Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DNMT FAMILY INDUCES DOWN-REGULATION OF NDRG1 VIA DNA METHYLATION AND CLINICOPATHOLOGICAL SIGNIFICANCE IN GASTRIC CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: Chang et al investigate the correlation of NDRG1 with DNA methyltransferases in patients with gastric cancer. The authors utilize publicly available patient data and cell lines to support their findings.

Nevertheless, there are several parts that need significant improvement.
Experimental design: First of all, the authors in a biased approach, focus their efforts on the correlation of NDRG1 levels with the DNMT family, due to the importance in DNA methylation of CpG islands.

Given that the DNMTs are not solely responsible for the DNA methylation of CpG islands, the authors should also include their analysis in several components of PRC complex, expecially KDM2B.
Validity of the findings: The authors should include more publicly available datasets from GEO to improve the power of their study since the statistical significance of their findings are very weak.

More importantly, in the cell line data (Figure 2), the authors should use shRNA and inhibitors (decitabine) of DNMT1,3a,3b (and probably KDM2B) and overexpression vectors of the same proteins, in order to demonstrate the direct effect of DNMTs in the expression of NDRG1.

With their data they provide correlation, not causation.

Furthermore, in the same cells the authors should show the mRNA levels of NDRG1. To further prove the effect of DNMTs in NDRG1, the authors should use luciferase constructs of NDRG1 promoter and transfect them in shRNA against DNMT or DNMT inhibitors.

These data will support the claims of the authors.
Additional comments: Chang et al proposed the possible correlation of NDRG1 with DMNT DMA methyltransferase in patients with gastric cancer. The manuscript needs significant revision. I would be delighted the review the revised version of this interesting work. I would like to wish the authors good luck with their efforts.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DNMT FAMILY INDUCES DOWN-REGULATION OF NDRG1 VIA DNA METHYLATION AND CLINICOPATHOLOGICAL SIGNIFICANCE IN GASTRIC CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, the reporting is scientific, clear and unambiguous. Sufficient background and references have been provided as well. However, here are some suggestions,
1. There are some grammatical and sentence construction errors that can be changed by getting it reviewed professionally. For example, line 39, 46, 117, 178 and so on.
2. The use of abbreviations are commendable and makes reading an article more pleasant. However, please introduce abbreviations in their first occurrence and following which only the abbreviations should be used in the rest of the article.
3. Only a couple of sentences are missing relevant references. For example, line 56, 174, 202.
4. Consider inserting a reference to the figure number when discussing it in the discussion for more concise understanding for the reader.
Experimental design: The experimental design is sound, relevant and meaningful.
Validity of the findings: The findings have been clearly stated along with underlying data have been provided. However, here are a few suggestions to consider for the discussion,
1. Consider addressing the claim in line 187 to address the challenges that can arise from comparison of in-vivo to in-vitro experimental assays. Consider expanding on probable future directions for the project as explained in brief in line 261.
2. Additionally, adding more relevant discussion on the role of DNA methylation can add value to discussion overall.
Additional comments: Overall, this article communicates the aim of the study, methodology and results very well to its readers.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DNMT FAMILY INDUCES DOWN-REGULATION OF NDRG1 VIA DNA METHYLATION AND CLINICOPATHOLOGICAL SIGNIFICANCE IN GASTRIC CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: Chang et al., provide a revised manuscript of their recent study on the regulation of NDRG1 by DNMT mediated DNA methylation in gastric cancer. The authors include several of the proposed experiments and fix issued noted. In my opinion, the revised manuscript is profound and strong. I would support the publication of this interesting concept.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Congratulation to the authors for their efforts.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: STRUO2: EFFICIENT METAGENOME PROFILING DATABASE CONSTRUCTION FOR EVER-EXPANDING MICROBIAL GENOME DATASETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well structured, straightforward and easy to read. Enough background is provided to contextualize the work. Figures are clear and references are up-to-date.
Experimental design: Methods are well-described both in the manuscript and in the associated GitHub repository. The authors have done a very thorough effort of describing each individual step to ensure reproducibility.
Validity of the findings: The conclusions regarding the speed of the tool and the effect of assembly quality on taxonomic classification are well supported. As detailed below, I would be interested in understanding why database “b100-r20-c10” in their simulation showed reduced performance in the Kraken2/Bracken test (Fig. 3D) compared to the other sets (even though there were others of lower assembly quality).
Additional comments: Youngblut and Ley describe an updated version of Struo, a software tool for constructing custom databases for commonly used metagenomic profilers. Their newer version is more than 3 times faster and has additional flexibility to update existing databases. This works represents a very useful contribution to the metagenomics field, as there is an increasing need to develop bioinformatics tools that can scale with the hundreds of thousands of microbial genomes that are now available in the public domain. In addition, given that the Genome Taxonomy Database is becoming the gold-standard approach for genome classification, it is especially useful to be able to generate custom databases using this updated taxonomic framework.

I did not review the initial version of the manuscript, but I believe the authors have appropriately addressed the reviewers’ comments and suggestions. I commend the authors for the extensive documentation provided in the GitHub repository, as it will be particularly useful for others to be able to easily run and test their tool. I only have two suggestions/comments:

1) Would be important to discuss in the text any useful pre-processing that should be done on the genomes prior to database building. The authors evaluated the effect of assembly quality on classification accuracy, but do all the tools cope with a highly redundant genome database, or should genomes be dereplicated (e.g. at 95% identity) for increased performance? Dereplication at the protein level appears to already be embedded within the HUMAnN3 database construction. What effect would different genome clustering thresholds have on taxonomic profiling?

2) Their Kraken2/Bracken benchmarking with the simulated databases is quite useful. However, I am wondering why database b100-r20-c10 showed the worst performance (Fig. 3D), even though it was of better assembly quality than some of the other sets? Could the authors elaborate on which specific genomes affected the results and why?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: STRUO2: EFFICIENT METAGENOME PROFILING DATABASE CONSTRUCTION FOR EVER-EXPANDING MICROBIAL GENOME DATASETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have carefully reviewed the manuscript from Youngblut & Ley entitled "Struo2: efficient metagenome profiling database construction for ever-expanding microbial genome datasets", which came to me already after its exhaustive response to reviewers, which I also assessed. I feel that authors answered satisfactorily and exhaustively all previous reviewer's comments, and the questions I personally also had (mainly about the inclusion of Metaphlan in the Struo2 pipeline) were also raised by some reviewers and answered satisfactorily by authors. This manuscript is strong and provides a much needed tool aiding the analysis of metagenomes, very importantly so for the easy inclusion of GTDB definitions in commonly accepted metagenome analysis pipelines.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE IMPACT OF PEER REVIEW ON THE CONTRIBUTION POTENTIAL OF SCIENTIFIC PAPERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is clear but the authors should explicitly mention, from the abstract and the introduction, that the work is only dealing with manuscripts accepted and published.

Raw data for LDA preprocessing and sentiment calculation is not available.
Experimental design: I am concerned about which h-index is used for the analysis: the "current" one or the one the authors had when the paper was first submitted. From your interpretation in Section 5.1, I guess the latter would be the right one but, if Dimension API does not provide it, you would need to calculate it from the citations received before the paper submission.

Sentiment is calculated using TextBlob's default configuration (i.e. NLTK Naive Bayes classifier on the whole text). The authors could check that the results hold for other more sophisticated approaches (e.g. weighting positive/neutral/negative sentences using SentimentR or following a neural network based approach as in Stanford CoreNLP).

More information is needed on how added words are collected for measuring manuscript modification. How can this process be affected by the fact that new words may appear as a result of text changes or deletions? How does this process deal with word changes such as making a term plural, using different variations of the same root (e.g. see fig and figure in Topic 9 of Figure 2) or having different hyphenation from the PDF? What text cleaning and preprocessing has been applied after text extraction?

Two unbalanced research areas are considered in this study (i.e. 4917 vs 165 articles in Table 1). I wonder how this can affect the models in Tables 1 and 4.
Validity of the findings: The models in Table 2 should also look at the number of reviewers reviewing an article and at the level of agreement of their reports or recommendations, since the editorial decision may be also driven by these variables. The relationship between the polarity of authors and reviewers might be further analyzed to better understand the dialogue established during the peer review process.

The selection of the number of topics (i.e. 13) in Figure 2 should be further justified, as well as the effect of the threshold set for the minimum frequency (i.e. 50). The manual grouping of words should be explained into more detail and it should be numerically analyzed, maybe beyond the Top 10.

