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: GENOME-WIDE IDENTIFICATION OF HSF FAMILY IN PEACH AND FUNCTIONAL ANALYSIS OF PPHSF5 INVOLVEMENT IN ROOT AND AERIAL ORGAN DEVELOPMENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The author analyzed the HSF gene family of pear genome, and studying the functional of hsf5  in regulating root length development.
Experimental design: Authors analyzed 18 members of HSF gene family, promoter elements and further analyzed the negative regulatory effect of HSF5 on root development
Validity of the findings: Authors analyzed 18 members of HSF gene family, promoter elements and further analyzed the negative regulatory effect of HSF5 on root development
Additional comments: 1, Compared with other species, which members of HSF gene family in Pear genome had evolved under the pressure of artificial selection?
2, Previous studies have found that HSF gene family are mainly involved in the regulation of gene expression under stress conditions. In the paper, author only focuses on the temperature sensitivity pear shoot growth, but does not involve other stress response. However, author selected hsf5 gene for functional identification is also aimed at the regulation of root length. Why? The stress treatment is too little, so little informations can be got from the process of stress response to HSF gene family.


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: GENOME-WIDE IDENTIFICATION OF HSF FAMILY IN PEACH AND FUNCTIONAL ANALYSIS OF PPHSF5 INVOLVEMENT IN ROOT AND AERIAL ORGAN DEVELOPMENT
Review round: 2
Reviewer: 3

Output:
Basic reporting: The level of the English language used throughout the manuscript is of a low standard, to being very poor. Many sentences are so poorly worded that they do not make any sense to the reader.

Background to the area is average at best. A lot background is required for ease of understanding for the reader.

Figures are also of an average standard. Tables add little.

Not a self-contained article as not enough background information is provided, or is too poorly explained to adequately understand.
Experimental design: Very standard largely bioinformatic based study with little supporting experimental work provided.

Research question is defined, but not overly relevant or meaningful.

Investigative rigour is lacking in this article. 18 gene family members identified, yet only one experimentally validated - not acceptable rigour and why this family was selected for further analysis over the other family members is also lacking. Only two Arabidopsis transformant lines generated and analysed - not acceptable scientific rigour.

Bare minimum methodological detail provided - one would struggle to reproduce many aspects of this study due to lacking detail.
Validity of the findings: Not an appropriate level of experimental validation performed to support the provided bioinformatic data.

RT-qPCR assessment of one gene family member from 18 identified is not acceptable

Required number of transformant lines are also lacking for the Arabidopsis work.

Discussion and Conclusion are far too general in their nature and do not tailor accordingly to the specific of the study.

Some claims made by the authors are not supported by the experimental evidence and/or rigour of the reported results.
Additional comments: See above comments addressing criteria 1 to 3.


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: GENOME-WIDE IDENTIFICATION OF HSF FAMILY IN PEACH AND FUNCTIONAL ANALYSIS OF PPHSF5 INVOLVEMENT IN ROOT AND AERIAL ORGAN DEVELOPMENT
Review round: 3
Reviewer: 1

Output:
Basic reporting: no
Experimental design: no
Validity of the findings: no
Additional comments: 1. Most Arabidopsis plant of Hsf transgenic showed dwarf and develop slowly, but thermotolerances were enhenced (Ogawa D et al. J Exp Bot, 2007, 58: 3373–3383). Meanwhile，From the Heatmap of transcript levels of HSF genes in peach in this paper (Fig. 3), with the growth stages of temperature-sensitive peach shoot development, PpHSF5 were remarkably upregulated, which means PpHSF5 is strong heat-response gene, so the thermotolerance function of gene must be performed firstly.
2. In Fig.6, the legend was not coherent with the photos. To ensure the consistence, the seedlings of different transgenic lines should be planted in one culture pot.To validate the experimental results, more seedlings than one should also be cultured in every lines. The data of Figure 6B/D/F/H is no sense, the better phenotypic photos can exhibit the results.


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: GENOME-WIDE IDENTIFICATION OF HSF FAMILY IN PEACH AND FUNCTIONAL ANALYSIS OF PPHSF5 INVOLVEMENT IN ROOT AND AERIAL ORGAN DEVELOPMENT
Review round: 3
Reviewer: 2

Output:
Basic reporting: Tan et al. elucidated the rationales behind analyses at multiple places, which makes the logic flow clear and coherent.

Most of my concerns have been handled except for a few minor points. There is no response to my comments in the rebuttal doc. I suggest accepting this manuscript.
Experimental design: The authors rearranged the figures as I suggested, which are currently easy to follow.
Validity of the findings: The study is more complete with the newly added functional experiments.

The authors may want to add the name of statistical tests and their power for the relative expression analysis. For now, I can't tell if some conclusions are qualitative or quantitative, and even if some of the claims hold true statistically.
Additional comments: There are missing caption for newly added figures, e.g. Fig 6E-H.


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: GENOME-WIDE IDENTIFICATION OF HSF FAMILY IN PEACH AND FUNCTIONAL ANALYSIS OF PPHSF5 INVOLVEMENT IN ROOT AND AERIAL ORGAN DEVELOPMENT
Review round: 4
Reviewer: 1

Output:
Basic reporting: no
Experimental design: no
Validity of the findings: no
Additional comments: My concerns have been handled.


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: EFFECT OF BACILLUS SUBTILIS ON ANTIOXIDANT ENZYME ACTIVITIES IN TOMATO GRAFTING
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: •	It was not clearly stated how the grafted plant sample was processed to obtain the enzyme extract for measuring enzyme activity and measuring phenol quantity.
Validity of the findings: •	There was so much variation in enzyme activities between control and Bacillus treated grafted plants according to the scion/root stock combination as well as time of sampling, that no conclusion regarding the effect of Bacillus treatments affecting the free radical scavenging activity on the grafted samples can be drawn.

•	Frequently, authors make erroneous or misleading statements such as,
-	In line 37 for Abstract, It is stated that “For all combinations, the quantity of total phenols decreased”.  But in Table 3.  For Day 1 in RGPep, Day15 in RGCh and Day 28 in RG1ber, total phenol was significantly increased in Bacillus treatment.
-	In line 477-8 in discussion, it is stated that “In general, B. subtilis decreased the activity of SOD, CAT, POD and PAL as well as the quantity of total phenol, on day 1 on the compatible grafts”.  But Table 2 shows no significant difference in POD activity between control and Bacillus treatment.

•	It is not stated/ discussed whether these different enzyme activities originated from plants or bacteria or whether Bacillus is successfully colonized in the grafted plants or whether Bacillus treatment promotes the grafting performance and plant growth.
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: EFFECT OF BACILLUS SUBTILIS ON ANTIOXIDANT ENZYME ACTIVITIES IN TOMATO GRAFTING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study is innovative and clear technically. They expressed and presented well.
You took three rootstocks, third one is totally different and practically it has no use.
Experimental design: Experimental design is well illustrated.
Validity of the findings: Work is novel and useful data they have generated which would be quite beneficial for the future studies.
Additional comments: This needs some English improvement. Otherwise, Its a good study. I would recommend for publication as full 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: EFFECT OF BACILLUS SUBTILIS ON ANTIOXIDANT ENZYME ACTIVITIES IN TOMATO GRAFTING
Review round: 1
Reviewer: 3

Output:
Basic reporting: English needs improvement, especially with spelling and punctuations. I corrected many spelling errors and placed commas throughout, but not in whole document. authors are advised to learn from my suggestions and make similar changes.

nevertheless, the article was well researched and the results were well presented.
Experimental design: no comment
Validity of the findings: conclusions are well stated and referenced (compared) to prior research
Additional comments: please review and study my comments and make necessary corrections


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: EFFECT OF BACILLUS SUBTILIS ON ANTIOXIDANT ENZYME ACTIVITIES IN TOMATO GRAFTING
Review round: 1
Reviewer: 4

Output:
Basic reporting: The structure is well represented by professional data, tables, and figures. Sufficient literature used with enough background justification. But the usage of English is very poor esp sentence formation. Many typographical errors, improper use of spacings, superscript/subscript, etc. Needs to revise English in many parts esp in the discussion.
Experimental design: The objective of the research is clear with a proper hypothesis. The experimental design is appropriate. The investigation is okay, but it would be valid it is provided with the grafting success rate in the treatments at the end of the experiment based on the visual observation.
Validity of the findings: Finds are well presented with appropriate statistical analysis. Again, it would be great if it is supported by the grafting success percentage.
Additional comments: Kindly revise the article with proper English and rectify the errors.

