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 CHARACTERIZATION AND EXPRESSION OF THE TLP GENE FAMILY ASSOCIATED WITH COLLETOTRICHUM GLOEOSPORIOIDES INOCULATION IN FRAGARIA × ANANASSA
Review round: 1
Reviewer: 3

Output:
Basic reporting: It is nit clear why this specific study was needed? What specific knowledge gap existed?
Experimental design: Methods are not described with sufficient detail.
Validity of the findings: No comment.
Additional comments: Manuscript is well written and authors have demonstrated good methodology in addressing the research object. However, there are several issues that the authors need to address before this manuscript can be published. Here are my comments:
- Line 72 - 77 (Paragraph 4 sentence 1 to 3) - Here, just before the Objectives statement, there should be a concise Rationale statement (Why this specific study was needed?). What specific knowledge gap existed?
- Line 77 to 80 (paragraph 4 sentence 4 to 6) - This reads like “Materials and methods” when it should be a statement of objectives.
- Line 123 – 126 – preparation of C. gloeosporioides cultures, this is not correct as stated in this section, kindly revise this method. Revise this section clearly. It is not clear in what form was the C. gloeosporioides provided. The culture medium is supposed to be potato dextrose broth.
- Line 128 – What was the basis of using 60-day old plants as the age of plants for fungal inoculation?
- Line 130 - Control plants were inoculated with sterile water – spraying with sterile water cannot be referred to as inoculation. In addition, why did the authors use sterile water instead of spraying with non-pathogenic C. gloeosporioides as controls? The authors would have autoclaved the C. gloeosporioides suspensions and use as controls.
- Significant sections of the methods of the MS are profoundly unclear
- Lines 175 – 177 - What was the basis of selecting these plant species Arabidopsis thaliana, Oryza sativa, and Fragaria vesca to determine the evolutionary relationship of TLP gene families.
- Lines 272 – 277 - So, what new have you discovered then? You are basically confirming what others have already found. This corroboration with a previously study is not that important.
- Lines 280 – 286 – This is literature review and not discussion of your results. This section should be transferred to the introduction section.
- Lines 303 – 305 - What are the implications of all this in the control C. gloeosporioides? The information you are talking about is already present in Figures. Discussion of implications is more important.
- Lines 312 – 314 – You are not sure. This is speculation! We need to know the implications of the findings for the control C. gloeosporioides program.
- In the Discussion, you have been emphasizing more on your agreement with other studies than on the novelty of your own findings. How does your agreeing with others help your program to control C. gloeosporioides? You should highlight what new you have discovered.

- Lines 323 – 324 – The statement is not correct since the authors did not determine differences in TLP gene expression patterns among different resistant strawberry cultivars. It was between one susceptible and one resistant.
- The quality of figures are low and needs to be revised.


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 CHARACTERIZATION AND EXPRESSION OF THE TLP GENE FAMILY ASSOCIATED WITH COLLETOTRICHUM GLOEOSPORIOIDES INOCULATION IN FRAGARIA × ANANASSA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript contributes to the knowledge of C. gloeosporioides infection in F×ananassa. The introduction and discussion section is improved as compared to the earlier version. Appropriate information about the earlier study is presented well for the readers to follow the rationale and findings of the study.
Experimental design: The experimental design for this manuscript is robust and acceptable for publication
Validity of the findings: The results presented in this manuscript will help in understanding the role of TLP against C. gloeosporioides.
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: GENOME-WIDE CHARACTERIZATION AND EXPRESSION OF THE TLP GENE FAMILY ASSOCIATED WITH COLLETOTRICHUM GLOEOSPORIOIDES INOCULATION IN FRAGARIA × ANANASSA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have have made the necessary changes as advised in the previous version.
Experimental design: The experimental design has been added in more detail now and is satisfactory.
Validity of the findings: The comments and required references have been incorporated in the discussion part.
Additional comments: The authors have done a good job of revising the manuscripts as per the instructions.