Table 3 should look at the reviewer recommendation as previous research has found the relationship between the sentiments of reviewing reports and the reviewer recommendation (e.g. https://elifesciences.org/articles/53249, do not need to cite, I am sorry for the self-reference). Indeed, previous research has found that reviewers providing a positive recommendation (mainly "accept" or "minor revisions") are those more willing to sing their reviews, which would also be in line with your findings of more subjective and positive reports.

Table 4 concentrates on the number of citations but other Altmetrics could have also be considered (e.g. number of reads).
Additional comments: Congratulations for this very interesting and nice work.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE IMPACT OF PEER REVIEW ON THE CONTRIBUTION POTENTIAL OF SCIENTIFIC PAPERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Needs to be improved to be more reader friendly. Literature needs to be expanded.
Experimental design: Research questions are defined 3 times differently, a clear list of outcomes and exploratory variables is needed.
Validity of the findings: Until authors address my questions, I am unsure of the validity of the findings.
Additional comments: Thank you for the opportunity to review this manuscript. I would like to offer my suggestions for its improvement:
Introduction
1.	Reviewers are also able to leave feedback, which enables the authors of the submitted paper to revise the paper to improve upon or extend the paper. – this is redundant, u mention that with minor and major revision
2.	to improve the submitted work – you failed to mention here the role of peer review in improving the reporting not the work itself, those two aspects should be clearly differentiated
3.	You goal says that you look at contribution potential, yet this is not described in the abstract. And in the methods and results, you mix this with number of revisions. According to your definition this should be citations of a paper or Altemtric scores. Aditionally, if a journal has like peer j, has an impact factor, and this represents the average number of citations of paper for that journal – how likely is it that you would find any correlations with the things you investigate when most papers should fall closely around that citation number.

Background

1. The first paragraph of your background says nothing on the contribution potential as you define it.
2. we wonder to what extent the increased efficiency translates to the equability and quality of a paper’s eventual contribution potential. – this does not belong to lit. review section
3. You should include the following study and its findings in your lit. review – missing this, it is not clear how your lit. search was conducted: 10.1186/s12874-019-0688-x
4. we propose to explore a use of objective measurement for accessing peer review quality and their impact on paper contribution potentials. – If this was your goal it should not be under lit. review section, also it is not mentioned in the abstract, nor do I see it anywhere in results.

PeerJData

1. typically invited by the editors of the journal – please elaborate, how many are not invited by editors? How many of those are in your sample?
2. Please specify if reviewers make recommendations (minor, major, rejection)
3. Table 1, please include data on number of all articles, and then percentage of those with open reviews. Additionally, what if one review is open and another is not for the same paper – how is this counted?

The Impact of Revisions
1. We show that the number of revisions does not correlate to the paper’s contribution potential,
we note here that as we were unable to obtain a data set that includes the revision history of rejected papers, our conclusions only hold true for accepted and published manuscripts. – this is results, should not be in methods.
2. If you are correlating number of revisions and citations why not state so in abstract and methods, instead of saying contribution potential – the former is clearer for the readers.

Results

1. Please start results with a clear description of the number of analyzed manuscripts, how many reviewers they had, and how many revisions, what were the most common decision after round 1 stage, and how many had more than 2 review rounds.
2. Table 2, please provide Odds ratios
3. In all models, we observed that the h-index of the authors decrease the probability of receiving request for additional revision(s).  Please quantify this and be clear do you mean after review 1 round – how many papers were accept in initial stage?
4. status in the academia – how is this defined, and how different is it from h index?
5. Please provide in the methods table of all collected variables, and specify why did you not control for all of them in your models? You seem to have review decision, field, number of reviewers, sentiment, number of revisions, gender of authors (female ratio, female last authors, female first author – and these are very likely intercorrelated – how do you account for that), polarity of comments and rebuttals, citations and so on – so please list all in a table, and indicate how they were included in each model.
6. How many of the manuscripts had the same authors? Or overlapping authors?

Topic modeling
1. this analysis insinuates that during the manuscript modification process, authors add additional analysis
313 and evidence and may alter the implications of the results from their original papers. – I don’t think you have proof of this, please take 10% of the sample and manually compare have there actually been changes in analysis – also clearly define what you mean by change in analysis ? for example adding OR is that a change in analysis or change in reporting? Increasing or decreasing decimal points? Moving things from or to supplement?

2. Figure 2 clearly shows that the majority of the top words are words related to analyzing the results – nothing clear there when I look at it  – most common blue word is study – it could be found in intro, in discussion, in abstract – what has that word have to do with analysis – how do you know it is used as a verb and not a noun?

Overall, I find the results poorly written as there is too much talk of models, and less on findings. Please make the language understandable to readers – e.g. please start the results (after descriptors I asked above) with: we found that h-index of the authors decrease the probability (OR XY) of receiving a requests for additional revision(s) (finding confirmed by 4 models, see Supplementary fie) and remove unnecessary text of modeling, do the same for all other findings. In the supplement, please provide n for the models, and it isn’t clear how many manuscripts with more than 1 revision you had. Additionally, its seems you only looked at acceptance after first revision, what about characteristics after 2nd or 3rd revision, were all those lumped together as control?

Discussion

1.Please start first with your main findings. In our study of xy PEER J manuscirpts, we found that….
2. We find that the number of revisions requested over the peer-reviewing process does not have a
346 correlation with the contribution potential of the paper. – it isn’t clear here what is the contribution potential of the paper, as in methods you said it’s the number of revisions?

Final comments:

1. It is not clear what relations authors have to PeerJ, and what is PeerJ policy on data scraping. Was PeerJ contacted to provide info or participate in the study?
2. Please use STROBE to report the study and make it clearer.

In hopes may comments can help you improve your manuscript,
Kind regards,
Mario Malicki


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE IMPACT OF PEER REVIEW ON THE CONTRIBUTION POTENTIAL OF SCIENTIFIC PAPERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors report about a study on open reviews of the journal PeerJ. The go into the details of the written reviews. They analyse communications between reviewer committees and authors regarding the revisions on specific paper.

The paper is well-written.
For a journal paper, the amount of references seems not too comprehensive. Introduction and related work are easy to read but could be extended.
The structure of the paper follows the typical format.
Experimental design: The authors write: "our study provides insight into the content of peer reviews and the subsequent modifications ..." ... "Therefore, we propose to explore a use of objective measurement for accessing peer review quality and their impact on paper contribution potentials" This claim holds true: They provides some insights, ... they explore ... but they do not provide clear evidence of a critical process during peer review.

A concrete RQ with an clear answer is missing. This is the major weakness of this paper.

In general their experimental design is sound.

The methods and techniques used in the study make sense but I miss a concrete discussion why the authors used this and that tool. E.g. they used TextBlob for the measurement of polarity of texts. Why this tool? Why LDA in the described configuration (e.g. Words that appear less than fifty times)? We lean not enough on this in the paper.

I am not sure why the authors excluded the rejected papers. Sure, desk rejected papers can be excluded, but paper which got 1 or more revision and are then reject could have been a nice control sample. Authors could report about this.
Validity of the findings: The regression analysis to generate the results looks correct.

E.g. they say "In all models, we observed that the h-index of the authors decrease the probability of receiving a request for additional revision(s)."

This result sounds logic but is not really discussed. Of course, experienced authors with a reasonable h-index, produce papers that a readable and structured in the understandable manner.

I am also not too happy with the inclusion of bibliometric metrics into the analysis. High h-index of authors can be a side indication but should not over interpret.
I suggest to perform more analysis on the validity of the reviewer comments and the straightness of the revision and readability of the rebuttals.
Additional comments: In general, a very nice paper with a good contribution.
See 3 for the weaknesses.

As this is early reseach, I miss the main 3+ take aways and reasonable next steps.

The paper seems like a short paper for me. Most parts could be extended.
In my view, the paper takes not the most logic approach and slips away with the inclusion of bibliometrics metrics and sentiments.

"We use the h-index of the author as a proxy to quantify his or her prestige or reputation in academia" ok, but why? I do not believe that reviewers check the h-index of an author before starting the review. Or do they? If yes, please provide evidence that this is standard.

Including these metrics is too early. Too many things are unclear and undisputed, to start with indicators here.

Again, the topic and the dataset of the paper and study seem to be very precious and sound. But explain better why you use which technique and boil it down to the most fundamental questions and answer.

I like to review a 2nd version of the paper.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE IMPACT OF PEER REVIEW ON THE CONTRIBUTION POTENTIAL OF SCIENTIFIC PAPERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I found tables in the Appendix difficult to follow because they are not uniquely identified (eg. three different Table A1).

Table A2 (the alternative to Table 3) is not analyzed within the text.

Can you please report on the time window covered by the PeerJ Data? Collection date is specified but I am afraid I have missed the date of the earliest submission. If it was far from the collection date, it might also be far from the current h-index values and have an influence on the interpretation of the models in Table 1.

