie the viability of ndc80-14D SAC-deficient mutants is not consistent with the published observations. A thorough examination by the authors of the phenotype of ndc80-14D cells in their hands should be carried out to support these conflicting conclusions. If authors find that ndc80-14D cells actually die because of chromosome mono-orientation, then this would highlight an important function for some or all the six additional phosphorylation sites, relative to the ndc80-8D mutant, for chromosome biorientation in vivo.
3. The conclusion that Spc105 phosphorylation by Mps1 is not required for the Mps1-mediated weakening of kinetochore attachments in vitro is based on the comparison between kinetochore particles bearing wild type, untagged Spc105 and particles bearing non-phosphorylatable Spc105-6A tagged at the C-terminus with twelve myc epitopes. Thus, the presence of the tag could obliterate the effects of the mutations in the phosphorylation sites by destabilising kinetochore-microtubule attachments in the presence of ATP. Consistent with this conclusion, Spc105-6A-12myc-bearing kinetochores withstand lower rupture forces than Spc105-bearing kinetochores upon ATP addition. Furthermore, Spc105-6A-12myc kinetochore particles show an interacting protein at MW above 150 KD that is not present in wild type particles (Fig. S2A), suggesting that either the tag or the mutations might affect kinetochore composition. Thus, this set of experiments should be repeated using Spc105-6A kinetochore particles lacking the tag.
4. In general, it would have been informative to complement the data presented here with a mass spec analysis of the composition of kinetochore particles, at least for the experiments that are most relevant to the conclusions. For instance, the composition of the Ndc80-8A kinetochore particles is assumed to be similar to that of wild type kinetochores based on gel silver staining (Fig. S4A; note also that ndc80-8A particles are compared to ndc80-8D particles and not to wild type particles). However, the authors previously showed that kinetochore particles purified from dad1-1 mutant cells (affecting the Dam1 complex) have an apparently identical composition to particles purified from wild type cells by silver staining, yet they display significantly lower resistance to the rupture strength in vitro (Akyioshi et al., 2010). What is the status of the Dam1 complex (or other kinetochore subunits) in kinetochores purified from ndc80-8A/-8D or spc105-6A cells relative to wild type kinetochore particles?

Minor comment:

I believe that the right reference for the sentence in the Discussion "If Aurora B is defective, for example, the opposing phosphatase PP1 prematurely localizes to kinetochores" is Liu et al. 2010.

Significance

Although the experiments are well designed and the conclusions are mainly supported by the data, the question arises as to what extent the in vitro assays recapitulate, at least partly, what happens in vivo. An emblematic example is the involvement of Spc105 in the error correction pathway. The Biggins lab previously showed that Spc105 phosphorylation by Mps1 and subsequent Bub1 recruitment is not only essential for the spindle assembly checkpoint, but is also crucial for chromosome segregation in vivo, as shown by slow-growth phenotype and aneuploidy of the spc105-6A non-phosphorylatable mutant (London et al., 2012). Additionally, a recent paper showed that Spc105 is a crucial Mps1 target in chromosome biorientation (Benzi et al., 2020).

In sharp contrast, the ndc80-8A mutant, which in vitro completely erases the ability of Mps1 to destabilise kinetochore-microtubule attachments, displays no growth defects in otherwise wild type cells and only modestly enhances chromosome mis-segregation in a mutant affecting an intrinsic correction pathway (stu2ccΔ). The N-terminal part of Ndc80 (aa 1-116) containing the aforementioned eight phosphorylation sites can even be deleted altogether without any consequence on cell viability (Kemmler et al., 2009). Thus, although the in vitro assays presented here produced clear-cut and reproducible results, their physiological relevance in vivo remains unclear.

Left apart this criticism, the manuscript has several merits outlined above and will be of interest for people working in the fields of chromosome segregation, kinetochore assembly, spindle assembly checkpoint, etc.

Expertise of this reviewer: mitosis and related checkpoints

该版本同测试网的不兼容，到6月21日上主网了才能用。需要BZZ代币。

Reviewer #3 (Public Review):

The authors modified a previously reported hybrid cytochrome bcc-aa3 supercomplex, consisting of bcc from M. tuberculosis and aa3 from M. smegmatis, (Kim et al 2015) by appending an affinity tag facilitating purification. The cryo-EM experiments are based on the authors' earlier work (Gong et al. 2018) on the structure of the bcc-aa3 supercomplex from M. smegmatis. The authors then determine the structure of the bcc part alone and in complex with Q203 and TB47.

