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: SHORT AND LONG-TERM EFFECTS OF A SYNBIOTIC ON CLINICAL SIGNS, THE FECAL MICROBIOME, AND METABOLOMIC PROFILES IN HEALTHY RESEARCH CATS RECEIVING CLINDAMYCIN: A RANDOMIZED, CONTROLLED TRIAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1) Introduction and rationale: Although the rationale can be assumed from the abstract and introduction, the authors do not present it in a linear, focused manner. The authors start out their intro by providing the rationale that antibiotic-associated GI signs (AAGS) is prevalent, and that this is a reason for non-compliance, and immediately bring up the use of probiotics without providing any kind of data that supports the efficacy of probiotics. Example, (lines 58-59): What does “the efficacy of probiotics in preventing AAGS does not appear to hinge on the antibiotic used” mean? They use this as a rationale to support the hypothesis that “dysbiosis” is a contributor to AAGS by altering the metabolome, which is not really a logical line to that rationale. This is all in the first paragraph. The next paragraph discusses antibiotic resistance following antibiotic exposure in detail, which is not the context of the manuscript at hand. Although the next couple paragraphs link this concept back to compliance, it is not immediately clear what the relevance of doing metabolomics in this study is, to cats or humans. I would suggest re-focusing the introduction to provide a clear line of thinking that lays out the rationale for doing metabolomics on antibiotic-treated cats, and how this relates to solving issues with AAGS. What is the purpose of doing metabolomics if you can just compare the clinical results of synbiotic/antibiotic combinations? I would also add some of these concepts and rationale, particularly in relation to synbiotic use, in the ‘Background’ section of the Abstract—as it currently stands, the reader is left wondering why synbiotics were used, and how this relates to metabolomics.
Experimental design: 1) Some of the methods need clarification so that these results can be compared to work of others. The commercial kit used to extract the DNA needs to be directly listed in the manuscript and not just cited. The synbiotic used needs to be listed directly, not just referenced (what substance was used?). What is MR DNA? What similarity cutoff was used to cluster the OTUs? From the way the methods are written, it is unclear if sequences were directly binned taxonomically (which would be classified using a database, and does not require OTU clustering) or if the sequences were actually clustered into OTUs using distance, then classified to a database (using a representative sequence from that OTU). Also, where were the raw sequences deposited? What database was used to identify the untargeted metabolomics profiles? Some detail should be added here. Finally, even though the general experimental timeline is given, there is no mention of how many samples were collected during the defined time periods in methods or throughout the manuscript. One suggestion is to add a “figure 1” that illustrates the study design and indicates sampling. More importantly, what “synbiotic” was used for the study? The authors never discuss relevancy of the synbiotic, and which microbes are capable of utilizing this product, or how it is purported to work.

2) Results: Some of the described data needs some clarification and more rigorous analysis.
--According to Table 1, one of the largest differences in the clinical scores of cats with or without synbiotic treatment at baseline is vomiting in synbiotic-treated cats,which then decreases by week 3, at which point placebo-treated cats had less. How exactly did the authors determine that “there was no difference between group”? In looking at the data, differences between the groups at each time point seem significantly different (example, 0+/-0 compared to 1.8+/-5.1 at baseline).
--What is the point of Figure 1? Although it does rationalize subsampling to a level of 35,000 sequences per sample, if the point is to compare differences between the groups, there are better ways of making this point, such as a diversity metric, which the authors reference in the text (line 234) but do not actually show in Figure 1. Instead of having a separate table and a rarefaction plot, Figure 1 could show Shannon diversity, goods coverage, or chao1 (or all) to make this point more efficiently.
--(line 238): The authors state that beta diversity was significantly altered, but then state that there was no difference between groups? Since the data is grouped both by a) timepoints and b) synbiotic vs. placebo groups throughout the manuscript, the authors should specify which groups (a or b) are being referenced to when referencing “groups”.
--(line 238 and Table 2): What is a “dysbiosis index”? In addition to citing this, the authors should explain what this is, and what it means. What are the superscript a’s and b’s in Table 2?
--Metabolomics results: How was change over time in the assayed compounds done (line 250)? The statistical tests/methods should be called out in the text. While organizing the samples by group makes the changes in metabolites easy to visualize, was there any unsupervised clustering that revealed shared patterns between the groups (time periods compared to each other, or between the treatment groups during antibiotics)? Similarly, the information conveyed by both Figure 4 (heatmap) and Figure 5 (PCoA from metabolites) basically convey the same information.
--The authors call out 1 metabolite out of many differences (N-acetylglutamate) that differs between placebo and synbiotic groups during antibiotics. While this molecule may indeed be relevant to the biology of the treatment, was this the only one? In the current way that the analysis and text is organized, it is difficult to distinguish which “groups” are being compared to each other (particularly without referencing what type of statistical method was used), and what cutoffs were employed to identify the specific metabolites called out. Part of this problem is using group-wise vs. longitudinal analyses interchangeably. This is a rich dataset, and some more attention could improve the analysis and interpretation of the data. One suggestion could include actually graphing significantly altered metabolites over time per cat, colored by treatment group (synbiotic vs. placebo), over time. This would demonstrate significant changes in context with the study design within the different groups, as well as be appropriate for longitudinal data visualization.
--Related to the above point, the authors also call out specific metabolites (cholesterol, CDCA) in the text as being unchanged. What is the relevance of calling out these particular metabolites? Another way to demonstrate context to the rationale would be to mention why these are metabolites of interest.
Validity of the findings: Discussion and conclusions:
--(line 298): Does the data suggest that “stimulation of the motilin receptor had minimal impact on the incidence of vomiting”? What is the motilin receptor, and how was it exactly stimulated in this study?
--The authors state that abx administration was associated with marked changes in the fecal microbiome and metabolome that persist at followup (line 302; 330-333), but figures 1 and 2 would argue otherwise. The authors also state that resolution was found at followup later on (lines 344-359). Any changes observed in the metabolome during followup sampling suggest that the metabolomics profiles are also markedly differnet from baseline samples, not just during antibiotics, suggesting a major shift over time in general independent of treatments.
--(line 307): The authors do not define a “dysbiotic state”—what would a normal cat gut microbiota community look like? What does a dysbiotic one look like? How does the authors’ data compare to other cited data in terms of community members identified, healthy or otherwise? These types of broad statements would be greatly strengthened by having some detailed definitions or comparisons.
--(line 310-313): What does the statement “AAGS can be delayed in onset by as much as 8 weeks with recurrence of antibiotic-associated C. diff infection in up to 50% of individuals” mean? C. diff infection is a very specific type of antibiotic-associated diarrheal case, and if there is a recurrence, you are not experiencing AAGS, you are experiencing recurrent CDI, which is an entirely different disease definition.
--(line 317-320): This section is unclear. Why would “efficacy of probiotics in preventing AAGS suggest that disruptive effects of antibiotics on the microbiome are a primary contributor to AAGS”? The data on probiotic efficacy against anything are complicated and in some cases controversial. While the prospect of probiotics is of interest, the authors should be careful and detailed in their statements concerning probiotics. On a similar note, the authors should be careful in over-interpreting published work in their discussion points about AAGS, “dysbiotic” microbiota, and pathogen colonization/infection (lines 324-333).
--All of the discussion in lines 344-596 (note: this is 8 pages) is either out of the scope of the current manuscript, listings of different types of microbiota profiles found in various animals, or actual data that was never referenced in the results (lines 334-343).  The discussion topics range from data modeling human CDI (a very different clinical definition), to detailed discussion about particular metabolite groups, to antibiotic resistance found in probiotics. The authors need to put their data into the context of their rationale, original aims, and the relevant work, preferably in 2, 3 pages maximum.
--(lines 599-609): One of the concluding statements by the authors is that “antibiotic-induced changes in bacterial diversity and taxonomic bundances closely mirror those found in people”, yet this is not what is obvious by the discussion points. It is also not clear what the actual conclusions are, other than re-stating the results. Why exactly does this warrant “whether persistent antibiotic-induced dysbiosis is a risk factor for AAGS secondary to repeated antibiotic exposure”? There are many themes present throughout the manuscript (preventing AAGS with synbiotics, using cats as models of human interventions, identifying metabolic biomarkers from metabolomics data that might indicate microbiota-induced GI infections). My suggestion would be to focus on one or two themes/questions out there (provide rationale initially), assess what can be concluded from the study at hand, and how this changes or advances our ability to treat the stated problem. This would strengthen the manuscript as a whole.
Additional comments: General Comments
-	In the Abstract results section, there needs to be some context. For instance, what does “normalization by days 631-633” mean? Day relative to what—antibiotics? Synbiotic treatment? Baseline?
-	The manuscript uses a combination of citation with a superscript letter, and full citations (author and year) throughout the paper. In most cases, footnotes should be citations, or the facility can just be listed as is. Whatever the correct format is for PeerJ, consistency is necessary.
-	(in methods and lines 213-214) The authors can remove mention of cats that were removed from study, and just concentrate on the screening for the 20 cats actually included, for simplicity.
-	Pertaining to the ethics statement, there should be a statement about welfare and enrichment of the cats in the facility.
-	(lines 219-220): should specify that none of the cats had abnormal clinical scores at RECOVERY time (if this is the case).
-	Statistical tests applied to data should be listed in their respective text and legends. This is important.


