In regular Ruby, `` executes in a shell, but obviously there is no shell of that sort in JS, so it makes sense that they could (and should) repurpose that syntax for something that makes sense in context of JS -- like running native JavaScript -- prefect!

This examplifies how brands can drop people who don't supporttheir image

It was pretty entertaining i must say, especially when they had like 25 women on the magazine covers.

This reminds me of one of Apple's policies; The villain in a movie/TV show is not allowed to use an Apple device. This is because they don't want a negative connotation with their brand.

intresting

This is a definition of a term that could be useful later

The amount of money being spent to be able to achieve a colony on Mars seems to be the biggest drawback that we face in order to achieve this mission. I think that people have to look at the mission as a whole instead of pinpointing reasons why going there isn't the best use of our resources.

1. 阅读
2. 笔记转移到obsidian中的高亮笔记文件夹
3. 编辑笔记，用自己的语言表达
4. 打标签，想到什么打什么？基于目的的想象

Reviewer #3 (Public Review):

Using high fat diet (HFD)-fed male mice and a variety of experimental approaches, the authors demonstrated the efficacy of xanthohumol (XN) and tetrahydro-xanthohumol (TXN) in attenuating weight gain and hepatic steatosis independently of calorie intake and identify inhibition of PPARγ as a mechanism. A strength of the study design was the incorporation of the test compounds into isocaloric, ingredient matched high-fat diet (HFD) formations and inclusion of a LFD control group. A weakness of the study, although minor, is that the dose of compound consumed will vary between mice and from day-to-day depending on how much food each animal consumes. The lower dose of XN (LXN, given as 30 mg/kg of diet) was found ineffective compared to the higher dose of XN (HZN, 60 mg/kg of diet) and TXN (30 mg/kg of diet) was most effective in attenuating weight gain and reversing HFD-induced liver steatosis. TXN almost completely suppressed hepatic lipid vacuole accumulation and showed greatest reduction in liver mass relative to body weight. TXN increased fasting plasma triglycerides compared to all other groups, but explanation is uncertain. Fecal excretion of TAG between groups was similar and therefore could not explain the decreased weight gain or improved liver phenotypes in XN- or TXN-treated groups. Whole body energy metabolism suggested that XN and TXN supplemented mice were more physically active then HFD-fed mice. HXN and TXN supplemented mice showed less accumulation of subcutaneous and mesenteric fat mass, but these groups had somewhat higher levels of epididymal fat mass.

After 16 weeks on diets, RNAseq performed on murine liver tissues. Compared to HFD group, TXN group had 295 differentially expressed genes (DEGs), HXN group had 6 DEGs, and LFD group had 212 DEGs. TXN supplementation upregulated 6 and down regulated 25 KEGG pathways. SVM was used to identify signature genes that significantly differentiated HFD and TXN group transcriptomes. Of 13 identified genes, 8 showed significant, differential hepatic expression between TXN and HFD groups. Of these 8 genes, 3 genes (Ucp2, Cidec, Mogat1) were identified as known target genes of PPARγ with roles in lipid metabolism. qPCR of liver tissues was used to verify these RNAseq results.

XN or TXN were shown to inhibit murine preadipocyte 3T3-L1 differentiation and adipogenesis and lipid accumulation in a dose dependent manner. In a second dose escalating experiment, TXN or XN were shown to block the ability of rosiglitazone (RGZ), a PPARγ agonist, to promote adipogenesis of 3T3-L1. These data suggested that XN and TXN may interfere or compete with binding of RGZ to the PPARγ receptor. qPCR of 3T3-L1 cells confirmed that TXN or XN could inhibit gene expression of RGZ-induced PPARγ target genes (Cd36, Fabp4, Mogat1, Cidec, Plin4, Fgf21) and further supported the hypothesis that TXN and XN are PPARγ antagonists. To further test this idea the authors performed a competitive PPARγ TR-FRET binding assay and showed that XN and TXN could displace a labelled pan-PPARγ ligand in a dose-dependent manner. Finally, molecular docking experiments confirmed the putative binding pose and position of XN/TXN and estimated the relative binding affinities of various ligands for PPARγ. XN and TXN may serve as scaffolds for the development of more potent therapeutics in structure-activity relationship (SAR) studies. Overall, this work contributes compelling preclinical data to support future clinical investigations to determine dosing, efficacy, and safety of XN and TXN as therapeutics for diet-induced NAFLD.

本书作者王骥是迄今为止中国唯一的艺术家手作书藏家，十几年来，他斥巨资收藏了200多本艺术家手作书，却始终蜗居在30㎡的小房子里。

2020年末，《书之极》一出版，旋即获选2020年“中国最美的书”。

书中呈现了24位艺术家的作品：有常玉、赵无极、萨尔瓦多·达利、安迪·沃霍尔、亨利·马蒂斯……甚至还有诺贝尔文学奖获得者君特·格拉斯。它展现了“书”这个载体做到极致能做成什么样子。

Congratulations on this research and best wishes for a helpful and robust peer review experience! I offer the following feedback, which I hope you can consider:

1. Given the involvement of a patient author and the recommended reporting guidelines for patient involvement, would you be able to include the GRIPP2 Short Form in your manuscript? See EQUATOR Network/BMJ/Res Involvement and Engagement for GRIPP2. By sharing insights via this best practice reporting guideline we can all learn from each other. The table could be added in your existing Methods section in the Patient and Public Involvement paragraph.

2. To assist with meta-research on patient authorship and facilitate listing and searchability in PubMed via the 'Patient Author' affiliation tag, would Laurie Proulx be able to add 'Patient Author' to her existing affiliation? We are working with publishers now to help boost use of this affiliation tag as trying to identify patient-authored publications right now relies on hand searching. A quick, free, and easy alternative is possible via PubMed if the 'Patient Author' affiliation is used. Wide use of this tag would help answer the Table 7 question re, has your journal published a paper with a patient author? We are presenting research on the use of this tag at a US conference this year and would be happy to share our results with you.

3. It is a concern that almost 1 in 5 of the respondents were not sure whether their journal adhered to the ICMJE criteria for authorship (Table 7). It could be useful from a hypothesis-generating perspective to see if the answers from this cohort influenced the broader results.

4. While not peer reviewed, there is reported documentation from ICMJE about patient authorship in the grey literature from Peter Bates "anyone (including patients) who meet all the criteria for authorship should be provided the option / opportunity to be so. Our document says so, and you should point to that if others think patients who meet all 4 criteria should not be authors.

In short, we believe that our statements apply to patients just as they do to anyone involved in a study and its reporting.

Personal communication from the Secretary of the ICMJE, December 2018"<br> https://peterbates.org.uk/home/garden-shed/payment-for-authors/

1. We have partnered with patients to conduct and author the first systematic review on the benefits and risks of patient authorship. It has been published in the peer-reviewed journal, Research Involvement and Engagement. I thought this might be of interest to your team for this or future work you do in this area. https://researchinvolvement.biomedcentral.com/articles/10.1186/s40900-020-00190-w

2. We have also partnered with patients to create a free and online plain language guide to help patients review the rights and responsibilities of authorship...before they commit to authorship. It is CC BY 4.0 so can be shared easily https://figshare.com/articles/poster/Plain_Language_Summary_of_Good_Publication_Practice_Guideline/11292047

3. Patient authorship is likely to increase as more patients lead robust research projects. One wonders who the 33 editors (who don't believe patients can be authors) would expect to author the resulting publications. If the researchers involved can't author publications, then could this lead unethical guest authorship?

4. Relevant to the advocacy from patients for authorship, we are working with WECAN to develop the first, online, co-created 'Publication Training Course for Patient Advocates'. This course will be released under a CC BY 4.0 license to facilitate free sharing and re-use. We would be happy to speak with you about the course.

Again, I hope these comments are useful and I look forward to seeing this preprint progress to a peer-reviewed publication.

Professor Karen L. Woolley Twitter: @KWProScribe

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

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Reply to the reviewers

We wish to thank the reviewers for their detailed and constructive comments on our manuscript. This valuable feedback has resulted in substantial improvements to our paper. A detailed list addressing the reviewers’ comments and the changes to our manuscript since the first submission is outlined below:

Reviewer #1:

The manuscript by Martens et al investigates the mechanisms of Bnip3-mediated cell damage during hypoxia. The Authors show that modulation of prostaglandin (PG) E1 signaling with misoprostol prevents cardiac dysfunction, mitochondrial impairment and cell death induced by hypoxia. In addition, they show that the effect of misoprostol is dependent on PKA-mediated Thr181 phosphorylation. The Authors also suggest that there is a possible interaction between Bnip3 and 14-3-3beta that prevents ER Ca2+ release and mitochondrial Ca2+ overload. The Authors conclude that Bnip3 phosphorylation plays a key role in the regulation of cardiac and metabolic dysfunction and identify misoprostol treatment as a therapeutic intervention to prevent hypoxia-induced cardiac injury.

**Major Comments:**

1.Results presented in Figs. 1, 2 and 3 pertaining to hypoxia-induced changes in Bnip3 expression, changes in mitochondrial function, cell death, Bnip3-dependent Ca2+ transfer from ER to mitochondria and the effect of misoprostol have partially been demonstrated in a previous publication from the same group (PMID: 30275982).

Thank you for noting our previous work using predominantly the HCT-116 cell line and a rat model of neonatal hypoxia published in the journal Cell Death Discovery. The work in the current manuscript builds on our previous papers, and extends these findings utilizing a neonatal mouse model, primary neonatal ventricular myocytes, human iPSC-derived cardiomyocytes, and H9c2 cells. These models not only demonstrate the robust nature of the effects of misoprostol treatment on the hypoxic neonatal cardiomyocytes, but they also allowed us to utilize powerful genetic models, such as the Bnip3 knockout mouse, and knockout mouse embryonic fibroblasts (MEFs), which phenocopy many of the effects of misoprostol treatment. These findings strongly implicate Bnip3 as a primary target of misoprostol treatment in the hypoxic neonatal heart in rodents.

In addition, Figure 1 contains very important in vivo endpoints that we have not previously utilized, including echocardiography to assess neonatal cardiac function, transmission electron microscopy to assess mitochondrial ultrastructure, cardiac ATP and lactate levels, an array of gene expression, and HMGB1 immunofluorescence (Fig.1 I in the current version of the manuscript) implicating a necro-inflammatory phenotype that is modulated by misoprostol treatment. Moreover, in Figures 2 and 3 we confirm previous observations related to hypoxia- and Bnip3-mediated mitochondrial function, and calcium signaling, but also extend these observations to include the impact of hypoxia, Bnip3 and misoprostol treatment on mitochondrial morphology and necro-inflammatory markers, in additional to utilizing human cardiomyocytes and knockout MEFs. Finally, Figures 4-7 of our manuscript describes a novel mechanism by which misoprostol treatment can therapeutically target Bnip3 function both in vivo and in cell models (see below).