The manuscript is well written and the obtained results are presented in a concise, clear-cut manner. In general, the data support the conclusions drawn.

To this reviewer, the following points are unclear:

1. The purified enzyme elutes from the gel filtration column as one peak, but there seems to be no information given on the subunit composition and the enzymatic activity of the purified hybrid cytochrome bcc-aa3 supercomplex.

2. It is unclear what is the conclusion of the structure comparison (Fig 6) is regarding the affinity of Q203 for M. smegmatis.

SciScore for 10.1101/2021.06.16.448640: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

• tag : fission, webnative

pure function components

Reviewer #1 (Public Review):

The manuscript by Chakraborty focuses on methods to direct dsDNA to specific cell types within an intact multicellular organism, with the ultimate goal of targeting DNA-based nanodevices, often as biosensors within endosomes and lysosomes. Taking advantage of the endogenous SID-2 dsRNA receptor expressed in C. elegans intestinal cells, the authors show that dsDNA conjugated to dsRNA can be taken into the intestinal endosomal system via feeding and apical endocytosis, while dsDNA alone is not an efficient endocytic cargo from the gut lumen. Since most cells do not express a dsRNA receptor, the authors sought to develop a more generalizable approach. Via phage display screening they identified a novel camelid antibody 9E that recognizes a short specific DNA sequence that can be included at the 3' end of synthesized dsDNAs. The authors then showed that this antibody can direct binding, and in some cases endocytosis, of such DNAs when 9E was expressed as a fusion with transmembrane protein SNB-1. This approach was successful in targeting microinjected dsDNA pan-neuronally when expressed via the snb-1 promoter, and to specific neuronal subsets when expressed via other promoters. Endocytosed dsDNA appeared in puncta moving in neuronal processes, suggesting entry into endosomes. Plasma membrane targeting appeared feasible using 9E fusion to ODR-2.

The major strength of the paper is in the identification and testing of the 9E camelid antibody as part of a generalizable dsDNA targeting system. This aspect of the paper will likely be of wide interest and potentially high impact, since it could be applied in any intact animal system subject to transgene expression. A weakness of the paper is the choice of "nanodevice". It was not clear what utility was present in the DNAs used, such as D38, that made them "devices", aside from their fluorescent tag that allowed tracking their localization. Another potential weakness is that the delivered DNA is limited to the cell surface or the lumen of endomembrane compartments without access to the cytoplasm or nucleus. In general the data appeared to be of high quality and was well controlled, supporting the authors conclusions.

• doing what one believes is best for community

• number of simultaneous connections (peak and average)

• the message rate/payload size.

SciScore for 10.1101/2021.06.09.447527: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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).

To avoid this therapeutic limitation, we screened and selected the antibodies showing neutralization activity against several variants of SARS-CoV-2. We took advantage of the mouse immunoglobulin repertoire 18 through the hybridoma technology to isolate monoclonal antibodies neutralizing live authentic SARS-CoV-2 virus. The in vitro and live virus screening assays allowed us to identify two neutralizing antibodies, AX290 and AX677, each of them neutralizing live authentic SARS-CoV-2 virus circulating in Europe in the spring of 2020 and two fast-spreading authentic variants of concern B.1.1.7 and B.1.351. It is important to emphasize that the antibodies were resistant to the E484K mutation, which has been recently shown to endow the S-typed VSV pseudovirus with resistance to several human convalescent sera 33. The authors have suggested that the repertoire of antigenic sites on RBD is limited in some individuals and that this amino acid is a part of a dominant neutralizing epitope. The use of a live authentic SARS-CoV-2 virus to identify the escape mutations, as opposed to other studies that employed an S-typed pseudovirus 10,33,35–38, allowed to eliminate a potential bias from an inadequate infection and replication machinery of a model virus. It has been noted that only some of the spike escape mutations identified in SARS-CoV-2 S-typed infectious vesicular stomatitis virus were found so far in the context of authentic SARS-CoV-2 virus isolates, it is possible that those not...

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.

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

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

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.

"tag": 🦋Biocolores

SciScore for 10.1101/2021.06.07.447437: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

In Moz's example of their Title Tag, they have their name and what their role is as well as what service they provide. Aside from a company name and possibly a role or action, what other key words would be suggested?

SciScore for 10.1101/2021.06.05.447221: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

SciScore for 10.1101/2021.06.06.446826: (What is this?)