Line 46: Cucurbitaceae and Solanaceae
Poor writing style and unnecessary spacing: eg: line 97, 104, 159, 179, 220, etc. in using spaces. 269-280, unnecessary spacing between paragraphs: Line 300, 311.
Improper usage for superscript and subscript eg: Line 107:  H2O2, Line 159: 117umol s-1 m-2
Line 139: Kindly give the tray size and the dimensions used for the experiment.
Line 140: What is the media composition used to raise the seedlings?
Line 140: Which part of the scion was treated? or the entire scion? It was done before the cut or after the cut? Not clear.
Line 153: What is the height of the rootstock and scion with how many leaves?
Line 159: 117umol s-1 m-2) to 117 umol: Spacing and superscript (s–1m–2).
Line 94, 241: Unnecessary italics.
In many places letter “O” was replaced with the number “0” in H202 Vs. H2O2.  Eg: Line 363, 365.
Rewrite the results with better English with clear sentences.
Kindly justify why you take three different species of the rootstock. (Two Solanaceous and one Cucurbitaceous).
It is worth to mention the grafting success percentage of graft union at the final stage.
 Kindly check the references and use punctuations appropriately eg: full stop at the end of the references. Some have and some missing. Also, different styles used. Kindly change it to the journal style. & is missing in many references before the last author (line:588, 569). Some references instead of &, “and” are used (line 616).

Fig.1.: Provide space before unit nm.


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

Input:
Title: HIGH THROUGHPUT MICRORNAS SEQUENCING PROFILE OF SERUM EXOSOMES IN WOMEN WITH AND WITHOUT POLYCYSTIC OVARIAN SYNDROME
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear outline of the paper. Sound description of aims and objectives. Citations are relevant and recent.
Experimental design: Authors have employed a reasonable experimental design. The subject selection us clear. The manuscript provides detailed research methodologies although authors need to include  some more details on statistical analysis.
Validity of the findings: This is an interesting and informative study; and the findings would contribute to the current knowledge in the field.
Additional comments: Manuscript submitted by Zhang et al., describes characterization of the miRNA profiles from serum exosomes isolated from patients with polycystic ovary syndrome (PCOS) and the healthy individuals. The study identifies 54 miRNAs that show statistically significant difference in the expression. Authors further confirm some of the identified candidates by biochemical assay (RT-qPCR) and show the evidence of their differential expression. The study is interesting and well designed. The role of miRNAs in exosomes as potential biomarkers is an exciting field in research investigation. The findings from this paper would contribute to the growing body of the evidences for the same. Here are my comments that would help authors to improve the quality of the manuscript and benefit general readership.

1) What are the downstream targets of miRNAs that authors identified to be differentially expressed by both sequencing AND RT-qPCR. Can authors provide any evidence of changes in expression of the target mRNAs (by reporter assays and/or Western blot)?
2) Can authors include a model depicting one their differentially expressed miRNAs in exosomes and the potential implications in disease development. It would be really helpful.
3) Authors need to mention how many times each experiment was repeated (e.g. Figure 1A: Western blot, Figure 3: RT-qPCR assay etc.).
4) For sequencing data: Authors need to comment on the data reproducibility across the four biological replicates (e.g. Pearson’s correlation and/or Principal component analysis).
5) Figure 1A: Did authors confirm the purity of exosomes by immunoblot analysis using any non-exosome biomarkers? Also, why is there a difference in the expression of both HSP70 and CD9 in PCOS samples as compared to control? Is this statistically significant and/or biologically relevant?
6) Line 157: Authors need to provide little more details of statistical analysis of the sequencing data.
7) Lines 164-166: These statements wrongly describe the results. Figure 3 shows significant changes (represented by *) in the expression of ONLY miR-1299 (up) and miR-192-5p (down) and NOT miR-6818-5p or miR-145-5p. The lines 190-192 in the discussion and the lines 222-223 in conclusions also wrongly describe these results. This needs to be corrected. Also, the legends (data-symbols) on Figure 3 need to be clearer. I assume circles represent ‘normal samples’ and squares represent ‘PCOS samples’. This needs to be clarified.
8) Figures 1B and 1C: Are these data from normal individuals or PCOS patients? It would be helpful to show the data from both the groups and comment on the general morphology of the exosomes, if any.
9) Line 154: Authors mention that the study identified 1475 miRNAs. What was the read-count cutoff used?
10) Figure 2B: What do the numbers in round brackets represent?
11) The fold-change criterion described on line 159 is not the same as described in Figure legends for 2B.
12) Line 77-78: Authors write: “Although some studies have reported alterations in miRNA expression in PCOS..” This needs relevant citation/s.
13) Figure 2C: X axis label is missing.
14) Legends for Supplementary Figures are missing.
15) There are a few grammatical and sentence construction errors that need to be corrected (e.g. line 71: ‘inhibits’ should read ‘inhibit’, line 74: “…follicle fluid exosomes was also has been..” should read “…follicle fluid exosomes has also  been..”, line 78: “…expression in PCOS, there are still need to further study..” should read “…expression in PCOS, there is still need to further study…”).


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

Input:
Title: HIGH THROUGHPUT MICRORNAS SEQUENCING PROFILE OF SERUM EXOSOMES IN WOMEN WITH AND WITHOUT POLYCYSTIC OVARIAN SYNDROME
Review round: 1
Reviewer: 2

Output:
Basic reporting: The structure of the whole article is clear, but there are many formatting errors in the article. To sum up, the content is of interest and innovation, so this manuscript is recommended for publication before some issue modification.
Experimental design: This work started with PCOS and normal human serum exosomes, using miRNA sequencing technology to reveal the difference in miRNA expression and predict the novel molecular biomarkers for clinical diagnosis. It provides scientific help for clinical identification of PCOS.
Validity of the findings: All underlying data have been provided，but it needs to be added some more.
Additional comments: 1. Line 159，P＜0.05 should be in italics，There were many similar formatting errors in the article, please correct.

2. Line 159,  |FC|>1.2 or log2|FC|>1.2?

3. Line 171, repeated punctuation.

4. Line173-174, these contents should be written in the method.

5. Figure2B legend,  |log 2(fold‐change)| ≥1 or ≥1.2 ？

6. The legend in Figure 2B shows that the up-regulated microRNA is red and the number is 777, but it looks like there are only a few dozen. please explain.

7. Figure2C: Please indicate the unit of horizontal axis and vertical axis in detail.

8. I noticed that in your original data, the expression level of the hsa-miR-497-3p gene in the NC group is 0, but the log2FC value is 4.39. Please explain the calculation method to get the value of 4.39.

9. Please complete the legend in Figure 3

10. Figure 4A: What is the unit of the vertical axis? Target genes are enriched in many pathways. Why choose these pathways as the result display? What is the basis? Are these pathways significant? Please give the logical closeness between the pathway result and PCOS.

11. There are several errors in the legend in Figure 4A, please correct them.

12. Human_Participant_Consent_Form is an empty form, please complete it.

13. Because many miR expression levels are very low, I want to look at the quality control data of mirna sequencing to prove the accuracy of mirna sequencing.

14. Is it possible to add a Principal Component Analysis chart (PCA chart) based on the miRNA gene expression data of different samples to visually display the differences between and within groups.


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

Input:
Title: HIGH THROUGHPUT MICRORNAS SEQUENCING PROFILE OF SERUM EXOSOMES IN WOMEN WITH AND WITHOUT POLYCYSTIC OVARIAN SYNDROME
Review round: 1
Reviewer: 3

Output:
Basic reporting: Zhang et al have studied the differential regulation of miRNAs from serum exosomes in PCOS with matched controls using miRNA sequencing. They have characterized the exosomes and profiled the miRNAs and performed enrichment analysis.
The formatting of the authors' affiliation has to be checked and presented in conventional ways.
Conclusions section in the abstract is missing.
The language is clear and professional except for a few places where there are grammatical errors. The introduction is sufficient and discusses the relevant literature. The quality of the figures needs to be improved. Detailed comments on the figures are given in the general comments.
Experimental design: The scope of the work matches with that of the PeerJ and the research question is well defined.
However, the experimental methods are insufficiently explained with critical information missing making it difficult to assess the findings.  The missing information has been explained in the general comments.
Validity of the findings: The study is novel and there is no known study reporting the exosomal miRNAs in PCOS compared to matched controls. The rationale and benefit to the literature have been clearly stated.
However, the data and methods which have been provided are inadequate to support the findings of the study.
Additional comments: For high throughput studies, it is always recommended to conduct the analysis with correcting for multiple testing and generally false discovery rate (FDR) corrected p-values is considered appropriate to define statistical significance. The study has used p-values to define differentially expressed miRNAs throughout the manuscript and have used those miRNAs for downstream analyses. While checking the raw data from this study (Suppl. Table S3), only 6 miRNAs passed multiple testing (FDR <0.25) and just 3 miRNAs passed more stringent criteria (FDR <0.05). The authors have chosen to proceed with the uncorrected p-values for enrichment analysis. Please justify this,
Methods
In methods under section 2.3,  most of the critical information in methods are missing. No information has been presented on the library preparation, illumina instrument used for sequencing, depth of sequencing, sequence alignment, mapped reads, how differential expression of miRNAs was quantified and GO analysis was performed. All these critical information needs to be included in the methodology section.
Section 2.5
Details of PCR including the temperature and no of cycles, primers is missing.
The sentences in section 3.4 169-171 and 172-174 should ideally be placed in Methods rather than in results.
Results
Figure 1: The loading control is missing which is critical to ensure equal loading.
Figure 2: The information on how the heatmap was constructed is missing. Were the values normalized and scaled? Legend is missing. Also, please check the discrepancy in the volcano plot which mentions 777 up-regulated and 698 downregulated miRNAs while the results section lines 158-160 indicate that 54 miRNAs were differentially regulated.
Figure 3: Please check the missing legends
Figure 4: Please provide the tool used to perform KEGG pathway enrichment. In Figure 4A, the Y-axis is poorly defined. In Figure 4B, dot plot has been wrongly mentioned as scatter plot. In addition, a part of the legend, “Pathway enrichment is measured by Rich factor, P value and the number of genes enriched in this pathway. Rich factor refers to the ratio of the number of differentially enriched genes to the number of annotated genes in the Pathway. The larger the Rich factor, the greater the degree of enrichment” is identical to the legend from Hu et al 2020 – Figure 6. Please rephrase the legends.
Discussion
The observations from the studies can be put together in a better manner. There is a list of miRNA showing their links with other conditions. The possible links between the newly identified miRNAs and PCOS have to be discussed. The major 6 miRNAs and their targeting genes can be checked against the known RNA markers of PCOS. Also, it is important to discuss about the enriched pathways with respect to PCOS. The observation of high Anti-Müllerian hormone in PCOS has not been discussed with respect to the differentially expressed miRNAs.
The conclusive statements are difficult to understand.