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: DEVELOPMENT OF A JAPANESE VERSION OF THE PSYCHOLOGICAL OWNERSHIP SCALE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The structure of the article is required further improvement.
Experimental design: Research question is meaningful, and methods are sufficient.
Validity of the findings: Conclusions were well stated, however, limitations were not well written.
Additional comments: Thank you for the opportunity to review your manuscript.
Authors conducted development and validation of the very important questionnaire.
I would appreciate some clarification on following issues.

Many contents are described in different sections.
Ex. Study procedure or ethical comments were written in discussion. You need to note the things about material and methods in the ‘Methods’ instead of ‘Discussion’.

Introduction
Line 165-185
These contents should be included in the method.

Material and Methods
It was not described statistical analyses at all like statistical soft, statistical criteria. The criteria to judge the validity and reliability as satisfied also should be noted here.

Results
You need to explain other demographic data like Income, job, and education including missing value. You used the crowdsourcing service, including people have various background.

Line 292-322
Basically, statistical analyses conducted in the research are required to explain in the method prior to results. Many statistical words are found firstly found in Results, not introducing in Methods such as KMO, Bartlett, Cronbach’s α, AGFI, and so on.

P294-296
The following description is not ‘Results’, but ‘Methods’.
the usage of principal components analysis

Line 306-308
The following description is not ‘Results’, but ‘Methods’.
Exploratory factor analysis, KMO, and Bartlett’s test

Discussion
Line 358-542
In discussion section, following contents are included.
materials and methods, participants and design, materials, measurements, procedures, results, discussion, general discussion.
This made readers confusing. Ex. Material and methods section should be material methods section not discussion section. Thorough amendments should be needed to be easily to read.
You should focus on the content of discussion and conclusion.

Line 325-351 Validity testing
You firstly need to explain about how to prove the validity and reliability testing of your questionnaire in the Methods.
For example, validity testing contains content validity, criterion/criterion-referenced validity, construct validity such as convergent validity and discriminant validity.

Yang, H., Shi, L., Lebrun, L. A., Zhou, X., Liu, J., & Wang, H. (2013). Development of the Chinese primary care assessment tool: data quality and measurement properties. International journal for quality in health care, 25(1), 92-105.
Dullie, Luckson, et al. "Development and validation of a Malawian version of the primary care assessment tool." BMC family practice 19.1 (2018): 1-11.

Line 353 Test-retest reliability
Which correlations analysis was selected in these analyses?
You should also explain in the Methods.

Line 359
You need to say how you prove the high level of reliability

Line 374-391
These contents are mostly ‘Methods’ or ‘Results’.

Line 394-397
This section is discussion. Mistake? Why do you describe here?
In addition, ethical contents were already described in the previous ‘Methods’ section, too.

Line 400-406
These are mostly ‘Results’ contents like participants, missing value. Sample size and exclusion criteria are ‘Methods’.

Line 409-412
These are ‘Methods’ since this is a procedure you adopted how to measure.

Line 415-419
This needs to be noted in the ‘Results’.

Line 421-434
Some descriptions are in the ‘Methods’ like how participants look and evaluate the blanket for the hepatic imagery and how to evaluate, others are in the ‘Results’ like the number of participants.

Line 437-451
These descriptions should be noted in the ‘Results’. Based on the ‘Results’, you need to discuss.

Line500-505
Please discuss the failure which your results are different from previous studies more. For example, participants’ recruitment.

Limitation
Authors should also describe about the limitation such as selection biases and generalizability.
Ex. Authors conducted this study using crowdsourcing service.


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: DEVELOPMENT OF A JAPANESE VERSION OF THE PSYCHOLOGICAL OWNERSHIP SCALE
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this article, the authors developed and validated a Japanese version of the well-established Psychological Ownership Scale in two studies focusing on owned and non-owned objects, respectively.
In my opinion, the article is very well-written, and the quality of the English is excellent. Tables are clear and raw data shared.

I have some remarks/suggestions for the introduction:

Line 41:  I'm not convinced that psychological ownership is exclusively recognized by the individual and not by society, please clarify

Lines 66-68: Is there any causal relationship between the self and owned object evaluation?. I see that correlational evidence is reported. Please clarify

Lines 73-76: The superior evaluation of owned vs. others object is modulated by the evaluation of oneself and cultural factors? please clarify

Line 89-911: The authors cite the study by Peck & Schu (2009) on the role of touch in object ownership. In my opinion, it would be helpful to provide a brief explanation of why this is the case.