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

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

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.

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

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

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.

new tag?:

• extract reusable functions to reduce duplication / allow elegant patterns elsewhere

new tag?:

• understand why it is correct

SciScore for 10.1101/2021.06.04.447160: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

SciScore for 10.1101/2021.06.02.21258073: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

Same feedback as the Atelier. In addition,

• The job title and "self-employed" sound inconsistent. Consider a more powerful title. I'd get rid of the self-employed tag. It's got a bit of a bad reputation on LI
• The company description needs to add some points of differentiation. Currently it doesn't impress.

SciScore for 10.1101/2021.06.02.446468: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

SciScore for 10.1101/2021.05.26.21256092: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

Should this be in an annotations property of the Canvas, Manifest, or AnnotationPage?

Reviewer #1 (Public Review): 

The paper by Sim et al describes phospho-proteomic analysis of ATR kinase-dependent pathway in mouse spermatocytes. By administrating an ATR inhibitor, AZ20, to mice and using Rad1 (a component of 911 DNA damage clamp) conditional knockout mouse (cKO), the authors isolated testis from these mice, isolated phospho-peptides and analyzed with Tandem Mass Tag (TMT). The analyses identified 37,180 phosphorylation sites and created the data base for them. Importantly, in-depth analysis of the phosphorylation sites revealed an unique consensus site of the ATR-dependent phosphorylation; S/TPXK, whose kinase has not been identified yet. In addition, the authors showed new ATR-dependent phosphorylation sites in proteins in RNA metabolisms including piRNA biogenesis for transposon silencing and showed ATR-dependent localization of two RNA processing enzymes, SETX helicase, and RANBP3 in meiosis sex chromosome inactivation (MSCI). This is an important body of work as a good resource for phosphorylation in mouse germ cells. The study had been done in a great care with proper controls. The data set in the paper is very much useful and of great interest to researchers in meiosis and DNA damage response (DDR) field.

SciScore for 10.1101/2021.05.26.21257441: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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).

While our study aims to provide an overview of many different aspects of pre-existing immunity against VOC, there are some limitations. In this study, we present a vaccinee population that consists of primary health care workers that are generally of middle age, with only four (8%) participants being older than 60 (Table 1). However, we found no correlation between age and titers in convalescent patients against any of the VOC tested. Moreover, we have focused on the neutralization potential of immune sera and did not examine antibody effector functions which have been implicated previously in the control of SARS-CoV-2 infection (41). Similar to other studies, our convalescent and vaccinee sera samples were collected four to six weeks after infection and four weeks after vaccination (i.e. at the peak of immunity), respectively. While we present findings that may have implications for additional booster vaccines and the monitoring of VOC, we did not examine the aspect of waning antibody titers and how those might influence VOC binding and neutralization. Finally, we have merely examined an early line of defense against infection with SARS-CoV-2 (i.e. serum antibody levels), while we expect memory B cells to play an important part in re-infections with SARS-CoV-2 VOC. Indeed, swift re-activation of memory compartments may lead to reduced transmissibility, a milder course of disease and a more potent immune response. In conclusion, we observed a substantial reduction in binding ...

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.

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

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

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.

SciScore for 10.1101/2021.06.01.446555: (What is this?)

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

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

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.

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

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

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.

• not worth saying
• not necessary to say/write
• useless information

Suppress-Script

unstated-but-realized

Why was it not free???

select ... over

database: query builder

like ActiveRecord for node

SQL: experimenting with table-free data

SciScore for 10.1101/2021.05.29.446300: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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).

At present, the limitations for selecting POI for Antibody Display such as protein shape and size remain unclear; further studies will be required to address these questions. Comparing with conventional Fc fusion proteins in which the POI is fused to the N terminus of Fc usually including the flexible hinge region, we reasoned that the Antibody Display, which grafts the POI onto the β-strands G and F of the heavy chain, provides several potential advantages: (1) design and production of a more conformationally constrained chimeric protein, (2) reduction of proteolysis with the POI inserted into a stable antibody Ig fold, and (3) extension of the current antibody engineering toolkit for generating bispecific and chimeric antibodies. Further studies will be needed to explore these potential advantages. An intriguing feature of using the Antibody Display for Tspan12EC2 is with a better yield of protein production than Tspan12EC2. Our functional assays demonstrated that Tspan12EC2 bound to Norrin directly, in agreement with previous genetic studies (Lai et al., 2017). As Tspan12 playing critical roles in CNS vascular development (Junge et al., 2009; Zhang et al., 2018) and tumorigenesis (Knoblich et al., 2014; Otomo et al., 2014), Tspan12EC2-AD may be a useful reagent for these studies. Moreover, Tspan proteins are well known for their important roles in regulating tumor migration, invasion and metastasis (Charrin et al., 2014; Hemler, 2014; Vences-Catalan and Levy, 2018). Antibo...