Supplemental figures
The figures are missing legends.


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

Input:
Title: HIGH THROUGHPUT MICRORNAS SEQUENCING PROFILE OF SERUM EXOSOMES IN WOMEN WITH AND WITHOUT POLYCYSTIC OVARIAN SYNDROME
Review round: 2
Reviewer: 1

Output:
Basic reporting: No additional comments.
Experimental design: No additional comments.
Validity of the findings: No additional comments.
Additional comments: In the revised manuscript authors have addressed many of my comments but some of them still remain unresolved.
1) In response to my previous comment regarding miRNA downstream target gene expression analysis – authors provided western blot data for miR-1299 target gene FANCC. The data clearly show decreased levels of protein and convincingly demonstrate the effects of increased levels of miRNA-1299. But I did not find these data included in the revised manuscript. I think biochemical validation of the findings is a significant addition to the existing data and it strengthens the claims. Please include these data and add relevant text in pertinent sections of the manuscript.
2) My second comment has not been addressed satisfactorily as well. I was anticipating a more thorough and comprehensive summary of the findings including possible target mRNAs (and their possible biological role in disease). I agree with authors that there remains much need for further elucidation. But based on the available data, can authors include a speculative (and more comprehensive than they showed in the rebuttal letter) schematic model? Also, authors need to make sure to include the model in not only the the rebuttal letter but also the manuscript as well (may be as a supplementary figure; if not the main data).
3) Lines 81 – 84 [File: peerj-49119-Manuscript_(tracked_changes)]: Authors write – “Although other studies have reported alterations in miRNA in human follicular fluid (HFF) exosomes and mesenchymal stem cells exosomes, there is still a need for further research in promising therapeutic and prognostic serum biomarkers in PCOS”. I had commented in my earlier review that this needs relevant citation/s of the published literature. Authors claim (in their rebuttal letter) that they added the citations. But I do not see the relevant literature is cited here. Please do the needful.
4) I am still not convinced the way authors have defined “differential regulated microRNAs” and “significant differentially regulated miRNAs”. If I understand correctly, the ONLY difference between these two groups is – for the former, the fold-change cutoff is 2-fold while for the latter it is 2.3-fold. Why did authors employ this arbitrary cutoff to define latter set of miRNAs? It is surprising (but not practically impossible) to get the number restricted from 1475 to only 54 when the cutoff stringency is just marginally increased. Please clarify this discrepancy.
5) Upon reading the manuscript again, I realized that a statement on depositing the raw sequencing data (data availability) is missing. Have authors deposited their raw sequencing data to a publicly available repository? To ensure reproducibility, these data need to either be cited or deposited into an appropriate repository and a clear statement needs to be included in the manuscript.


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

Input:
Title: HIGH THROUGHPUT MICRORNAS SEQUENCING PROFILE OF SERUM EXOSOMES IN WOMEN WITH AND WITHOUT POLYCYSTIC OVARIAN SYNDROME
Review round: 2
Reviewer: 2

Output:
Basic reporting: The structure of the whole article is clear, many formatting errors in the article have been corrected. To sum up, the content is of interest and innovation and this manuscript is recommended for publication.
Experimental design: This work started with PCOS and normal human serum exosomes, using miRNA sequencing technology to reveal the difference in miRNA expression and predict the novel molecular biomarkers for clinical diagnosis. It provides scientific help for clinical identification of PCOS.
Validity of the findings: All underlying data in the article have been provided.
Additional comments: To sum up, the content is of interest and innovation and this manuscript is recommended for publication.


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

Input:
Title: HIGH THROUGHPUT MICRORNAS SEQUENCING PROFILE OF SERUM EXOSOMES IN WOMEN WITH AND WITHOUT POLYCYSTIC OVARIAN SYNDROME
Review round: 2
Reviewer: 3

Output:
Basic reporting: The revised version of the manuscript seems improved.
The language is professional with clarity.
The background has been sufficiently described.
Article structure and tables are clear enough and the raw data look convincing.

I have a comment here in Figure 4 on how the heatmap was constructed. Can you please explain how can one plot the log2foldchange of expression values? As we would calculate the log2(fold change) with respect to control, one would be left with only values for PCOS.
And it is not possible to plot this graph with values for both the control and PCOS. Ideally, the values should be normalized and scaled for all the 8 samples and the heatmap should be constructed. Even the difference does not look convincing. The profiles of NC1 and NC2 do not look much different from PCOS samples.
Experimental design: The experimental design is well-defined and they have made the revisions as suggested improving the details of methods
Validity of the findings: The study is novel and there is no known study reporting the exosomal miRNAs in PCOS compared to matched controls. The rationale and benefit of the literature have been clearly stated.

The methodology and validity of Figure 4 needs to be checked.
Additional comments: The modifications are quite convincing except for Figure 4.

I have a minor comment.

The article has to be checked for grammatical errors.


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

Input:
Title: HIGH THROUGHPUT MICRORNAS SEQUENCING PROFILE OF SERUM EXOSOMES IN WOMEN WITH AND WITHOUT POLYCYSTIC OVARIAN SYNDROME
Review round: 3
Reviewer: 1

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


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

Input:
Title: HIGH THROUGHPUT MICRORNAS SEQUENCING PROFILE OF SERUM EXOSOMES IN WOMEN WITH AND WITHOUT POLYCYSTIC OVARIAN SYNDROME
Review round: 3
Reviewer: 2

Output:
Basic reporting: No new comments
Experimental design: No new comments
Validity of the findings: No new comments
Additional comments: 1. The question of figure 2 is still not answered satisfactorily. Please explain how the heatmap was plotted. Were the expression values normalized and what method was used for clustering? Also please include a title for the legend (Expression or normalized expression).

2. The quality of Figure 4 (A and B) is poor. Please provide figures with better resolution.


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

Input:
Title: HIGH THROUGHPUT MICRORNAS SEQUENCING PROFILE OF SERUM EXOSOMES IN WOMEN WITH AND WITHOUT POLYCYSTIC OVARIAN SYNDROME
Review round: 4
Reviewer: 1

Output:
Basic reporting: No additional comments
Experimental design: No additional comments
Validity of the findings: No additional comments
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: SCREENING AND IDENTIFICATION OF ENDOMETRIAL PROTEINS AS NOVEL POTENTIAL BIOMARKERS FOR REPEATED IMPLANTATION FAILURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: A very worthy topic which merits publication as papers in this field are generally lacking.

I find the syntax and general English usage to be a bit incoherent and feel the document, while obviously translated from the native language, could benefit from further work by a native English speaker.
Line 57 onward.  The proteomic references are incomplete and should include the work of Alonso and J Horchadas on the European continent.

The selection of the candidate proteins out of 6000 plus proteins should be fully elucidated.  Should more WB's of other candidates been performed to make the argument more convincing?
The reviewer is not clear why CBG selected above other candidates present in a cluster, is there an introduction of Bias in this selection?
Experimental design: I'm a little bit disappointed with the number of patients selected for this pilot study, I feel the numbers could be higher to allow more robust conclusions.  I also note some Male factor patients in the RIF group and wonder why these patients did not make inclusion into the "control" group.
Are controls used by merit of the fact that they happened to get pregnant while part of the IVF program.  They may have had conditions not representative of true population controls which may thus influence the results.
Is it possible to aquire endometrial samples for this protocol which are not related to IVF in any way?

Use of LC MS and WB is a nice way to definitively assess the proteins involved rather than the heretofore accepted methods of mRNA evaluations, ultrasound, immunohistochemistry and flow cytometry.  In this aspect the paper is unique and merits publication.

Ln78.  The WOI should be defined specifically and how it was evaluated. e.g. LH+7.  Clarity should be given that WOI may be variable in approx 30% of cases and this may be the origin of the RIF.
Ln82.  Do I take this as patients were batch processed rather than processed individually?
Fig 1.  it is unclear what Control 1 and 2 and RIF 1, 2 and 3 are..are these individuals selected as examples?
All figure legends need to fully fleshed out with explanatory annotations.
Validity of the findings: Repeat implantation failure is by no means limited only to endometrial receptivity and other factors, other than the rather obvious embryo quality, which lead to this negative outcome should be explored fully to fully enlighten the reader.  Examples would be a "hostile" immune profile.