Section: Purpose of the study:

In my opinion, this section should start by stating the purposes of the study. I see that the authors report them starting from line 151. Please consider restructuring this section
Experimental design: The research question is clear, and the authors provide a solid background for the need to develop a validated instrument in the Japanese population

The methodology and the statistical approach sound, and the results are reported in detail and accurately.

I have one suggestion. Please report the nationality of participants (although I assume they were all Japanese?)
Validity of the findings: The authors performed confirmatory and controlled analyses, showing the validity of their results.
About the impact, this article may be useful for subsequent studies conducted in Japan  as a validated scale is here provided
Additional comments: no 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: METAGENOMIC INVESTIGATION OF THE EQUINE FAECAL MICROBIOME REVEALS EXTENSIVE TAXONOMIC DIVERSITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dear editor and authors,

This is a novel and interesting study that adds to the current knowledge on the intestinal microbiota of horses. I have a few suggestions to improve this already good study.

Introduction gives too much emphasis on the importance of the horse, but lacks a deeper justification on the rationale and on the importance of the methods used in the current study. Authors should describe in more details the limitations of short reads sequencing and how the use of more comprehensive methods have aided the characterization of the microbiota of other environments such as soil and the human intestines.

While the discussion is extremely concise, many parts of the results are actually discussing and interpreting results. I recommend you to combine those two sessions.

Interesting aspects of the study deserve more attention, as the inter-horse variability, as evidenced in Figure 2.

>65% of reads were classified as unassigned. Most studies using V4 region find around 10% of unclassified bacteria that are frequently deemed to truly unknown organisms, but impossible to distinguish from sequencing errors. How likely your results are due to limitations of the methods used in this study?

Other limitations of the study need to be discussed. Maybe I am wrong, but I learned that sequencing based genome assemblies needed to be confirmed by culture based studies to prove the existence of those species. Isn’t necessary to culture a bacteria before you can definitively name it?
Experimental design: The study design is adequate to support the conclusions. I am not a bioinformatician, but data analysis seems to be performed by an expert in the field.
Validity of the findings: This is the first study using shotgun sequencing to analyze the equine microbiome and therefore is extremelly novel. Conclusions are supported by the results, but the article lacks further discussions about the possible limitations.
Additional comments: Other suggestions:
Line 86: please rephrase “reveal nothing about”
Line 90: Please consult a recent study related to this study by DiPietro et al. (2021):
Animals 2021, 11(10), 2859; https://doi.org/10.3390/ani11102859
Line 99: please define “same location”. Same barn, same county, etc.?
Line 102: because this is the first study of this nature, it is important to provide more details on factors influencing the microbiota to be used as comparison for future studies. For example, the time of the year, the type of pasture, any supplementation the foals were receiving, level of exercise, deworming, etc.
Line 103: how samples were shipped? How long between sampling and freezing?
Line 104: what do you mean with “samples were mixed”? Maybe homogenized would be better to avoid the impression of pooling.
Line 213: It is intuitive that the study is showing novelty, but why unexpected diversity? Are you actually showed higher diversity than previous studies?
Line 218: were the most abundant phyla found in this order?
Figure 1: the names in the phylogenetic tree are impossible to read.


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: METAGENOMIC INVESTIGATION OF THE EQUINE FAECAL MICROBIOME REVEALS EXTENSIVE TAXONOMIC DIVERSITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Gilroy et al. presents findings on the microbial community composition in horse gut/fecal microbiome. The authors evaluated the community compositions using shotgun metagenomics and assembled over 200 microbial and bacteriophage MAGs from five horse gut microbiome samples.
Overall, the manuscript is well written, and provides sufficient context for the work, knowledge gap being addressed, with suitable references where required. Language is clear for the reader to follow.
Raw data has been deposited in the SRA and accession numbers are provided.