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: SHORT AND LONG-TERM EFFECTS OF A SYNBIOTIC ON CLINICAL SIGNS, THE FECAL MICROBIOME, AND METABOLOMIC PROFILES IN HEALTHY RESEARCH CATS RECEIVING CLINDAMYCIN: A RANDOMIZED, CONTROLLED TRIAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have responded to the remarks made by the referees is a careful and correct way. I have no further remarks (except for one typo on line 129).
Experimental design: no comment
Validity of the findings: no comment
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: SHORT AND LONG-TERM EFFECTS OF A SYNBIOTIC ON CLINICAL SIGNS, THE FECAL MICROBIOME, AND METABOLOMIC PROFILES IN HEALTHY RESEARCH CATS RECEIVING CLINDAMYCIN: A RANDOMIZED, CONTROLLED TRIAL
Review round: 2
Reviewer: 2

Output:
Basic reporting: Results within the manuscript are improved by adding a figure demonstrating the study design and collating the reported metabolomics data into figures rather than a long table. Overall, the authors are very detailed in their reporting.
Experimental design: The methods have been largely clarified by the authors in this resubmission. In the description of the dysbiosis index, the authors state "the dysbiosis index is a quantitative PCR based assay that quantifies the abundances of 8 major bacterial groups". However, I do not see a description for quantitative PCR in the methods?

The lack of control groups (cats without antibiotics and cats receiving the synbiotic alone) made some of the study conclusions about how probiotics might change the microbiome/metabolome difficult to make, but the authors have now included a statement addressing this in the discussion.
Validity of the findings: The discussion section is still long and in need of refocusing. The authors discuss a lot of concepts in a fair amount of detail. Given the results and analysis presented in the study, I am not sure that each compound needs its own paragraph detailing all the mechanisms/studies that have been published for that compound. One would expect that many metabolites will decrease following antibiotics, so it is unclear what a decrease in a specific group of metabolites means. Having such detail for each metabolite group is highly speculative, and the message of the study is lost. This section should be condensed to address the impact the authors' results have that pertain to their original study question. The authors also have some results in this section (lines 618-623) that were not presented in the results section.

Minor comment: I am not sure what this statement means (lines 613-615): "Consistent with other reports (Schubert et al. 2014), the return of diversity at a global level was not due to return to baseline abundances for individual OTUs". Additionally, the cited Schubert et al study was not a longitudinal study comparing baseline measurements.
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: INTEGRATED ANALYSIS OF DIFFERENTIALLY EXPRESSED PROFILES AND CONSTRUCTION OF A COMPETING ENDOGENOUS LONG NON-CODING RNA NETWORK IN RENAL CELL CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: This paper may not be considered as statistically sound. Student t-test may not be a suitable method for identifying differentially expressed lncRNAs, miRNAs, and mRNAs.
Additional comments: The authors have identified cancer- specific lncRNAs and constructed a ceRNA network for RCC. A survival analysis related to the RNAs revealed candidate biomarkers for further study in RCC. However, the student t-test used here may be problematic in identifying differentially expressed lncRNAs, miRNAs, and mRNAs. This major issue must be addressed and solved before acceptance.

Major revisions:

1). In the “Statistical analysis” section, the authors used Student’s t test to find differentially expressed lncRNAs, miRNAs, and mRNAs, this approach may be problematic. As we know student t-test only can be apply to those data which follows normal distribution, it cannot be applied to those expression counts since those counts are most likely follow negative-binomial distribution. That’s why some DEG tools, e.g. DESeq and edgeR are more accurate in identifying DEGs in RNA-seq data. In my opinion, even we do not really sure about the distribution of these data, then a non-parametric method, e.g. Wilcoxon Rank-Sum Test will be more suitable and safer to use in this case. The authors need to give solid explanations that why they can use student t-test here, otherwise the differentially expressed lncRNAs, miRNAs, and mRNAs which obtained in this step will be very questionable for the downstream analysis.

2). In lines 120 and 121, could authors explain what’s the reason for choosing these different cutoffs of p-value, FDR, and FC for lncRNAs, miRNAs, and mRNA? I suggest to give people more details in the method section.

Minor revisions:

line 87, lincRNA -> lncRNA

For the titles of subsections of “Materials and methods”, e.g. “Patients and samples”, the space in the front may need to be removed

Line 99, remove “inclusion”


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: INTEGRATED ANALYSIS OF DIFFERENTIALLY EXPRESSED PROFILES AND CONSTRUCTION OF A COMPETING ENDOGENOUS LONG NON-CODING RNA NETWORK IN RENAL CELL CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript from Qianwei Xing, and colleagues used existing sequencing data from TCGA to investigate the differential expression profiles of a cancer-specific lncRNA and ceRNA coexpression network in RCC, and their work would be very useful for elucidate the functions of lncRNAs in RCC.  Overall, I think the authors supplied useful approach and theoretical basis of mechanisms for RCC research. Besides, I think it is a good idea to investigate data on public database and make full use of the existing data.

I have several minor concerns about this paper.
1.	Are the p values in line 118 are adjust p valuse? Is yes, please mark them clearly; if not, please clarify what kind of p value they are.
2.	I think it would be better to add GSEA analysis to this study.
3.	The resolution of figures need to be improved, especial fig2 and fig6.
4.	Paragraph transitions need to improve in the introduction part
5.	I suggest to add reads or counts data used in this paper as a supplemental table.


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: INTEGRATED ANALYSIS OF DIFFERENTIALLY EXPRESSED PROFILES AND CONSTRUCTION OF A COMPETING ENDOGENOUS LONG NON-CODING RNA NETWORK IN RENAL CELL CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am satisfied with the revision. I agree with reviewer 2 about adding GSEA results to this paper to make it more scientific solid. GSEA is a software of gene set enrichment analysis, not a database similar to TCGA. It should be relatively easy to add it to this 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: INTEGRATED ANALYSIS OF DIFFERENTIALLY EXPRESSED PROFILES AND CONSTRUCTION OF A COMPETING ENDOGENOUS LONG NON-CODING RNA NETWORK IN RENAL CELL CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: All the text in figures should be the same font, please check carefully and modify.
The data to show the quality of the libraries used in this paper should be added.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised manuscript is much better than previously one. The introduction and methods were improved and more detail were added. I am satisfied with the replies to all my concerns except one. The reason why I suggested to add raw counts data was to show the quality of the libraries. A very simple table with total reads, mapping rate should be totally fine. Good libraries are very import base for accurate analysis. So I insist to add QC of libraries used in this paper.

After go through carefully of the revised manuscript, I still find some minor problems, such as line 173 "of2,650", a space should be added between "of" and "2,650". One more thing, the  text in figures should be the same font. I suggest author check the manuscript carefully and try your best to reach all the publication standards of Peer J.


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: WHOLE-GENOME AMPLIFICATION IN DOUBLE-DIGEST RADSEQ RESULTS IN ADEQUATE LIBRARIES BUT FEWER SEQUENCED LOCI
Review round: 1
Reviewer: 1

Output:
Basic reporting: good. most of my comments are in the general section. Minor errors or requested clarifications are noted there.
Experimental design: I would like to see a little bit more analysis done, as noted below, but this should not be too big a problem. I hope that the questions about clone fragment filtering can be dealt with.
Validity of the findings: Good. Some comments on the discussion, phrasing of conclusion at end of abstract that I thought was a bit overly strong given what was presented.
Additional comments: Overall, the paper seems reasonable and I would say that results are broadly in agreement with my own experience.

Was there no process carried out in the analysis for the removal of clones? If so, I missed it. This is fairly standard, I think. This is going to be particularly important for low DNA starting material in the context of this study.

A main point is that the number of loci recovered with MDA is not expected to be lower based on Blair et al, but in Shortt et al (which you cited elsewhere) there was a clear redistribution of lengths of the loci recovered with MDA towards shorter length, which directly predicts that the number of loci that would achieve some depth of sequencing would also shift (as you found). This is worth commenting on, and also potentially worth describing the shift in length of loci recovered in your data (including how many sequences are wasted on loci that are too short to included in the analysis). This point is in addition to, and possibly much larger effect than the stochastic shift (cited Sabina and Leamon).

The last sentence of the abstract (“Overall, we find 37…) would be better with some kind of qualifier about the relative development, study design, and utilization costs of “target enrichment” in terms of method development and producing less data. This is especially because the current paper does not include a direct comparison of this other method. I don’t disagree with most of the results, but I am not convinced that I would or should even consider switching to target enrichment.

I am not too fond of the numbered analyses format. Is that by author choice or PeerJ?

On line 94, are the authors implying that they can make libraries regularly off of a few ng of DNA without amplification? I am quite impressed if so. The Andrews et al paper cited mentions 50-100 ng of DNA “can often be implemented”, which seems more reasonable in my own experience.