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.

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

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

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.

SciScore for 10.1101/2021.05.31.445667: (What is this?)

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

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr style="background-color:#FF0000"><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT811</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Trial number did not resolve on clinicaltrials.gov. Is the number correct?</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NA</td></tr></table>

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.

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

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

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.

SciScore for 10.1101/2021.05.31.446386: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 TrialIdentifier: No clinical trial numbers were referenced.

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

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

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

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

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.

SciScore for 10.1101/2021.05.28.446179: (What is this?)

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

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

Although several articles and preprints in the past year74,79–84 have reported simulations of this structure, a review of their results shows similar limitations, with abbreviated timescales,80–82 reliance on secondary-structure restraints,74 elevated RMSDs,74,80 and/or dewetting in the absence of electric fields.74 Instability of the open structure may reflect underdetermination of the starting protein or membrane models, unresolved interactions with the C-terminal or other domains, or other factors yet to be identified. Taken together, we have shown that recombinantly expressed full-length E protein from SARS-CoV-2 is capable of independent multimerization, possibly as a tetrameric or smaller species. We also confirmed that the protein localizes intracellularly, similar to its predecessor from SARS-CoV, with no evidence of ion channel properties at the cell surface. Our coarse-grained simulations further support a role for the E protein in viral budding, as the presence of the protein bends the surrounding membrane. Reduction of intracellular Ca2+ in E-protein-transfected cells may further promote viral replication. However, our atomistic simulations and permeation calculations based on previously reported NMR structures resulted in unstable proteins incapable of Ca2+ conduction. We emphasize the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insight of the E protein, and enable future drug-screening effort...

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.

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

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

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.

• supporting those you like by sending business to them
• less formal and more approachable

.

SciScore for 10.1101/2021.05.26.445185: (What is this?)

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

Table 1: Rigor

Table 2: Resources

Fitting was carried out in MATLAB by non-linear least-squares regression using the Trust-Region algorithm.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MATLAB</div><div>suggested: (MATLAB, RRID:SCR_001622)</div></div></td></tr></table>

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

Results from 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.

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

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

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.

art factores

definicion referencia oportuna

I've noticed that Dan Mackinlay has some public notebooks with an interesting system for indicating knowledge process too.

His Twitter tag line is: "The Spectator's Wiki Man."

There are a number of bloggers who have to some extent, specifically used their blogs for this purpose though. I've documented several at https://boffosocko.com/tag/thought-spaces/

cherry-picking only some commits from a PR

where did it come from? don't remember

after a while, something that came from another starts to feel like your own

you make it your own

happy middle/medium?

I wonder if this insulation will be applicable to other gram negatives? (or other gram positives too?)

SciScore for 10.1101/2021.05.21.445201: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

SciScore for 10.1101/2021.05.23.445348: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

This as since been renamed to updateless decision theory%20is,which%20it%20makes%20its%20decisions.&text=It%20acts%20as%20though%20its,some%20dualist%20free%2Dwill%20theories.)

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

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Summary:

In the manuscript „Intra-helical salt bridge contribution to membrane protein insertion" the authors investigate the effect of salt bridge formation between positively and negatively charged amino acids on the insertion behavior of α-helical protein segments into the membrane. Generally it is believed that polar or even charged residues prevent stable membrane insertion of α-helical protein segments, but some of these authors had already shown in a previous paper that such residues are more frequent than expected in transmembrane helices. In the current study, the authors investigate in detail the role of intra-helical salt bridge formation on stable membrane insertion. Using an in vitro membrane insertion assay based on the E. coli leader peptidase (Lep) protein, they found better membrane insertion for helical segments with opposite charge pairs placed at positions compatible with intra-helical salt bridge formation (positions i→i+1; i→i+3 and i→i+4). Furthermore, the authors performed a database screen which revealed that oppositely charged residues are overrepresented at these positions. Finally they picked two candidate membrane proteins from the database (Halorhodopsin and calcium ATPase 1) and proved the presence of an intra-helical salt bridge and determined the contribution of the salt bridge to the apparent free energy of membrane insertion (ΔGapp), which was in the range of 0,5-0,7 kcal/mol.