The authors use the phrasing metagenomic species and MAGs interchangeably to reference the dereplicated metagenome assembled genomes. This makes the text a little difficult to follow at times, so I would recommend selecting one and using that throughout.
While the authors have extensively evaluated the taxonomic breakdown of the microbial community in the equine gut microbiome, the functional evaluation appears to be a bit preliminary and not comprehensive. The study could benefit from a more thorough assessment of the functional gene profiles of the microbes present in the gut microbiome to better understand the functional contribution of the microbes to equine health and fitness. It is also unclear what the motivation was to look into viral diversity and if there is apriori information on their importance in equine gut microbiomes, so additional background/references on this would be useful to include.

Line 242: The authors mention that the clustered MAGs span ten phyla. This is inconsistent with the referenced figure which shows just 8 phyla.
Line 245 can be rephrased.
Line 247: It is unclear to a reader who may not be familiar with GTDB what alphanumerical designations mean and/or its significance.
Line 264 & 265: Unclear which part of the figure represents this result.
Line 272: Missing citation for referenced study

Fig 1: The figure caption mentions “Hemicellulose substrates have been clustered and highlighted in red text” but there is no text highlighted in the figure, so it is unclear what is being referenced.
Fig 2a: Caption mentions abundance of MAGs, but the data displayed is relative abundance of MAGs in the community, so I would recommend rephrasing this.
Fig 2b: It is unclear how the species accumulation curve was constructed (methods are sparse)..
Fig 2c: Axis and legend can be more descriptive.
Supplementary Table 1 provides a summary of sequenced samples and the number of MAGs obtained from each. However, some of the numbers do not add up as a reader might expect e.g.  columns R, S & T should add up to the total no. of bins (column Q). However, this is not the case since only bins above a certain threshold have been categorized. A better description of the columns/column headers would be helpful for the reader.
Supplementary Table 3: It is unclear what column Y represents. Descriptive column headers would be helpful.
Experimental design: The experiment is well designed to answer the questions of microbial community structure and function in equine gut microbiomes.
The authors have described the methods comprehensively to allow easy replication of the work.
The taxonomic evaluation performed here was based on just shotgun metagenomic sequencing and binning to retrieve metagenome assembled genomes (MAGs). A summary of  how many total MAGs were retrieved from each sample, before dereplication, and what proportion of total reads assembled into MAGs is not included in the main text. This would be good information to provide to the reader and provides context on how comprehensively the sample was sequenced. (This is only available in the supplementary table – may consider moving Table S1 to main text.)
A few points where additional clarity may help:
-	Fecal microbiota may be influenced by diet. It is unclear if all the horses sampled were fed the same kind (and which) diet. This can be clarified in the text.
-	Unclear if any kind of host DNA removal was performed
-	The authors may consider using an alternative reference database to run their Kraken taxonomic classification against (E.g. maxikraken2 https://lomanlab.github.io/mockcommunity/mc_databases.html). Since a large fraction of the reads remained unclassified using the RefSeq database, the use of a more comprehensive database comprised of sequences from non representative genomes and environmental isolates would help with improved classification of the raw reads.
-	The authors compute and assign a quality score to the MAGs. May be good to describe the significance of that and why that is an additional criterion used, in the text.
- MAG level coverage is reported, however it would be useful to also report relative abundances of the MAGs in the different samples for sample-specific trends.
-	Method used to compute species distribution and data used to generate Fig 2b is unclear.
-	It is unclear which contigs were included in the the viral contig classification. This should be clarified, since it is not clear if the MAG contigs were excluded from this analysis.
Validity of the findings: Overall, I found the evaluation and description of the results from the MAGs to be not comprehensive.
This study and data generated provides ample opportunity to assess how many of the most/least abundant taxonomic lineages the authors were able to retrieve good quality genomes for, and if there were sample specific trends to what was captured in the bins. Additional evaluation of the lower quality bins could have provided additional hypotheses into why they the bins were prone to higher contamination (increased strain-level diversity?).
The results were heavy on assigning Candidatus names to the MAGs but did not go into much detail to describe trends in observed functional potential of the MAGs and how they could contribute to equine health/fitness.
In Line 281, a newly named class is described which does not contain representatives from vertebrate gut in NCBI. It would be very intriguing to see trends in relative abundance of this microbe in the different samples, and additional description of the functional potential of this microbe and how it compares to publicly available representatives.
The results of bacteriophage abundance in the samples is interesting. It would be useful to elucidate why they observed varying trends in phage abundance between the samples (E4 has the greatest number of phages, E5 has the least). What are the different sample specific factors that could contribute to this?
The authors acknowledge that a detailed evaluation has not been performed in this study, however there are several unaddressed points that would be useful to elaborate on, if the goal of the manuscript is to provide a detailed report of the taxonomic and more specifically functional diversity of equine gut microbiomes.
Additional comments: I would recommend additional assessment of the functional diversity of these MAGs to complete the story since the manuscript is currently heavy on just providing an overview of the taxonomic diversity and doesn’t delve much into functional potential.