The discussion on lines 389-397 about potential problems with phylogenetics was not particularly clear to me. I’m not sure that “locus dropout” is the most straightforward term for differences in locus existences between species, as it is more suggestive of low frequency variants in a population. The switch from missing loci in samples to excluding data and that that is a problem with “less redundant” sampling in phylogenetic studies was particularly confusing. What point is being made? It seems clear that there will be fewer loci that can be compared between species than within populations, but still a lot. If a sample didn’t amplify well enough to provide reliable information, it isn’t that useful data is being left out, but rather limited and potentially misleading data. At any rate, please clarify the point.

In the discussion on biased recovery of different loci, (line 402 on), this was mentioned earlier, but I don’t see why the authors don’t look at the bias due to MDA in recovery, rather than speculating that it is a smaller difference than size selection. Further, the fact that thousands of loci align between datasets may trivially mean that they are overlapping, but it does not say clearly how much overlap there is as opposed to wasted sequencing due to non-overlap. This can be assessed directly, and should be.

Line 95: would be better without redundant “useful…use”. Suggest “MDA may enable the use of…”

Line 168: I don’t know what would make 7 “the minimal threshold”, which makes it sound like a necessary boundary, although it is probably a reasonable lower bound. Is this due to the program used to call, or is it based on some accuracy criterion? Please mention in either case.

Line 175: The minimum of 4 samples for a locus seems somewhat random and unmotivated as written. Perhaps you could justify it? I would think that a reasonable criterion would depend greatly on the number of samples per species, and if it is 4 across all the species, it would seem to arbitrarily depend on how close the species are. Please clarify how and why this cutoff was (and should be, for future studies) chosen.

Line 183: “we have counted” makes it sound like you did it once, maybe not related to this paper. Better to use direct past tense, as in, “To test, …we counted…” The recommended change is also the same format as e.g., Line 197, making it consistent.

Line 275: fix “are have” and plural consistency, use of “higher”. For example, “MDA libraries have greater genetic distances among themselves than the gDNA library has…”

Line 277: change “higher number of” to “more”

Line 278: change “have also observed” to “also observed” for tense consistency and readability.

Line 290: change to “loci in common”

Line 311: the first part of the sentence just repeats what was said in the previous two sentences.

On line 311-315, this is a question where the analysis of clonality among the reads would impact interpretation of the results.

On line 331, please go through the manuscript and eradicate the use of “higher number”. The correct term here is “larger numbers” or just “more”, where appropriate. Also, I believe the intended plural usage would make this “these proportions decrease with larger numbers of loci”.

On line 332, “in fact” is a colloquialism that doesn’t belong in a scientific paper.

Change on line 394 to “…should not be…”

On line 440, was this meant to be “on the order of 1 ng”?

Figure 2 is quite hard to interpret. Would it be better if it were two figures? As is, it is quite confusing and doesn’t easily get the point across. An improvement in this figure would be helpful. Figure 3 and 4 are also quite messy.


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: WHOLE-GENOME AMPLIFICATION IN DOUBLE-DIGEST RADSEQ RESULTS IN ADEQUATE LIBRARIES BUT FEWER SEQUENCED LOCI
Review round: 1
Reviewer: 2

Output:
Basic reporting: See attached pdf
Experimental design: See attached pdf
Validity of the findings: See attached pdf
Additional comments: See attached pdf


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: META-ANALYSIS OF TRANSCRIPTOMIC RESPONSES TO BIOTIC AND ABIOTIC STRESS IN TOMATO
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript requires major English language copyediting. The abstract requires major overhaul as with regards to using the English language professionally.

In Figure 5, the font size is too small to read the various organisms listed.

Figure 3 seems to be very crowded and hard to comprehend.

The authors should also gather more information for review of related literature related to this study. There seems to be a number of studies related to this one.
Experimental design: The authors should be clear in the goals of this study. They should also be able to clearly express the novelty and significance of this study in the introduction.
Validity of the findings: It is unclear as to how this study was validated. Did the authors provide training and validation sets to verify the findings? For the training set, how were the results cross-validated?
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: META-ANALYSIS OF TRANSCRIPTOMIC RESPONSES TO BIOTIC AND ABIOTIC STRESS IN TOMATO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing quality is somewhat subpar and the manuscript has grammatical errors that are too numerous to address individually. This hampers the flow of the paper and makes it difficult to understand key results and insights from the study.

Literature references are sufficient and an appropriate background is provided
Experimental design: The research aims are well-defined and are relevant. In this study, the authors use meta-analytic statistical techniques and bioinformatics tools to identify and characterize genes in tomato that are differentially expressed during responses to stress conditions. The analysis is based on publically available microarray tomato gene expression data. The analysis meets the appropriate level of rigor and the results are laid out in sufficient detail.
Validity of the findings: The data is robust and the analytical technique is statistically sound. However, the conclusion is stated in poor English that is difficult to understand. The Conclusion section is minimal, and the authors do not provide a discussion about how their findings connect with the study aims and how their work fits into the larger framework of the related literature. The manuscripts reads too much like a report
Additional comments: The Results section is not well organized and difficult to read. Also, it includes a lot of material that is better suited for the Discussion/Conclusion section. The reviewer suggests summarizing the findings in the Results in 1 or 2 tables, so as to provide a compact summary that readers can easily understand. The reviewer also suggests expanding the Conclusion section to discuss the implications of the findings.

Below are some specific suggestions:

Page 5, Line 95: The sentence that begins with "Our analysis revealed...." is out of place here. Do not state results in the introduction section. This should be moved to the Results section.
Page 6, Lines 98-100: this sentence is unclear, please reword.
Page 6, Line 115: Citations should be given for Fisher's method and maxP
Page 7, Line 120: The authors state the maxP statistic follows a beta distribution with K degrees of freedom, what does K represent? Also, the authors state beta=1 under the null, but don't define what beta is. Is beta a parameter of the beta distribution? If so, this needs to be stated clearly
Page 7 line 121: "A gene to be detected by this method if has small P-values in all studies", please correct the grammatical errors so that it is clear to readers what the procedure is.
Page 7 line 136: the sentence is ended prematurely, "Database" should not be capitalized
Page 8 lines 151-155: Only the results using Fisher's statistical method are stated, but not maxP results
Page 10 lines 197: The sentence beginning with "Forasmuch as,..." should be reworded correctly
Page 16/17 lines 346-348: this statement of this result is critical but it is incorrectly worded. It is difficult to understand what is being stated here


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: META-ANALYSIS OF TRANSCRIPTOMIC RESPONSES TO BIOTIC AND ABIOTIC STRESS IN TOMATO
Review round: 1
Reviewer: 3

Output:
Basic reporting: The work is written in fairly good English, excluding ambiguous interpretation. Terms are used correctly and to the place.

References to the literature are sufficient to understand the relevance of the key problem. However, in my opinion, the authors overly briefly cite the work on meta analysis of expression data. I recommend improving the fragment of 77-82 with sufficient details. Meta transcriptome analysis in various variations is actively used for plants before, for example:

Rest, J. S., Wilkins, O., Yuan, W., Purugganan, M. D., & Gurevitch, J. (2016). Meta-analysis and meta-regression of transcriptomic responses to water stress in Arabidopsis. The Plant Journal, 85 (4), 548-560.

Shaar-Moshe, L., Hübner, S., & Peleg, Z. (2015). Identification of conserved drought-adaptive genes using a cross-species meta-analysis approach. BMC plant biology, 15 (1), 111.

Figures are relevant to the content of the article, but their improvement is necessary to better understand the results. For figures 2 and 5, it would be good to have a higher resolution. However, for Figure 3 this is strictly necessary. Also, figure 3 is desirable to provide a graphic legend of the colors of nodes and links. The perception of the picture is complicated by the presence of a links of different meaning and importance - for example, the "co-expression" for the experimental biologist is treated less unambiguously than the "empirical protein-protein interaction data", for example, and "homology" is of little relevance to the regulation networks. Perhaps it is worth putting out blocks of protein-protein and regulatory interactions into a separate section of the picture.

The work is called "Comprehensive meta-analysis focused on the transcription factors involved in responses to biotic and abiotic stresses in tomato", but in a detailed way it treats the genes associated with a nonspecific response to stress (biotic together with abiotic). In connection with the fact that the authors left out the detailed analysis of all types of stresses  - I recommend changing the title, adding the word "non-specific" or "simultaneously" in it.
Experimental design: In the light of current trends in the use of new technologies to improve crop plants, the work looks profitable and certainly contains interesting data. The urgency of the work does not raise any doubts and the topic fits into the journal field.
In this work, the integration of expression data on various stress factors on tomato plants is carried out. For a tomato, such a large-scale work is carried out for the first time and will certainly be of interest to tomato geneticists.
When analyzing transcriptomic data, quite good statistics were used, which makes it possible to unambiguously understand where the strong influence of individual experiments is observed, and where there is a general trend. Methods are described with sufficient information to be reproducible by another investigator.