Could normalization of the CBG expression profile be demonstrated with supplemental progesterone administration?

I think the conclusions could be more fully fleshed out and the clinical implications discussed
Additional comments: Meritorious work and well conducted using suitable techniques.  I'd like to see better use of English if possible and more coherent arguments used for selection of the protein candidates used for WB and ultimately decided as being directly involved or otherwise in the process of RIF


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: SCREENING AND IDENTIFICATION OF ENDOMETRIAL PROTEINS AS NOVEL POTENTIAL BIOMARKERS FOR REPEATED IMPLANTATION FAILURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Specific comments:
In the abstract, we have no idea of the compared groups with the proteomic approach
Line 20: what do you mean by 'poor endometrial receptivity ?
Lines 56-57: insufficient literature (PMID:15602768, PMID:19714818, PMID:18793766, PMID:21137015, PMID: 26760977...)
Lines 78: What is the exact timing for hysteroscopybiopsy. How was determine the WOI ? How much time after the LH surge or ovulation was performed the hysteroscobiopsy?
In the general and clinical data of RIF patients (n=40) and pregnant groups (n=80), some data presented as the mean ± SD are issued from too small clinical data. For example, the mean for endometrial thickness the day of embryo transfer included only 3 patients in each group (RIF and pregnant group); therefore, these data are not representative of the cohort.
In the same way, there are only 28/40 and 55/80 patients for the AMH concentration in the RIF and pregnant group respectively, etc. Moreover, I do not find certain values of means ± SD using the clinical database provided in the excel file (eg. [P] = 0.61 ± 0.88 ng/ml in the RIF group , and not 0.47 ± 0.27 ng/ml).
Do you perform a normality test before choosing a parametric test ?
The number of samples used for protein extraction is very low, only 2 for the pregnant group and 3 for the RIF group. We recommend increasing the number of samples per group.
The total number of peak detected must be supplied.
How much peaks as significantly differentially expressed were identified between the 2 groups ?
The differences in peaks intensity volume must be supplied
how much peaks were selected for protein identification ? all peaks between 350 to 1800 m/z?
How much samples used for the first-dimensional fractionation procedure ? Please, clarify
How was selected candidates for validation by western blot ?
How to explain that very few protein candidates were consistent with prior studies ?
A workflow for the protein detection and identification will be appreciate
Lines 161-162: as some clinical data are not representative of the entire cohort, it cannot be said that there were no significant different in clinical data between groups
In the western blot results, bands must be quantified and fold changes provided.
Fig. 2D, unreadable
In the fig. 3, I'm not sure that KEGG pathways comprising only 1 protein were really significant pathways. It is not relevant. Same remark for the EggNOG analysis.
Lines 216-218: Not totally agree with this comment as Domínguez et al identified the 40S ribosomal protein SA and the L-Plastin proteins, while in the present study the 60S ribosomal protein L18 and Plastin-1 were identified.
Validity of the findings: no comment
Additional comments: The manuscript of Wang et al. analyses the difference in endometrial protein expression profile during the implantation window between pregnant vs. RIF patients using the iTRAQ labeling coupled with 2D LC MS/MS technique. The idea is not novel and several articles have already been published in this field using omic technologies. However, this is an interesting article that may have potential implications in the understanding of molecular mechanisms associated with implantation failure. However, experiments and experimental design were not presented in a satisfactory manner to judge the present study. In addition, the literature referred is incomplete and the authors should conduct a more careful review of the data. Therefore, there are a number of items that should be addressed to improve the quality of the manuscript (see specific 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: SCREENING AND IDENTIFICATION OF ENDOMETRIAL PROTEINS AS NOVEL POTENTIAL BIOMARKERS FOR REPEATED IMPLANTATION FAILURE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript needs to be thoroughly revised for the appropriate use of the English language. Word choice and unclear sentences make the text difficult to read at times. Words, expressions, and sentences that should be revised have been highlighted on the PDF version of the manuscript, which is attached to this review. Two supplementary files were provided in a language other than English; one of them seems to have an English version. Figure 4 legend should be improved to provide more information. There is no indication of sample size. Otherwise, the manuscript meets the expected journal's criteria.
Experimental design: The authors have clearly stated a relevant and meaningful research question, as the lack of clinically relevant markers of improved endometrial receptivity is a major limitation in the field. Methods are well-described and adequate to answer the proposed question.
Validity of the findings: The question addressed by the authors is not novel, but it still remains relevant. The data are robust and statistically sound; however, the conclusion, although liked to the main question, is not supported by the results. The authors report the CBG protein as a novel and objective indicator of endometrial receptivity, which has not been shown by the data. Substantial experimentation should be performed to validate these results. Given the relatively large sample size, authors could try to predict RIF according to CBG protein expression, for example, which would provide further support to the observed relationship.
Additional comments: The manuscript entitled "Screening and identification of endometrium proteins as novel potential biomarkers for repeated implantation failure" used endometrial biopsy samples of RIF and pregnant patients and a proteomic approach to try to identify endometrial markers of receptivity, and validated two identified candidates by western blotting. The authors propose that Cortisol Binding Globin is a marker of endometrial receptivity. Overall, the manuscript has a relevant question and an appropriate experimental approach; however, it needs a thorough language review and needs to revise its conclusion, which is not fully supported by the data. In its current format, the manuscript is not adequate for publication.


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: SCREENING AND IDENTIFICATION OF ENDOMETRIAL PROTEINS AS NOVEL POTENTIAL BIOMARKERS FOR REPEATED IMPLANTATION FAILURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: Thanks you for the revisions, I think they have clarified the content and improved the paper.


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

Input:
Title: SCREENING AND IDENTIFICATION OF ENDOMETRIAL PROTEINS AS NOVEL POTENTIAL BIOMARKERS FOR REPEATED IMPLANTATION FAILURE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The use of professional English was much improved. Few observations made in that regard.

Please revise:

Throughout the text, authors use abbreviations to begin sentences; Is that adequate?
L149 - skimmed milk powder
L232-233 - specify "higher" and "lower" than what?
Experimental design: L146 - use g force rather than rpm;
L160-161 - how can any of the variables from the dataset be analyzed by a paired t-test? As far as I can tell those are not paired samples;
L170-176 - What is the purpose of comparing the clinical data of the 120 patients between RIF and pregnant individuals? Considering that molecular data are compared within a subset of samples, it seems more useful to know how groups compare within each subset of samples;
L212-216 - It would significantly increase the power and impact of the data if Western blot was performed on all available samples;
Validity of the findings: The study's conclusions are still not adequate. There is not a functional experiment and the large dataset was not explored to validate the differentially expressed markers. There is excessive speculation being used to make a conclusion.
Additional comments: The manuscript was significantly improved; however, major issues still remain to be addressed before the study can be considered for publication.


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: SEGMENTAL ANALYSIS BY SPECKLE-TRACKING ECHOCARDIOGRAPHY OF THE LEFT VENTRICLE RESPONSE TO ISOPROTERENOL IN MALE AND FEMALE MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is clear and unambiguous, with a few grammatical and spelling errors that need to be corrected before acceptance.

The authors have cited relevant literature; however, some important references addressing sex differences in isoproterenol-induced cardiac dysfunction are missing e.g. the work of Angela Woodiwiss group. Also the reference Grant et al. 2020 should be updated to the published peer-reviewed article, instead of the preprint version.

Article is structured professionally with relevant data shared.
Experimental design: The primary research focus is within the scope of the journal.

The manuscript addresses an important gap in knowledge. The global effect of chronic isoproterenol administration on the heart has been reported previously, but reporting the segmental effects of isoproterenol is new. The authors addressed this gap in knowledge rigorously and described their methods in sufficient details.
Validity of the findings: Data presentation and interpretation:
1- The tables should show the interaction p value in addition to the Sex and Iso effects.
2- It seems that isoproterenol increased the LV mass in male mice only (from 86 to 95 mg, almost a 10% increase). Although this was not significant with this sample size and high variability, this needs to be addressed.
3- The data and the statistical analysis in Figure 1 do not address the sex differences. The data for each segment should be compared between males and females in parallel and the Two-Way ANOVA should be used here, similar to the Tables. Sex differences cannot be properly addressed without Two-Way ANOVA.
4- The authors used the Pre- to Post- comparison in the Figures, in contrast to the Tables where the used control vs. treatment. There should be an explanation.
Discussion and Conclusions:
The Discussion has a lot of repetitions from the Results section. The authors should use the Discussion to compare their findings with prior research and critically evaluate/speculate on the differences and similarities.
Additional comments: In general, the manuscript advances this particular area of research by showing segment-specific differences in the response to isoproterenol. The major limitation, as stated by the authors, is the lack of any tissue-driven data e.g. molecular markers of remodeling, histopathology,...etc. It is not hard to repeat the mouse experiments for the purpose of obtaining these tissues (these are regular C57Bl/6J mice, not very expensive). Without these tissue-level data, this reviewer thinks that the amount of data in this manuscript commensurate with a short communication, not a full length 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: SEGMENTAL ANALYSIS BY SPECKLE-TRACKING ECHOCARDIOGRAPHY OF THE LEFT VENTRICLE RESPONSE TO ISOPROTERENOL IN MALE AND FEMALE MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author gave a clear and straightforward background introduction. The structure of this manuscript is professional. The results provided by the author was almost self-contained. But some sentence in the manuscript is a little bit confusing, like "Anterior segments were also reduced in females.". Please revise them and give more detailed descriptions.
Experimental design: The author continuously infused the mice with ISO for 3 weeks and found that there were decreased systolic and diastolic cardiac function and there was a gender difference. The research design was good and the description of the methods was detailed enough.
Validity of the findings: According to the results, the author found that the strain analysis could identify regions of LV that was more negatively affected by ISO. They also mentioned that continuously infused the mice with ISO could build a model for Takosubo cardiomyopathy.
All underlying data have been provided and conclusions are well stated.
Additional comments: 1. The range for HR (400-550 BPM) is relatively large. Please provide the HR for speckle tracking analysis (male and female, before and after ISO). HR might also affect the strain and strain rate.