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: METAGENOMIC INVESTIGATION OF THE EQUINE FAECAL MICROBIOME REVEALS EXTENSIVE TAXONOMIC DIVERSITY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, this manuscript was very well-written and easy to follow. The language is nearly perfect with only minor grammatical errors and sometimes confusing syntax. I have listed these instances below.
Statements are well-cited and sufficient background information is provided. The article, figures, and tables are professionally structured and complete with raw data available. As no other literature, that I am aware of, regarding the equine gastrointestinal metagenome have yet been published, this article fills an important knowledge-gap. Though primarily descriptive, versus hypothesis-driven or mechanistic, given the scope of their work, I find this a valuable contribution to the field.

Please see suggestions for improvements below, as organized by line number:
-	Line 59-60: “spread of human populations” - The point is understood but I believe this could be phrased differently to more clearly highlight your point. Perhaps “extension of human settlement”
-	Line 64 - The jump from discussing equine therapy to slaughter is a bit jarring, I would recommend separating out these two ideas.
-	Line 75 – The way this sentence is structured implies that the same number of human pathogens are just as likely to be harbored in the equine gut as equine pathogens, which is not the case. I would instead qualify human pathogens “equine and several human pathogens”
-	Line 103 – Please define "bijous" for those not familiar
-	Line 129 and 130 – Please define or describe SAM and BAM files for those not familiar
-	Line 230 – Update tense, change show to showed
-	Line 245 – Species is redundant in this line:  ‘fourteen species of bacterial species’ – instead ‘fourteen of the bacterial species’?
-	Line 263 – Missing end of parentheses
-	Line 295 – Better characterize ‘each consecutive sample’ – unclear if this is multiple samples from one horse or each horse sample processed in succession
-	Line 326 – Correction needed of "who’s" to "whose"

Figures:
- Figure 1a – Obviously challenging with so much information to include, but the text is very small in phylogenic tree. Is it possible to change layout so it is more legible? Also, it was unclear why the number of phyla included on the graphic (8) versus that stated in the text (10) were different -  please address this discrepancy.
-	Figure 2 – Please try to enlarge text, for all subsets of the figure, if possible.

-	Supplemental:
- Line 74-76: The  syntax and punctuation of this section is confusing, please reformat to make more clear.
- Section beginning Line 569/Description of Candidatus Darwinibacterium: missing closed end of parentheses
- Section beginning line 589 - also missing closing parentheses.
- Line 1052 – Italicized ‘this’ – please provide more explanation if intentional
- Section beginning line 1321 – phrasing and sentence structure is confusing for Candidatus Treponema, please reorganize to make more clear (similar issue to sections beginning line 569 and 589)
- Section beginning line 1331 – Similarly unclear syntax and appears misplaced period in line 1337.
- Section beginning	 line 1342 – Same issues as above. Please revise this section into complete and more clear sentences
- Section beginning line 1352 – Same issues as above re syntax and punctuation.
Experimental design: This paper fits well within the Aims and Scope of the journal. The exploratory research questions is well-defined, relevant, and as the first assessment of its kind, meaningful. This application of metagenomics to the equine fecal microbiome definitely fills a knowledge gap. The metagenomic methods applied are rigorous and sufficient detail is provided such that these steps are reproducible.
Validity of the findings: All underlying data have been provided. A larger sample size, as always, would have added significance to the findings but the cost of metagenomics makes this limitation understandable.
The conclusions are well-stated and generally supported though I do find them slightly over-reaching in one area (see comment below). Otherwise, findings are presented very well.