It should be noted that the results of the GO enrichment analysis revealed the three most common related components - "cellular process", "metabolic process", "response to stimulus". These results are given even in abstract lines 35 - 36. These terms are very broad and not very constructive, at the same time in Table S6 as well as S7 there is a rich list of reliable terms containing more specific terms. Data on GO associated terms should be discussed in detail, possibly below in conjunction with the analysis of associative network.
The results of the analysis of expression experiments and the analysis of GO terms enrichment are supported by sufficient additional materials, which can not be said about the analysis of associative networks.

At the beginning of the network analysis, the number of genes is postulated as "hub role genes", but it is not explained in detail on what basis such a conclusion is drawn. A natural addition to this work is the analysis of node connectivity in the network by various connections separately and together. These data should be welcomed and discussed. Also optional, but recommended This analysis brings a more detailed understanding of the results.
Validity of the findings: The article operates with competent methods of setting tasks, revealing regularities and relies on statistical reliability. References to all used data, just like the main intermediate results are given in the Appendix. The results concerning the found transcription factors are discussed step by step in great detail, which adds to the article a review value and will certainly be of interest to specialists.
Conclusions are clearly formulated and they are justified by the results of the work done.
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: META-ANALYSIS OF TRANSCRIPTOMIC RESPONSES TO BIOTIC AND ABIOTIC STRESS IN TOMATO
Review round: 2
Reviewer: 1

Output:
Basic reporting: The work is still written in fairly good English, excluding ambiguous interpretation. Terms are used correctly and to the place.

Due to the fact that the editorial board decided to send me an article for re-reviewing, I will make some important remarks.
Here and further I will concentrate my consideration on the recommendations that the authors did not follow. I will try to explain in detail what is required to improve the work to the required level.

(1) Strict

Previous recommendation 1:
« I recommend improving the fragment of 77-82 with sufficient details. Meta transcriptome analysis in various variations is actively used for plants before, for example: Rest … (2016) …. Shaar-Moshe …. (2015)»

The authors added the references I mentioned to the article.

Line 79-81
«… and find out some genes that their products are key molecules in the response to stress (Tseng, Ghosh & Feingold, 2012; Shaar-Moshe et al., 2015)»

However, no additional words were given to the description of the results obtained in these papers. It is not clear for what species it is done and what is found. Why is it important? It is important to emphasize the specific value of the analysis that you carried out. This is not seems clearly in the introduction and does not follow from the conclusion. If the authors of the works of 2012 - 2016 managed to find «key molecules in the response to stress» and « figured out interactions between biotic stresses (BS) and abiotic stresses (AS)», then what did you find new?

This article does have scientific value, but it is not visible to the reader without a clear comparison with previous works.

(2) Optional

Previous recommendation 2:
«…  For figures 2 and 5, it would be good to have a higher resolution. However, for Figure 3 this is strictly necessary…»

The illustrations are improved and now they are perceived much better. However, the resolution of image 3 is extremely low and may appear careless when printing in a magazine. I recommend to correct it.
Experimental design: (3)
Previous recommendation 3:
«… The results of the analysis of expression experiments and the analysis of GO terms enrichment are supported by sufficient additional materials, which can not be said about the analysis of associative networks.
At the beginning of the network analysis, the number of genes is postulated as "hub role genes", but it is not explained in detail on what basis such a conclusion is drawn. A natural addition to this work is the analysis of node connectivity in the network by various connections separately and together. These data should be included and discussed…»

Still in the text:
200- 201
«Some of these gene possess hub roles in DEGs shared by AS and BS networks (Table S7).»
What is the criterion of choosing the Hub genes?
It would be logical to take a network connectivity into account, but the hubs # 1 and #4 are loosely connected to the network nodes (Fig. 3). This part in results (203-218) and discussion needs to be improved by adding a proper network analysis as recommended before.
Validity of the findings: (3) relates to this part too.
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: META-ANALYSIS OF TRANSCRIPTOMIC RESPONSES TO BIOTIC AND ABIOTIC STRESS IN TOMATO
Review round: 3
Reviewer: 1

Output:
Basic reporting: The structure of the new version of the article does not have significant weak points.
Experimental design: Acceptable
Validity of the findings: Acceptable
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: COMPARATIVE CHEMICAL ANALYSIS OF ARMY ANT MANDIBULAR GLAND VOLATILES (FORMICIDAE: DORYLINAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: See comment about literature reference under point 3, validity of the findings, perhaps to be included in the introduction as well.
Experimental design: Materials and methods are not clear on the point of sample size and origin, and thus do not provide sufficient detail and information to replicate:

Despite stating the number of colonies per species from which majors were collected, the number of workers per species included for analyses is not mentioned at this point. The information is eventually provided in Table 2, but still is ambiguous. There is no indication as to how many of the majors that were included in the analysis came from the same and/or different colonies.
As it stands, sample selection seems arbitrary: on which basis were the 5 workers selected from the different colonies for species in which 2 or 4 colonies were sampled?
Could there be differences between colonies (not expected, but it might be)?
Also, it is not clear whether the "colonies" really are different colonies (how was this ascertained?) as samples are reported to have been collected over 2 months and in a restricted area.

Please provide the respective information in the Materials and methods chapter.
Validity of the findings: You do not cite Torgerson and Akre (1970, Interspecific responses to trail and alarm pheromones by New World army ants), who report on interspecific reactions to alarm pheromones in several of the species that are analysed in the present study. Despite the fact that they do not report the chemical composition of mandibular glands, I believe at least the discussion would benefit from including the findings of that study, especially in regard to shared compounds as reported in the present manuscript.
Additional comments: lines 8-9: "little is known about their chemical communication" - this manuscript does not provide any further insight in that direction, either, and thus is somewhat misleading. I would rephrase.

line 130: the reference for R is not correct, please change to "R Core Team, 2017"

line 175: why is it remarkable that a communication signal does not follow phylogeny? If there are reports of chemical communication signals of ants that actually are determined by phylogeny, please provide the relevant references.


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: COMPARATIVE CHEMICAL ANALYSIS OF ARMY ANT MANDIBULAR GLAND VOLATILES (FORMICIDAE: DORYLINAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is good and understandable. But I miss some biological background. As it is now, the study seems to have a very narrow focus – although the evolution of chemical bouquets is a field of high interest to people from many biological fields! The authors should put their study into a broader context and e.g. write more about chemical evolution, and phylogenetic signal, in other taxa and other substance classes. There are some studies on phylogenetic constraints in beetle pheromones and in ant cuticular hydrocarbons, for example; probably there are many studies on the evolution of floral scents or other plant secondary metabolites.
Minor comments.
line 31: remove semicolon after “sensu”
lines 31-32: the two sentences are not really connected, make sure their content is linked better.
in line 33, you mention “ecological selection”, in line 179 you write “natural selection”. I guess the same is meant in both cases; you should use a consistent term to avoid confusion.
line 76: no differences… chemical differences or differences in the behaviour they evoke?
Experimental design: The research question (lines 48-52) is well-defined and relevant. However, it seems narrow as it is presented. The authors should try to make it more general and put it into the context of chemical evolution.
The chemical methods are clearly described and understandable, and the same is largely true for the statistical analysis. However, I suggest that the authors provide the detailed classification for each compound that entered the analysis. This could be easily done with an additional column in Table 1. Furthermore, the authors should describe how the determined which substances significantly contributed to data separation ( in Fig. 1B), and how the arrows were calculated. In fig. 1B, the authors may consider adding the species initials (“Ne”,”Eb”,…) to each dot so that the figure can be interpreted from a black-and-white copy as well. Did the relative abundances of the compounds also enter the analysis?
line 80: Spelco or Supelco?
line 113: what do you mean by “weighted in”?
line 128: I guess you did not randomly rotate the nodes, but had a goal… so I would rather write “… by rotating certain tree nodes…”
line 130: the reference for R is incorrect.
Validity of the findings: The data are robust and statistically sound. The authors write only little about the implications of their findings, this should be expanded. For example, they suggest that ecological effects may shape alarm pheromone blends (lines 180-182) – here they could become more concrete and suggest more precisely which effects may promote pheromone diversification. Also, put the results in the context of other studies on the evolution of chemical blends.
I suggest that the authors re-run the Mantel test with other types of classification (e.g. only chain length, only substance class) to see whether there is phylogenetic signal in one of the chemical traits. It should also be mentioned that the statistical power is low – given an n of 7 species, the Mantel test is less likely to become significant anyway.
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: COMPARISON OF VENTRICULAR TACHYARRHYTHMIA RECURRENCE BETWEEN ISCHEMIC CARDIOMYOPATHY AND DILATED CARDIOMYOPATHY: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English language used needs revision to correct grammatical and spelling errors.