2. Please provided the BW and/or TL corrected LV wall thickness, LV diameter/volume, CO, and SV, which would make the data more persuasive for mice with significantly different BW.

3. How about the systolic and diastolic cardiac function when using the ISO (before stop)? Did the systolic function increase or decrease after continuously infusing the ISO after 1 week, 2 weeks, or 3 weeks?

4. Have any other experiments been done to prove the decrease of systolic and diastolic cardiac function, like hemodynamic analysis?

5. When doing the speckle tracking analysis, please provide the time to peak for strain and strain rate and the standard deviation of time to peak for different segments. This parameter is critical for evaluating cardiac synchronization and represents the systolic function as well. The asynchrony could be proved in videos S1 and S2.

6. The intra- and inter-observer variability should be added for both speckle tracking and conventional echo analysis.

7. In line 106, the author mentioned that “2D echo B-mode loops were acquired from the LV PSLAX”. Should it be “PSLAX and SAX”?

8. In line 182, the author mentioned that “sometimes showed features of an infarct in the posterior wall near”. Please give more description about that, and please provide more evidence, like ECG, for infarction, if possible.


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: SEGMENTAL ANALYSIS BY SPECKLE-TRACKING ECHOCARDIOGRAPHY OF THE LEFT VENTRICLE RESPONSE TO ISOPROTERENOL IN MALE AND FEMALE MICE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is mostly well written, but suffers from numerous typological errors, including in the conclusion.
Experimental design: The echo recordings are of high quality and the animal model seems well maintained and may have clinical relevence.

The mice were not euthanised and so no morphological or biochemical data were collected.

The research described are interesting, but very similar in nature to those of Grant et al 2020. That paper used a mouse ISO infusion model for 14 days in female and males. They find functionally similar differences in both sexes.

Grant et al conclude that mice do not replicate the sexual dimorphism of humans and so may not be a relevant model for the disease. There may be a slight reduction in ejection fraction, but this was opposite to human, where males were are more severely affected.

It is unclear therefore what additional information is gained by this study.
Validity of the findings: No comment
Additional comments: The data are of high methodological quality, are analysed to a high standard data and are statistically valid. The animal model seems to replicate that previously published. The novelty of the data are the main reservation.


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: SEGMENTAL ANALYSIS BY SPECKLE-TRACKING ECHOCARDIOGRAPHY OF THE LEFT VENTRICLE RESPONSE TO ISOPROTERENOL IN MALE AND FEMALE MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is clear
Experimental design: Good experimental design
Validity of the findings: Data are clearly presented
Additional comments: The authors have responded to most of the raised critiques, The additional follow-up data are interesting and added novelty to the manuscript. The dependence on imaging as the only technique used in the manuscript is the main drawback, but it was addressed by the authors as a limitation.


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: SEGMENTAL ANALYSIS BY SPECKLE-TRACKING ECHOCARDIOGRAPHY OF THE LEFT VENTRICLE RESPONSE TO ISOPROTERENOL IN MALE AND FEMALE MICE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is clear and unambiguous after revision. No further comment.
Experimental design: No further comment.
Validity of the findings: No comment.
Additional comments: For the paper “Segmental analysis by speckle-tracking echocardiography of the left ventricle response to isoproterenol in male and female mice”, the authors addressed my concerns in my review by performing more data analysis and giving more explanation and discussion.  I recommend acceptance for publication.


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: IN SILICO CANDIDATE VARIANT AND GENE IDENTIFICATION USING INBRED MOUSE STRAINS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper presents a tool (MouseFM) that can extract genome-wide all variants that are different between two user specified groups of inbred mouse strains. They downloaded Variant data from Mouse Genomes Project and other public websites and filtered the results.

Major: The bioconductor package doesn’t work and so it could not be tested.
Experimental design: The author needs to provide comparison statistics about MouseFM with others
.
Validity of the findings: They don’t have enough data to support their claim that their tool outperformed others.
The results need to be experimental validated.
No future work was mentioned
Additional comments: Minor:
There is no description for sub-figure 4C.


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: IN SILICO CANDIDATE VARIANT AND GENE IDENTIFICATION USING INBRED MOUSE STRAINS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The presentation of the material is fine
Experimental design: The design and analysis are straight forward
Validity of the findings: Although there is some replication of previously identified candidate genes, there is no way to know if the novel candidates are any thing more that an association. This makes it difficult to tell if the novel findings are meaningful.

The approach seems to be one that is already used and not necessarily novel.

The conclusions are based on assumptions that predictions made through Vep are accurate and this may not always be the case.

The work would be strengthened if there were some independent means to demonstrate that the new fine-mapped SNP are indeed functional.  This is of course a big ask - If the authors pointed out these limitations in the discussion this would certainly be an improvement
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: IN SILICO CANDIDATE VARIANT AND GENE IDENTIFICATION USING INBRED MOUSE STRAINS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The quality of writing is generally good, with a few minor errors, and the article is formatted well. One important omission is that Panel C of figure 4 is not mentioned in the legend.
Experimental design: The aim of the manuscript is well described, and the research gap is pointed out, i.e. that genotypes from large sequencing datasets and large phenotype datasets have not been well integrated.
I have concerns about the methods, and how they were shown to be significant, which I have described in my general comments.
Validity of the findings: The results presented are valid, but I have concerns about how generalizable they are, as outlined in my general comments. The underlying data and code is available, and the R code is easy to use.
Additional comments: Dear Authors,
The manuscript is interesting, and provides a good interface for looking for variants between inbred strains. As the authors point out, there is now good phenotype and genotype data for many of these inbred strains, and these two resources should be taken advantage of. The manuscript is well written, with the authors points being clearly made.

That being said, I have a few concerns that I feel need to be addressed.

It is clear to me that this method works for mendelian traits, such as albinism used by the authors. It is much less clear how well this will work for quantitative traits, especially those that have several causal variants of similar effect size. If it is used as a second-line after QTL mapping (i.e. just looking at between strain differences within the QTL region) then I see how it would work, but as presented, it appears to be looking genome-wide from the start and clustering the strains into two groups, which would simply not work for cases with several large effect loci.

I think rather than just looking at three examples, a more convincing argument would be made if a whole dataset (e.g. all of the JAX mouse phenome data with > X sequenced strains, or a large gene expression dataset) was used.

Linked to this is a concern about power. Given the small number of strains (e.g. for interfrontal bone formation 9 strains) and the large number of variants (up to 74,873,854) how many variants would we expect to see segregating between the two groups by chance? A simple way of testing this would be to look at the number of variants called by your method in each combination of four and five strains, and how many of these meet the criteria that you used to prioritize genes for presence of the interfrontal bone. My worry is that you will get nearly as many candidate variants using a random split of strains as you got by splitting strains by phenotype.

These concerns are somewhat demonstrated in the authors’ Dystrophic cardiac calcification use case. In the previous QTL mapping, four loci were identified, whereas using the current ‘fine mapping’ method, 13 loci were identified. It is unclear to me how the authors prove that the additional 9 loci are not simply false positives.

Another issue is that only SNPs are used. Although these are easier to find in commonly used short-read sequencing data, it leaves all potentially larger variants (e.g. insertions and deletions), that could also alter the phenotypes, unaccounted for. In QTL mapping this is not such an issue, since the markers/SNPs will tag nearby large variants, but in this case, where variants (and therefore genes) are being prioritized based on their predicted effects then this is not the case.

Linked to this, variants are being prioritized based on their protein coding effects (e.g. ‘high’ impact). GWAS data in humans shows the importance of gene expression regulating variants. These variants would be missed by the current method. As a demonstration of this, one wonders how this method would perform in providing candidate variants for gene expression as a phenotype.

Finally, there does not appear to be any consideration of potential kinship between inbred strains. Given the relatedness among many inbred strains, it is possible that situations occur where strains share many variants simply due to this kinship. This becomes increasingly problematic as fewer strains are used for phenotyping. The inverse of this is that if closely related strains show different phenotypes, then there will be fewer variants between the two (and examples of this have been used for reduced complexity crosses). This effect of kinship should be mentioned.