Please, double check for typos (eg. The results of our study __suggests__ ..., Number of __author__, polarity of __reviwers__, etc.).
Experimental design: When comparing the results for the h-index (Table 2) with those for the average number of affiliations (Table A2), I am worried about them being inversely correlated with the editors decision.

Also, when comparing Table 2 with Table A1, I am concerned about the different effect of the variance of polarity of reviewers’ comments. Can you further elaborate on this?

Does the variable "Number of revision" in Table 3 and Table A2 mean the number of review round (as defined in Table A3) or the number of reviewers involved? More reviewers are expected to provide more feedback, which might generate more manuscript modifications and affect the contribution potential.

Regarding text preprocessing, I wonder if you are running any kind of word stemming (e.g. see fig and figure in Topic 9 of Figure 2) and if that would have any effect on your results.
Validity of the findings: I wonder whether the interpretation "that the h-index of the authors decrease the probability of receiving a request for additional revision" (line 349) could be seen all the way around. Could the results be actually saying that authors receiving more positive editorial decisions end up having a higher h-index?

Do the models in Table 4 account for the reviewer recommendations? Do reviewers sign because they are actually recommending "accept or "minor revision" and, in turn, the type of recommendation is ruling the values of polarity and subjectivity?
Additional comments: The authors have mostly solved my previous concerns and I am happy with their responses.

Still, I have some minor comments shown above.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE IMPACT OF PEER REVIEW ON THE CONTRIBUTION POTENTIAL OF SCIENTIFIC PAPERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Paper structure needs to be improved. Methods section and ordering must be the same as in the results, with all variables and their definitions clearly stated.
Experimental design: Most of the methods are clearly described, but variables need to be better defined. Additionally, authors continue to use the term contribution potential, when in fact they are only talking about papers citations - and in Table 2 they call it number of citations. There is no need to talk about contribution potential unless they specify how that differs from number of citations measured by Dimensions.
Validity of the findings: The authors do not report 95%CI, and it is my opinion that unless they manually compare differences between two versions for a sample of manuscripts and their revisions, they can not use the LAD method to claim that "authors add additional analysis and evidence" during revisions. LAD has not been validated as a method that can prove this in any previous studies.
Additional comments: Thank you for the opportunity to re-review this work. I find it improved, but I still find many issues that I suggest addressing:

Introduction
1) - Although the peer-reviewing process often feels like a heavy burden to many scientists, - citation is missing for this part of the sentence
2) During the review process, reviewers decide whether to accept,  request a major or minor revision or reject a submitted paper. – this is not true for all journals, some do not ask reviewers for decisions
3) The results of our study suggest – this paragraph should not be in the introduction – leave results to results and discussion
4) influence the editor’s final decision – this study is observational, you are not looking at influence, but association

Literature review
1) Therefore, we aim to explore a use of
90 objective measurements for assessing peer review quality and their impact on paper contribution potential. – this is a different goal then the one you mentioned in the introduction

2) In our study, we operationalize “contribution potential” as the citation metrics and a paper’s
93 citation count. – what is the difference between metric and count? Finally, in intro u state: We define contribution potential as the 30 subsequent contribution and impact metrics that a paper amasses over time – Please have one clear definition of what contribution potential is.

Methods
All list of options for 4.1.3 and 4.1.4. should be listed (e.g. minor, major revisions, or type of changes for 4.1.4.)

Results
1) Table 2 – is missing 95%CI for Odds ratios, and with so many odds ratios being close to 1, the conclusions from these models are questionable.

2) Table 2 on the left column has a much clearer list of variables, than any you described in the methods section. I would strongly urge the authors, to have a list in the methods of variables, using the exact names as were used in Table 2 – and one sentence descriptions of their meaning. This would me much clearer for me as a reader to follow, then trying to decipher the meanings in the long paragraphs of current methods – and constantly going back to detect mismatches between what was written in methods, vs what is in the Table 2.

3) 5.1.2. We used publicly available Altmetric scores – this paragraph should be in methods not results

4) 5.3 My previous comment stands, authors should manually compare a sample of randomly chosen articles to demonstrate that authors add additional analysis and evidence. Without that, they must greatly tone down their results and can only claim indications of changes. Additionally, this section of results requires a statement on what percentage of manuscripts based on this method indicates additional analysis or evidence. Otherwise the usefulness of methods is very debatable.

 Overall structure of results:
Please make the paper easier to follow, if your 4.1.1 is Peer Review Content, then results 5.1.1. should be about peer review content, not about acceptance timeline. Use the same numbering order as is presented in the results in the methods. In other words, if 4.2. is Peer review feedback, than 5.2. needs to have the same title.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE IMPACT OF PEER REVIEW ON THE CONTRIBUTION POTENTIAL OF SCIENTIFIC PAPERS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors improved their paper largely.
Experimental design: The authors also improved the experimental design.
Validity of the findings: Findings look good!
Additional comments: Thank you for the successful revision.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE IMPACT OF PEER REVIEW ON THE CONTRIBUTION POTENTIAL OF SCIENTIFIC PAPERS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have very much improved the manuscript, which now looks complete and clear.
Experimental design: The experimental design is well defined and analyzed.
Validity of the findings: Findings are very interesting and valid. Some of them confirm previous work while other shed more light on the study of peer review.
Additional comments: Congrats, I like your work very much.

One final question, I wonder whether the conclusion " the process of peer-reviewing plays a role in a paper’s eventual contribution potential" is in line with the analysis in section 5.1.2 or with the sentence in the discussion referring to the "process does not have a correlation with the contribution potential of the paper".


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE IMPACT OF PEER REVIEW ON THE CONTRIBUTION POTENTIAL OF SCIENTIFIC PAPERS
Review round: 3
Reviewer: 2

Output:
Basic reporting: Biased interpretation of results.
Experimental design: Methods do not align with the statements made.
Validity of the findings: Conclusions overinterpret the data.
Additional comments: Thank you for the opportunity to again review this manuscript, but as my requests from the previous round were not met, I will only mention few things, and not provide a full review:
1) I may be now repeating myself for the 3rd time, but I find it strange that authors still do not clearly define some aspects of their study to the readers, e.g., why is there a need in the introduction to only partly define contribution potential. Specifically, the authors stated: (“e.g. the number of citations or altmetrics”), yet in the rebuttal letter they say that it is: “We used the number of citations, Altmetrics and the number of readers from reference manager software, such as Mendeley. More precisely, the paper’s citation count refers to the number of citations a paper receives and the citation metrics refers to Altmetrics and the readership numbers. We define the contribution potential of a given paper as how much recognition that article earned, which is calculated by both the citation metrics (readership counts) and a paper’s citation count.”
I have not once criticised your definition, only asked that it is clearly spelled out what it entails – so please do so for the readers. Comparatively, this is your definition in the literature section: “we operationalize “contribution potential” as the citation metrics and a paper’s citation count”; and in the methods section 3.3. you say: “Additionally, we used publicly available Altmetric scores and the number of readers from reference manager applications such as Mendeley to calculate the contribution potential of papers, which were retrieved through the Altmetric API.” (Why say such as Mendeley, clearly specify all u used, and the exact formula)
So I ask again, that there is somewhere only one singular definition of this concept, and a clear example how it is calculated – perhaps it would be best if the authors had a box with an example what is the contribution potential of one single paper, and what is the reproducible score/formula that anyone can use to get that number. As currently, it still escapes me how it was done.
2) Sentence: “Therefore, we aim to explore a use of 90 objective measurements for assessing peer review quality and their impact on paper contribution potential.” is not needed in the liter. review, you specify your aims in the introduction
3) Finally, the abstracts and discussion states: “We show that the exchanges between the
peer review committee and the authors, regardless of the number of revisions, implement a check for a baseline robustness of the final publication…. in response to peer reviewer feedback, authors add additional analysis to their papers” – I find these statements to be increadibly wrong, as there is no proof in this paper for them. Neither was robustness defined or measured, nor was adding of additional analyses – claiming that topic modeling proves this is completely wrong in my opinion as no comparison to manual detection of changes has ever been done. Therefore, authors should only report on associations they found and not make this kind of statements. In the rebuttal letter they stated this nicely: their results demonstrate that most revisions include a modification of these three topic categories, not of the actual changes.
4) In the rebuttal the authors said: We revised our manuscript so that the general section order and titles of the results section better reflects those of the methods. – and again, the order does not match, 4.1.1 and 5.1.1 are nor related - I would strongly encourage the editor to make the authors comply with this simple structuring request, and I will not bother with reviewing the rest, as it requires too much effort on my part again.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CONSTRUCTION OF COMPETING ENDOGENOUS RNA INTERACTION NETWORK AS PROGNOSTIC MARKERS IN METASTATIC MELANOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Generally well written manuscript, but some of the English language is poor and would benefit from further proof-reading. There are a number of typographical and grammatical errors in the manuscript that should be addressed during revision.