In this single centre prospective observational study, Fang et al investigated the prevalence of ventricular tachycardia and ventricular fibrillation in the patients with ischaemic and non-ischaemic cardiomyopathy with ICD in situ for secondary prevention. They also look at the demographic and echocardiographic predictors of VT/VF.
Experimental design: This is a thought-provoking single-centre prospective observational study. The research questioned are well defined but considering the design of the study and the sample size, the results are reports of observations and would not change the practice in the field.
Validity of the findings: Conclusions are not in line with the nature of an observational study.
Additional comments: Major comments:
1.	Line 60 of the introduction requires clarification. The authors claim that cardiac arrhythmias are associated with a significant rise in sudden cardiac death. I am assuming the authors were meant to say ventricular tachyarrhythmias instead of cardiac arrhythmias. The most common cardiac arrhythmia, atrial fibrillation, is not associated with significant cardiovascular mortality.
2.	Line 65 of the introduction is reemphasising on the beneficiary role of implantable cardioverter-defibrillator as a secondary prevention. In the following sentence, the authors’ emphasis on the improvement in the survival of the patients with heart failure thanks to improved optimal medical therapy makes the reasoning of the authors more supportive of primary prevention ICDs rather than secondary prevention ICD. Definition of primary and secondary prevention ICD needs to be elaborated on in the introduction.
3.	If all these ICDs implanted for secondary prevention, the primary presentation of the patients and their presenting rhythm should be presented as part of the study group characteristics.
4.	The Methods is very brief and needs elaboration. The authors refer to a separate published study for their methods which appears insufficient. The authors needs to expand this section by explaining their methods in more detail.
5.	The diagnostic methods used in the diagnosis of non-ischaemic cardiomyopathy are not described. I would recommend describing the diagnostic methods utilised to rule out ischaemic causes, i.e. coronary angiogram, computed tomography angiogram, myocardial perfusion scan, etc.
6.	The primary end of the study requires explanation. It is not clear what the authors meant by recurrence of VT and ICD shock. Was any non-sustained VT considered a primary endpoint? Were inappropriate ICD shocks excluded? What happened to VTs terminated with anti-tachycardia pacing? Were they considered as the primary endpoint of not?
7.	The follow up duration is better to be presented in years rather than days. Although the mean follow up is very close to the standard deviation, it is still better to be presented in years.
8.	Fig 2 depicted the Kaplan-Meier curve of mortality showing a divergence between ischaemic and non-ischaemic cardiomyopathy starting after 3 years. Considering the small number of the patients with non-ischaemic cardiomyopathy with follow up of 3 years (10 pts) and very small number at 5 years (5 pts), this study is not powered to detect any difference in the mortality rate between ischaemic and non-ischaemic cardiomyopathy and P-value, in this case, would not bear any weight.
9.	Line 204 of discussion, refers to the controversies around ICD implantation in non-ischaemic cardiomyopathy by referring to DANISH study (reference number 8). The authors follow by discussing their findings in risk stratification of VT/VF in this study. It needs to be highlighted that DANISH was a primary prevention ICD trial and cannot be referred to in this study as the current study is a prospective observational study of secondary prevention ICD.

Minor Comments:
1.	There are multiple grammatical and spelling errors that need to be corrected.
2.	Under Echocardiography, line 113, “biplanar” is a typo and should spell biplane.


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: COMPARISON OF VENTRICULAR TACHYARRHYTHMIA RECURRENCE BETWEEN ISCHEMIC CARDIOMYOPATHY AND DILATED CARDIOMYOPATHY: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors have employed professional English through out. The introduction of the article has highlighted the importance of ventricular arrhythmia as a cause for sudden death and the improvement achieved with AICD. They have identified a lacuna in the literature which is, a comparison of predictors for recurrent VT in patients with ischemic as opposed to the dilated cardiomyopathy patients.
Experimental design: Fang et al have performed a retrospective analysis of consecutive cardiomyopathy patients who had an AICD implanted as secondary prevention strategy. They classified the patients into ischemic {ICM} and dilated {DCM} with an attempt to highlight the predictors for recurrent ventricular arrhythmia. All events recorded by the device were interrogated and appropriately adjudicated by the investigators. Various parameters were studied including the medications and echocardiographic variables.
Validity of the findings: Appropriate statistical analysis was used, and ROC curves employed to determine the optimal cut off for the echocardiographic parameters.

There was significant difference between the two groups at baseline. The ischemic cardiomyopathy patients were significantly older with higher prevalence of coronary risk factors as compared to those with dilated cardiomyopathy. Additionally, the use of ACE inhibitor was lower in the former. The DCM group had lower EF, larger LV volumes that resulted in increased prevalence of ventricular arrhythmias as compared to the ICM. However, when patients with similar ejection fraction and LV volume were compared between the groups, there was no significant difference in the prevalence of ventricular arrhythmia.

The conclusions are well stated but not discussed sufficiently.
Additional comments: 1.	Although it is a retrospective study, it sheds light on recurrent ventricular arrhythmia in cardiomyopathy patients. The authors have employed sound statistical methods. However, the discussion of their findings can improve. There is no reasonable explanation given for the findings of increased prevalence of arrhythmia in DCM as compared to the ICM. The fact that when similar ejection fraction and LV volumes were compared, there was no increase in the prevalence of arrhythmia suggest that these are indeed the confounders and probably hearts are more diseased to start with.
2.	When the authors described that the patients with “similar LV volume and mean EF were compared”, they did not explain further the variation that was allowed.
3.	The VT occurrence is similar at first year. It is not clear when they diverge. It was not obvious from the study if the follow up echocardiogram at this time revealed a larger LV and lower EF compared to the baseline.
4.	It is interesting to note that in spite of the greater prevalence of ventricular arrhythmia in DCM patients, the all cause mortality and cardiovascular mortality has not changed. A discussion of this finding was lacking in the manuscript provided.
5.	A few studies have been quoted but not linked appropriately to the study during the discussion.
6.	The spelling of small – line 236 needs to be corrected.


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: COMPARISON OF VENTRICULAR TACHYARRHYTHMIA RECURRENCE BETWEEN ISCHEMIC CARDIOMYOPATHY AND DILATED CARDIOMYOPATHY: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors present a retrospective analysis of predictors for ventricular arrhythmia in patients implanted with a secondary prevention ICD, comparing patients with ischaemic and non-ischaemic cardiomyopathy.The study is single center, inclusion and follow-up are done between 2004-2015. Clinical predictors including cardiac risk factors and medications, echocardiographic parameters including LV function and dimensions were investigated.

The manuscript is well structured and clearly written.
Experimental design: The methods section is very brief referring to the 2016 paper. I would recommend at least mentioning the inclusion criteria, the patient selection (time period of inclusion and follow-up period), the timing and method of data collection including baseline, clinical and echo parameters in relation to follow up (eg was echo from prior to device implantation?) and method of determining endpoint (was there a core-lab analysis of the ICD interrogation to account for inappropriate therapies?)

Statistics
the term 'significant' is used a number of times, it would be better to replace that with 'statistically significant' or omit the term and list the P value for the comparison.
The number of decimal places used should be reevaluated (eg age just use one decimal point, and for the standard deviation don't use a decimal).
Median with confidence interval is more appropriate than mean with SD for non-normal distributed parameters (that should be apparent from the test for normal distribution)
In one of the regression models both LV diameter and LV diameter >158ml were used, please check with your statistician if it is appropriate to include both at the same time in the regression model.
Validity of the findings: See also the comments in 2.

There is continuity in structure regarding research question, study design, results, discussion and conclusions.
Additional comments: 32: consider listing follow-up in months or years instead of days
87: as previously mentioned, the methods should be described in this paper as well
102: which hospital?
103: how was coronary disease ruled out (angiography, CT)?
111: when was echo performed and analyzed (at inclusion to registry prior to device implantation, were all patients on 3 months of maximally tolerated heart failure medication?)
116: what duration of VF/VT was considered significant? How were events evaluated for inappropriate device therapy? Was a core-lab analysis method used with independent reviewers?
119: did all cause mortality only include the listed causes or also others (eg death from any cause)
122: see comments with statistics.
140: please correct 'valve'
146, 149,150,161, 166,174 etc see comments regarding significance
173 see comments with statistics
229: is lack of ACE the reason for increased VT/VF or is it a marker of decreased renal function or increased disease severity and hypotension that do not allow for addition of ACE?

Figures & tables: please consider the comments regarding decimal point, and perhaps use months rather than days.