Although based on our previous observations, we feel the work in the present manuscript is highly novel and original, and adds substantially to our knowledge of both Bnip3 function, and neonatal hypoxic injury, which currently represents a world-wide health crisis that is underrepresented in the biomedical literature.

2.The very same publication from 2018 shows that misoprostol treatment of pups exposed to hypoxia for 7 days is able to prevent the increase in Bnip3 protein levels. Yet, in the present manuscript misoprostol treatment had no effect on Bnip3 protein levels in the same model (Fig. 1E). This raises some concerns regarding the solidity and soundness of the results presented. Thank you for noting this in our previous work. In our 2018 paper, we treated hypoxic neonatal rats with misoprostol and observed a complete repression of Bnip3 expression in the gut and hippocampus, but only a partial repression in the heart. This observation prompted us to explore other mechanisms by which misoprostol could inhibit Bnip3 function. We have increased our sample size for the data in Figure 1 J and K to be more statistically conclusive. The evidence is now stronger that misoprostol only the partial represses Bnip3 expression in the neonatal mouse heart. In addition, we provided a representative western blot (Fig. 1 J), which is consistent with the result in Figure 3C in MEF cells.

3.The validation of the custom antibody against p-Thr181 needs to be shown. Fig. 4E shows that p-Bnip3 band is quite strong in H9c2 cells, despite total endogenous Bnip3 levels are barely detectable. In addition, phosphorylation of the Bnip3 Thr181 residue in cells and/or in vivo should be confirmed by mass spectrometry.

Our plan in the next revision, we will be to provide additional validation of the p-Thr181 antibody, as we have done previously (PMID: 33044904). In addition, we will re-run the western blots noted above to improve their quality, as the difference between total Bnip3 and p-Bnip3 in H9c2 cells is likely due to different exposures of the two blots. Confirmation of Bnip3 phosphorylation using mass spectrometry in extracts from intact cells was previously published (PMID: 26102349), however, the nature of the signaling pathways leading to phosphorylation was not determine, nor was the mechanism of Bnip3 inhibition previously determined.

4.Fig. 4L shows that misoprostol treatment of H9c2 cells leads to an increase in Bnip3 phosphorylation, but this does not seem to be the case in normoxic conditions in vivo (Fig. 4N). Moreover, shouldn't this presumable increase in phosphorylation induced by misoprostol in normoxic conditions lead to Bnip3 accumulation in the cytosol thereby reducing its colocalization with mitochondria (Fig. 6B)? The results obtained with the colocalization method should be corroborated using different methods, such as cell fractionation.

In the previous version of the manuscript, we reported that misoprostol treatment increases Bnip3 phosphorylation in H9c2 cells following acute exposure (Supplement 5 B). In the updated version of the manuscript, we confirmed this in vitro observation in PVNC’s, demonstrating that in culture, acute misoprostol drug treatments during normoxia result in Bnip3 phosphorylation (Supplement 5 C). However, when we increased our N in the revised manuscript this difference remained not statistically sustained after 7 days of misoprostol treatment in vivo (Fig. 4 M, N). Importantly though, our observation that hypoxia exposure resulted in reduced Bnip3 phosphorylation, and that misoprostol drug treatment was sufficient to restore it is particularly novel, and ties together with our new colocalization data (Fig. 4 M, N). What is very intriguing about the data in Figure 6B is that myc-Bnip3 did not colocalize with the mitochondrial matrix-targeted mito-Emerald under normoxic conditions, and thus was not impacted by misoprostol treatment in normoxia. However, in hypoxic cells the colocalization coefficient between myc-Bnip3 and mito-Emerald increased and was abrogated by misoprostol treatment. This observation suggests that Bnip3 is actively translocated deeper into the mitochondria ultrastructure during hypoxic stress, and that misoprostol treatment can prevent this phenomenon. This observation is consistent with the pBnip3 data shown in Figure 4M. In the most recent version of the manuscript, we have performed additional confocal experiments to substantiate this novel observation. New data clearly demonstrates that hypoxia exposure in vivo increases the colocalization of Bnip3 with the inner mitochondrial membrane protein Opa1 (Fig. 6 C). However, when mice are treated with misoprostol, the colocalization with Opa1 is reduced and the colocalization of Bnip3 with 14-3-3b increases (Fig. 6 M). We have also shown in H9c2 cells that expression of Opa1 prevents Bnip3-induced mitochondrial fission (Fig. 3 J), and that when both Bnip3 and 14-3-3b are ectopically expressed, misoprostol treatment can increase their colocalization (Fig. 6 N, O), suggesting that this is regulated by post-translational modification and not alterations in Bnip3 expression due to hypoxia. Our plan is to include additional fractionation experiments in the next revision of the manuscript; however, this approach may not be as sensitive as confocal microscopy.

5.In relation to Fig. 4 M, N (page 19), the Authors concluded that the reduction in Bnip3 phosphorylation suggests an increase in Bnip3 activity in the hypoxic neonatal hearts. Nevertheless, this has not been demonstrated.

Thank you for pointing this out. At this time, we do not have data to suggest that a reduction in Bnip3 phosphorylation increases its activity in vivo. In the revised manuscript, we have new confocal-based colocalization experiments using fixed sections from hypoxic and misoprostol treated hearts that provide insightful information into the subcellular localization of Bnip3 (Please see above; Fig. 6 C, M). Importantly, based on our data in figure 5, particularly Fig.5F using Bnip3-null MEFs, the protective effect of misoprostol is completely prevented by reconstitution of the T181A mutant, but not wild-type Bnip3, suggesting phosphorylation at T181 is an important mechanism by which misoprostol inhibits Bnip3-induced mitochondrial depolarization. Finally, we have also been careful not to overstate our conclusions is the most recent version of the manuscript, have been more specific with our language, and have avoided vague terms like ‘activity’.

6.Along that line, the Authors concluded that misoprostol-induced cytoprotection is dependent on PKA Thr181 phosphorylation. Nevertheless, this dependence has not been convincingly demonstrated in hypoxic cells and in vivo.

The new data described outline above, in point #4 and #5, have provided assurances regarding the role of Bnip3 phosphorylation on its subcellular location in vivo and in cultured cells. To further address the dependency of PKA on T181 phosphorylation, we have performed experiments using the PKA inhibitor, H89, in cellular experiments and evaluate whether the protective effect of misoprostol is lost in the presence of this inhibitor. This new data has been added to the most recent version of the manuscript (Fig. 4 G).

7.Previous studies showed that Bnip3 induces mitochondrial fragmentation and mitophagy (PMID: 16645637, 20436456). What is the hypothesis for the inhibition of mitochondrial fragmentation induced by misoprostol in the present study? Does it prevent Bnip3 interaction with Opa1 or is this event downstream of ER Ca2+ release and mitochondrial Ca2+ overload? Does misoprostol affect mitophagy?

We have added new experimental data to address the hypothesis that Bnip3 colocalizes with Opa1 to induce mitochondrial fragmentation (as noted by the reviewer, they were previously shown to physically interact), and that this is inhibited by misoprostol treatment (Fig. 6 C). We have also added new data to the supplemental material demonstrating that misoprostol inhibits hypoxia- and Bnip3-induced mitophagy. Based on our data, we proposal that misoprostol inhibits both Bnip3-induced ER-calcium release and Opa1-dependent mitochondrial fusion. This is based on our data that misoprostol prevents Bnip3 accumulation at both the ER and mitochondria, respectively.

8.The link between Bnip3 interaction with 14-3-3 and Bnip3 Thr181 phosphorylation, if there is any, is not clear. The Authors mention that Thr181 lies within the 14-3-3 binding domain. Is Thr181 phosphorylation required for 14-3-3 binding or are these events unrelated? What is the significance of these events in hypoxia, does 14-3-3 binding to Bnip3 occur in vivo? Is Bnip3 localization affected by hypoxia, 14-3-3 binding and/or misoprostol treatment in vivo?

Previously, we described the role of phosphorylation of Bnip3L (Nix) at Ser-212 how this regulates the interaction with 14-3-3 (PMID: 33044904). This phosphorylation site is conserved in Bnip3 as T181. Interestingly, phosphorylation of Nix by PKA was not required for interested with 14-3-3b, but the interaction between Nix and 14-3-3b was enhanced by phosphorylation. Our plan is to perform similar experiments with Bnip3 and 14-3-3b to determine if this mechanism is conserved. However, as noted above we have new in vivo data showing that misoprostol increased the colocalization of Bnip3 and 14-3-3b in the hypoxic heart (Fig. 6 M).

9.Fig. 6P shows the presence of myc-tag after IP for HA-tag, even when HA-14-3-3 was not expressed (middle lane). How is this possible?

This appears to be a small amount of non-specific interaction between the HA antibody and myc-Bnip3. This is relatively small compared to the band in lane 3, which demonstrates specificity, and the importance of including this control condition. Our plan is to re-run this CO-IP to improve the western blot quality.

**Minor Comments:**

1.Please co-stain with the cardiomyocyte marker in Fig. 2A (such as alpha-actinin).

Yes, good suggestion.

2.The Methods are not sufficiently detailed. For instance, it is not clear what is the Ca2+ concentration used for Ca2+ pulses in the CRC experiment. The fact that cardiac mitochondria are able to uptake only two Ca2+ pulses raises some concerns regarding the quality of mitochondrial preparation. What is the reason for isolating mitoplasts instead of intact mitochondria?

We have provided more detail in the revised manuscript.

3.TMRM fluorescence should be measured before and after FCCP administration, to account for the difference in plasma membrane potential (the results should be expressed as F/FFCCP).

We can provide some additional control experiments in the revised manuscript, if necessary.

4.Measurement of extracellular acidification is mentioned in the methods, but the relative results are not shown.

Thank you, this has been removed.

5.RNAi experiments targeting Bnip3 are also mentioned in the methods, but the results are not described.

Thank you. This has been fixed.

Reviewer #1 (Significance (Required)):

Previous studies have demonstrated that Bnip3 is upregulated by hypoxia and plays a key role in inducing mitochondrial dysfunction and PTP opening that eventually results in cell death (PMID: 12169648, 10922063). Along that line, misoprostol has been shown to prevent damaging effects of hypoxia by repressing Bnip3 and promoting the expression of pro-survival alternative splicing isoforms (PMID: 30275982). Indeed, the same study showed that misoprostol treatment prevents loss of mitochondrial membrane potential, ROS formation and impairment in mitochondrial oxygen consumption caused by hypoxia in primary neonatal cardiomyocytes. The present manuscript recapitulates these previously published findings. The truly novel findings concern the identification of Bnip3 residue Thr181 as target for PKA phosphorylation and the possible interaction of Bnip3 with 14-3-3. However, the role and/or involvement of these events has not been thoroughly investigated in relation to hypoxia and misoprostol treatment in cells or in vivo.

Thank you for noting our previous work and identifying the novelty in our present work. As stated above, for Reviewer #1 comments 4, 6, and 8. We have provided additional mechanistic and in vivo data to more fully describe the role of T181 phosphorylation and the interaction with 14-3-3 chaperones in the revised manuscript.