Suggested Improvement:
-	Line 358 – I believe this statement is a bit over-reaching. Instead I would update to "... preliminary census of the horse gut microbiome specific to yearling thoroughbreds"
Additional comments: I enjoyed reading this manuscript. While I was not surprised to read that the equine fecal microbiome is very diverse and home to many yet-to-be-characterized species, this group's demonstration of these facts using advanced sequencing is a very useful addition to the equine gut microbiome research field. The writing is clear with only minor grammatical and syntax suggestions. The figures are understandable and applicable, though are a bit difficult to read in their current 'print' format and thus could benefit, as much as possible, from slight adjustments to text size. Thank you for the opportunity to review this manuscript and please let me know if there is any further information I can provide.


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: METAGENOMIC INVESTIGATION OF THE EQUINE FAECAL MICROBIOME REVEALS EXTENSIVE TAXONOMIC DIVERSITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: All my questions have been answered.
Experimental design: The experimental design was not changed.
Validity of the findings: The same as previous.
Additional comments: No 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: METAGENOMIC INVESTIGATION OF THE EQUINE FAECAL MICROBIOME REVEALS EXTENSIVE TAXONOMIC DIVERSITY
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have addressed the comments provided in the first round of review. One of the biggest points raised was that the manuscript did not provide a comprehensive assessment of the functional potential of the equine gut microbiome. The authors have clarified this and the manuscript has been restructured to focus on a comprehensive description of the taxonomic diversity. This makes the manuscript more focused and sets up the study as a resource for future exploration in this field.
I have one additional clarification - relative abundances of each of the obtained MAGs in the respective samples are provided in Table S7. Relative abundances add up to values > 1. This seems incorrect and should probably be double checked and/or clarified and results in line 497 corrected accordingly.


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: METAGENOMIC INVESTIGATION OF THE EQUINE FAECAL MICROBIOME REVEALS EXTENSIVE TAXONOMIC DIVERSITY
Review round: 2
Reviewer: 3

Output:
Basic reporting: I enjoyed reading this revised manuscript and believe that the suggested revisions have been well-integrated. The writing is now perfect apart from one missed comma (see below) and very easy to follow. References and background information have been enhanced for this revision as have the figures, tables, and general structure of the paper. The raw data remains accessible and the results are relevant to the exploratory hypothesis.

Suggested change:
Line 90: Suggest changing "vast only superficially" to "vast, only superficially"
Experimental design: This paper continues to fit well within the Aims and Scope of the journal. The exploratory research questions is well-defined, relevant, and, as the first assessment of its kind, meaningful. The metagenomic methods have been enhanced, in response to other reviewers' comments and, with additional detail regarding methodology added to this version, the work is reproducible.
Validity of the findings: These findings, and their processing, are valid and accessible to those who may wish to apply their own evaluations.
Additional comments: I thank the authors for their revisions and congratulate them on the work they have done. No further 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: REFERENCE VALUES FOR BONE DENSITY AND BONE MINERAL CONTENT FROM 5 TO 80 YEARS OLD IN A PROVINCE OF CHILE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Much of my review includes editorial revisions (i.e., statements that can be streamlined, unclear, grammatically incorrect phrasing etc.) as well as some more major issues in terms of discussion of findings. See below for details:

This manuscript “Reference values for bone density and bone mineral content from 50 to 80 years in the province of Chile” offers important and much-needed insight onto skeletal health across global populations, for which there are limited data available. Reference values that are regionally specific are crucial for guiding health policy and practice that target the population in question. The sample size in this manuscript is massive, across many critical age cohorts, and though this is a cross-sectional study, it sheds light on patterns of bone accrual and loss across the life course. Ultimately, this manuscript can make an important contribution to skeletal health literature, in addition to the website they created which may serve as a useful reference for international scholars.