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: COMPARISON OF VENTRICULAR TACHYARRHYTHMIA RECURRENCE BETWEEN ISCHEMIC CARDIOMYOPATHY AND DILATED CARDIOMYOPATHY: A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: This article is revised and reads better now with less grammatical errors.
Experimental design: This is a retrospective, single centre, and observational study. In limitations of small observational studies, this study is well designed and well conducted. The endpoints are clear and as far as one could ascertain from the article, the follow-up and data collection was performed meticulously.
Validity of the findings: In limitations of a small single centre study, data is well presented and analysed. The conclusion is linked to the original research question
Additional comments: The manuscript is nicely revised and is more comprehensible, however, it could benefit from another revision. The language and grammar could benefit from another 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: GENETIC DIVERSITY AND POPULATION STRUCTURE OF THE ENDANGERED BASAL ANGIOSPERM BRASENIA SCHREBERI (CABOMBACEAE) IN CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	Clear and unambiguous, professional English used throughout. – ok
2.	Literature references, sufficient field background/context provided – I missed more references on genetic diversity and structure in macrophytes. Since Brasenia schreberi is an aquatic plant, I think this is crucial
3.	Professional article structure, figs, tables. Raw data shared – ok
4.	Self-contained with relevant results to hypotheses – I don’t reckon the work is self-contained yet. I think the results of other molecular markers in the manuscript are important.
Experimental design: 1.	Original primary research within Aims and Scope of the journal. – ok
2.	Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap. – ok
3.	Rigorous investigation performed to a high technical & ethical standard. – I reckon that the discussions and conclusions that the authors provide will become more sound if the work had used some other molecular marker, for instance, a plastidial one. Nuclear Microsatellites are excellent tools to solve more recent issues in historical time and along with such markers may contribute greatly to the understanding of older evolutionary aspects. To draw sound conclusions about the phylogeography of plants during glaciations periods a plastid marker is crucial. If we analyze the studies quoted by the authors in the Discussion most of them used at least one plastidial marker, none used only SSR. Moreover, the vast majority of these works were done by having trees or shrubs as focus of the study. Almost no comparison was made with other aquatic plants by the authors of the manuscript. This is also critical to the work's soundness. I should mention also that according to prevailing ethical standards, information on submission of DNA sequences to an appropriate repository (such as GenBank) must be given. Voucher specimens information must also be clearly provided in text (herbarium code). And these informations were not clearly quoted in the manuscript.
4.	Methods described with sufficient detail & information to replicate. – As regards SSRs markers, since incomplete information were provided by the authors, it is not possible to reproduce the experiments.
Validity of the findings: 1.	Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated. – ok
2.	Data is robust, statistically sound, & controlled. – the data were not sound enough to support the conclusions.
3.	Conclusion are well stated, linked to original research question & limited to supporting results. – The conclusions are not limited to those supported by the results.
4.	Speculation is welcome, but should be identified as such. - ok
Additional comments: I believe the authors have a potentially excellent work at hand. Studies that assess genetic diversity in aquatic plants are lacking. And the conservation of these plants is a primary issue. Microsatellites are excellent markers that provide a wealth of information capable of answering many questions about plant biology. However, I believe that it would be important to adapt the discussion and conclusions to the questions that microsatellites can resolve with soundness. In order to assess the phylogeographical issues that authors wish to discuss, using another marker would be important. I strongly recommend that the authors make the necessary adaptations and resubmit the 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: GENETIC DIVERSITY AND POPULATION STRUCTURE OF THE ENDANGERED BASAL ANGIOSPERM BRASENIA SCHREBERI (CABOMBACEAE) IN CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors present a paper on the population genetics of Brasenia schreberi. The article is very well written and I have no hesitation in recommending it to be published, providing that typos and inconsistencies in spelling and formatting are corrected.
Experimental design: See below
Validity of the findings: The experimental design and analysis of the data set is thorough and I found the results interesting.
Additional comments: Line 39 I think this should be ‘Microsatellites’
Line 51 leaves during food gathering is confusing. Do you mean leaves harvested for food?
Line 65 decrease in the size of
Line 96 The CTAB reference should be Doyle & Doyle (1987). It is correct in the References.
Line 116-128 The styling of genetic diversity measures is inconsistent, e.g. line 116 Na vs. line 121 NA
Line 121 Did you mean ‘number of private alleles (Ap)’?
Line 121 If the explanation of NA is included the other measures should be as well, or only the ones that are not listed in the earlier paragraph
Line 139 Did you mean ‘with 70%’?
Line 145 I think it would be clearer as: (K) was tested from K=2 to 20 clusters, based on…
Line 148 structure harvester is confusing as this is a name of a program. It might be clearer to delete structure harvester, i.e. was verified based on the
Line 187-188 You mention one subgroup, what about the other subgroups?
Line 210 The P value and the test of significance in AMOVA relates to the probability of the Fst value being different from random, but does not test biological significance. I think it would be best to replace significant with substantial in the sentence.
Line 288 ‘among populations in contact’ would be better
Line 300 evidenced instead of evidence
Line 309 Delete ’in’ i.e. facilitate the development
Line 316 Should be: subgroups are located
Line 328 Did you mean 20 cpDNA regions in total? In which case total is not necessary.
Line 333-335 The last sentence is near verbatim repeat of line 309-310. I think extra data both in terms of sampling and NGS methods could lead to more robust conservation strategies.
Table 2 Headings are inconsistently formatted, some in subscript some not
Table 3 I have not found *** significance in the table. Either the asterisk is missing in the table or there is no need to list it in the caption.
Table 4 whitin is misspelled. Among groups appears the second time without capitals. It would be useful to indicate how this table is actually for the results of three separate AMOVA’s. Maybe some form of subdivision of the table could help?
Figure 1 Some labels on the map do not match the text of the paper e.g. COSZ/CQSZ, XGLH/XGLHT
Figure 3 I don’t think there is any point starting the X axis at -1000 km. Maybe start is just below 0 so no dots fall on the Y axis. The X axis label is missing a space between distance and (km)


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: GENETIC DIVERSITY AND POPULATION STRUCTURE OF THE ENDANGERED BASAL ANGIOSPERM BRASENIA SCHREBERI (CABOMBACEAE) IN CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript describes the genetic diversity and population structure of the endangered basal angiosperm Brasenia schreberi (Cabombaceae) This a very important conservation effort and good scientific merit.
Experimental design: The aim of the study is fulfilled.
The intent and the composition of the paper are good:
- The sampling strategies are correct to reach the desired goals (21 populations and 376 individuals).
- 12 microsatellite markers used provide good amount of information.
- Statistical analysis is satisfied.
- Figures and table are representative.
- The conclusion is also meaningful.
Validity of the findings: The paper is nearly ready to publish. Furthermore there are some points for consideration.
Additional comments: The authors could consider the following suggestions:

1- In the summary statistics for B. schreberi populations in China (Table 3) you must provide confidence interval for Ar, Ho, He and Fis. Standard deviations is not the best statistical approach to compare populations using its parameters.
2- Provide more information about null alleles.
3- What is the effect of the low bootstrap nodes in NJ dendrogram?
4- BOTTLENECK is flexible and results depends on the chosen parameters. Cite and provide more information about 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: GENETIC DIVERSITY AND POPULATION STRUCTURE OF THE ENDANGERED BASAL ANGIOSPERM BRASENIA SCHREBERI (CABOMBACEAE) IN CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1.	Clear and unambiguous, professional English used throughout. – ok
2.	Literature references, sufficient field background/context provided – revised!
3.	Professional article structure, figs, tables. Raw data shared – ok
4.	Self-contained with relevant results to hypotheses – explained!
Experimental design: 1.	Original primary research within Aims and Scope of the journal. – ok
2.	Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap. – ok
3.	Rigorous investigation performed to a high technical & ethical standard. – explained! It seems ok for me now.
4.	Methods described with sufficient detail & information to replicate. – revised!
Validity of the findings: 1.	Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated. – ok
2.	Data is robust, statistically sound, & controlled. – explained! It seems ok for me now.
3.	Conclusion are well stated, linked to original research question & limited to supporting results. – explained!
4.	Speculation is welcome, but should be identified as such. – ok
Additional comments: In this version of the work the authors clarified many important things, which I missed in the previous version. I believe it can be recommended to publish now. I just want to highlight a few simple points for consideration below:
Line 41 - Bertin, 1993 is¬ missing in References.
Line 76 – Martínez instead of Martinez.
Line 77- Nuphar submersa instead of Nuphar submerse.
Line 116 - Yeh et al., 1999 must be in italics.
Line 139 - check the punctuation after “IAM”.
Line 153 - why “e.g.”? Is there any other locus in which null alleles were observed?
Line 156 – the 5.08 value doesn’t match with the value observed in Table 02.
Line 158 – the 0.414 value doesn’t match with the value observed in Table 02.
Line 159 – the value 0.223 doesn’t match with the value observed in Table 02.
Line 160 – Table 02 instead of Table S1?
Line 166 – It is mentioned “HO…0.310 ± 0.120”. But, what about TWYL population? HO = 0.354(0.137).
Line 176 – Isn’t it nine populations instead of eight?
Line 179 - Isn’t it two subclusters instead of four?
Line 180 – I think it is cluster II instead of cluster 2.
Line 182-183 – I think the sentence “It is possible that the clusters were correlated with geographic location and different levels of introgression among various populations” fits better in Discussion.
Line 222 - Wang & Ge 2006 is missing in References.
Line 226 – “Qiu et al., 2009”. Is this Qiu et al. 2011? Or a work that is missing the References?
Line 243 – Instead of Cluster I is Cluster II, at least according to Fig_1.
Line 263-264 - Ming, 1987 is missing in References.
Line 269-270 - Chen et al., 2008 is missing in References.
Line 270 - Li et al., 2012 is missing in References.
Line 290 - Subcluster I-2 according to Fig_1.
Line 291 - Subcluster II-2 according to Fig_1.
Line 304 – “Shao et al., 2009”. Is it Shao et al. 2015 (mentioned in References) or another work?
Line 304 - Hu et al., 2010 is missing in References.
Line 307-309 – “Compared to other endemic aquatic plants, this species shows low genetic diversity and significant genetic differentiation.” It would be nice if this comparison were better discussed in Discussion section.
Line 376 – This reference is not mentioned in the text.
Line 459-461 – This reference is not mentioned in the text.
Line 498- The populations SZDSZ and YNGLG are incorrectly typed in the Figure.
In the References Section some of the references are out of order and there are some typos.