Reviewer #2:

Systemic hypoxia, a major complication associated with reduced gestational time, affects more 60% of preterm infants and is a known driver of hypoxia-induced Bcl-2-like 19kDa-interacting protein 3 (Bnip3) expression in neonatal heart. At the level of the cardiomyocyte, Bnip3 activity plays a prominent role in the evolution of necrotic cell death, disrupting subcellular calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests both a cardioprotective role for protein kinase A (PKA) through stimulatory prostaglandin (PG) E1 signalling during prolonged periods of hypoxia, and a cytoprotective role for Bnip3 phosphorylation, indicating that post-translational modifications of Bnip3 may be a point of convergence for these two protective pathways. Using a combination of in vivo and multiple cell models, including human iPSC-derived cardiomyocytes, the authors tested if the PGE1 analogue misoprostol is cardioprotective during neonatal hypoxic injury by altering the phosphorylation status of Bnip3. Here we report that hypoxia exposure significantly increases Bnip3 expression, mitochondrial-fragmentation, -ROS, -calcium accumulation and -permeability transition, while reducing mitochondrial membrane potential, all of which were restored to control levels with addition of misoprostol, despite elevated Bnip3 protein expression. Through both gain- and loss-of function genetic studies, the authors show that misoprostol-induced protection directly affects Bnip3, preventing mitochondrial perturbations. They demonstrate that this is a result of PG EP4 receptor signalling, PKA activation, and direct Bnip3 phosphorylation at threonine-181. Furthermore, when this PKA phosphorylation site within Bnip3 is neutralized, the protective misoprostol effect is lost. They also provide evidence that misoprostol traffics Bnip3 away from the ER through a physical interaction with 14-3-3β, thereby preventing aberrant ER calcium release and MPT. In vivo studies further demonstrate that misoprostol treatment increases Bnip3 phosphorylation at threonine-181 in the mouse heart, while both misoprostol treatment and genetic ablation of Bnip3 prevented hypoxia-induced reductions in contractile function. Taken together, these results demonstrate a foundational role for Bnip3 phosphorylation in the molecular regulation of cardiomyocyte contractile and metabolic dysfunction and identifies EP4 signaling as a potential pharmacological mechanism to prevent hypoxia-induced neonatal cardiac injury. While this work is interesting, a number of issues remain.

1.English expression needs some attention. For example, the first sentence of the abstract - "more than 60%...."; Page 20, line 9 "We observed that misoprostol's ability to to". Many sections should be broken into 2 or 3 sentences.

Thank you, we have made these changes and have fully proof-read our manuscript.

2.Evidence from In vivo studies such as those described in section 3.7 is minimal. Much more in vivo evidence is needed. It is unclear the authors established this in vivo model of hypoxia - supposedly gestational hypoxia should be considered. Consider citing these reviews on maternal over- and under-nutrition for postnatal heath (PMID 33181042; 22982026).

Thank you, we will cite these papers and have clarified our in vivo model, related to comparable human gestation time, in the Methods section. We have also revised the Introduction and Discussion to be more consistent with our in vivo model. In addition, and noted above, we have preformed addition in vivo experiments in both the hypoxia/misoprostol model, and in Bnip3 KO mice to more fully support our conclusions. Additional HMGB1 immunofluorescence has already been added to Figures 1 and 7, and we have include additional confocal-based colocalization experiments from fixed tissues (described in more detail above; Fig. 7 D).

3.Which one does misoprostol exactly execute its action? Phosphorylation through PKA or trafficking Bnip3 away from the ER through a physical interaction with 14-3-3β?

This is a very good question. Based on our previous work on Bnip3L (Nix; PMID: 33044904,) PKA-induced phosphorylation of the transmembrane domain increased the physical interaction with 14-3-3b, which acts as a chaperone to translocate Nix away from the mitochondria and ER/SR. We will preform similar experiments with Bnip3 and 14-3-3b for the next revision to provide additional support for this conclusion.

4.More in vivo proof of concept studies are needed to validate the signaling mechanism - this is an invitro-based study (hypoxia challenge occurs in vitro).

We have already included additional in vivo immunofluorescence to Figure 1 and 7, and have performed additional colocalization experiments to validate the signaling pathway in this revision. Many of these experiments are described above.

5.Quality of figures is somewhat poor.

Our images are the highest possible resolution within the confines of the figure size limit. Perhaps the reviewer received a web-optimized version of the figures for review.

Reviewer #2 (Significance (Required)):

Relatively high - although in vivo evidence is needed.

Thank you. This is provided in the revised manuscript.

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

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Referee #1

Evidence, reproducibility and clarity

The manuscript by Martens et al investigates the mechanisms of Bnip3-mediated cell damage during hypoxia. The Authors show that modulation of prostaglandin (PG) E1 signaling with misoprostol prevents cardiac dysfunction, mitochondrial impairment and cell death induced by hypoxia. In addition, they show that the effect of misoprostol is dependent on PKA-mediated Thr181 phosphorylation. The Authors also suggest that there is a possible interaction between Bnip3 and 14-3-3beta that prevents ER Ca2+ release and mitochondrial Ca2+ overload. The Authors conclude that Bnip3 phosphorylation plays a key role in the regulation of cardiac and metabolic dysfunction and identify misoprostol treatment as a therapeutic intervention to prevent hypoxia-induced cardiac injury.

Major Comments:

1.Results presented in Figs. 1, 2 and 3 pertaining to hypoxia-induced changes in Bnip3 expression, changes in mitochondrial function, cell death, Bnip3-dependent Ca2+ transfer from ER to mitochondria and the effect of misoprostol have partially been demonstrated in a previous publication from the same group (PMID: 30275982).

2.The very same publication from 2018 shows that misoprostol treatment of pups exposed to hypoxia for 7 days is able to prevent the increase in Bnip3 protein levels. Yet, in the present manuscript misoprostol treatment had no effect on Bnip3 protein levels in the same model (Fig. 1E). This raises some concerns regarding the solidity and soundness of the results presented.

3.The validation of the custom antibody against p-Thr181 needs to be shown. Fig. 4E shows that p-Bnip3 band is quite strong in H9c2 cells, despite total endogenous Bnip3 levels are barely detectable. In addition, phosphorylation of the Bnip3 Thr181 residue in cells and/or in vivo should be confirmed by mass spectrometry.

4.Fig. 4L shows that misoprostol treatment of H9c2 cells leads to an increase in Bnip3 phosphorylation, but this does not seem to be the case in normoxic conditions in vivo (Fig. 4N). Moreover, shouldn't this presumable increase in phosphorylation induced by misoprostol in normoxic conditions lead to Bnip3 accumulation in the cytosol thereby reducing its colocalization with mitochondria (Fig. 6B)? The results obtained with the colocalization method should be corroborated using different methods, such as cell fractionation.

5.In relation to Fig. 4 M, N (page 19), the Authors concluded that the reduction in Bnip3 phosphorylation suggests an increase in Bnip3 activity in the hypoxic neonatal hearts. Nevertheless, this has not been demonstrated.

6.Along that line, the Authors concluded that misoprostol-induced cytoprotection is dependent on PKA Thr181 phosphorylation. Nevertheless, this dependence has not been convincingly demonstrated in hypoxic cells and in vivo.

7.Previous studies showed that Bnip3 induces mitochondrial fragmentation and mitophagy (PMID: 16645637, 20436456). What is the hypothesis for the inhibition of mitochondrial fragmentation induced by misoprostol in the present study? Does it prevent Bnip3 interaction with Opa1 or is this event downstream of ER Ca2+ release and mitochondrial Ca2+ overload? Does misoprostol affect mitophagy?

8.The link between Bnip3 interaction with 14-3-3 and Bnip3 Thr181 phosphorylation, if there is any, is not clear. The Authors mention that Thr181 lies within the 14-3-3 binding domain. Is Thr181 phosphorylation required for 14-3-3 binding or are these events unrelated? What is the significance of these events in hypoxia, does 14-3-3 binding to Bnip3 occur in vivo? Is Bnip3 localization affected by hypoxia, 14-3-3 binding and/or misoprostol treatment in vivo?

9.Fig. 6P shows the presence of myc-tag after IP for HA-tag, even when HA-14-3-3 was not expressed (middle lane). How is this possible?

Minor Comments:

1.Please co-stain with the cardiomyocyte marker in Fig. 2A (such as alpha-actinin).

2.The Methods are not sufficiently detailed. For instance, it is not clear what is the Ca2+ concentration used for Ca2+ pulses in the CRC experiment. The fact that cardiac mitochondria are able to uptake only two Ca2+ pulses raises some concerns regarding the quality of mitochondrial preparation. What is the reason for isolating mitoplasts instead of intact mitochondria?

3.TMRM fluorescence should be measured before and after FCCP administration, to account for the difference in plasma membrane potential (the results should be expressed as F/FFCCP).

4.Measurement of extracellular acidification is mentioned in the methods, but the relative results are not shown.

5.RNAi experiments targeting Bnip3 are also mentioned in the methods, but the results are not described.

Significance

Previous studies have demonstrated that Bnip3 is upregulated by hypoxia and plays a key role in inducing mitochondrial dysfunction and PTP opening that eventually results in cell death (PMID: 12169648, 10922063). Along that line, misoprostol has been shown to prevent damaging effects of hypoxia by repressing Bnip3 and promoting the expression of pro-survival alternative splicing isoforms (PMID: 30275982). Indeed, the same study showed that misoprostol treatment prevents loss of mitochondrial membrane potential, ROS formation and impairment in mitochondrial oxygen consumption caused by hypoxia in primary neonatal cardiomyocytes. The present manuscript recapitulates these previously published findings. The truly novel findings concern the identification of Bnip3 residue Thr181 as target for PKA phosphorylation and the possible interaction of Bnip3 with 14-3-3. However, the role and/or involvement of these events has not been thoroughly investigated in relation to hypoxia and misoprostol treatment in cells or in vivo.

SocArXiv. (2020, May 30). You can always see the latest SocArXiv papers on COVID-19 topics here: Https://t.co/pzqftUqY81. You can comment using the @hypothes_is tool, and endorse using the @PlauditPub button. And add your own work, using the covid-19 tag. Https://t.co/owGxoaDfsJ [Tweet]. @socarxiv. https://twitter.com/socarxiv/status/1266796731527806983

SciScore for 10.1101/2020.10.27.357558: (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.

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 10.1101/2020.11.04.355842: (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.

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">ISRCTN66726260</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NA</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.

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 10.1101/2021.01.11.21249265: (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).