Smaller, minor suggestions are below:

In the abstract, ‘In addition, lately, also’ does not read well, ‘In addition, recently’ would work better.

Line 35: ‘favourable animal model’ should be plural, ‘favourable animal models’

Lines 38-40. It would perhaps be better to refer to this strain as Black 6J. C57BL/6J, C57BL/6N, C57BL/6NJ and C57BL/6NCrl are all sometimes called Black 6, and indeed, you have C57BL/6N among your inbred strains.

Lines 47-50: This idea of in silico fine mapping has certainly been used since then, even if not using that exact phrase. Examples include Hall and Lammert, 2017 (10.1007/978-1-4939-6427-7_21) and Mulligan et al., 2019 (10.3389/fgene.2019.00188).

Line 109: End of parentheses missed

Lines 137: The sentence should start with ‘The’, i.e. ‘The function finemap takes’.

Line 149: “albinisim” should be “albinism”

Figure 4: Panel C is not mentioned in the legend.

Line 166: Should read ‘we applied our algorithm to the phenotype’


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: IN SILICO CANDIDATE VARIANT AND GENE IDENTIFICATION USING INBRED MOUSE STRAINS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Well written throughout, with no major errors.
Experimental design: The aim of the manuscript is well described, and the research gap is pointed out, i.e. that genotypes from large sequencing datasets and large phenotype datasets have not been well integrated.
The authors have addressed or at least acknowledged previous concerns about significance and power, so that they are now clear to the reader.
Validity of the findings: Again, the authors have addressed my concerns abut the validity of the findings, and pointed out the caveats in a way that is clear to the reader.
There is still some question about the novelty of the biological findings (as they are not validated), but I consider this to be secondary to the main point of the paper: the authors have produced an easily used code for looking for variants between strains that they can expand in the future, and shown that it gives useful outputs.
Additional comments: Dear Authors,
I feel that you have addressed the review comments well, in that you have made clear to the reader what the short-comings are, and how to minimize false discoveries.

Smaller, minor suggestions are below:

Lines 62-63: The preprint Ashbrook et al was recently published, PMID: 33472028 https://t.co/gDeKS50ANt?amp=1

Lines 69-70: I think it would read better if the sentence was “Several of the aforementioned resources allow the user to interactively query genotypes for 70 user-selected inbred mouse strains for input genes or genetic regions”.

Lines 70-72: The Variant browser in GeneNetwork has some of this functionality (http://www.genenetwork.org/snp_browser and http://gn1.genenetwork.org/snpbrowser.html), however it does not seem to be programmatic accessible, limiting its usefulness compared to MouseFM.

Line 181: Should read “we used” rather than “we use”.

Expression quantitative trait loci section: It would be interesting to know how many of the novel (i.e. not found in Mostafavi) variants are possibly cis (e.g. with ~5-10Mb of the gene)? I would expect many of the these large effect variants to be in cis. This might be another quality metric, or another way to determine the best candidate gene for expression of that gene.

Lines 263-272: Looking at the Zimmerman et al 2019 paper, it seems that they don't give confidence intervals (e.g. 1.5 LOD drop), but rather just gave the peak position. Eyeballing their QTL maps (without having the data to reanalyze it), it seems like your Chr7, and certainly your Chr11 SNPs overlap with the QTL detected in Zimmerman et al.
To me, it would be a plausible suggestion that you have identified variants underlying those two QTL. It is also possible that the two QTL that you do not see in this study are driven by the other strains that you did not use.

Lines 289-290: It seems odd that you detect the same QTL region on chr7 for Dystrophic cardiac calcification and Interfrontal bone. Is this due to chance? Biological overlap (e.g. something in the calcification/bone pathway)? Or similar strains, and therefore regions that are divergent between the groups appear often?

Line 301: “a setting” is not needed.

Line 310-311: If the data for the eQTL mapping was available, you could redo it, using only your subset of strains, to test this hypothesis.

Line 379-380: the expression datasets are also an example of quantitative traits.


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: RAPID AND COST-EFFECTIVE NUTRIENT CONTENT ANALYSIS OF COTTON LEAVES USING NEAR-INFRARED SPECTROSCOPY (NIRS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript introduced a nice study to use NIR as a rapid tool for the analysis of macronutrients and micronutrients contents of cotton leaves.  The experiment was performed on the leaf samples of three states, Fresh Intact, Fresh Removed, and Dried Ground. The study compared the predictive accuracy of NIR between macronutrients and micronutrients, between fresh and dried leaves. The manuscript was well prepared and easy to follow. NIR analysis of plant leaves for nutrients was done on dried and fresh samples.  This study did both and compared the results, which would be adding new information for the research community.  The discussion on the ability of NIR to predict low-concentration micronutrients in fresh state was relevant and insightful. A few comments for the authors to improve.
Line 109-111.  Recently there are papers on NIRS analysis of plant leafs of field crops such as corn and wheat. Encourage the authors to search for the recent literature.
Table 1 is not easy to understand.  Your description in the text was clear on the numbers of samples from each scanning state. Consider removing Table 1 to avoid redundancy and confusion.
Figures 4, 5 and 7 are not clear to me. First, I cannot understand what “wavelength importance” was and how it was derived. Second, I did not understand “Level of Importance”, First, Second and Third. Could you please add more information in this regard?
Table 3, if I understand correctly, the fresh and removed cotton leafs had 114 samples and dried cotton leafs had 114+146 samples. Did this comparison run only on the 114 common samples for both sets?  Because if not, then the result of favorance to “Dried and Ground” for most leaf attributes could be due to the difference in the datasets rather than the scanning state?
Experimental design: Experimental design is appropriate.
Validity of the findings: Findings are valid and supported by the data presented.
Additional comments: Very good work.  Please see my basic reporting for improvement.


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: RAPID AND COST-EFFECTIVE NUTRIENT CONTENT ANALYSIS OF COTTON LEAVES USING NEAR-INFRARED SPECTROSCOPY (NIRS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Thank you to the authors for the manuscript titled “Rapid and cost-effective nutrient content analysis of cotton leaves using near-infrared spectroscopy”. Overall, I believe that the project conducted is important and it would be helpful to many readers. This is a good, self-contained study that will be helpful for many readers who intend to use near infrared spectroscopy. The study provides a good overview of the literature, the discussion was well-written and the authors have provided the raw data and supplementary information that will also be helpful to readers.

Most of my comments relate to the readability of the manuscript and highlight where some clarity is needed. I suggest that the overall manuscript will need to be heavily edited to improve overall readability of the manuscript and the figures legends. Authors will need to fix grammatical errors and restructure some of the paragraphs. Specifically, many of my comments come from the results section. While the results are informative, I believe that the results do not flow together as there are many “paragraphs” with only one sentence. The manuscript would be greatly improved if authors spend some more time rewriting this section. Furthermore, while the authors reported the R2 for various macro- and micronutrient models, there was no mention of the RMSE for each of the models. Please add the RMSE values for each model as it is important for readers to know the amount of error (RMSE) associated with predicting various traits.
Experimental design: This original primary research manuscript appears to follow the Aims and Scope criteria of PeerJ. The overall research question was well-defined and provides evidence for why this project is needed, both from a scientific and industry view point. Furthermore, it is an interesting paper as the authors have used a less common technique, Cubist regression models, for developing the NIR models. There are some issues regarding the clarity of their methods which I have highlighted in "General comments for the author".
Validity of the findings: The findings of the manuscript seem reasonable given the samples the used and the plant traits they measured. Further, the manuscript provides information on potential issues that can arise when moving from laboratory-based measurements to field-based measurements. This is helpful for readers across many different research fields who wish to use NIR.
Additional comments: I have provided some comments below regarding the readability of the manuscript.