Major comments

1. It seems that the data in Fig. 3b has been mixed up, making it difficult to judge the conclusions. The bars with forward slash seem to represent the "same charge" data and the bars with backward slash seem to represent the "opposite charge" data (exactly contrary to the figure legend). In general the forward and backward slash representation is not easily distinguishable, and for the position i+4 both bars contain a forward slash (making it impossible to discriminate same and opposite charge). Please use filled and unfilled bars instead. Furthermore the bar diagram in Fig. 3a is stacked for opposite and same charge, whereas in Fig. 3b the respective bars are placed next to each other. Additionally the label of the y-axis in Fig. 3c is misleading, as it is not the "Frac. of opp. charged pairs" but the fraction of oppositely charged pairs that form salt bridges.
2. The authors don´t give details no how the log odds ratios and the respective p-values have been determined. Please include this in the Materials and Methods section. What does a p-value of 0.00e+00 mean (see Table 2, Spacing: +3, "All Log odds")?
3. What is the proof that for the isolated helix A from the calcium ATPase 1 the membrane embedded part is identical to the full-length protein? The authors investigated two different helix A peptides, the full-length helix ranging from L49-F78, and one short fragment ranging from L49-A69 containing the more hydrophilic N-terminal region, which is the membrane-embedded region in the full-length protein. The authors state: "In contrast, when only the membrane-embedded sequence was included, the Lep chimera was mainly doubly-glycosylated (Fig. 5c, lane 3), suggesting that helix A is properly inserted when the full helical sequence is present." In my opinion this conclusion cannot be drawn from the data presented. The authors used an isolated helical segment, so in my opinion it is much more likely that the isolated full-length helix inserted via its hydrophobic C-terminal part (L60-F78) into the membrane. The authors themselves state in their manuscript: "It has been previously shown that the position in the membrane of TM helices in protein folded structures does not always correspond to the thermodynamically favored positions in the membrane of the isolated helices." Also the i→i+5 mutant points into that direction, because the effect of disturbing the intra-helical salt bridge for the helix A is much less pronounced compared to the similar data in Fig. 4f for the Halorhodopsin protein. In my opinion this shows that most probably only one charged residue (R63) is embedded inside the membrane (with a membrane embedded part of L60-F78).

Minor comments:

1. line 151: ",see Figure 2)" Typo: Bracket missing.
2. line 172: "Other known structural features can also be hinted at, including aromatic ring stacking by His-Trp pair [20] at i→i+6." Please give some more examples of important structural features of membrane proteins, which can be seen in your analysis (e.g. I think that also the glycine zipper can be seen in the i→i+4 data set).
3. line 255: "The salt bridge contributes approximately ~0,5 kcal/mol to the apparent experimental free energy of membrane insertion." Please explain that this value was derived from the comparison of the ΔGexp between the wt and the i→i+5 mutant. Please comment also on the large difference between the predicted (ΔGpred) and the experimental values (ΔGexp), even if no salt-bridge is involved (e.g. for the DD mutant).
4. line 348: "Asp-Lys pairs at position i, i+4 and Glu-Lys pairs at position i→ i+3 are the most prevalent as seen previously in Figure 2. They are both among the most prevalent oppositely charged pairs and the charged pairs that form the highest number of salt bridges in membrane protein structures. This is in stark contrast to Glu-Arg pair at position i→ i+1 that although as frequent in pairs as Asp-Lys and Glu-Lys at positions i→i+4 and i→i+3 respectively, only form salt bridges in one-fourth of the cases." Fig. 2 shows that each charge pair has a different prevalence depending on the order (e.g. Asp-Lys and Lys-Asp pairs). I think for this statement the sum of both prevalences should be taken into account, and as the sum is not easy discernible from Fig. 2, it would help to include a table containing the sums. Furthermore, it would be good to refer also to Fig. 3, which also contains a part of the discussed data.
5. line 402: "c-myc tag (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, EQKLISEEDL) was added in Ct in hanging with de PCR primer before cloning." Please revise the sentence and I think the one letter code for the c-myc tag is sufficient (please correct this also in line 428).
6. line 420: "A region's total ΔG is the sum of these individual scores weighted on where in the region the residue, a residue in the middle of the helix has a higher weight than residues at the ends." Please revise the sentence, the meaning is unclear.
7. line 436: "Total protein was quantified and equal amounts of protein submitted to Endo H treatment or mock-treated, followed by SDS-PAGE analysis and transferred into a PVDF transfer membrane (ThermoFisher Scientific)." Please revise the sentence.
8. line 498: "Topological files with sequence and membrane topology are created with the help of the RCSB secondary structure file and only membranes annotated as pure α-helices were retained." I assume that the description contains a typo (membranes annotated as pure α-helices?)
9. line 507: typo "..., but we did not clustered the proteins" 14: line 560: "The individual value of each experiment in represented by a solid dot being represented as a green square the experimental ΔG value for the L4/A15 sequence from [2]." Please revise the sentence. 15: line 562: "The wt and simple mutants are shown in white bars." Typo: single mutants 16: line 563: "Charges at compatible distances with salt bridge formation (i→i+1; i→i+3; and i→i+4) are shown in yellow. Not compatible distances with salt bridge formation (i→i+2; and i→i+5) are shown in dark gray. Compatible distances but not compatible amino acid pair (i, i+4 DD pair) is shown in clear gray." The given colors don´t match with the figure (i→i+1 = brown; i→i+3 = orange; i→i+4 = yellow and i→i+4 DD pair = white) 17: line 597: "The different monomers are shown in transparent blue, purple and indigo." The different colors are hardly distinguishable in the figure. 18: Figure 4a: The table could be simplified. I think the column "charges" can be removed, as it contains not really charges and the names of the peptides already contain the same information. The column "Å" contains only a value for the wt (and not for the DK i,i+5 mutant) and as the distance for the wt is also given in Fig. 4g, this column can be also removed.
10. Fig. 4f: The marker lane is hardly visible (completely dark lane)
11. Fig. 5b: The column "Å" contains only values for the wt sequences (long and short). See also comment 18.
12. Fig 5d: Why is in the SDS gel a mass shift between the wt and the i→i+5 mutant visible, even though the peptide mass is equal.
13. There are several changes of font type or format changes (e.g. line 210-214). Please correct this.

Significance

As a structural biologist with a focus on membrane-proteins, I understand that the study is concerned on intra-helical salt bridges, but the implications of inter-helical salt bridges should also be discussed, at least in the introduction or outlook. The authors propose that their results are important for the improvement of membrane protein topology prediction methods, so for this aim it is also necessary to take any potential inter-helical salt bridges into account. In this context, it would be relevant to point point out that there even exist extended rows of salt bridges between transmembrane segments (charge-zippers), which serve an important structural element in several membrane proteins.

The article is well written and most of the conclusions drawn from the experimental results are convincing. I agree with the authors that their results are relevant for future improvement of membrane protein topology prediction software, which so far does not take the possibility of salt bridge formation into account. Therefore, I recommend publication after clarification/revision of the abovementioned points.

SciScore for 10.1101/2021.05.19.444889: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 TrialIdentifier: No clinical trial numbers were referenced.

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

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 10 and 21. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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

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

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.

SciScore for 10.1101/2021.05.16.444369: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

SciScore for 10.1101/2021.05.15.444222: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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

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.

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

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

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.

SciScore for 10.1101/2021.05.13.444010: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

IMHO #go == [[go]]. In that sense we should probably support all conventions that clearly demonstrate an [[intent]] to trigger an action, I'm fine adding support for these.

DOI: 10.1016/j.celrep.2021.109151

Resource: (Thermo Fisher Scientific Cat# 26183, RRID:AB_10978021)

Curator: @Naa003

SciCrunch record: RRID:AB_10978021

Curator comments: HA Tag Monoclonal Antibody (2-2.2.14) Thermo Fisher Scientific Cat# 26183

What is this?

This is an example of spaced repetition being done as retrospective and hiding some of the value of making the important things stand out and reviewing them for better long term retention.

Tag:RSS,ColorAposematico

https://pubmed.ncbi.nlm.nih.gov/rss/search/1-7FJ7kkHKrEcV2x8GNHBU9h3RdKSg9-Zlru1eJQISJRXlEg5e/?limit=100&utm_campaign=pubmed-2&fc=20210518104625

https://twitter.com/bugsnstuffcom/status/1301445545299505153

Tag:ColorAposematico

https://www.wikidata.org/wiki/Q619751

"Tag:"ColorAposetamitco

TAG:CicloVida

Tag:Colage

**Tag:"Aposematismo,ColorAposematico,Query

Tag:Aposematismo,ColorAposematico,Query

Tag:Aposematismo,ColorAposematico,Query

https://doi.org/10.1111/jeb.12676 TagColorAposematico

https://doi.org/10.1093/beheco/arl046

Tag:ColorAposematico

https://doi.org/10.1007/BF00384948

Tag:ColorAposematico

https://doi.org/10.1016/j.tree.2005.07.011

Tag:ColorAposematico

https://doi.org/10.2307/3544135

Tag:ColorAposematico

SciScore for 10.1101/2021.05.06.21256766: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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).