Article is suitably referenced and well structured.
Experimental design: The delineation of RNA-networks in cancer of of great interest. Here the authors analyse existing RNA-seq data from TCGA and attempt to construct ceRNA relationships between lncRNA-miRNA-mRNA and then extend these data to clinical relevance. These are bold aims and the approach used, while simple, is clearly described.

The authors have not investigated the relative expression of their main targets, however, it is unclear if they have investigated whether inverse expression is seen for the miRNA and mRNAs? Also, in terms of Kaplin HS, is there any advantage to including lncRNA and miRNA, are the mRNAs not the end point here and the only differential in terms of expression that contributes to risk? If not, what are the implications? Are other unidentified targets also important?
Validity of the findings: Conclusions are generally supported by the data, however, the authors need to address several key points:

1. Are the lncRNA investigated nuclear or cytoplasmic? The majority of lincRNA localise exclusively to the nucleus, limiting their potential as ceRNA. The authors do not consider this and should discuss this point.

2. Why do A375 cells transfected with miR-3662 expression construct display lower levels of miR-3662 (Fig 5A). Is this construct actually an inhibitor of miR-3662? The methods section is unclear here and this needs addressing. What happens to PKIA and ZNF831 expression in 5B when the RP11 lncRNA is also expressed? Does this rescue knockdown of the target by the miRNA? This is an essential experiment if the authors wish to test the validity of their ceRNA triplet.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CONSTRUCTION OF COMPETING ENDOGENOUS RNA INTERACTION NETWORK AS PROGNOSTIC MARKERS IN METASTATIC MELANOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some more recent references on melanoma treatment should be cited (see comments to the authors);
English would benefit a revision throughout the text
Experimental design: The study was logically thought and state of the art methods were performed. Howver it lacks a more thorough assessment in the melanoma cell model to validate the lncRNA action as ceRNA before the network can be considered of real value.
Validity of the findings: As specified above, the functional part in the cell line model needs to be more thoroughly developed
Additional comments: The authors performed an in silico analysis to build ceRNA interaction networks that could have prognostic value for metastatic melanoma.
The rational is valid and the approach in silico is powerful counting on large published data sets and state of the art tools. However, without further experiments it is quite hard to verify whether the identified network have indeed functional relevance.
The authors should at least test the protein levels of PKIA and ZFN831 upon miR-3662 modulation in the selected melanoma cell line and ideally also testing whether the selected lncRNAs indeed acts as ceRNAs in the cellular model.
The manuscript would also greatly benefit a round of English scientific revision.

Listed below are a few minor comments.

In the intro the authors should update to the most recent available references and mention the role of immunotherapy which has been paradigm changing for metastatic melanoma.

Abstract: Change ‘affects’ to ‘affect
Abstract: Change to ‘The prognostic risk model was used..’
Abstract: in the following sentence explain better how mir-3662 is linked to the ceRNA network: ‘Four groups of ceRNA interaction triplets were finally obtained, which miR-3662’…
Abstract: please clarify the following sentence which is ambiguous: miR-3662 acts as a suppressor in the treatment of melanoma.

Intro, lines 2-4: please edit the sentence (in the skin’ is repeated three times)
Intro lines 22-26 please rephrase sentence on miRNA action; it is unclear at present.
Intro line 36 define SKMC at first mention
Methods line 53. Do you mean shRNA (expressed through plasmid -DNA vector) rather than shDNA?
Results lines 144-148 when describing DE RNAs please clarify in with the total number you mean both those upregulated and downregulated
Results line change to ‘by down-regulating PKIA and ZNF831’
Discussion lines 253, 282 what do you mean by metastaticred?
Figure 1. In panel A should the hub box contain DEmRNA, DEmiRNA, DElncRNA rather than ‘DEmRNA DEmRNa DEmRNA’?
In figure 1D and 3d legends: please clarify what  you mean by transfer sample?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: CONSTRUCTION OF COMPETING ENDOGENOUS RNA INTERACTION NETWORK AS PROGNOSTIC MARKERS IN METASTATIC MELANOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed my previous comments relating to basic reporting in a satisfactory manner.
Experimental design: The authors have addressed my previous comments relating to experimental design in a satisfactory manner.
Validity of the findings: The authors have addressed my previous comments relating to their experimental findings in a satisfactory manner.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A RANDOMIZED TRIAL TO EVALUATE THE IMPACT OF COPRA MEAL HYDROLYSATE ON GASTROINTESTINAL SYMPTOMS AND GUT MICROBIOME
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript describes a clinical trial to evaluate the impact of CMH (two doses) on gut health in healthy adults.  The major limitations are  that the sample size is quite small and the age of subjects was significant different in 3gCMH group compared to others. Several of the key finding was found with 3gCMH intake. It is therefore extremely important to have an appropriate statistical model to analyze the data.  In addition, the manuscript is quite lengthy and the data presentation and language need to be improved.  Please add Faecal calprotectin as marker for gut immune status.
Introduction
1)	Line 48-49, please update the definition of prebiotics “The current scientific definition of a prebiotic was developed by a panel of experts in microbiology, nutrition, and clinical research convened by ISAPP in 2016.
2)	For line 51-52, please revise. Prebiotics are frequently equated with dietary fibers, not include many others. SCFA is just one of the mechanism.
Materials and method
1)	Line 82, please describe the MOS using degree of polymerization.
2)	The description of the study design (line 94-100) is confusing, please simplify.
3)	Please provide more details and reference for stool sample preparation.
4)	Suggest to use more appropriate statistical model, such  linear mixed effects models to compare among three groups (line 193-194)
Results
1)	Table 2: Does the carbohydrate exclude sugar? Please clarify.  Although the P value is not significant (0.062), 3CMH and 5CHM seems to have higher carb. Please discuss how this could impact your results.
2)	Move Table 3 into supplement and show statistical analysis in Figure 3.
3)	Figure 2, please discuss the higher  drop out in CMH groups.
4)	Please move Figure 4 into supplement and add the p value. Please als provide beta diversity analysis.
5)	Please use Table instead of Figure to present data in Figure 5.
6)	Missing Figure legend to point out the representative symbol for each group.

Discussion: Please 1) simplify the discussion, and don’t describing result repeatedly; 2)  discuss why many effects were observed in 3g CMH group but not 5g CMH groups; 3) provide explanation why many effect on gut microbiota was observed during wash out , but not intervention.
1)	This statement is not accurate (line 293): there is age difference.
2)	Line 318: at family level


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A RANDOMIZED TRIAL TO EVALUATE THE IMPACT OF COPRA MEAL HYDROLYSATE ON GASTROINTESTINAL SYMPTOMS AND GUT MICROBIOME
Review round: 1
Reviewer: 2

Output:
Basic reporting: Given two dosage of daily consumption were tested, it would be helpful to provide a brief background and rationale to test two different dosages in the introduction (and elaborate more in discussion section).

Tables 2 and 3 – were means ± SD presented? Please clarify in a footnote. Figures 6 and 7, the meaning of superscripts “a” and “b” next to some bars should be described in the footnote. This was not made clear in Table 1 either.

Why figure 3 presented only the significant differences in flatulate/bloating and size of stool? What about the changes of the other 6 indicators on defecating conditions and gastrointestinal symptoms? Would it be fair to conclude that 3g CMH improved defecating conditions if only two out of 8 indicators showed a significant improvement?

Figures 4 and 5 did not indicate statistical significance at all. Was the reduction in the Firmicutes/Bacteroidetes ratio statistically significant? The information seems to be missing in the results section. P-values should be consistently reported.

A discussion of potential limitations is missing. As no study is perfect, acknowledging limitations is important. For example, could self-reported stool chart and dietary record be free of measurement errors? Were all participants consume the distributed supplementation as indicated? Could differences in fiber or carbohydrate intake not influence the results? Could the results be generalizable to larger population with different health status and other demographic characteristics?

The sample was predominantly female, given the biological difference, it may be helpful to comment on potential gender differences.
Experimental design: More details on subject selection should be provided, including recruiting methods, location, and procedure etc.

It would be helpful to comment on the validity of stool size and characteristics instruments (i.e., stool record chart), their clinical implications, and potential reporting bias etc.

It would be helpful to provide the details of sample size calculation, the main outcome measure(s), the effect size, alpha level, power etc. to allow replication of the calculation.