Line 29 – 30. Please edit R2 to either r2 or R2.
Line 35. I am not sure what you mean by the word “effective”. Please consider using a different word or provide some clarification in how it is more effective.
Line 78 – 81. Please consider editing or provide further clarification in the sentence. I am not sure how unreliable measurements of plant available nutrients can be an issue for soil analysis. Is it a separate issue or is there a link that is missing in the sentence?
Line 77 – 88. Overall this paragraph will need some editing from the authors. It is not clear if the methods and issues described relate to measurements on soil or plant. Perhaps add some details to clearly state soil analyses look at overall nutrient content in soil and this overall nutrient content does not necessarily equal the amount of nutrients available for a plant. To make this point clearer, please provide an example(s) of this and include a reference(s). The He et al. 2007 doesn’t seem to best reference for this paragraph. While it does indicate that one can measure nutrients in soil using NIRS technology, it doesn’t highlight that soil nutrient content doesn’t equal nutrient availability to plants.
Line 100 – 102. Consider editing this sentence to make it more concise.
Line 121 – Remove “possibly”.
Line 123 – 124. I am not sure if the last sentence of this paragraph is required.
Line 132. What are nutrient decision support tools, how are they typically used and why is it necessary to link leaf nutrient status information to them?
Line 137/183. Please provide timeline of data collection, from when were leaves collected to when samples were scanned. The sequence of data collection is not clear.
Line 141. Consider inserting paragraph about the spectrometers (Line 195-204) before this paragraph on spectrometer set up and calibration. This would help link set up protocol to actual instrument as this can vary across spectrometer brands.
Line 160. Were the exact same leaves used for the “fresh” scan and then picked for the “fresh and removed” and “dry and ground” scans? If different leaves were used, please include this in your methods and provide a sentence about the comparability of these samples.
Line 169-174. Please revise the order of these sentences and edit for clarity. I assume that petioles were removed prior to grinding and that scanning was performed on the ground leaf samples. Please ensure that the sequence of these sentences reflects this. Line 173 can be interpreted that the petioles were removed then processed and scanned, is this correct or did you analyse the leaf section only? Also, it states “scanned with a similar method to the dried and ground leaf samples” but this section is designated for the dried and ground leaves”. Please clarify.
Line 175. Replace “poured” with spread.
Line 231. Edit to “analysis are shown in Table 2”.
Line 243 – 249. How/why did you choose this particular combination of treatments? Did you try multiple combinations of pretreatments and this performed the best or did you use a specified pretreatment from another project (and if so, please reference).
Line 250. I assume Figure 1 shows the raw NIR spectra and perhaps should be mentioned somewhere in this paragraph.
Line 253. Do you mean “(section 2.3) and the nutrient analysis data (section 2.2)”?
Line 259 – 267. This comment is to highlight that I am not familiar with Cubist regression tree models and so I am unable to provide advice on the set up for this type of analysis. I appreciate that models using partial least squares regression (PLSR) were also developed and compared with the Cubist models. Given that these PLSR models were developed and compared, it would be helpful to provide information on these methods in the supplementary materials. For example, were the same pre-processed spectra used for the two types of models? Also please explain why the Cubist approach was better, for example did you compare R2 and RMSE?
Line 269 – 274. This section of the first sentence makes this paragraph misleading to readers; “the other 25% was used as validation data to assess the predictive accuracy of the model”. In order to assess the predictive accuracy of a model, one wants to see how the model performs on new samples that have not been used to train the model. As soon as validation samples are introduced to the model, they are no longer considered to be independent/validation samples and therefore cannot provide information on the predictive accuracy of the model for new samples. Because of Line 272-73 “The 75:25 data split and calibration processes were repeated 50 times to gain an average...”, I interpret this as the initial validation samples were reintroduced to the train the model at some point during the 50 x 75:25data splits. This resembles cross-validation of the model, rather than predictive accuracy. Please consider rephrasing the paragraph to either a) describe the cross-validation methods and provide reasoning for why there are no validation samples for the model or b) redo the analysis where the 25% validation samples are left aside for prediction tests and never included during model calibration. If option a is chosen, randomly splitting a dataset for cross-validation may not necessarily be the best technique to assess how the model will perform for new samples, it simply tells you how good it can perform for your dataset. To get a better understanding on how it will perform for new samples, it is likely that there is some nested structure in the data set; samples were collected across different days/different plots etc. Splitting up the dataset using nested structure during cross-validation will provide more information on the feasibility of using NIR to assess nutrient content across cotton leaves. For more information on cross-validation and prediction techniques: Au, J. et al. 2020, Sample selection, calibration and validation of models developed from a large dataset of near infrared spectra of tree leaves. Journal of Near Infrared Spectroscopy.
Line 288 – 290. What NIR models were compared and what were the different treatments? Do you mean the different leaf treatments, intact vs dried or do you mean different spectra treatments?
Line 307. Ensure to add a description of a nutrient decision support system and how it is used. This will help the reader better understand the methods in Line 314-319.
Line 323 – 329. To me, this paragraph seems unnecessary but could be useful for other people.
Line 332. What is the “statistical distribution of the average R2 values of the…”
Line 335. What does “used as standards” mean? Is that in reference for comparing the different treatments?
Line 332 – 338. This appears to describe methodology and I don’t believe it was included in the methods section. Please consider rephrasing or moving it to the methods section.
Line 342 – 346. It is not clear if there was only one validation set or multiple validations used in Figure 3.
Line 381 – 383. This looks like it should be in the discussion.
Line 417 - 419. Why isn’t this critical? Should it be introduced in the discussion rather than the results?
Line 424 – 426. This sentence is confusing, please consider rephrasing it.
Line 430 – 434. The numbers appear to be incorrect as 32.7% = high, whereas 42.0% = low. Also, how is the error rate of nutrient classification performed? Was this written in the methods?
Line 436 – 439. I am not sure how “this shows that the error of the models is within an acceptable margin”.
Line 450. If this is an example for the previous paragraph, please keep the sentences together.
Line 533 – 534. Please add this floating sentence to a paragraph.
Line 592 – 593. If known, please provide a reference to support that the prediction of micronutrients is negatively affected by the water in fresh leaves.
Line 665. Please ensure that all references are checked and all follow a consistent format.
Figure 2. It appears that the same example of spectra is shown in Figure 1 and 2. I suggest that this is not necessary and to remove Figure 2b.
Figure 6. It is not clear what “a lower NIR reflectance spectrum” means.
Table 1. If a left-aside validation set is used (i.e. one that is not used for calibration) please add information in this table to describe how many samples were used for calibration vs. validation.


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: PMPARSER AND PMDB: RESOURCES FOR LARGE-SCALE, OPEN STUDIES OF THE BIOMEDICAL LITERATURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Thank you very much for an excellent manuscript describing this useful tool. The language is clear and the reporting is concise.

General comments

The quality of the tool is well described, and as an advanced user of PubMed for text mining applications, the value is also clear. However, I worry that "casual" readers may be a little unsure of the utility, as you offer no use cases. I don't think is necessary to carry out advanced analyses for this short report, but a discussion (beyond the very brief overview in the introduction) of some of the analyses that this tool facilitates would be extremely useful context.

On a related note, I find figures 1 and 2 and the results they show to be a little out of place. They do not seem to be related to the discussion of the tool and I am not convinced that these are relevant results. Is the increasing number of authors relevant to the complexity of the database? Is the relatively low number of ORCID authors obstructing analyses using ORCID?

Table S1 provides an overview of the fields in the database with a very sparse descriptor in the second column. This should be elaborated. Is there any information beyond the metadata listed in table S1? Could one extract citation structures? Co-author structures? Please clarify.

For the uninitiated, it would also be useful with a brief description or recommendation for accessing and analyzing the data in SQL format. I imagine many user will expect either a point-and-click style analysis option, which is of course not an option here (nor is it the purpose).

Minor comments

Line 22: "As biomedical researchers continue to push into the unknown" I think I know what you are trying to say here, but I think this sentence is a bit unclear. What does it mean to "push into the unknown"? I would exactly consider that part of my job description!

Line 29: "The definitive resource for the biomedical literature is PubMed/MEDLINE". I'm not so sure about this. Google Scholar comes to mind as a strong competitor. Perhaps find a different word that "definitive" - no one knows what will happen in 10 years.
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: PMPARSER AND PMDB: RESOURCES FOR LARGE-SCALE, OPEN STUDIES OF THE BIOMEDICAL LITERATURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Schoenbachler and Hughey describes a straightforward method of converting the data in PubMed from its native XML format to a single relational database and augmenting the PubMed data with citation information from the NIH Open Citation Collection (iCite), updated monthly. While all of the data is already publicly available, having a regularly updated data stream that links these data sources may lower the barrier of entry for researchers lacking the resources to process the XML themselves. The manuscript itself is direct, well-written, and adheres to PeerJ standards for article organization.

That said, the authors should address the following issues:

1.	Major issue: The authors are using only some of the citation information from snapshots of the iCite database (according to their code: get_citation.R) but have not included other metrics and metadata, including Relative Citation Ratio and Approximate Potential to Translate. For maximum utility to researchers, it would be beneficial to include the full set of iCite fields in the pipeline.
2.	Minor issue: The authors compare their work to another PubMed parsing project, pubmed_parser, with similar goals and a largely overlapping approach. The authors detail the some of the differences between the projects, but more elaboration would be helpful for understanding when pmparser would be the better option. Additionally, a search for other PubMed database mirrors returned a different python package called “medic” (https://github.com/fnl/medic), which hasn’t been updated for a few years but claims many of the same features as pmparser, including relational database storage and incremental updates. How does it compare to pmparser?
Experimental design: As a project meant to increase the accessibility of biomedical citation information, this manuscript is within the scope of PeerJ’s focus. The experimental design and methods underlying the overall pipeline are described sufficiently in the methods section, however additional description of how the two figures were generated is needed. The first figure needs more clarification that the MEDLINE author limits may be affecting the metrics, and that the results shown may not be an accurate representation of the maximum author count. The second figure appears to be incorrectly labeled: it is not reporting the percentage of authors with an ORCID identifier, but rather the percentage of author names, without the deduplication or disambiguation that would be required to extract unique authors. The methodology behind these figures, including the specific database fields used to generate them, should be better explained in the legend or text.
Validity of the findings: Overall, the pmparser pipeline and code described in this manuscript are well documented. However, the supplemental tables need more information to be useful. The authors present a list of tables and fields stored in the database, but no description of what those fields represent. At a minimum, a description of each of the database tables and data fields are needed to help users make sense of the wealth of analytical options available to them.
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: PMPARSER AND PMDB: RESOURCES FOR LARGE-SCALE, OPEN STUDIES OF THE BIOMEDICAL LITERATURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have adequately addressed reviewer comments in this revision.