Regarding the potential limitations, we did not collect skin swabs to control for the potential presence of SARS-CoV-2 in the skin [7,33]. However, all milk samples as well as the infants were negative for SARS-CoV-2 presence. Thus, although there are still limited data, it is accepted that breastfeeding does not represent a vehicle for vertical transmission of SARS-CoV-2 [9]. There are still many open questions: when are SARS-CoV-2 antibodies produced after maternal infection, when can they be detected in breast milk, and how long do they persist. To cover some of these questions, we aimed to determine the presence of antibodies in breast milk samples from COVID-19 women and to compare these with milk samples collected prior to the pandemic as reference controls. While different studies have reported presence of specific IgA antibodies against SARS-CoV-2 [7,12,13,36], limited information is available on IgG and IgM. Our results showed presence of anti-SARS-CoV-2 antibodies in milk, primarily IgA but also IgG and IgM targeting RBD. Furthermore, IgA- and IgG against non-structural MPro were analyzed for the first time in human milk samples. High intra- and inter-individual variability was observed in antibody presence and significant differences were observed for all three antibody classes when compared to prepandemic samples in agreement with previous data [26]. In our dataset we found that 82·9 % of the milk samples tested positive for the RBD antigen at least in one of the ...

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04768244</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">Impact of Maternal COVID-19 Disease on Breast Milk and Infan…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT03552939</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Completed</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">The Impact of Maternal Microbes on Infant Health Programming</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.

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

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

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.

这段主要解释嵌套匿名结构在 marshal or unmarshal 时，field key 的冲突情况。我把 untagged 域 json 化的 key 称为 field key，tagged 域 json 化的 key 称为 tag key。

注意：这里讲的 tagged 域是指设置了 key name 的 tag，没有设置 key name 的 tagged 域在 json 化中使用的还是 filed key。

本段涉及的域处在同一层级，且该层级至少是一层嵌套层。

# 至少是这样的嵌套
type A struct {
    Name string
}

type B struct {
    Name string
}

type C struct {
    Name string
}

type D struct {
    A
    B
    C
}

func TestFeature(t *testing.T) {
    d := D{A{"A"}, B{"B"}, C{"C"}}
    r, _ := json.Marshal(d)
    fmt.Println(string(r))
}

以上 D struct 的 instance 有多个相同名称的域 Name。在对其做 marshal or unmarshal 时应用以下规则：

1、json tagged 的域优先于 untagged 的域考虑。

# 如果 A 的域被 `json:"Name"`，则 B C 里的 Name 域都被忽略。
{"Name":"A"}

2、如果 tagged 域的 tag key 唯一，则被正常 marshal or unmarshal，否则冲突的 tagged 域都被忽略。如果 untagged 域的 field key 唯一，则被正常 marshal or unmarshal，否则冲突的 untagged 域都被忽略。

# 如果 A 和 B 的 Name 域都被 `json:"Name"`，则视为  tag key 冲突，所以两者都被忽略。并且 C 的 Name 域为 untagged，所以 C instance 在这场“Name 域 json 化争夺战”中完全处于下风。最终这场战斗没有人胜出。
# {}

# 如果 A 的 Name 域被 `json:"Nickname"`，则剩下 B C 两个 untagged 域争夺，根据规则，最终没有在 json string 中见到 Name key。
# {"Nickname":"A"}

首先要明白什么是 anonymous struct field，看这里。

看过上面文章，应该对 anonymous struct field 及其规则有大致的了解。

在对 anonymous struct field 做 json encoding 时，anonymous struct field 中的 exported field 默认会被当做 outer struct 的 field 来 encoding。