An important advantage of Kruskal-Wallis test and Mann-Whitney test is that they do not assume that the data are normally distributed. However, these non-parametric approaches, which substitute ranks for the original values, could be less powerful than their parametric equivalents. Moreover, practically ANOVA was not very sensitive to deviations from normality. Given that many of the outcomes were continuous measures, the blanket application of non-parametric tests to ordinal and interval/ratio outcomes may have sacrificed efficiency.

A few baseline characteristics appear to be unequally allocated, particularly, the placebo group was significantly younger compared with the treatment groups, and 5CMH group seem to have lower median body weight. With only 5 baseline characteristics measures included in Table 1, it is difficult to judge whether baseline characteristics were well balanced and how well randomization performed. Since BMI is a function of height and weight, Table 1 essentially just showed mean levels of 3 variables -- age, sex, and BMI -- by study groups.

Related to this, average consumption of dietary fiber was over 70% higher among 3CMH group compared with that among placebo group (9.27 vs 5.44 g). Although the omnibus Kruskal-Wallis test showed a p value slightly larger than 0.05, the difference is likely significant with Mann-Whitney test. Similarly, 3CMH group had 23% higher consumption of carbohydrate. Albeit at lower concentration, many foods have prebiotic effects, including garlic, onion, wheat, honey, banana, barley, tomato, soybean, cow’s milk, peas, beans, etc. These differences warrant a careful evaluation and discussion, given that unbalanced dietary patterns could be a strong alternative explanation to treatment effect.
Validity of the findings: The abstract should include a few important qualifications related to the sample characteristics, i.e., the number of participants, predominantly female, aged 18-40, normal BMI, and healthy status etc. I would also suggest toning down the general conclusion, especially the statement in the abstract that 3g CMH “could be considered a source for a future prebiotic product”, given the limited generalizability of the present study to a more diverse population (with a wide variety of age, health conditions, physical activity levels, dietary patterns etc.).
Additional comments: The manuscript assessed the potential prebiotic effect of copra meal hydrolysate. The authors reported that daily 3g/d CMH supplementation over a period of 21 days was beneficial to gut health. In general, the manuscript is well written, the analysis is rigorous, and the topic is of scientific interest.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A RANDOMIZED TRIAL TO EVALUATE THE IMPACT OF COPRA MEAL HYDROLYSATE ON GASTROINTESTINAL SYMPTOMS AND GUT MICROBIOME
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed my comments sufficiently. I have no further comments.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: NOVEL ACE2 PROTEIN INTERACTIONS RELEVANT TO COVID-19 PREDICTED BY EVOLUTIONARY RATE CORRELATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present their study of evolutionary rates and their correlations between the ACE2 protein and ~2000 mammalian proteins. The evolutionary rate correlation (ERC) approach has been validated by them and other groups previously and has been used to identify new functional interactions in the past. The authors set their sites on the ACE2 protein which is the entry key for the SARS-CoV-2 virus. By examining the genes that correlate with ACE2's terminal branch rates, they find significant enrichments for coagulation cascade pathway proteins and interferon proteins. These represent potential new functional interactions for ACE2, which is known for catalyzing a chemical reaction of angiotensin and thereby affecting blood pressure.

The study appears to be conducted well and the findings are summarized in a set of tables and graphs. One concern to address is the relatively small proportion of the genome (~2,000 genes) that were surveyed and thereby potentially leaving out many important interactions. Second, the interpretation of ACE2's correlations as being strictly related to viral activity is too narrow and should consider other selective pressures that could be acting on ACE2. These and other points of potential concern are itemized below.

Generally, the study has significant contributions to make and appears to be well conducted. It is worthy of publication and would be of interest to many outside the evolutionary field. Major concerns were with interpretation, length, and small gene set.
Experimental design: 1. Study would be strengthened by including more than 2,000 proteins. As is, it could likely be missing many key interactions. This low number also probably explains the low amount of overlap with the phylogenetic profiling study.

2. Reciprocal Ranks is an interesting idea, but the rationale behind it is speculative. Why not dig and find out why one gene ranks differently by examining their rate values and species representations? If all genes are constrained to the same set of species, does the rank discordance remain?

3. The idea of unidirectional networks is interesting but would need some support of the assertions they are making. Also, see alternative explanations above,

4. Line 303. It's not clear how the authors "sequentially extending branch lengths in different clades", so this analysis is hard to interpret.

5. Line 304. If the authors want the conclusion that rates increase as branch length to be a feature of their analysis, it would be necessary to determine if this is an artifact of phylogenetic methods. Simulate a set of genes under similar divergence times but with a uniform rate of change across branches and see if the same result is obtained.

6. Also, why not show any results for this (Lines 303+)? Plots, tables?
Validity of the findings: 7. Viral pressures are surely not the only thing affecting ACE2's rates. ACE2 is the entry point for SARS-CoV2 but the observed correlations may not be related to the downstream pathologies induced by the virus. These associations are interesting but of less certain functional linkage to pathology which involves inflammation and cell death caused by cell lysis.
ACE2's general role, outside of being exploited by SARS-CoV2, is to regulate blood pressure through hydrolysis of angiotensin II, a vasoconstrictor peptide, into angiotensin, a vasodilator. Perhaps these general roles in modulating circulation are also reasons for the observed ERC correlations, rather than being linked to coronaviruses. Even new general, unappreciated functions of ACE2 could be interesting given its role in maintaining circulatory homeostasis, even if those functions were not related to viral activity.
Relevant to Line 502, "this is very relevant to COVID-19...", and in the framing of the entire study.

8. Relatedly, have coronaviruses being using ACE2 throughout mammalian evolution? That might be a requirement for the ERCs to be relevant to SARS-CoV2 pathology.

9. Could the authors demonstrate that characterized viral-associated proteins show high ERCs with their known interaction partners? To what degree are these accurate? Or outside of viruses, how well do ERC predict interactions for other cell surface proteins?
Before asking the reader to embark on interpreting ACE2's ERCs, it would help to show likelihood of these being correct.

10. Line 314, the authors state that ERC is predictive of direct protein interactions. However, previous studies by (Juan Pazos and Valencia. PNAS 2008) and (Clark Alani and Aquadro. Genome Research 2012) found strong ERC values for non-interacting proteins as well, such as those participating in metabolic pathways, so direct interaction does not seem to be not required.

11. Subnetwork analyses. Are the known functions of these genes significantly enriched within the networks? The worry is that it's always possible to find genes that "make sense" given a list of genes.
Additional comments: 12. Large parts of Section C and C2 contribute relatively little to the manuscript and could be greatly truncated. The main findings were already presented in previous sections, and these repeat many of those points. If a journal needed a shorter format these could be shortened or left out.

13. These sections C and C2 are very descriptive and it is difficult to take away major conclusive points from them.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: NOVEL ACE2 PROTEIN INTERACTIONS RELEVANT TO COVID-19 PREDICTED BY EVOLUTIONARY RATE CORRELATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The experimental design is robustly structured
Validity of the findings: The findings are very important as they suggest possible mechanisms for what is going on in vivo and suggested ideas for other interested people to investigate the coevolved proteins in practical namely the cytokine signaling pathway.

The authors pushed the idea of their article using different bioinformatics approaches to make robust conclusion with the support of what is published about proteins implicated in COVID-19 pathogenesis. So the quality of data is very convincing.
Additional comments: I think the following conclusion is based on weak evidences
“Thus, connections of FAM3D to F13B, possibly through 541 physical binding as implied by their high reciprocal ranks, could be important in modulating the 542 dynamics of clot formation”

I think the following conclusion is not clear needs to be rephrased:
“their protein 691 evolutionary correlations suggest that ACE2 may be part of a signaling network (perhaps 692 modulated through XCR1) that is contributory”

It will be great if you add a clarification regarding  the correlation of ACE2 or any mediator with the plasma cell differentiation and antibody production such as Blimp-1, Predm1m  and PAX5 as a try to understand the vaccination efficiency.