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: GENETIC DIVERSITY AND POPULATION STRUCTURE OF THE ENDANGERED BASAL ANGIOSPERM BRASENIA SCHREBERI (CABOMBACEAE) IN CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK
Additional comments: I am happy with the corrections the authors made to the paper and recommend it to be published. I would change ‘significant’ to ‘substantial’ in line 209 to be consistent with line 191.


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: GENETIC DIVERSITY AND POPULATION STRUCTURE OF THE ENDANGERED BASAL ANGIOSPERM BRASENIA SCHREBERI (CABOMBACEAE) IN CHINA
Review round: 2
Reviewer: 3

Output:
Basic reporting: Many of the requested changes have been incorporated, but there are several key points in the reviewed manuscript:
What is the effect of the low bootstrap nodes in NJ dendrogram? Use your response in the discussion of NJ dendrogram.

Please cite the BOTTLENECK chosen parameters.

Line 205: “It is possible that the clusters were correlated with geographic location and different levels of introgression among various populations.”
Line 223: “that genetic differentiation in populations might not be the result of isolation by geographic distance”
It is contradictory, why?
Experimental design: The aim of the study is fulfilled.
The intent and the composition of the paper are good:
- The sampling strategies are correct to reach the desired goals (21 populations and 376 individuals).
- 12 microsatellite markers used provide good amount of information.
- Statistical analysis is satisfied.
- Figures and table are representative.
- The conclusion is also meaningful.
Validity of the findings: The paper is nearly ready to publish. Furthermore there are some points for consideration.
Additional comments: Many of the requested changes have been incorporated, but there are several key points in the reviewed manuscript:

What is the effect of the low bootstrap nodes in NJ dendrogram? Use your response in the discussion of NJ dendrogram.

Please cite the BOTTLENECK chosen parameters.

Line 205: “It is possible that the clusters were correlated with geographic location and different levels of introgression among various populations.”
Line 223: “that genetic differentiation in populations might not be the result of isolation by geographic distance”
It is contradictory, why?

In the summary statistics for B. schreberi populations in China (Table 3) you must provide confidence interval for Ar and Fis. Use diveRsity R package.


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: EFFECTS OF TWO-WEEK E-LEARNING ON EHEALTH LITERACY: A RANDOMIZED CONTROLLED TRIAL OF JAPANESE INTERNET USERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The basic reporting of this manuscript is good and sophisticated.
Experimental design: The author reported that the short-period e-learning has a positive effect on the health literacy in a randomized control study. The author evaluated the internet users’ health literacy with the eHealth Literacy Scale, which has been validated in the prior studies. The score change of the eHealth Literacy Scale in the training group was significantly higher than that of the non-intervention group. The author evaluated the other scales. The author concluded that the e-learning is useful to learn the health literacy on the web.
However, Several concerns in method and terminology remained as below.
1.    The reviewer had severely concerned that the score of Attitude. Firstly, the reviewer is in doubt as to whether this question about osteoporosis is appropriate. Secondary, the scoring system of attitude is not appropriate because the sharing the wrong information widely did not equal to investigate the scientific evidence. Third, the score of attitude has never been validated in a prior study. The score of attitude had several critical problems, so the reviewer recommended the attitude should be expunged in this manuscript.
2.    The author defined the subjects as having the evaluation skills person, who had selected the government’s website. Van Deursen and Van Dijk elaborated four types of internet skills in their prior study. These are termed operational, formal, information, and strategic internet skills (JMIR 2011). They mentioned as internet skills not only to evaluate information source but also to make the right decision to reach their goals. Why did the author ignore the other skills such as operational, formal and strategic skills?
3.    The HON criteria (Boyer C. Comput Bio Med, 1998), The DISCERN score (Charnok D. J Epidemiol Commun H, 1999) and JAMAcriteria (Silberg WM, JAMA. 1997) are known as the tools to evaluate the quality of health information of the internet. The reliability of the individual website did not depend on only the information provider but also authoritative, attribution, transparency and financial disclosure. However, the author evaluated the reliability of contents with only the address of the website. Could you show the evidence or reference that the subjects who selected the go.jp or co.jp website had the internet skills?
4.    The e-learning contents played the important roles in this study. Could you show the examples of the e-learning teaching materials? If possible, the author should demonstrate these contents translated into English as Supplementary data. If impossible, the author should describe the content more in detail.
5.    The author mentioned, “The HEL was employed to assess change to health literacy other than a change to eHealth literacy.” Could you explain the difference between the HEL and eHealth Literacy Scale? The author should demonstrate these scales as figures or tables because these scales were unfamiliar with the audience.
6.    If the e-learning contents did not have information about healthy eating literacy, it is natural that the value of HEL did not change after training. The author should show their e-learning contents, which also included the health eating literacy or demonstrate the reference, which the score of HEL can change after receiving some kind of e-learning.
7.    The total 37 subjects (27.6%) were not able to complete the e-learning program in the intervention group. This author should mention the fact in the result section because the rate of adherence would affect the results. It may prefer to analysis separately in each case.
8. Line 185-186
The meaning of the sentence is unclear. Please rewrite it.
9. Line 186-187
When the subject selected the governmental website, the subject had the evaluation skill. In results, the author divided the three groups, “better”, ”no change” and “worse” in table 3. There is no definition of the groups in this manuscript. The author should define these groups.
10, 11. Line 203-208
10. Was the primary outcome the change of the eHealth Literacy scale? Similarly, were the second outcomes the change of the HEL, the attitude and the skills?
11. What kind of the statistical test did the author use when the author compared the change in scores? (Student t-test?  Mann-Whitney U test?) . Please describe the details of statistical analysis.
Validity of the findings: This study could demonstrate that e-learning education had a positive effect on eHealth literacy for Japanese internet users. The conclusion is redundant. It need to rewrite it.
Additional comments: Abstract
 Line 27-32 and 35
 　“There were no large differences at baseline between the intervention and control groups in the eHealth literacy scale or skill level for evaluating search results.”
1. Is “in the eHealth literacy” in this sentence “the Healthy Eating Literacy Scale” right?
2. What did the author mean skill “level”? Please define the skill “level” in this manuscript.

 Line 32-33
There is no data to improve both outcomes at the follow-up.

 Line 34-37
In this manuscript, the author demonstrated the significant statistical difference of the change for eHealth Literacy scales. However, it is unclear whether the statistical difference has clinically meaningful. In addition, the author could not show the difference of the secondary outcomes. Please rewrite this section.

Results
 Line 238-240 and Line 247-249
The reviewer did not understand the results of the relative risk ratio (RRR). Because the meaning of “no change” had two patterns as follows, 1) the subject who had had no skill from the beginning to the end, 2) the subjects who had had enough skill from the beginning to the end. How did the author treat the “no change” group in their analysis?

If the author would like to demonstrate the relative risk, the author should use the data at follow-up point.
    Getting the skill    No skill    sum
Intervention    70    64    134
Control    46    102    148

Relative risk: (70/134) / (46 /148)=1.68 [95% confident interval 1.26-2.24],
Fisher exact test: P=0.0004

Discussion
Limitation and Generalizability were written well.

Conclusions
Line 300-302
This sentence,“Although the e-learning~” is tediousness. This content of a sentence is merely a repetition of what was already mentioned in Introduction.

Line 304-314
It seems that these sentences are not a conclusion. The author should move these sentences to the Discussion section.

Line 314-315
This sentence is very assertive. This study provided with the interesting results. However, There are several limitations at the same time.
“This study could demonstrate that e-learning education had a positive effect on eHealth literacy for Japanese internet users.”

Figure and Tables
Table 1
 1. It is preferable that P value between intervention group and control group is described.
 2. The reviewer cannot understand the meaning of “Self-rated health”. Please explain the term in this manuscript.

Table 2 and Table 4
 “a score change of intervention~”  made no sense. Where is the “a” in this table?

Minor comment
Line 132 “In a previous study”
Please describe the reference.


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: EFFECTS OF TWO-WEEK E-LEARNING ON EHEALTH LITERACY: A RANDOMIZED CONTROLLED TRIAL OF JAPANESE INTERNET USERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Thank you for the opportunity to review this easy to read manuscript it would benefit from additional revision to strengthen its contents. While this article adds to the literature regarding ehealth literacy acquisition by Internet users, some of the literature in the introduction is older than 5 years and use of more contemporary articles could strengthen this section of the manuscript, especially as they are not seminal work. Update of references would improve relevancy to readers. Removal of the colloquial language and explanation of acronyms would also improve readability.  Please see general findings for specific examples. The conclusion needs to be re-written to ensure it does not contain any new information that requires referencing, the importance of this information needs to be included earlier in the manuscript.
Experimental design: The experimental design, including recruitment pool, limitations and issues with  generalisability are mentioned and justified.
Validity of the findings: This study is clearly described and could be replicated by others.