There are important limitations to this study. The use of the exact cut-off of VFS to categorize samples into clinical groups may not universally apply. Nonetheless, the basic premise of the argument that more intact virus would reflect in more longer fragments in a clinical sample would universally apply. As the study’s main conclusions are based on NGS, with some comparison to RT-PCR results, without evidence from virus culture studies it cannot be ruled out that some of the observed infectivity measures are correlated and result from a feature of the specific NGS method. In conclusion, we have applied NGS to comprehensively characterize longitudinal samples collected from different sites. NGS is an enabling tool that provides sequence-related information for which it is primarily designed, and also information from size and length dimensions. Based on this, we identify fragment length differences among clinical samples which are correlated to clinical features of infectiousness of SARS-CoV-2, quantification of which could be incorporated as relevant and straightforward measure to determine infection potential.

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.

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 10.1101/2021.02.05.21250127: (What is this?)

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

Table 1: Rigor

Table 2: Resources

No key resources detected.

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).

Our study has several limitations. First, although our third-party data provider reported that about 70% of posts were from the US, we do not know the location for most posts according to our direct geotagging methods, which were only able to tag about 80% of posts (eTable 2). As a result, we cannot make international comparisons, but our dataset is more representative of the US than of any other country. Second, the number of posts included in our dataset was much lower than previous studies, likely due to the types of data sources used, which excluded sites such as Twitter in order to exclude noise that might have obscured signals in social media data, and our methodology, which included removing posts not relevant to our more refined taxonomy. We used a stringent exclusion criterion with a list of prespecified keywords that may also have led to a smaller sample size, but our approach aimed to create a sample with high specificity. Finally, there is no demographic information available from the data posts directly due to privacy considerations and data use agreements. Thus, we cannot determine whether our data sample contains biases due to the demographics of the people who post. For instance, Reddit, which was the most common forum source for our data sample, has been found to be used by a younger, male audience.[45,46]

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.

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 10.1101/2020.06.17.157982: (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.

To some degree, these caveats are universal of experimental studies, as even sophisticated animal models are imperfect proxies for true fitness (Louz et al., 2013)—but they are especially true for basic biochemical phenotypes like the ones we measure. However, on a hopeful note, our measurements correlate well with cellular entry by spike-pseudotyped viral particles expressing sarbecovirus RBD homologs (Figures 1D) and single mutants of the SARS-CoV-2 RBD (Figure 4E). Furthermore, fitness ultimately arises from the concerted action of biochemical phenotypes, which are in turn determined by genotype (Dean and Thornton, 2007; Harms and Thornton, 2013; Russell et al., 2014). By making the first link from mutations to biochemical phenotypes, we have taken a step towards enabling better interpretation of viral genetic variation. One important area where our maps do have clear relevance is assessing the potential for SARS-CoV-2 to undergo antigenic drift by fixing mutations at sites targeted by antibodies, as occurs for some other viruses such as influenza (Smith et al., 2004). The RBD is the dominant target of neutralizing antibodies (Cao et al., 2020; Ju et al., 2020; Pinto et al., 2020; Rogers et al., 2020; Seydoux et al., 2020; Shi et al., 2020; Wu et al., 2020; Zost et al., 2020), and so any antigenic drift will be constrained by its mutational tolerance. Our results show that many mutations to the RBD are well-tolerated with respect to both protein folding and ACE2 binding. H...

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.

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 10.1101/2020.07.30.229377: (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.

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 10.1101/2020.08.24.264333: (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.

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 10.1101/2020.09.14.20194308: (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).

A limitation is that we were not able to study infections occurring more than six to seven weeks before enrollment, as community transmission of SARS-CoV-2 in the region started only about six to seven weeks before the study. Participants were not questioned about present or prior symptoms, but the hospital rules were clear that employees with symptoms should not be at work and we had, by design, decided to use only sick leave data to avoid possible recall bias. Hospital rules state that also employees working from home that develop COVID-19 symptoms must report this as sick leave. We conclude that antibody testing is a powerful tool for identification of subjects who have had prior virus exposure and are protected against future disease. Although it is commonly assumed that antibodies mark immunity, it is important that it has now been shown in a large cohort study that seropositive subjects have no excess risk for future sick leave. We would like to caution that there is a large number of serology tests currently on the market and the extent of their validation may vary. Also, none of these tests have been specifically validated for the indication proposed here (to separate exposed subjects who are protected from future disease from exposed subjects at risk to develop disease in the future). In summary, the present study has found that validated antibody testing may be helpful in SARS-CoV-2 screening strategies as antibody-positive subjects were found to have no excess risk...

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">NCT04411576</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">Current and Past SARS-CoV-2 Infection and COVID-19 in Health…</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.

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 10.1101/2020.06.14.151357: (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.

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 10.1101/2021.02.03.429555: (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.
• No funding statement was detected.
• No protocol registration statement was detected.

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 10.1101/2021.02.05.429566: (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).

We understood the limitations of our work in the number of cases analyzed and the experimental strategy used; however, it provides information for some laboratories with limited resources to apply, exceptionally, basic research tools for monitoring COVID-19 cases during a pandemic.

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">NCT04384588</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">COVID19-Convalescent Plasma for Treating Patients With Activ…</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.

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 10.1101/2021.01.28.21250577: (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.

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 10.1101/2021.02.16.431318: (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.

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 10.1101/2020.08.01.20166553: (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.

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 10.1101/2020.09.09.20191205: (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 52 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.

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 10.1101/2021.02.17.21251933: (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 the association of lithium therapy and reduced risk of COVID-19 across three health systems is both signficant and intriguing, observational studies have many limitations. A variety of factors with potential biases cannot be measured even after comparing the cases and controls in a manner that accounts for known confounding factors using a rigorous matching algorithm. For instance, details on medicine usage were derived from records of prescription orders, but information on compliance before SARS-CoV-2 PCR testing is not available. In addition, the collection of a non-random sample population can create a collider bias and lead to distorted associations. For example, the COVID-19 test was restricted particularly in the early pandemic to symptomatic patients so that many asymptomatic patients in the EHR were not tested. These findings should therefore be interpreted carefully and deeper investigation is required in a cohort with a larger sample size. Prior work has shown that lithium and the GSK-3 inhibitor Kenpaullone inhibit N phosphorylation and reduce viral titers in SARS-CoV and JHMV infected Vero6 cells8, 9 and GSK3 knockdown also impairs replication of IBV in Vero cells15. We also show that multiple small molecule GSK-3 inhibitors, including CHIR99021, BIM-I, AR-A014418, Enzastaurin, and Sotrastaurin block SARS-CoV-2 phosphorylation. These pharmacological studies are compelling evidence that GSK-3 is a critical host kinase for N protein, but these drugs may have ...

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.

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 10.1101/2021.02.22.432402: (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: 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">NCT04275245</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Clinical Study of Anti-CD147 Humanized Meplazumab for Inject…</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.

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

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 10.1101/2020.09.24.311027: (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: 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">NCT04405908</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">SCB-2019 as COVID-19 Vaccine</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.

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 10.1101/2020.10.09.20209858: (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.

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 10.1101/2020.08.25.256339: (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 number of alternative molecules are being developed to complement and partially overcome this limitation of antibodies. Here we describe the generation of DARPin molecules - one prominent alternative to antibodies(30) amongst others(31–34) - that bind the SARS-CoV-2 spike protein. We tested a library of one trillion DARPin molecules and identified multiple DARPin molecules with different functionalities and binding specificities. By molecular linkage of individual DARPin molecules, we developed multi-DARPin molecules with low picomolar neutralizing activity and demonstrated their protective efficacy against SARS-CoV-2 infection in a hamster model. In particular, reduced lung tissue damage and reduced virus replication in the lower and upper respiratory tract were observed, the latter also being important for reducing virus shedding and transmission. The most advanced of those multi-DARPin molecules, MP0420 or ensovibep, is currently being studied in Phase 1. The most potent neutralizing mono-DARPin molecules were found to target the RBD, blocking the spike-ACE2 interaction necessary for infection. This finding is congruent with the identified epitopes of potent neutralizing antibodies obtained from COVID-19 patients(20, 35-39). Thus, the in vitro approach using DARPin molecules independently confirms that the ACE2 interaction site on the SARS-CoV-2 spike protein is one of the most vulnerable sites to block virus infection in cell culture. The single-chain binding domain nat...

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 14 and 30. 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.

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 10.1101/2020.08.26.266825: (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.

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 10.1101/2020.12.09.417741: (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.

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 10.1101/2020.04.25.20079103: (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).

Strengths and weaknesses: A strength of this review is that we used clear definitions and separated review questions to distinguish between SARS-CoV-2 infections that remain asymptomatic throughout their course from those that become symptomatic, and to separate proportions of people with infection from their contribution to transmission in a population. This living systematic review uses methods to minimise bias whilst increasing the speed of the review process [5,6], and will be updated regularly. We only included studies that provided information about follow-up through the course of infection, which allowed reliable assessment about the proportion of asymptomatic people in different settings. In the statistical synthesis of proportions, we used a method that accounts for the binary nature of the data and avoids the normality approximation (weighted logistic regression). Limitation of the review are that most included studies were not designed to estimate the proportion of asymptomatic SARS-CoV-2 infection and definitions of asymptomatic status were often incomplete or absent. The risks of bias, particularly those affecting selection of participants, differed between studies and could result in both underestimation and overestimation of the true proportion of asymptomatic infections. Also, we did not consider the possible impact of false negative RT-PCR results, which might be more likely to occur in asymptomatic infections [116] and would underestimate the proportion of a...

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.

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 10.1101/2020.05.31.20118554: (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.

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 10.1101/2020.07.07.20148106: (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.

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 10.1101/2020.07.17.20155937: (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.

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 10.1101/2020.07.21.214759: (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.

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 10.1101/2020.07.30.20163824: (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.

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 10.1101/2020.05.21.108506: (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.

However, we acknowledge this approach has some limitations. We have, for example, relied on admittedly arbitrary choices concerning the number of minimal observations and nodes required to conduct statistical testing. While it seems unlikely this would change our overall conclusions, which are highly consistent for two tested alignments, results for particular mutations should be considered in light of this caveat and may change as more genomes become available. Further, our approach necessarily entails some loss of information and therefore statistical power. This is because our motivation to test independent occurrences means that we do not handle “embedded homoplasies” explicitly: we simply discard them (Figure 2), although inclusion of embedded homoplasies does not change the overall conclusions (Figure S11b). Finally, while our approach is undoubtedly more robust to demographic confounding (such as founder bias), it is impossible to completely remove all the sources of bias that come with the use of available public genomes. In addition, it is of note that the SARS-CoV-2 population has only acquired moderate genetic diversity since its jump into the human population and, consequently, most branches in the phylogenetic tree are only supported by very few mutations. As a result of the low genetic diversity, most nodes in the tree have only low statistical support [41]. This prompted us to apply a series of stringent filters and masking strategies to the alignment (see Meth...

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.

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 10.1101/2020.06.17.156455: (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.

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 10.1101/2020.09.27.316174: (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.

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.

Fascinating how human minds are almost like computers themselves.

This is very popular on social media, comical videos surrounding the internet, youtube creators that rely on the humor to help fuel their views

训练一个源头，只要某种类别的信息

DOI: 10.1074/jbc.RA120.015400

Resource: (Abcam Cat# ab1187, RRID:AB_298652)

Curator: @Naa003

SciCrunch record: RRID:AB_298652

What is this?