Do you have an idea about any coevolutionary correlation of ACE2  to IRF4 and IRF8? if you can make a clear conclusion about them please include it.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: NOVEL ACE2 PROTEIN INTERACTIONS RELEVANT TO COVID-19 PREDICTED BY EVOLUTIONARY RATE CORRELATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made clear the scope of their study in their responses. The analysis was not expanded to more genes, which in the end limits its impact. However, the results they provide based on analysis of 2000 genes are useful and could indicate new functional interactions of ACE2 with previously unexpected proteins.
Experimental design: I appreciate the textual edits and clarifications. It was also noted that the authors performed a new analysis on a gene pair to examine how species set representation affects reciprocal ranks.
Validity of the findings: My overall review is that the response was partial and declined to add analyses that would have strengthened the findings. For example, the authors still do not know if the new reciprocal ranking method is more informative than other potential ranking systems. However, the authors’ response argues that their ERC predictions are useful now and should be published as such. I agree and find the results to be sound. I recommend publication.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: NOVEL ACE2 PROTEIN INTERACTIONS RELEVANT TO COVID-19 PREDICTED BY EVOLUTIONARY RATE CORRELATIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: I had no comments
Validity of the findings: Novel and helps to predict the correlation between covid 19 and post infection complications
Additional comments: The authors tackled all the comments that I was mentioned in the first reviewing process


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF EXPRESSION PROFILES OF NEUROPEPTIDES AND THEIR GPCRS IN THE SWIMMING CRAB, PORTUNUS TRITUBERCULATUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Well organized and written manuscript. Figures are good quality and clarity.
Experimental design: Very solid experimental design. Replication of transcriptomes and tissue expression patterns is sufficient for a qualitative analysis (identification of transcripts), which is what is done in this manuscript.
Validity of the findings: The finding are well supported by supplemental data. Phylogenetic analysis identifying homologues is strong. High quality figures support the data.
Additional comments: The manuscript is a basic description of peptides and peptide GPCRs in a single species of decapod crustacean, the  swimming crab, Portunus trituberculatus.

Strengths include strong experimental design, clear writing and figures. Includes sequences and patterns of expression in various neural and other tissues.

The text does not stray far from the empirical descriptions. Discussion is short, without much phylogenetic analysis save identification of homologues.

Two relevant references not cited are the following. They are relevant and should help with the discussion. For example, both papers identify considerably more GPCRs (especially type A GPCRs) in a cumulative five species, compared with the number of GPCRs identified in the swimming crab. While the number of species studied so far is relatively small, might the lower number of transcripts in swimming crabs compared to other species be real, and if so, is it significant? :
Tran, N.M., Mykles, D.L., Elizur, A. et al. Characterization of G-protein coupled receptors from the blackback land crab Gecarcinus lateralis Y organ transcriptome over the molt cycle. BMC Genomics 20, 74 (2019). https://doi.org/10.1186/s12864-018-5363-9
Rump, M.T., Kozma, M.T., Pawar, S.D., Derby, C.D. G protein-coupled receptors as candidates for modulation and activation of the chemical senses in decapod crustaceans. PLoS ONE 16(6), e0252066 (2021). https://doi.org/10.1371/journal.pone.0252066

Also, the authors might consider submitting their databases to CrustyBase, an interactive online database for crustacean transcriptomes ...: https://crustybase.org/ and Hyde, C.J., Fitzgibbon, Q.P., Elizur, A. et al. CrustyBase: an interactive online database for crustacean transcriptomes. BMC Genomics 21, 637 (2020). https://doi.org/10.1186/s12864-020-07063-2
Minor comments:
In all the figures, the inset (not the legend) misspells two words: “singal” (should be “signal”) and “cleveage” (should be “cleavage”)
5. Confidential notes to the editor


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF EXPRESSION PROFILES OF NEUROPEPTIDES AND THEIR GPCRS IN THE SWIMMING CRAB, PORTUNUS TRITUBERCULATUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is also well written. Clear and relatively professionl English used throughout.
Experimental design: This paper reports on the identification and characterization of expression profiles of neuropeptides and their GPCRs in the swimming crab, Portunus trituberculatus. In general, the work presented in this paper is relatively large, and has been performed to relatively good standard.
Validity of the findings: To identify the neuropeptides and their GPCRs may help to understand some physiology and behavior, such as gonad maturation and molting of decapod crustaceans.
Additional comments: 1. In this paper, the author did not verify the results of the transcriptome assembly. Because there are some errors existed in sequences of genes obtained by the Next Generation Sequencing (NGS), it may be better and necessary to detect the accurate sequences of the screened neuropeptides and their GPCRs using normal PCR and sequencing.
2. L52: Which six species of GPCRs have been reported? Please list.
3. The format of the references are not consistent, such as the abbreviation of the name of the journals.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF EXPRESSION PROFILES OF NEUROPEPTIDES AND THEIR GPCRS IN THE SWIMMING CRAB, PORTUNUS TRITUBERCULATUS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript has been written in good, unambiguous English (in the main)- I have some comments to make regarding style, which should be helpful.  References are fairly comprehensive, but rather incomplete, and often incorrectly cited, which is a failure of scholarship. Several transcriptomes of portunid crustaceans have now been published, and since Portunus trituberculatus is also a portunid, these MUST be referenced and referred to in the text. In particular the omission of Oliphant et al (2018) BMC Genomics 19:711 is unfortunate, since this gave a reasonably complete neurotranscriptome of a closely related species. The presentation of the Figs. and Tables in the text is fine (a few repetitive typographical errors though (see my comments). Sharing/availability of raw data. I have a real problem here. Obviously the conceptually translated sequences are available, but the raw data is not. The transcriptome reported must be firstly deposited at DDBJ/EMBL/GenBank. The raw read sequence files should be archived in the NCBI SRA archive under a Bioproject number. Finally, it would be most useful if Trinotate annotation reports were included in some supplementary files- others may want to mine these for nucleotide as well as conceptually translated sequences. The results are self-contained and relevant, but there is a problem with the descriptions given, which often include references (and not always the most appropriate ones!). As the results section stands, it has a rather unusual structure- whilst being inevitably descriptive, there is also an element of discussion.  I would strongly suggest that the authors completely rewrite this section as a “Results and Discussion” section. This would be a real improvement, and make the manuscript much more readable.
Experimental design: The research question is relevant, and reasonably well defined. The methods are described in sufficient detail, and the pipelines used to extract ands assemble transcriptomes are now pretty well defined in quite a number of papers. I do, however, have a problem with the PCR experiments (results on Figs 10, 11). The authors have performed many PCRs on cDNA extracted from a good variety of tissues. However, there are no RT controls! These are essential. It is  extremely difficult to remove gDNA contamination, and certainly just doing a Trizol extraction and DNase treatment will never be sufficient- normally further purification to extract mRNA is needed, and  since touchdown PCR was used (35 cycles), I wouldn’t be at all surprised to find that some of the fainter bands might well represent gDNA contamination. Also please show the   DNA marker size (ladder) on the figures!
Validity of the findings: The data provided, findings, and the analysis seem reasonable in the main. It is good to see that there is pretty good congruence between the neuropeptide families, diversity within transcripts (where a single transcript encodes several different, but related neuropeptides), putative receptor identities in the published decapod (and particularly crab) transcriptomes. I was surprised to see that there were no eclosion hormone-like transcripts. There are two of these in decapod crustaceans, one  is only expressed in the eyestalk, and is undoubtedly a neurohormone, whilst the other is expressed just about everywhere. Similarly, I was surprised to see that no AST CC transcripts were found, or the four PDHs which seem to be found in most crabs.  The authors refer to one peptide as “ETH”.  This seems unlikely, since it is found in nervous tissues, whereas the authentic ETH of insects is found in non-neural tissues, ie the Inka cells.. There is, as yet, no experimental evidence to suggest that this peptide has any role in molting, except that its expression changes dramatically over the molt cycle. Since it has a PRL-amide motif, and seems to be identical in all decapods (unlike the situation in insects where a wide variety of structures are found), it would be more prudent to call it “Carcikinin” for the time being!
Additional comments: Some suggestions and things that caught my eye.