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

Input:
Title: THE ASSOCIATION BETWEEN PRIOR PHYSICAL FITNESS AND DEPRESSION IN YOUNG ADULTS DURING THE COVID-19 PANDEMIC—A CROSS-SECTIONAL, RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an original manuscript with no main problems at all. My main concern is the confusion when no detailing OR values.
Experimental design: This is an observational study.
Validity of the findings: The findings are interesting. They add something new since there is nothing related to physical fitness during the pandemic.
Additional comments: This is an interesting manuscript dealing with a hot topic. Indeed, little is known about physical fitness assessments associated to mental health in the Covid-19 pandemic. However, there are still several points that should be addressed.
Abstract
-Indicate percentage of women and mean age of participants
-Why don´t you report ORs for fitter individuals instead of a p value
Introduction
-Lines 102-105. There are prior research showing inverse association between mental health and physical activity during the Covid-19 pandemic, which should be commented, at least, in the introduction. Otherwise, it seems as there has been no revision of the current literature on the topic:
https://www.frontiersin.org/articles/10.3389/fpsyt.2020.00729/full
Later, you can add the comment that your study is the first examining the association between physical fitness and mental health during the pandemic.
-You have stated no hypothesis
Methods
-Some words stating that the sample has been selected by convenience sampling.
-How did you link information of the questionnaire with information from the dataset (fitness)
-I am missing the point in statistics. If you conducted binary logistic regression, you got ORs as a result.
Results
-Lines 196-197. I suggest avoiding interpretations in the results section.
-Line 220. Points or ORs?
Discussion
-Since your sample is not representative, it should be stated the possibility of an information bias.
Tables
-Table 2 values are ORs. Aren´t they? You should indicate this.


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

Input:
Title: THE ASSOCIATION BETWEEN PRIOR PHYSICAL FITNESS AND DEPRESSION IN YOUNG ADULTS DURING THE COVID-19 PANDEMIC—A CROSS-SECTIONAL, RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is generally well written and clear. It is well structured with no fatal flaws. generally is it well references though I question the use of some references in my other comments
Experimental design: The design is generally consistent with the aims. The knowledge gap this study fills is understaed and more could be done to highlight the significance of the study, especially in the introduction
Validity of the findings: All relevant data appear to be provided, however a statistical review is recommended. Some conclusions are overstated and may required revising.
Additional comments: Overall comments
This is a useful study that differs in important ways from other covid-related works. There are some suggestions requiring attention.

Abstract
Conclusions. The second point that improving physical fitness may represent an effective approach to help prevent/reverse depression cannot be confirmed from the available data and should be revised.

L91. This sentence is very long, contains too many elements and should be revised.
L103. Sample studies should be cited on the association between COVID-19 and changes in mental health, being cautions that the timing of data collection, population sampled, social restrictions and other factors vary between studies.

L106. The introduction does not provide a basis to address the first aim of the study, as there are multiple papers already on this topic, and stated limitations of previous work is the single factor approach to fitness. The logic link missing in this introduction is most studies have examined the association between exercise and / or physical activity participation and mental health during the pandemic , NOT fitness and mental health during the pandemic. This latter point is the real novelty of this study, and has not been sufficiently emphasised.

L127. Please explain how written informed consent was provided when data were collected using an online survey, and how data were match between timepoints.

L146. It is not clear if BDI and stress where administered only at baseline, or during May, presumably using the online survey system. Please clarify.

L153. Please provide a reference for the cutpoints for depression severity scores..

L156. Please explain what tools were used to measure health behaviours (exercise/PA, smoking, etc) including the validity and reliability of these measures. Presumably these measures were collected at baseline and in the online survey in May?
L210. Please confirm how normality of distributions were determined
L212. Are the mean depression score here baseline or during pandemic scores? The indication from the heading is during the pandemic but it is not explicit in text. Clearly depression score did not confirm to normal distribution

L274. The description that depression and depressive symptoms are ‘weakening disorders’ is highly stigmatising and are inappropriate. Many people with depression live highly productive lives and make significant contribution to communities. This must be redressed in your manuscript. Symptom severity varies and while at times may impact functional capabilities, it is not constant.

L282. This statement needs references since some studies show the prevalence of various aspects of psychological distress to be not different to ‘normal’ population severity levels.

L301. Please check the reference cited here. Based on the article abstract there is no mention of depression being examined. If this statement is true then you have used second referencing and the primary reference should be cited. Also 1 reference from a single study is not ‘well documented’

L307. The tests of muscular fitness are markedly different (isometric back extensor and handgrip strength) to those used in the present study. Are there studies with more comprehensive evaluations of muscular fitness (not musculoskeletal, which is not the correct term) for comparison?

L324. The authors seem to interchange the terms exercise and physical activity. The definitions are different and the authors must differentiate between them, especially when considering pulmonary capacity and flexibility.

L348. The reference to a average public effect is overstated since the sample is confined to students at 2 Universities in China


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

Input:
Title: THE ASSOCIATION BETWEEN PRIOR PHYSICAL FITNESS AND DEPRESSION IN YOUNG ADULTS DURING THE COVID-19 PANDEMIC—A CROSS-SECTIONAL, RETROSPECTIVE STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an interesting investigating the association between physical fitness during quarantine and depression. Although the clinical importance is well stated, some points need to be addressed and further discussed to make it suitable for publication. The professional use of the English language should be checked by a native English speaker.
Experimental design: 1.	The statistical methods need to be revised.
a.	The authors wrote that ‘The primary outcome of the present study was depression during COVID-19 pandemic’, however, further details need to be provided. Please add if depression would be treated as a categorical or continuous outcome, and the way to define it.
b.	The authors stated that Wilcoxon signed-rank tests were used for assessment in mean difference between sexes of categorical variable. However, this is not appropriate. Wilcoxon signed-rank test is a non-parametric test used to compare two correlated samples. To compare categorical variables between gender, chi-square test or fisher’s exact test should be performed.
c.	The authors mentioned that ‘The independent relationships of prior physical fitness factors and BDI-II depression score or prevalence of depression during the pandemic were assessed using multivariate linear and binary logistics regressions accordingly.’ Firstly, it is suggested that the authors to add the definition of outcome(eg., is the linear regression based on the score change or absolute number), otherwise it is confusing how the models were performed; Secondly, the authors only added ‘prior physical fitness factors’ as covariates, can the authors confirm if physical fitness factors during the COVID-19 was added? Thirdly, if the authors run two sets of models, it should be described separately to avoid confusion. Lastly, ‘logistics’ should be revised as ‘logistic’.
d.	The authors mentioned that ‘a p value <0.20 in the univariate analyses were included in the multivariate linear and binary logistic regression analyses.’ However, no description of univariate analyses were included in statistical section. This should be added.
e.	‘Potential nonlinear effects of decreases versus increases in each variable were evaluated by modeling changes in indicator categories’, here the statement is different from the above ‘prior physical fitness factors were added’. Was  the physical fitness and patient characteristics measured in ‘mean change’ or ‘absolute numbers’? Also the ‘modelling changes’ is not specific enough.
f.	‘multivariate’ should be revised as ‘multivariable’, since multivariable is more used for the analysis with one outcome and multiple independent variables, while multivariate is used for the analysis with more than 1 outcomes and multiple independent variables.
g.	‘a two-tailed alpha level of 0.05 was considered significant’, it is suggested to revised it as ‘was considered to be statistically significant’.
2.	All the ‘binary logistics regressions’ should be revised as ‘logistic regression’ throughout the paper.
3.	It wasn’t clear if the BDI-II was measured once or twice. Was the questionnaire performed only in May 2020? It is suggested for the authors to add a flowchart.
Validity of the findings: 1.	In the abstract-results section, the authors stated that ‘fitness were independently and inversely associated with depression across sexes’. However, this statement is not accurate, when stating something is different across a group, statistically we normally mean the interaction term is significant. Here it seems like the authors actually mean fitness were associated with depression for the overall population, so it is suggested for the authors to revise this statement throughout the paper.
2.	In table 1, for the ‘mean difference’, the direction of calculation should be specified.
3.	In table 2, the coefficients obtained from univariate linear regression model should be reported instead of mean. The digits of p-values should be consistent to 3 digits. Same for table 3 and table 4.
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: THE ASSOCIATION BETWEEN PRIOR PHYSICAL FITNESS AND DEPRESSION IN YOUNG ADULTS DURING THE COVID-19 PANDEMIC—A CROSS-SECTIONAL, RETROSPECTIVE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Tha authors have properly addressed my requests.
Experimental design: It is fine the way it is.
Validity of the findings: This has been improved.
Additional comments: Tha authors have properly addressed my requests.