<small><cite class='h-cite via'>ᔥ <span class='p-author h-card'>Cory Doctorow</span> in Pluralistic: 16 Feb 2021 – Pluralistic: Daily links (<time class='dt-published'>02/25/2021 12:20:24</time>)</cite></small>

It's interesting to note that there are already two other people who have used Hypothes and their page note functionality to tag this article as to read, one with (to read) and another with (TODO-read).

This is an excellent overview of how to take notes for academic research and creating writing output.

Others on the page here (specifically Dpthomas87's A, B, C) have done a great job at outlining their methods which I'm generally following. So I'll focus a bit more on the mechanics.

I rely pretty heavily on Hypothes.is for most of my note taking, highlights, and annotations. This works whether a paper is online or as a pdf I read online or store locally and annotate there.

Then I use RSS to pipe my data from Hypothes.is into a text file in OneDrive for my Obsidian vault using IFTTT.com. I know that a few are writing code for the Hypothes.is API to port data directly into Roam Research presently; I hope others might do it for Obsidian as well.)

Often at the end of the day or end of the week, I'll go through my drafts folder everything is in to review things, do some light formatting and add links, tags, or other meta data and links to related ideas.

Using Hypothes.is helps me get material into the system pretty quickly without a lot of transcription (which doesn't help my memory or retention). And the end of the day or end of week review helps reinforce things as well as help to surface other connections.

I'm hoping that as more people use Hypothesis for social annotation, the cross conversations will also be a source of more helpful cross-linking of ideas and thought.

I prefer to keep my notes as atomic as I can.

For some smaller self-contained things like lectures, I may keep a handful of notes together rather than splitting them apart, but they may be linked to larger structures like longer courses or topics of study.

If an article only has one or two annotations I'll keep them together in the same note, but books more often have dozens or hundreds of notes which I keep in separate files.

For those who don't have a clear idea of what or why they're doing this, I highly recommend reading [[Sönke Ahrens]]' book Smart Notes.

I do have a handful of templates for books, articles, and zettels to help in prompting me to fill in appropriate meta data for various notes more quickly. For this I'm using the built-in Templates plug-in and then ctrl-shift-T to choose a specific template as necessary.

Often I'll use Hypothes.is and tag things as #WantToRead to quickly bookmark things into my vault for later thought, reading, or processing.

For online videos and lectures, I'll often dump YouTube URLs into https://docdrop.org/, which then gives a side by side transcript for more easily jumping around as well as annotating directly from the transcript if I choose.

I prefer to use [[links]] over #tags for connecting information. Most of the tags I use tend to be for organizational or more personal purposes like #WantToRead which I later delete when done.

When I run across interesting questions or topics that would make good papers or areas of future research I'll use a tag like #OpenQuestion, so when I'm bored I can look at a list of what I might like to work on next.

Syndicated copies: https://forum.obsidian.md/t/research-phd-academics/1446/64?u=chrisaldrich

Common sense, so I'll tag as TYCO (Thank you, Captain Obvious).

argumentación

puntos que son un llamado a la acción, vinculado al propósito

citas, fuentes de consulta

DOI: 10.1016/j.nbd.2020.105141

Resource: (James Trimmer, University of California at Davis Cat# 12CA5, RRID:AB_2532070)

Curator: @Naa003

SciCrunch record: RRID:AB_2532070

Curator comments: Anti-HA tag mouse monoclonal antibody 12CA5 James Trimmer, University of California at Davis Cat# 12CA5

What is this?

rare in computer science using category theory directly in computer science What other areas of math can be used / are rare to use directly in computer science?

In other words, you can build on this software in your proprietary software but can't change the Trailblazer source unless you're willing to contribute it back.

loophole: I wonder if this will actually just push people to move their code -- which at the core is/would be a direction modification to the source code - out to a separate module. That's so easy to do with Ruby, so this restriction hardly seems like it would have any effect on encouraging contributions.

Reviewer #2 (Public Review):

In this manuscript, the authors generated transgenic zebrafish reporter lines that allow observation of cytoplasmic lipid droplets in vivo. They knocked in GFP or RFP in the endogenous loci of perilipin 2 and 3, and showed that the reporter genes exhibited similar temporal and spatial expression in the intestine in response to acute high-fat feeding as the endogenous perilipin 2 and 3 transcripts. They also characterized the reporter gene expression in the liver, adipocytes, and around neuromasts. These tools open up new opportunities to study lipid droplets dynamics in live zebrafish that is not feasible in mouse models. Overall the manuscript is well written. The authors have discussed in details the strength and caveats of these reporters. The weakness is the descriptive nature of the study - many interesting observations but no mechanistic study. I have the additional comments:

1) It is curious that in plin2 and plin3 reporter fish, the fluorescent tags were inserted at the 5' and 3' of the open reading frame, respectively. The authors did not provide any explanation. Does the location where the fluorescent tag is inserted affect the expression of the reporter genes?

2) GFP and TagRFP-T are not fast folding fluorescent proteins and are very stable, which may not be the best options for studying the formation and degradation of lipid droplets. How the fluorescent tags affect the stability and clearance of the protein should be carefully characterized.

3) Was there any indel being introduced by TALENs in these knockin fish? Is there off target effects of the TALENs?

4) The authors also generated transgenic fish overexpressing human PLIN2 and PLIN3 fluorescent fusion proteins. Is the subcellular localization of these fusion proteins similar to the zebrafish knockin under nofed and fed conditions? In other words, do human PLIN2 and PLIN3 proteins behave similarly as the zebrafish orthologs?

This one I do not understands, likes can result in it becoming viral, retweets cause it to spread, why replies? You can tag specific people in the replies? Concern the person posting will be harassed? Where is the open dialogue to discuss the post more as encourage above?

NexusFont 是 Windows 下一款轻量好用的字体管理器。

NexusFont 不但能快速预览自定义的文本，还可以给字体打上 tag 进行分类，极大地提高了工作效率。

SciScore for 10.1101/2021.01.31.428824: (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).

There are several limitations to this work as currently described. First, a structure showing the atomic details of 3A3 complexed with spike would provide additional insight into the mechanism of binding and neutralization. However, structures of antibodies bound to S2 are generally challenging to obtain with just one structure available of an antibody binding near the HR2 stem (28). It is possible that 3A3 binding distorts spike structure, disturbing otherwise ordered regions. Accordingly, additional efforts to better understanding the molecular underpinnings of 3A3/ spike interactions are underway. Second, while we have shown that 3A3 binds spike from all three highly pathogenic coronaviruses with similar affinities, we have only demonstrated its ability to neutralize SARS-2 spikes in vitro. Demonstration of broad neutralization in addition to broad recognition would increase the potential relevance of this epitope for future therapeutics. The 3A3 epitope is highly conserved, with pairwise comparisons showing between 56% and 100% identity to the SARS-2 epitope for MERS and SARS-1, respectively (fig. S14). Since 3A3 affinity for the least similar MERS spike is comparable to that for the SARS-2 spike and greater than for HKU1, it seems likely that binding and neutralization depend primarily on RBD position epitope accessibility. The most concerning emerging SARS-2 variants have one conservative substitution in this epitope in B.1.1.7, identified in the United Kingdom, and has...

Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

Results from scite Reference Check: We found one citation with an erratum. We recommend checking the erratum to confirm that it does not impact the accuracy of your citation.

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.

Reviewer #3 (Public Review):

In this study from the Selimi lab, Gónzalez-Calvo and colleagues investigate the role of the uncharacterized complement family protein SUSD4. SUSD4 is expressed at the time of cerebellar synaptogenesis and localizes to dendritic spines of Purkinje cells. Susd4 KO mice show impaired motor learning, a cerebellum-dependent task. Susd4 KO mice display impaired LTD and facilitated LTP at parallel fiber (PF)-Purkinje cell (PC) synapses, indicating altered synaptic plasticity in the absence of Susd4. Climbing fiber (CF)-Purkinje cell synapses show largely normal basal transmission, with the exception of larger quantal EPSCs. Immunohistochemical analysis shows a small decrease in the proportion of CF/PC synapses lacking GluA2. As their data indicates a role for SUSD4 in regulation of postsynaptic GluA2 content at cerebellar synapses, they next explored the molecular mechanism by which SUSD4 might do so. Activity-dependent degradation of GluA2 does not occur in the absence of SUSD4. Affinity purification of proteins associated with recombinant SUSD4 identifies ubiquitin ligases as well as several proteins involved in AMPAR turnover. Finally, the authors show that SUSD4 can bind GluA2 in HEK cells, and that SUSD4 can bind the ubiquitin ligase NEDD4, but that these two interactions are not dependent on each other.

This study provides novel insight in the uncharacterized role of SUSD4 and provides a detailed and well-performed analysis of the Susd4 loss of function phenotype in the cerebellar circuit. The exact mechanism by which SUSD4 affects GluA2 levels remains unclear. However, their findings provide leads for further functional follow-up studies of SUSD4.

Specific comments:

1) Localization of SUSD4. The authors demonstrate localization to spines in distal PC dendrites (Fig. 1C). Does SUSD4 also localize to CF/PC synapses? This is important to establish given the phenotype of increased quantal EPSCs and decreased proportion of synapses without GluA2 at the CF/PC synapse.

2) Figure 4B: There seems to be considerably less surface GluA2 in Susd4 KO cerebellar slices. Is the difference in surface GluA2 between WT and KO significant? Although the mean reduction in surface GluA2 in Susd4 KO following cLTD is similar to WT, the difference with control is not significant. This should be pointed out in the text because it can not be definitively concluded that endocytosis of GluA2 is not altered in Susd4 KO on the basis of this experiment.

3) Figure 4D: The colocalization of SUSD4 with GluA2 is difficult to see in this image. An inset with higher zoom could help. Quantification of colocalization using e.g. Manders coefficient would help.

4) Figure 5B: The negative control used here, PVRL3alpha, lacks an HA tag. This therefore does not control for non-specific interactions of highly overexpressed membrane proteins in co-transfected HEK cells. The authors should use an HA-tagged membrane protein as a control here to demonstrate that the interaction of SUSD4 and GluA2 is specific for SUSD4.

5) Figure 5D: The level of GluA2 recovered in the IP appears normalized to HA-SUSD4. This does not control for the variations in GluA2 input levels shown in Fig. S11. Statements on amounts of GluA2 recovered for various SUSD4 mutants should be adjusted to take this into account.

6) Line 357: binding of SUSD4=is likely meant to be binding of NEDD4.

Pace Grandmaster Flash.

See annotations with the build-phases tag.

Why are the build phases not enumerated in the Nix manual? If the instructions on how to create a derivation (and thus, a package) then why not go all in instead of spreading out information in different manuals, making the subject harder to grasp?...