The conclusion needs to be re-written to ensure it does not contain any new information that requires referencing, the importance of this information needs to be included earlier in the manuscript.
Additional comments: To improve readability and to clarify the definition of ehealth literacy line 13, could be improved by adding the word ‘health’ after ‘accuracy’. Line 24, were the score computed or analysed? Use of acronyms such as WELQ (line 50) US (line 59), eHEALS (line 131) needs to be described in full the first time used.  Removal of colloquial language, lines 51, 77, 131, 267 and 309 would improve readability. A range of tenses is used. I am unsure first person improves readability of the manuscript. Some of the references are quite old and are not seminal and more contemporary literature may strengthen the article. For example lines 41, 43, 49, 59-61, 71.

The innovation of using an RCT that identified and uses a modified form of CONSORT is a useful addition to the literature.


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: EFFECTS OF TWO-WEEK E-LEARNING ON EHEALTH LITERACY: A RANDOMIZED CONTROLLED TRIAL OF JAPANESE INTERNET USERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for revising the manuscript “Effects of two-week e-learning on eHealth literacy: a randomized controlled trial of Japanese Internet
users ”
 The author almost appropriately responded the issues of the manuscript. However, The reviewer1 have one question for statistical process.
Experimental design: Line 215-219

The author used the linear regression analysis and multinomial logistic regression analysis in this study.
In this study design the repeated measured ANOVA is prefer to the linear regression analysis. Why did the author use the linear regression analysis?
Validity of the findings: no comment
Additional comments: The manuscript improved especially in "material and method".


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: EFFECTS OF TWO-WEEK E-LEARNING ON EHEALTH LITERACY: A RANDOMIZED CONTROLLED TRIAL OF JAPANESE INTERNET USERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have used the track change version to review this resubmission:

The revisions have improved readability and understanding of the research and findings. Clarity has improved and the authors have addressed the issues raised. Sharing of the elearning content in English is valuable. On revision, I also concur with removal of the attitude information as it strengthens the remaining information.
Line 71, about levels of health literacy needs referencing, currently it is a broad statement with no evidence. Alternatively the full stop could be removed and "as evidenced by a German survey" could be inserted instead.
Experimental design: Inclusion of the content in English is valuable.  Removal of the attitude information strengthens the article. Line 46 that ends with Hawthorne effect, needs another sentence to conclude the abstract.  I believe ending with the Hawthorne effect idea weakens the tenet of the abstract. Line 122 needs a comma after 'aware of, and'; line 138 is 'participant data', 'not participants'.
Validity of the findings: Clarity has been improved by deletion of the attitude questions and provision of supplementary materials.  This section has minor edits including removal of brackets around the participant withdrawal or non-completion numbers on line 306-307. The addition as suggested of the Cohen's d is noted.

The authors still need to provide a strong conclusion that represents their findings. They have understated the significance of the research as it is rare to have an RCT of this nature. While the research does have limitations they are stated above in the generalizability section.
Additional comments: The revisions have strengthened the article. Inclusion of information regarding the Hawthorne effect and rationales for use of HEL strengthens the article and improves readability. The inclusion of the elearning content is valuable to readers. Removal of the attitude information simplifies the study and strengthens the findings. Ending the abstract with a summative sentence and ensuring the conclusion reflects the findings and discussion would also strengthen the article.


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: SOCIOECONOMIC STATUS IS NOT ASSOCIATED WITH HEALTH-RELATED QUALITY OF LIFE IN A GROUP OF OVERWEIGHT MIDDLE-AGED MEN
Review round: 1
Reviewer: 1

Output:
Basic reporting: Introduction:
Lines 43 and 44: there are numerous studies that show the impact of socioeconomic status on the HRQL of adults,for example Alishiri et al and Ovayolu et al in their respective studies in 2008 and 2011 (Modern Rheumatology, 18(6), 601-608) (Clinical Rheumatology, 30(5), 655-664) indicated that precarious economic conditions were associated with a poor HRQOL. It is not necessary to provide evidence regarding the impact on adolescents when the focus is middle-aged adults

Lines 45-47: “Preliminary findings from our group indicate that among overweight middle-aged men, lower SES is associated with adverse  metabolic outcomes (unpublished data)”. This assertion should have theoretical support based on studies already published.
Experimental design: Line 57: in the results refer that excluded individuals who did not complete the assessment of HRQL, should also be defined as an exclusion criterion.

Line 76: cite reference on the study of SF 36 validation in New Zealand
Line 87 it is necessary to detail the form of measurement and interpretation of the IPAQ. What sense did you have in the research, the evaluation of physical activity?

Lines 100-105: the demographic differences between the two groups to be compared should be evaluated using the t-student test or Mann Whitney U test. The ANOVA test and its non-parametric counterpart apply when there are three or more comparison groups, in this same sense the post hoc analyzes are also not necessary.

Why was not a descriptive and comparative analysis made of the other clinical variables (blood pressure, hypercholesterolemia) that were taken into account for the study population?
Validity of the findings: Results:
“Pearson's correlation coefficient or Spearman's rank correlation were initially run. Associations between NZDep2006 scores and SF-36v2 outcomes were examined using general linear mixed  models based on repeated measures. Models adjusted for randomization sequence, timing of  assessment, and on-going use o cholesterol-lowering and/or antihypertensive medications, as well as participant's age, BMI, and IPAQ score”. No results are presented that demonstrate the application of the statistical analysis proposed.

Line 136: As previously corrected, univariate analyzes do not allow the identification of associations.

Discussion:
Line 187 As previously recommended in the statistical analysis plan, the post hoc analyzes are not necessary for the comparison between the study groups,in this sense, it is not a limitation of the study, on the other hand I emphasize the recognition of the other limitations of the study for the purpose of new studies and interpretation of the results and conclusions of the study.
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: SOCIOECONOMIC STATUS IS NOT ASSOCIATED WITH HEALTH-RELATED QUALITY OF LIFE IN A GROUP OF OVERWEIGHT MIDDLE-AGED MEN
Review round: 1
Reviewer: 2

Output:
Basic reporting: English language used was clear and unambiguous.

The motivation of the study was not strongly put across by authors.

Although the authors did mention the effect of SES on metabolic outcomes among overweight men using their preliminary findings, this evidence was not adequate enough to justify the reason for choosing middle-aged overweight men as the prime subject of focus and to study the impact of SES on HRQL among them.

Authors should cite past studies which have assessed this association in New Zealand or provide national statistics on the trend of obesity prevalence across gender over recent years to further stress the importance of  studying the association between SES and HRQL among overweight men.
Experimental design: Authors should incorporate past literature and/or theories related to the study to identify gaps that underpins the aim and novelty of the study.

It is better for authors to clarify the use of a cohort study in this study as cohort study usually requires a longer period of time and interventions to assess changes in outcome variables.

Authors also did not take into account of control factors such as behavioural risk factors (smoking, alcohol consumption and healthy diet intake) which confounds the association between HRQL and SES and is also associated with being overweight. If information on this are not available, it would be better to acknowledge it as part of the limitation of the study.
Validity of the findings: Overall the findings of the study is quite interesting as it shows no association between SES and HRQL among overweight men. However this finding could be biased due to small sample size and its non-randomized sampling design. Hence the findings are not representative of the general population.
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: SOCIOECONOMIC STATUS IS NOT ASSOCIATED WITH HEALTH-RELATED QUALITY OF LIFE IN A GROUP OF OVERWEIGHT MIDDLE-AGED MEN
Review round: 2
Reviewer: 1

Output:
Basic reporting: Article is written in clear and unambiguous English. Background of the study has improved from the previous version. Although the authors did mention that the study was not conducted in New Zealand, it would be better to acknowledge that such a study is limited internationally after an extensive review of the literature.
Experimental design: Research objectives and experimental design have improved from its previous version.
Validity of the findings: Findings are okay. Discussion of the study could be improved by elucidating more on the findings of the study as to why the authors have obtained a negative association.
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: SOCIOECONOMIC STATUS IS NOT ASSOCIATED WITH HEALTH-RELATED QUALITY OF LIFE IN A GROUP OF OVERWEIGHT MIDDLE-AGED MEN
Review round: 3
Reviewer: 1

Output:
Basic reporting: In this section I consider that the suggestions made in the previous version are followed
Experimental design: For this section I only have three minor recommendations regarding the form of reference in the description of the collection instruments: SF-36 and IPAQ (see comments in the manuscript), in addition to the analysis plan proposed for the baseline comparison between the stratified groups specifically for the variable "Consumed any alcohol" (see tables 1 and 2)
Validity of the findings: Meet with the evaluation items of the journal
Additional comments: In general I think that the authors did a good job of revision and correction