如果对匿名结构域使用 json tag 功能给予 key name，则在 encoding 时不会被当作匿名结构域应用上述规则。

如果匿名结构域是 interface{} 类型，则也不会被当作匿名结构域应用上述规则，interface{} 的名称会被当作 key name。

举例：

type Kitchen struct {
    NumOfPlates uint
}

type Door interface {
    echo()
}

type FrontDoor struct {
    Name string
}

func (frontDoor *FrontDoor) echo() {
    fmt.Println("I'm " + frontDoor.Name)
}

type House struct {
    Kitchen    `json:"kitchen"`
    NumOfRooms uint
    Door
}

func main() {

    h := House{Kitchen{10}, 3, &FrontDoor{"Gold Door"}}

    sp, _ := json.Marshal(h)
    fmt.Println(string(sp))
}
# {"kitchen":{"NumOfPlates":10},"NumOfRooms":3,"Door":{"Name":"Gold Door"}}
# 正常被当作匿名结构域 encoding json 时：
# {"NumOfPlates":10,"NumOfRooms":3}

struct field 的 json encoding 行为可以通过 json tag 自定义，格式为：json:"keyName,option1,..."

以下是一些使用情况介绍：

1、即想使用 json tag 定义行为又要使用 struct field name 来作为 json key：json:",option1,..."

2、使用 omitempty 选项可以忽略该 field 的 json encoding，如果该 field 的值是空值的话。

3、使用 json:"-" 的 field 在 encoding 时被忽略。如果想要使用 "-" 做为 json key，那么在 "-" 后加上一个 comma 即可：json:"-,"。

4、如果你想要一些非 string 的类型被 encoding 为 json string，使用 json:",string" 标签选项即可。

type Person struct {
    Age uint `json:",string"`
}

func main() {
    p := Person{
        23,
    }
    sp, _ := json.Marshal(p)
    fmt.Println(string(sp))
}
# {"Age":"23"}

这里的 key name 不知道指什么，如果指的是 json tag 里给的 key name，那么在上面已经介绍过了，这里难道只再提一下作为 key name 的规则吗？

SciScore for 10.1101/2021.05.12.443228: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 TrialIdentifier: No clinical trial numbers were referenced.

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

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33, 37 and 38. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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

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

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.

SciScore for 10.1101/2021.05.08.21256891: (What is this?)

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

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

A potential limitation of our proof of concept method is that the use of intercalating dyes can make target-specific real-time detection challenging44, and could pose as a barrier for use in resource limited areas. Future lower resource implementations of our method could substitute EvaGreen for 100-200 µM halochromic dyes, like Cresol Red, in pH 8.8 buffered reaction mix to cause detectable colorimetric change when amplification products have been produced47. Alternatively more advanced detection methods utilizing sequence specific methods based on the use of probes (e.g. LAMP-BEAC23, OSD48, DARQ49 and QUASR50) could also be utilized as end-point detection methods, and remain to be tested in future work. Such methods would be advantageous as they could be multiplexed for different targets in the same reaction. QUASR, for instance, has been shown to allow single step and close-tube multiplexing50. The versions of both M-MLV and BstLF enzymes used in this paper are in the public domain in Chile but some of the M-MLV mutations are still subject to protection in other jurisdictions e.g. in the US (patent no. US7056716B251) with anticipated expiration date of 15 March 2021. Future work will, therefore, explore the use of off-patent enzymes Open-MMLV or HIV RT15 from the E. coli expression toolkit from ReClone initiative52 in order to have freedom-to-operate in any country. The use of public domain enzymes would also permit commercial partnerships seeking to scale-up production lo...

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.

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

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

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.

SciScore for 10.1101/2021.05.11.443686: (What is this?)

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

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

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.

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

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

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.

SciScore for 10.1101/2021.05.10.443532: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 TrialIdentifier: No clinical trial numbers were referenced.

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

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 56 and 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.

• No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
• No funding statement was detected.
• No protocol registration statement was detected.

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

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.

SciScore for 10.1101/2021.05.10.443524: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 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.

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

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

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.

If you look at the incomplete graph.js implementation, you can see that it's actually using a class instead. This is good! However, I advocate for class names that happen to also be a hostname + resource (i.e., like URL, but omitting the http:// part).

(I suppose that, alternatively, if someone wanted to use a shorter class name, such as "w2g-tag", then graph.js could be configured to allow for that, but it would need to be explicit configuration on the author/publisher's part.)

SciScore for 10.1101/2021.05.09.442808: (What is this?)

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

Table 1: Rigor

Table 2: Resources

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 TrialIdentifier: No clinical trial numbers were referenced.

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

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 34 and 31. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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

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

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.