(By the way, it is documented in the Nixpkgs manual under 6.5 Phases; not sure why it is not called build phases when every page refers to them like that.)

this is another key topic. Also:

• substitute vs. substituter => this (I think)

See annotations with the substitute tag

binary caches tie in with substitutes somehow; get to the bottom of it. See annotations with the substitute tag.

Maybe this?

We can use required tag for html, for a simple check.

I see what he means, but what would you call this (tag)? "have to remember state"? maybe "have to remember" is close enough

Or maybe order is important / do things in the right order is all we need to describe the problem/need.

new tag?:

• code that is universally relevant/valuable
• non - _-specific logic

It seems like GIFs became more popular with social media. It's almost as if social media reinvented the GIF.

What is the purpose of this paragraph?

What is the main idea of this paragraph?

I think tagging the type of annotation that is being made is one of the best ways for students to be consciously aware of the moves they are making as readers.

Mount Judi, also known as Qardū, is Noah's apobaterion or "Place of Descent", the location where the Ark came to rest after the Great Flood, according to very Early Christian and Islamic tradition. The Quranic tradition is similar to the Judeo-Christian legend.

https://noahsfloodnoahsark.wordpress.com/tag/iran/

I thought this was just awesome. It really highlights the attitude that has developed among some people. Things like food, water, and air are so taken for granted that they have actually been reduced to "inconsequential" status because they are "marginal" in the scope of the over all economy. Like the only thing in the world that is real are dollars.

SciScore for 10.1101/2021.02.12.430998: (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).

The following limitations of our study need to be considered. We employed pseudotyped particles instead of authentic SARS-CoV-2 and we did not determine whether Y453F affects viral inhibition by T cell responses raised against SARS-CoV-2. Further, we did not investigate whether presence of Y453F in the SARS-CoV-2 S protein increases binding to mink ACE2. Nevertheless, our results suggest that the introduction of SARS-CoV-2 into mink allows the virus to acquire mutations that compromise viral control by the humoral immune response in humans. As a consequence, infection of mink and other animal species should be prevented and it should be continuously monitored whether SARS-CoV-2 amplification in other wild or domestic animals occurs and changes critical biological properties of the virus.

Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

Results from 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.

[Geisel, 2017] Using social bookmarking tools to bookmark, tag, annotate, and take notes on websites; "Better bookmarking"

• Diigo
• Hypothes.is

Summary: This study provides new information about how amyloid beta (Ab) oligomers (Abo) may contribute to hyperexcitability which is important because Abo and hyperexcitability have been suggested to occur early in the development of Alzheimer's disease (AD). The authors added Abo intracellularly (iAbo) using dialysis from a patch pipette. Their data suggest iAbo led to increased synaptic excitation mediated presynaptically by retrograde signalling of nitric oxide (NO). Furthermore, they present data suggesting that there is spread of this increase in excitation to neighboring neurons.

Major Comments:

1) The nature of the described effects of intracellular iAbo are quite unexpected, occurring within a minute of obtaining intracellular recording configuration, which contrasts with at least on previous study. While some controls for intracellular application of oligomers are provided, with reverse iAbo failing to reproduce the effect (Fig 2S1) and the effect being blocked by the antibody A11 (Fig 2S2), further controls are necessary to explain this rapid effect, which seems faster than that for the diffusion of the fluorescent tag into the cell (Fig 1S1). Note that Pusch and Neher (Pflug Arch 1988) determined diffusion time for different substances. That paper or others should be cited, and then some estimation of equilibrium time based on diffusibility of ab oligomers should be provided. Equations 17 and 18 in that paper provide some estimates based on molecular weight or diffusion coefficient. One point in Pusch and Neher is there is extreme variability between access times across cells and that it depends on access resistance, of course. Finally, the Pusch and Neher calculations were for small spherical cells - diffusion into spatially extended cells with long dendrites where the synapses are will take even longer. This is especially critical, as one of the major papers of precedent for this work is that of Ripoli, et al. 2014 (cited in the manuscript) in which the authors of that work examined effects of patch applied Ab42 over the course of 20 minutes, with internal controls showing differences between initial responses, right after break in, and 20 minutes later when the oligomer and/or monomers will have had a chance to equilibrate with the intracellular contents. It is not clear how such a rapid effect as indicated in the figures could be achieved by such a large molecule as Ab. The data suggest a time to effect of seconds to minutes, and the peak effect occurs before the fluorescence peaks, which seems hard to explain.

2) The data need reorganization in terms of their results using h-iAbo or iAbo. There needs to be a clear demonstration of why both were used if the results are generalized with both (or not) and if they can actually use both interchangeably.

3) The authors need to clearly indicate whether the experiments were done in culture or in slices. The authors need to provide a rationale on why specific experiments were done in culture and others in slices.

4) There are aspects of the observed phenomenon that have not been taken adequately into account. For example, the authors have not investigated the effects of application of oligomeric beta-amyloid to either the extracellular space or the presynaptic neurons, two other compartments of the synapse.

5) Aspects of the data raise questions: 1) Western blots appear to have multiple bands 2) evidence that the fluorescent probe accurately measures NO. 3) The bursts of activity are not quantified. What was defined as a burst? What was the burst frequency and did it change over the recording period? 4)The external solution for cultures contain 5.4 mM K+ which is quite high, and can induce hyperexcitability. Similarly, the use of 100uM AMPA and GABA seem very high. Justifying these high concentrations is important. They should lead to hyperexcitability and toxicity (AMPA) over time. Another point of concern is that the concentration of K+ for the slice work is 3 mM, much different than cultures. There are also differences in Mg2+ and Ca2+, making data hard to compare in the two preparations. 5) sample sizes are unclear 6) Intracellular Ab produces increases in both EPSCs and IPSCs. However, in Fig 3, the IPSC measures using a charge transfer quantification, did not show a significant change in response to iAbo, in contrast to EPSCs. 7) With regard to the inhibition, In the schematic on Fig. 10, I find this incomplete and slightly inaccurate since it shows one terminal releasing both glutamate and GABA with NO increasing both. While this is obviously an oversimplification, it's slightly inaccurate since NO was not directly shown to increase sIPSCs. Were NOS blockers able to disrupt the increase in sIPSCs? Moreover, there are many papers that have shown that PKC can also phosphorylate GABA receptors and increase their conductance. What could be the reason that this was not involved here? This needs to be discussed.

6) How this work relates to other studies is necessary. For example, how this study is related to others about Ab exposure is lacking. Also, regarding hyperexcitability, many possible causes exist. These should be summarized in the introduction and the authors should comment how their results fit with these studies. Regarding PKC and NO, PKC and NO have several known actions throughout the brain and body. How do the effects the authors have identified relate to all these other effects? For example, if PKC is activated by another mechanism, would it occlude effects of Ab? What are the changes in PKC and NO in AD? Regarding the ability of the data to address AD, a major issue is whether the results are relevant to AD or represent interesting pharmacological data about what Ab can potentially do in some of its forms in normal tissue.

Reviewer #2 opted to reveal their name to the authors in the decision letter after review.

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Reviewer #4

Evidence, reproducibility and clarity

We have reviewed "A specific regulator of neuronal V-ATPase in Drosophila melanogaster." by Dulac et al. The authors have identified VhaAC45L as a regulator of neuronal V-ATPase in Drosophila melanogaster. The authors have utilized multiple techniques to determine the localization of VhaAC45L in neurons and specifically in the synapse. The use of multiple approaches including determining RNA levels in different regions of the fly, and using CRISPR-Cas9 technique to insert V5 tag, makes a very convincing argument about the synapse-specific expression of VhaAC45L.

The combined use of co-immunoprecipitation technique and LC/MS to show that VhaAC45L co-precipitated with V-ATPase complex subunits is convincing that VhaAC45L is a subunit of V-ATPase. To determine the role of VhaAC45L in acidification of synaptic vesicles the authors have utilized pHluorins in combination with multiple RNAi lines. The authors have used a well-designed experiment to prove that VhaAC45L regulates acidification of the synaptic vesicles. Further, larval locomotion and quantal size determination using VhaAC45LRNAi which is known to be altered due to pH gradient of synaptic vesicles shows the functional role of VhaAC45L in synaptic vesicle acidification.

Minor comments:

1. For all graphs, please remove gridlines to make data points more visible.
2. Line 120-123: Authors indicate the VhaAC45LRNAi induced lethal phenotype when expressed in glutamatergic and cholinergic drivers but the figure is missing. Please indicate as "data not shown" if not included in Figure.
3. A diagram summarizing the role of VhaAC45L in V-ATPase enzymatic complex and specific role is recommended.

Significance

V-ATPase play a crucial role at the synapse by being responsible for acidification of the synaptic vesicles and identification of a synaptic vesicle specific regulator of V-APTase is important to understand the complex regulation of synapse function. The authors have used well-designed experiments to convince the localization and function of VhaAC45L in synaptic vesicle acidification.

Referees cross commenting

The summary of Reviewer#2 looks good!

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

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Reply to the reviewers

We would like to thank the editors and the four reviewers for their careful consideration of our manuscript. We are very grateful for their positive appreciation of our work and we believe that their suggestions, which have been included in the preliminary revised version of the manuscript whenever possible, have greatly improved the quality of the paper and have helped us deepen our understanding of the results.

We were happy to note that all the reviewers found value in our work, as stated in their general comments: “This is certainly a useful contribution to our understanding of neuronal V-ATPase functions in vivo” (…)” (Reviewer 1) – “Dulac et al report the very interesting discovery of a previously uncharacterized neuronal specific regulator of the V-ATPase. (…) The experiments are very well performed, the data presented very convincing and the paper is well written.” (Reviewer 2) – “The discovery of a neuronal specific regulator of the V-ATPase is very interesting (…) The work is therefore of great interest to researchers working on synaptic function in general and on synaptic vesicle biology in particular.” (Reviewer 3) – “The authors have used well-designed experiments to convince the localization and function of VhaAC45L in synaptic vesicle acidification.” (Reviewer 4).

In their remarks, the reviewers suggested additional experiments that could be done to improve our understanding of the role of this new V-ATPase regulator, as well as several minor issues. We have addressed all their comments in our answers below, in which the full text of the reviews is included in blue type, and the responses in black. The line numbers refer to the revised version of the manuscript.

Reviewer #1

Dulac et al. present a first in vivo characterization of the 'accessory' v-ATPase subunit vhaAC45L in Drosophila. The key findings are localization and association of the protein with v-ATPase complexes at synapses and a functional requirement based on lethality and reduced synaptic function. This is certainly a useful contribution to our understanding of neuronal v-ATPase functions in vivo. The main weakness of the study is a lack of depth. The study focuses on localization, co-IP of associated proteins, an analysis of acidification and reduced synaptic function in fly larvae, thus providing a baseline for mechanistic study. However, the mechanism of vhaAC45L is not addressed in this short report. How does is vhaAC45L function different from its homolog vhaAC45? Is it required for v-ATPase assembly? Is it required to localize the full v-ATPase complex (or just V0) to the synapse? Is the defect really due to partial loading of synaptic vesicles or does loss of vhaAC45L also affect endosomal and lysosomal function at synapses? The work as is certainly represents a publishable contribution without answering any of these questions - more as an invite for the community to study the role of vhaAC45L; however, I feel this is a bit of a missed opportunity to put the function of a new potential regulator of specific synaptic v-ATPase functions in the context of the most basic functions obvious in this field.