Line 17. Tone down. I suggest “…their receptors that might be involved in regulation of reproductive processes….”
Line 36 include molt-inhibiting hormone.
Line 37 and throughout. Always include a space before the reference brackets.
Line 42. The Tang et al reference refers to the draft genome of P. trituberculatus, so should go in the preceding line. Also, end this sentence with …. “are unknown”.
Line 50…do not share as high an overall…
Line 52. I think this needs a separate emphasis. Many putative neuropeptide receptors have been tentatively identified on the basis of homology, but only a handful have been functionally deorphaned (using heterologous cell-based reporter assays) in crustaceans. Here reference should be made to these.
Line 59. Please include the Carcinus maenas transcriptome reference (Oliphant et al, 2018).
Line 68 … no studies have reported the …
Line 72-74. I think this is a rather sweeping statement. Tone it down.
Line 79 Molt stage should be given.
Line 82 Use the correct term. Ventral ganglion, not thoracic ganglion (the ventral ganglion is fused sub-oesophageal, thoracic and abdominal ganglia in crabs).
Line 86… was removed using…
Line 100 Peptides
Line 101.  The reader should be able to easily understand this: one transcriptome was assembled from all the ganglia combined.
Line 121 (and elsewhere). The sequences obtained were from transcripts, not gene sequences, so avoid using this word- I do appreciate that many do though, but I don’t like it.
Line 134. The ovarian stage (s) (i.e.1-4) should be mentioned here.
Line 159… including cerebral, eyestalk and ventral ganglia.
Results section. The structure of this (as mentioned earlier) is rather unusual- I strongly suggest that the results section is combined with the discussion. This would be much easier to do, and is generally used in transcriptome papers, to keep the style concise, yet informative. Additionally, it is vital that references include those where the hormone was first discovered in crustaceans (for example, bursicon, DH31, MIH, CHH splice variants etc).
Line 270. As mentioned earlier, please avoid calling this peptide ETH, its role in molt control  in crustaceans hasn’t yet been established.
Line 329…. other crustaceans. Also line 373. And give references here.
Line 377… completely conserved
Line 410. I see what is meant here, but without MS analysis  it can’t be said with certainty whether these conceptually  translated peptides are sulfated
Line 426 P. clarkii. Also please be more inclusive- include other transcriptomes with Trissin annotated.
Line 438 Clumsy sentence.
Lines 479-481. This doesn’t make sense!
Line 493 delete insect…. Present in related crustaceans, such as…
Line 531. This might suggest…. Again, tone down.
Line 533…. And showed similar…
Line 553. Relaxin in a peptide that is included in the gonadulin peptide family. So far, the roles of gonadulins in insect reproduction remain a little obscure. Veenstra (2020) certainly did not implicate insulin related peptides in crustacean reproduction! Please be more accurate here, otherwise these sweeping statements will mislead those not familiar with the field.
Line 560 onwards.  Please use the correct refs here. The Oliphant et al (2018) paper identified three putative PDHRs in C. maenas (and four PDHs) Subsequently only two of these putative receptors turned out to be functional, by deorphaning (Alexander et al, 2019).
Line 571. The Vasopressin (or more correctly inotocin receptor) wasn’t identified in the Lin et al 2020 paper. Again, please be more accurate.
Line 573-575. This sentence is meaningless.
Please correct all figures… Signal peptide,  Cleavage site


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF EXPRESSION PROFILES OF NEUROPEPTIDES AND THEIR GPCRS IN THE SWIMMING CRAB, PORTUNUS TRITUBERCULATUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HIGH-RESOLUTION PREDICTION OF AMERICAN RED SQUIRREL IN INTERIOR ALASKA: A ROLE MODEL FOR CONSERVATION USING OPEN ACCESS DATA, MACHINE LEARNING, GIS AND LIDAR
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
In general, the writing style is clear. However, there are several inaccuracies in the use of the English language throughout the manuscript which make it difficult to understand sometimes. I suggest having the manuscript proofread by someone with a high level of skill in written English.

Literature references, sufficient field background/context provided.
I suggest the authors include in the Introduction some background on previous studies that have used LiDAR and GIS for assessing squirrel habitat preferences (please see #1 y #2 in the General comments section)
The introduction should also include some background on the use of machine learning (ideally for similar studies) and provide a justification for the use of this tool in this project (i.e. what are the advantages of using machine learning for this study?)

The manuscript is well structured and conforms to PeerJ standards.
In general, figures are relevant, high quality and well labelled & described. Figure 1 should include a map of the study area in the context of Alaska (for international readers) Figures 13 and 14 can be removed as they provide little or no information and are not visually appealing

Some GIS files were supplied. However, only raster files can be loaded into QGIS. Vector files (i.e. shp) could not be loaded as only part of the file was provided. A shapefile consists of several files, of which .shp, .shx and .dbf are mandatory. These should be located in the same folder or in a zipped container. Furthermore, apart from the file name, there is no way to know what each file is. The folder should contain a metadata file (i.e. readme.txt) containing a description of each of the files provided
Experimental design: I consider the original primary research to be within the scope of the journal.
The research question is relatively well defined but it is not clearly stated how the research fills an identified knowledge gap. Thus, it is difficult to judge whether or not the research question is relevant & meaningful. Authors should clearly state in the introduction what the research question is and why this is important (i.e. what’s the knowledge gap it fills). Research aim and objectives should also be clearly stated at the end of the introduction and then discussed (in the Discussion) in the context of the study results and findings.

The Methods are well described and all procedures seem to have been performed in a rigorous way. However, the accuracy of the LiDAR derived variables (tree´s height and canopy density mainly) has not even been mentioned or discussed in the manuscript. Is this information available? If not, can the authors explain how this issue (i.e. potential inaccuracies of LiDAR derived variables) could affect the results of the study? There are also a few more issues the authors should address (please see the notes and comments in the PDF file)
Validity of the findings: Although the study findings are very interesting and well explained they should be linked -in the Discussion- with the research question and with the knowledge gap this research question attempts to fill. Study findings should also be compared with other similar studies (please see the notes and comments in the PDF file)
Additional comments: Important: I have attached an annotated PDF where I have highlighted parts of the manuscript- each highlighted part has an annotation. Please refer to the Editor if you are unable to see the annotation.

The study is interesting and the authors have done a lot a good work. However, in addition to all the comments already provided in the previous sections and in the annotated attached PDF file, authors should do a thorough search of current literature. This would greatly improve your work by providing support to some of the statements in the manuscript (please see annotated PDF) and also it would also help to better identify the contribution of your research to the exiting knowledge.
I am providing some examples of literature you could include in your article:

#1 S.Flaherty, P.Lurz and G. Patenaude, “Use of LiDAR in the conservation management of the endangered red squirrel (Sciurus vulgaris L.)” journal of Applied Remote Sensing , Vol. 8, 2014
#2 R. Nelson, C. Keller, and M. Ratnaswamy, “Locating and estimating the extent of Delmarva fox squirrel habitat using an airborne LiDAR profiler,” Remote Sens. Environ. 96(34), 292–301 (2005)
#3 Elkins, Eric & Tyers, Daniel & Frisina, Michael & Rossi, Joao & Sowell, Bok. (2018). Red Squirrel (Tamiasciurus hudsonicus) Midden Site Selection and Conifer Species Composition. Environmental Management and Sustainable Development. 7. 15. 10.5296/emsd.v7i2.12674.
#4 Jennifer K. Frey – 2003 - Initiation of Red Squirrel (Tamiasciurus hudsonicus) Monitoring on Carson National Forest, New Mexico, USDA Forest Service (Report) https://www.fs.usda.gov/Internet/FSE_DOCUMENTS/fsbdev7_011893.pdf
#5 Selecting pseudo-absences for species distribution models: how, where and how many? (2012)  by Morgane Barbet-Massin et al
#6 S. Flaherty et al., The impact of forest-stand structure on red squirrel habitat use, Forestry:Int. J. For. Res. 85(3), 437444 (2012)


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HIGH-RESOLUTION PREDICTION OF AMERICAN RED SQUIRREL IN INTERIOR ALASKA: A ROLE MODEL FOR CONSERVATION USING OPEN ACCESS DATA, MACHINE LEARNING, GIS AND LIDAR
Review round: 1
Reviewer: 2

Output:
Basic reporting: I have provided all comments below
Experimental design: I have provided all comments below
Validity of the findings: I have provided all comments below
Additional comments: PEERJ-47121

In their manuscript titled ”High-resolution prediction of American red squirrel in Interior Alaska: A role model for conservation using open access data machine learning, GIS, and LIDAR” the authors developed a methodology for assessing land-use by red squirrels based on midden surveys and incorporating multiple ecological and anthropogenic variables associated with urban environments. The concept is of interest and I don’t have any substantive comments regarding the detailed methods or results. However, this manuscript was difficult to read due to the writing style, and a wealth of presumptive statements about the state of research on this species and small mammals in general. It was not evident to me that the authors actually know the existing literature on squirrel monitoring and management. I hope that this kind of methodological platform becomes better developed and employed for more effective management but the manuscript as it is needs significant adjustment. I have provided some associated comments below.

L22 - adaptation is an evolutionary process

L27 - please rewrite this more scientifically?  The sentence “Not much small mammal research is carried out.” is anomalous, and simply not true. This needs to be put into context. Even considering only red squirrels, there is an abundance of literature and published reports on red squirrel midden survey techniques (see work by Frey, JK) as well as surveys of abundance and characteristics of middens related to squirrel densities, age structure, associated communities, 20+ years of red squirrel research including aspects of midden dynamics in Kluane, and so on.

L50 - either the authors should remove “(excluding Hawaii)” or preferably should be more explicit about where in the US red squirrels occur. There are many areas of North America besides Hawaii where red squirrels do not occur. Also, it should be noted that although the TSN for red squirrels has not yet been updated, the taxonomy for these squirrels has changed. Both Steiner and Huettmann (2021) and the current authors have failed to recognize the updated systematic relationships among North American red squirrels. See for example Illustrated Checklist of the Mammals of the World (C. J. Burgin, D. E. Wilson, R. A. Mittermeier, A. B. Rylands, T. E. Lacher, and W. Sechrest, eds.). Lynx Ediciones, Barcelona.