My main concerns are:

1. clearly, vhaAC45L is required for SOME function of v-ATPase in neurons - but it remains entirely unclear which one. It is not even clear what compartments are affected. Reduced quantal size of single vesicle exocytosis events can be a direct or indirect consequence of problems in SV biogenesis and recycling.

Is exo- /endocytosis unaffected? (FM1-43 uptake!).

We agree that alterations in the synaptic vesicle release/recycling cycle could indeed contribute to the locomotion defect, in addition to the acidification impairment observed in VhaAC45L knockdown larvae. As suggested by the reviewer, we plan to carry out FM-dye assays to measure endocytosis and exocytosis at the neuromuscular junction of control versus VhaAC45L-KD animals. If successful, a new figure will be added to the final version of the paper.

What compartments are affected? (markers for synaptic vesicles versus lysosomal compartments!).

Finding out whether VhaAC45L is specifically involved in the acidification of synaptic vesicles, or if it also plays a similar role in other synaptic organelles, in particular lysosomes, would be very interesting indeed. However, we found that it was technically difficult to address this issue in the Drosophila nervous system. A good way would be to check whether the lysosomal pH is affected by VhaAC45L knockdown, as it is the case for synaptic vesicles.

Unfortunately, because lysosomes are not abundant in neurons, lysosome-specific pH-sensitive probes such as Lysotracker do not yield detectable signals at Drosophila larval synapses. So, whether VhaAC45L is specific for synaptic vesicles or involved in the regulation of V-ATPase activity in all neuronal compartments reminas an open question for now.

1. molecular function: is vhaAC45L required for v-ATPase assembly? (IP/Pull-downs of v- ATPase complexes in the presence or absence of vhaAC45L with other subunits!).

In accordance with the reviewer, we are also very much eager to learn more about the precise molecular function of VhaAC45L, and in particular whether it is required or not for assembly of the V-ATPase complex. Pull-downs of V-ATPase proteins in controls versus VhaAC45L-KD could be used to address this question, but this would require a large quantity of antibodies directed against subunits of the V0 and V1 domains, respectively. Unfortunately, there are no such antibodies commercially available against Drosophila V-ATPase proteins. We have tried several antibodies that recognize V-ATPase subunits from other species and were predicted to react against Drosophila homologs, but with no success. The only V-ATPase antibodies currently at our disposal were samples generously sent to us by other laboratories in insufficient quantities for carrying out such experiments. To our regret, therefore, we were not able to answer this question until now because of the lack of appropriate tools.

1. vha100 was proposed in Drosophila to function on synaptic vesicles and the lysosomal pathway, but, if I remember correctly, here quantal size was normal. I am missing a comparison between the two.

We thank the reviewer for this comment. A comparison with previously published results on subunit Vha100-1 has now been added (lines 458-469) in the discussion related to this topic in the revised manuscript.

1. The V5 knock-in is used both as a mutant as well as a tool to analyze protein localization. This is likely okay, but a little concern of course has to be that by creating a mutant protein through stop codon deletion its subcellular localization, turnover, etc. are not normal. Similarly, anti-V5 co-IPs will isolate proteins bound to the mutant variant of vhaAC45L. Minimally, IPs or pull- downs using other members of the V0 complex should be done to understand the role of vhaAC45L in direct comparison with vhaAC45 on complex assembly and possibly targeting to the synapse (or ideally targeting to specific compartments).

It is indeed a legitimate concern to question the physiological relevance of results obtained by studying V5-tagged VhaAC45L. However, the V5 tag is very small (14 amino acids) and we fused it in place of the stop codon to keep intact the whole sequence of the protein. In addition, we found that the V5 knock-in flies are viable and fertile as homozygous. Given that the null mutants, as well as strong RNAi knockdowns, are lethal at early developmental stage, this suggests that the V5 knock-in has limited negative effects, if any, on VhaAC45L function. This led us to believe that at least a good portion of the V5-tagged protein might be targeted to the right subcellular compartment, and associate to its physiological partners.

Significance:

There is significance to the reporting of an accessory v-ATPase subunit required for SOME function of the v-ATPase in neurons. There is some lack of significance in the absence of basic mechanistic insight as to what vhaAC45L does to the v-ATPase in neurons.

We agree that we did not elucidate here the precise molecular mechanisms by which VhaAC45L contributes to synaptic vesicle acidification. It is rather an initial description of a novel neuronal protein that appears to be essential for proper synaptic functioning, and we provide consistent evidence that its function requires specific interaction with the V-ATPase complex, and in particular with three subunits that reproducibly co-immunoprecipitated with VhaAC45L (namely Vha1C39-1, Vha100-1 and ATP6AP2). Please note that it took many years and many papers before the molecular mechanisms of action of comparable accessory subunits, such as ATP6AP1/AC45 or ATP6AP2, was better understood, and it is still nowadays a matter of investigation. It is therefore very demanding to expect that we describe the exact function of the previously uncharacterized VhaAC45L at all levels in a single first paper.

Reviewer #2

In this study and using Drosophila melanogaster as a model system, Dulac et al report the very interesting discovery of a previously uncharacterized neuronal specific regulator of the V-ATPase called VhaAC45L. They combine genetics, IHC, Mass spec and ephys to unravel the expression pattern and function of this protein. They find that it is required to acidify synaptic vesicles in glutamatergic neurons of the Drosophila larval neuromuscular junction, for appropriate synaptic transmission and for larval locomotion. The experiments are very well performed, the data presented very convincing and the paper is well written. Nonetheless, a few additional pieces of evidence and some level of expanded analysis would strengthen the conclusions and increase the depth of the work.

Major comments:

1. Figure 1F: the while the localization to the presynaptic terminal is convincing, where exactly the protein is localized to is not studied. The imaging in these experiments could use increased resolution and concomitantly colocalization studies with more specific synaptic vesicle markers.

We agree that it would be very good to show this additional result. However, confocal microscopy does not provide sufficient resolution to localize the protein at the membrane of individual synaptic vesicles. Another way would be to see if VhaAC45L immunostaining co- localizes with domains enriched in synaptic vesicle markers, but these organelles are rather ubiquitously distributed in the synaptic boutons at the Drosophila neuromuscular junction. To correctly perform this experiment, we would have to do immuno-electron microscopy, a technique we do not master in our laboratory and that we did not plan to implement for the present work.

1. Figure 3B-G: these experiments should be complemented by a rescue experiment, ideally of the null mutant using a UAS construct and a pan neuronal driver, or - if such animals are viable to the third larval instar stage - a glutamatergic driver. If possible, it would also be good to study the NMJ phenotype of the null mutant rescued to viability using a neuronal driver that does not express in motor neurons (e.g. Chat-G4).

Although a rescue experiment could potentially add a further evidence that Vha45ACL deficiency is responsible for the synaptic vesicle acidification defect described in Figure 3, we don’t think that it is a requisite here because we obtained similar results by knocking down the gene using two different RNAis. As described in the manuscript, the pan-neuronal expression of Vha45ACL could rescue the embryonic lethality of the null mutant, so it would be theoretically possible to check the acidity level of synaptic vesicles at the neuromuscular junction of the recued larvae. However, this would involve making rather complex genetic constructions to express VMAT-pHluorin in motor neurons in rescued mutant background. In addition, the conclusions we could draw from such experiment would be limited by the lack of comparison. Indeed, in Figure 3 the defect was observed in knockdown context, and the same experiment could not be performed in knockout larvae due to the early lethality. If we could measure the acidity level of rescued null mutants, we would not have any comparison point besides the knockdown experiments. As knockout and knockdown are not likely to produce identical phenotype (especially in terms of magnitude of effect), the ideal would be to compare the rescued phenotype to the null mutant expressing VhaAC45L in all neurons except motoneurons, as suggested by the reviewer. However, such genotype would certainly not be viable, since we observed that expression of VhaAC45L RNAis with a stronger motoneurons driver (D42-Gal4) was sufficient to induce lethality at early developmental stage.

1. Figure 5: the authors focus on quantal size which measures the postsynaptic response to spontaneous release from the presynaptic terminal. However, it is unclear how this directly relates to the locomotor deficit beyond signaling potential deficits in vesicle loading or fusion. It would be more convincing to also study evoked release, and expand the analysis of presynaptic properties (number of events, amplitude, frequency).

We fully agree with this comment shared by Reviewers 2 and 3 related to the electrophysiology experiments. Note that these experiments have been carried out in collaboration with another laboratory located in another city. The Covid-19 situation during the past year has prevented, and is still complicating, movements between labs, preventing us from going further with the electrophysiology analyses of VhaAC45L KD. If the situation in the near future allows it, we would very much like to add a more extensive electrophysiological analysis, including in particular the study of evoked release. In the revised manuscript, we have nevertheless completed Figure 5 by adding representative distributions of spontaneous mEPSP amplitudes in control and VhaAC45L knockdown larvae, as well as the results of new analyses showing lack of effects the KD on the mean EPSP frequency.

1. General: showing some level of genetic interaction with V-ATPase subunits in at least some of the assays would be welcome.

We are definitely in accordance with the reviewer on that point, but we think that this would involve a lot of work and be beyond the scope of the present initial description. Here we show by proteomic analyses that at least 12 proteins co-precipitate and so potentially interact with VhaAC45L, three of them being previously identified constitutive or accessory V-ATPase subunits. In our opinion, studying the interactions between VhaAC45L and these proteins through genetic and molecular studies will be the subject of future works. As stated by Reviewer 2 in the Referees cross commenting below: “further biochemical analysis is interesting but probably beyond the scope of this initial description and would take too much time”. We fully agree with this statement.

Minor comments:

Some of the images, especially those in Figure 3, should be larger for ease of visualization.

As requested, the images of Figure 3 have been enlarged.

Significance

The discovery of a neuronal specific regulator of the V-ATPase is very interesting. To my knowledge it is the first description of a neuronal specific V-ATPase related protein since the description of Vha100-1 by Hiesinger and colleagues in 2005. The work is therefore of great interest to researchers working on synaptic function in general and on synaptic vesicle biology in particular.

We are grateful to the reviewer for his very positive assessment of our work.

I note that I do not have in depth expertise in electrophysiology, although I am sufficiently familiar with basic NMJ physiology experiments to render the opinions stated above.