L51 - again, this is a myopic statement. Red squirrels are an indicator species chosen by the USDA Forest Service and for which extensive research and management has been conducted.

L52 - macro-ecological niche predictions based on WorldClim datasets were conducted by Hope et al. 2016 (MPE).

L55 - red squirrels can occur at elevations exceeding 3500m in some areas of their range. Authors must be more specific.

L62 - again, adaptation strictly adheres to selective evolutionary processes. plasticity would be more appropriate

L104-110 - again, I do not dispute that small mammals deserve much greater emphasis for conservation and management, but this sweeping broad-brush series of statements is not a rigorous reflection of the existing small mammal research. Small mammals have been the focal point of numerous public and management perspectives, sometimes leading to extended legal debate over wildlife management practices in an anthropogenic world (see work on Zapus in Colorado, prairie dogs, water shrews in Canada, and many other examples). I would think a better approach to this would be for the authors to actually assess the existing literature and summarize some of the prominent cases where small mammals have been an important consideration

L115 - citation is not consistent in text with references cited.

L128 - what does midden cruising survey mean?

L130 - who is RA?

L141 - Betula papyrifera

L152 - need to define old growth

L162 - most recent taxonomic revisions from Hope et al. 2016. Recognized by Natureserv and Illustrated Checklist of the Mammals of the World (C. J. Burgin, D. E. Wilson, R. A. Mittermeier, A. B. Rylands, T. E. Lacher, and W. Sechrest, eds.). Lynx Ediciones, Barcelona.

L163 - correct taxonomy and unitalicize “and” P. glauca and P. mariana (already mentioned previously)

L168 - Koprowski et al have published extensively on midden counts and characteristics. Density estimates of squirrels have been extensively covered in the literature by Boutin, Krebs, and others from the long term Kluane site.

L184-188 - this section does not provide any information on how the authors actually estimated ecological niche.

L202 - mittens?

L356 - refer to relevant figure

L367 - correct text for reference

L370 - explain rank of app.

L389  and onwards - it was not clear that any of these predictors were discussed in the methods section

L411 - rephrase these sentences and remove all encompassing parentheses

L431 - the European red squirrel is not closely related.

L441-443 - This set of statements is presumptive. Maybe it’s because trees nearer the lake are not growing on permafrost and therefore are more productive for cones..  There are many reasons why this observation might be the case, but jumping to the conclusion that correlation is causation is not appropriate. Also, given that red squirrels prefer high density canopy, again it is not surprising that they do not build middens near to trails where there is no canopy, and this may have no direct relationship to the presence of humans.

L475 - be consistent throughout the manuscript using decimal points or commas. Preferably points.

L480 - This sentence is unclear, throughout the manuscript refrain from using casual abbreviations for general words

L487 - So, this actually indicates that squirrel densities benefit from human proximity?  This is counter to the central argument brought forth by the authors.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HIGH-RESOLUTION PREDICTION OF AMERICAN RED SQUIRREL IN INTERIOR ALASKA: A ROLE MODEL FOR CONSERVATION USING OPEN ACCESS DATA, MACHINE LEARNING, GIS AND LIDAR
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HIGH-RESOLUTION PREDICTION OF AMERICAN RED SQUIRREL IN INTERIOR ALASKA: A ROLE MODEL FOR CONSERVATION USING OPEN ACCESS DATA, MACHINE LEARNING, GIS AND LIDAR
Review round: 2
Reviewer: 2

Output:
Basic reporting: Good. Seems like some of the figures could be moved to a supplement.
Experimental design: Good
Validity of the findings: Good
Additional comments: PEERJ-47121-v1

In their study “High-resolution prediction of American red squirrel in Interior Alaska: A role models for conservation using open access data, machine learning, GIS and LIDAR” the authors present a new combined predictive framework for assessing distribution and abundance of red squirrels in Alaska as related to human occupation. Fundamentally, this study provides a robust method towards the authors goals, and I appreciate that they addressed adequately all of the previous reviewer comments.

The major issues I still have with this article is the layout, sentence structure and content of the abstract and introduction which seem to have had less revision that the rest of the manuscript, maintaining some presumptive and broad brush statements as outlined by minor comments below. Additionally, the authors seek to provide a standardized applied methodology for global adoption by the scientific community, however, this set of methods relies heavily on identification and quantification of red squirrel middens. As such, I’m not sure how applicable these methods are for other species. I feel like they could be, but the authors have made no attempt to extrapolate how they might be employed for other species, to “be effective for being used by a global audience” as stated in the abstract. Red squirrels are only located in North America. So, it would I think be highly beneficial if the authors provided some examples of other species that produced diagnosable food caches for instance that may be assessed in a similar manner, or some other structural element of other species’ life histories that may lend to these methods being effective for the broader scientific community.

Minor comments:

L21 - Again, unless the authors are explicitly assessing evolutionary adaptation, this is not the most rigorous term to use. Perhaps “interacting” or “habituating”

L22 - why not state that squirrels build middens of cone debris to cache cone food resources and situate these middens central to their territories (if that is what the authors mean). This would provide more relevant information.

L47 - I do not think there is any direct evidence that avoidance is due to fear. In general the authors need to more carefully phrase their sentences to be scientifically objective and avoid making statements that weren’t explicitly tested or have been cited from the primary literature. Just state avoidance of human disturbance.

L74 - ubiquitous within North American boreal forests, or “within Alaskan forests”. Please check that all sentences are accurate. Given this is the start of a new paragraph, it requires more detail.

L79-81 - this statement should be cited. I am not sure how the authors can state this. If there is no literature published in journals, there may well still be government reports.

L96 - sentence structure.

L102 - sentence doesn’t make sense.

L374=376 - So, how do the authors discriminate between high density understory and high disturbance? It seems like these are opposite observations leading to the same predictive outcome, which would suggest ambiguity for predicting cause and effect of squirrel distributions. If “low trees” is the same as “low canopy height”, then the authors should use only one consistently throughout.

L401 - presumably tree height is also correlated to the maturity of trees, where no cones would be produced by trees under a certain size? As in, it may not be height per se, but age of tree.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GLUCOSYLCERAMIDE SYNTHASE REGULATES HEPATOCYTE REPAIR AFTER CONCANAVALIN A-INDUCED IMMUNE-MEDIATED LIVER INJURY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript “Glucosylceramide synthase regulates hepatocyte repair after concanavalin A-induced immune-mediated liver injury” by Jian Gan and collaborators was critically reviewed.

The manuscript is aimed to figure out the relevance of GCS in Con A-induced immune-mediated liver injury in mice.

Although the work presents interesting data, some concerns should be addressed.
Experimental design: Some control group is required, please see below.
Validity of the findings: Please see below.
Additional comments: The manuscript “Glucosylceramide synthase regulates hepatocyte repair after concanavalin A-induced immune-mediated liver injury” by Jian Gan and collaborators was critically reviewed.

The manuscript is aimed to figure out the relevance of GCS in Con A-induced immune-mediated liver injury in mice.

Although the work presents interesting data, some concerns should be addressed.

The authors must provide convincing data demonstrating that animals under the GCS siRNA transfection indeed decreased the GCS protein content and activity; GCS mRNA quantification is not enough.
A complete technical description of the procedure must be provided, including the siRNA sequence and the method for animal assimilation, particularly in the liver.
How was the siRNA transfected into liver cells? Please explain this aspect clearly in the article.

Some reporter gene or siRNA control should be used to demonstrate the efficiency of the transfection. Even more, it is mandatory to include an animal group subjected to a mock siRNA.
Figure 1D must include the reference (standard ruler marker to identify the target band); otherwise, the experiment has no sense.

Because the entire work is based on this animal treated with the siRNA, all the results are irrelevant until these controls and evidence that the experiment using siRNAs was conducted efficiently.

Findings in histology (H&E staining) must be indicated; for example, use arrows to show the necrotic areas. It would be beneficial to suggest inflammatory cell infiltration in the tissue to support the immune system participation. Please provide some greater magnification to observe changes in morphology. Please indicate some reference, for example central vein (CV) or Portal vein (PV).

Figure 3 depicts GCS determination by IF, but this should be supported by western blot of the GCS. The images are not clear some doubts remain because it could be confused by background fluorescence.

Apoptosis should be verified by an alternative method, for example Caspase 3 activity.

Western blot in Figure 6C must include at least two samples of each animal group.
Animal group label as control should be referred to as Not treated (NT) because other control groups are in the study.