Reviewer #3

In this study, Dulac and colleagues investigated roles of VhaAC45-like gene, which codes one of the V-ATPase accessory proteins in Drosophila, in synaptic transmission. First, they demonstrated that VhaZC45L transcripts are expressed selectively in neurons and that the gene products are addressed to synaptic areas. Second, they showed that VhaAC45L is co- immunoprecipitated with some subunits of V-ATPases, which is consistent with bio-informatics predictions. They further demonstrated that VhaAC45L-knockdown (KD) resulted in defects in synaptic vesicle acidification as well as a reduction in quantal size of glutamate, indicating that VhaAC45L play a key role in regulating neurotransmitter release by modulating the driving force for transmitter uptake. Last, not least, they demonstrated that VhaAC45L-KD in motoneurons attenuated larvae locomotor performance, indicating its physiological relevance. Overall, this study is rigorously executed and nicely presented, and adds one more component of the V- ATPase that is responsible for neurotransmitter uptake into synaptic vesicles. However, since this study simply confirmed an established notion from other species such as yeast and mammals that AC45 is one of the accessory proteins of the V-ATPase complex, a conceptual novelty beyond the previous knowledge is relatively poor in its present form. Thus, this reviewer would suggest several issues as following to improve the comprehensiveness as well as novelty of the current manuscript.

1. The reason why the authors focused on VhaAC45-'like' is somewhat obscure, and therefore should be explained. How different VhaAC45 and VhaAC45L are in terms of amino acid sequences, tissue distributions, and KO phenotypes. It seems more comprehensive if the authors provide some experimental evidence on VhaAC45; e.g. whether it is also expressed in neurons or not (Fig. 1), and, if VhaAC45 is neuronal, whether it can rescue the phenotypes of VhaAC45L- KD to certain degree (Figs 4 & 5).

Following the reviewer’s request, we have added a sequence alignment of VhaAC45 and VhaAC45L, as well as a graph showing tissue distributions of both genes in Supplementary Figure 1 of the revised manuscript. To our knowledge, there is no published functional study of VhaAC45 in Drosophila, so we can only make assumptions derived from studies on predicted homologs in evolutionarily distant species. For that reason, it is difficult to compare VhaAC45 to VhaAC45L, as it would first require an entire new study of VhaAC45 function in flies. Since our interest is to study neuronal physiology, we focused on VhaAC45L because compelling evidence indicates that this subunit is specific to the nervous system, as described in our manuscript, rather than on VhaAC45 which seems to be expressed in all tissues. In addition, homologs of VhaAC45L have never been functionally characterized to date in any species, making it very interesting to study this new protein in a genetically tractable organism.

1. What is the mechanism of Ac45 in regulating V-ATPase activity? In mammals, it has been suggested that Ac45 is essential for proper sorting of the V-ATPase to the destined organelles (e.g. Jansen et al., Mol. Biol. Cell., 2010; Jansen et al., BBA, 2008). In this context, it should be examined whether VhaAC45L-KD would affect the synaptic localization of other V-ATPase subunits.

We thank the reviewer for pointing out these very interesting references. We have indeed tried to determine the relative abundance of two other V-ATPase subunits at the larval neuromuscular junction in control and VhaAC45L knockdown contexts. However, because the tested subunits are not specific to neurons, and are expressed at relatively low levels in synapses, it was not possible for us to properly separate the synaptic signal from the background immunostaining in surrounding muscles. This unfortunately prevented us from performing an accurate and reliable quantification.

1. Given that a rodent brain SV contains a few copies of the V-ATPase on average (Takamori et al., 2006, and some newer papers by others), it is interesting that >80% reduction of Ac45 showed moderate effects on quantal size. If SVs under study also contains 1 or 2 V-ATPase per SV, there must be some SVs lacking VhAC45L upon KD. In this context, it is interesting to see how VhaAC-KD (RNAi1~3) affect the frequencies of minis.

The reviewer’s valuable comment prompted us to undertake new analyses on our electrophysiological recordings. We have now added in Figure 5E graphs showing the mean EPSP frequency for larvae expressing VhAC45L RNAi1 and RNAi2, which are the ones that were used in the quantal analysis. Both of these RNAi apparently decreased the frequency compared to controls, but this difference was not statistically significant. As detailed in the Discussion (line 458-469), this may suggest that VhaAC45L does not influence the abundance of the V-ATPase complex at nerve terminals, but rather its efficiency.

1. In general, decrease in mini amplitudes is accounted for by changes in postsynaptic sensitivity for neurotransmitters. Although acidification deficits would support that decrease in quantal size is due to the decrease in the driving force for glutamate uptake, it should be examined whether the postsynaptic receptor fields are not affected by VhaAC45L-KD by recording postsynaptic response upon application of non-saturable concentrations of glutamate.

Testing for potential postsynaptic receptor field alteration by glutamate application would be an interesting experiment indeed, but, as we believe, not a critical control for the present manuscript. Because we expressed RNAis presynaptically, any modification in the postsynaptic receptor field would have to be an indirect consequence of VhaAC45L downregulation in motoneurons, and so, likely to be related to the synaptic vesicle acidification defect. It would not change, therefore, our conclusion that VhaAC45L deficiency in motoneurons induces a decrease in quantal size. Because electrophysiology experiments were carried out in collaboration with another laboratory located in another city, the current sanitary context has so far prevented us from performing this test (please refer to our answer to comment 3 of Reviewer 2 for more details).

1. Related to 4, it is also interesting to see if evoked responses are also attenuated as a result of VhaAC45L-KD, which is more physiologically relevant for locomotor activity phenotype than minis.

We also agree with this comment, shared by Reviewer 2, to which we already responded above in our answer to comment 3 of Reviewer 2.

Minor points:

1. Quantal size of glutamate is not affected by reduced expression of DVGLUT (Daniels et al., Neuron, 2006), which highly contrasts with VhaAC45L, expression of which defines quantal size. Distinct regulation of quantal size by the transporter and the V-ATPase subunit should be discussed.

As suggested by the reviewer, a discussion of this point has been added (lines 458-469). and Daniels et al. 2006 is now cited in the revised manuscript.

1. For electrophysiological experiments, respective sample traces should be shown in Figure 5.

Quantal size is not directly visible in sample traces, so we added instead representative distributions of spontaneous mEPSP amplitudes in control and VhaAC45L knockdown larvae in the new Figure 5C.

1. <![endif]>Only RNAi1 and RNAi2 lines were examined for SV pH estimation and mini analysis. The results from RNAi3 should be presented, or at least mentioned in the text.

These experiments were performed using two different RNAi constructs to ensure that similar effects were observed and to exclude the possibility of potential off-targets. Knocking down VhaAC45L in neurons with RNAi1 and 2 was lethal at pupal stages, suggesting that they give similar levels of inactivation. RNAi3 systematically induced lighter phenotypes, producing viable adults, which led us to believe it had a lower efficiency. Because the results on synaptic vesicle acidification and electrophysiology were very consistent with RNAi1 and RNAi2, we considered that it was not necessary to repeat the experiment with RNAi3.

Significance

As mentioned above, as it stands, the authors merely confirmed the pre-existing bioinformatic knowledge on one of the AC45 homologues in Drosophila. The audience of The EMBO Journal might be interested in how different/similar VhaAC45 and VhaAC45-like are, and their functional relevance. In particular, is VhaAC45 also mandatory for the V-ATPase functioning in neurons? Adding some basic information of VhaAC45, e.g. tissue distribution, KO phenotypes, and ability to rescue the VhaAC45-like-KD phenotypes, will certainly improve the comprehensiveness of this study, and capture audience's attention.

As mentioned in our response to point 1 of the reviewer above, we have added more data comparing the structure and distribution of VhaAC45 and VhaAC45L in the revised manuscript. VhaAC45 appears to be ubiquitously expressed whereas VhaAC45L is neuron-specific.

Comparing VhaAC45 to VhaAC45L would require a completely new study of VhaAC45 function, because it has never been done before in Drosophila to our knowledge. This would require repeating all the experiments with this other gene, probably involving two more years of work, and would make for a much longer and very different manuscript. It is understandable that this cannot be envisaged. Because homologs of VhaAC45L have never been functionally characterized to date in any species, we considered that it was worth studying this new protein on its own.

Reviewer #4

We have reviewed "A specific regulator of neuronal V-ATPase in Drosophila melanogaster." by Dulac et al. The authors have identified VhaAC45L as a regulator of neuronal V-ATPase in Drosophila melanogaster. The authors have utilized multiple techniques to determine the localization of VhaAC45L in neurons and specifically in the synapse. The use of multiple approaches including determining RNA levels in different regions of the fly, and using CRISPR- Cas9 technique to insert V5 tag, makes a very convincing argument about the synapse-specific expression of VhaAC45L.

The combined use of co-immunoprecipitation technique and LC/MS to show that VhaAC45L co- precipitated with V-ATPase complex subunits is convincing that VhaAC45L is a subunit of V- ATPase. To determine the role of VhaAC45L in acidification of synaptic vesicles the authors have utilized pHluorins in combination with multiple RNAi lines. The authors have used a well- designed experiment to prove that VhaAC45L regulates acidification of the synaptic vesicles.

Further, larval locomotion and quantal size determination using VhaAC45LRNAi which is known to be altered due to pH gradient of synaptic vesicles shows the functional role of VhaAC45L in synaptic vesicle acidification.

Minor comments:

1. For all graphs, please remove gridlines to make data points more visible.

We found that gridlines can be helpful for the readers to assess approximate values on the graphs. So, we have not removed them but rather changed the colour to a light grey so it does not affect any more visibility. We have also placed the points over the error bars in all the graphs, so they become more apparent.

1. Line 120-123: Authors indicate the VhaAC45LRNAi induced lethal phenotype when expressed in glutamatergic and cholinergic drivers but the figure is missing. Please indicate as "data not shown" if not included in Figure.

This mention has been added in the manuscript (line 125).

1. A diagram summarizing the role of VhaAC45L in V-ATPase enzymatic complex and specific role is recommended.

We believe that it is too early in this first report to draw an accurate diagram summarizing the role of this new protein in the V-ATPase complex.

Significance

V-ATPase play a crucial role at the synapse by being responsible for acidification of the synaptic vesicles and identification of a synaptic vesicle specific regulator of V-ATPase is important to understand the complex regulation of synapse function. The authors have used well-designed experiments to convince the localization and function of VhaAC45L in synaptic vesicle acidification.

We thank the reviewer for his very positive appreciation of our work.

Referees cross commenting

(Written by Reviewer 2)

There seems to be overall consensus among the reviewers on 3 issues:

1. A somewhat more precise understanding of the role of vhaAC45L in the synaptic vesicle cycle through better localization studies and some classic assays (like FM dye uptake).

—See our answers to comments 1 of Reviewer 1 and Reviewer 2.

1. A little more characterization of the transmission defects (e.g. studying evoked responses) would be welcome.

—See our answers comment 3 of Reviewer 2.

1. Ascertaining the validity of the alleles with rescue experiments, perhaps in the V5 mutant background to allow localization analysis in a rescued background.

—See our answers to comment 2 of Reviewer 2.

I think further biochemical analysis is interesting but probably beyond the scope of this initial description and would take too much time.

We fully agree with this statement.

The minor issues are easy to address

We have addressed all of them in the preliminary revised version of the manuscript.