That being said, there are major revisions must be made before being considered for publication. Most of the important revisions that I suggest are particular to the framing, results and discussion. This includes but is not limited to: 1) Framing the importance of this study more directly (i.e., emphasizing why local population-specific references are needed); 2) Performing a statistical analysis that directly compares US and Chilean mean values for BMC and BMD; 3) Clearly defining BMC and BMD in the manuscript; and 4) Streamlining the discussion to more clearly reflect the results of this study. Specifics as well as other suggested revisions are highlighted below:

INTRODUCTION:
•	First paragraph:  This opening paragraph is framed in a way that suggests children and adolescents are going to be the main focus of this paper. Yet this paper doesn’t emphasize subadults any more than adults. I think a more compelling way to open the paper is to note the importance of regionally/population specific reference values and the dearth of this information for Chilean and South American populations more broadly.
o	The authors may also highlight that there are relatively few studies that examine skeletal health, using the same methodology, across subadults and adults. I think this is one of the most valuable contributions of this current manuscript.

•	Line 56-57: The terms bone mineral content and bone mineral density need to be defined early on in this paper in the same place. Here BMC is mentioned but not BMD; why not?
o	 Also the sentence beginning with “This stage…” is inaccurate in that growth is a process rather than a stage. Please clarify.

•	Line 60: It’s better to be specific and note that accumulation of bone density and content establishes the foundation for skeletal health throughout the lifespan. Simply stating that these are “keys for skeletal health” is vague.

•	Line 62-65: Consider revising the wording here. Suggestion: “Furthermore, research indicates that more than half of the peak bone mass…”
o	Also, I understand the term “maximum bone mass” but it’s more commonly referred to as “peak bone mass” (PBM). Moreover, the abbreviation for maximum bone mass (MBM) is introduced in the discussion in line 265. The abbreviation should be introduced first here, in the beginning of the manuscript.
o	The last sentence beginning with “In addition,…” is confusing and it may be worthwhile to revise without the 7 to 11%. Suggestion: “The remainder of adult bone mass is acquired after linear growth ceases”

•	Line 66-70: Consider revising the wording here as it can be simplified for clarity. Suggestion “The specific age at peak bone mass achievement is unclear as data are inconsistent”.
o	The statement that other studies “argue that [peak bone mass] is reached in an individual’s third decade or beyond” is not a contrasting result from the previous sentence where the authors note peak bone mass is achieved between 25-30 years old. 25-30 years old is the third decade of life. So consider revising this sentence (line 69). Suggestion: “However, other studies conclude that peak bone mass may be reached beyond the third decade of life”.

•	Line 71-74: This seems out of place and should be moved to the beginning of the paper when peak/maximum bone mass is first mentioned (right after line 65).

•	Line 75: delete “the” before maximum bone mass; also, instead of “independent of the age where it presents” which is a confusing statement, consider revising to “regardless of age of achievement”

•	Line 77-78. Instead of “from one individual to another”, consider revising to “between individuals” to reduce wordiness. More importantly, the authors mention “distinct patterns according to age and sex” of bone loss. An example should be presented here such as (for i.e., female loss is accelerated compared to male loss) or something similar. I realize the authors can’t present all the patterns here but one example, at least, is warranted.

•	Line 84-87: Instead of “threats to bone mineral measurements”, consider “disruptions to skeletal health (BMD, BMC). Again, these need to be defined earlier in the paper. Furthermore, if bone area is not directly studied as a variable in this paper, it would be good to remove it from this paragraph altogether.

o	The sentence beginning with “The importance of assessing these variables during growth…” is not clear and is excessively wordy (which may be why it’s unclear). Consider revising to more simply say “Examining these variables across the life course may be critical for understanding and attenuating fracture risk in advanced ages”.

•	Line 88-92: I don’t think this paragraph is needed. It doesn’t add anything to the manuscript. I think the important thing to note is that there are these international references available but local, regionally-specific or population-specific references are more valuable for guiding health policy and practice.

•	Line 95-97: It’s unclear what the authors mean in this sentence beginning with “However, it seems that…” In particular, what is meant by “a higher proportion of reference values for pediatric populations”.  Again, I think it’s necessary to emphasize that local references are ideal because of the heterogeneity of global bodies (for example US NHANES data isn’t ideal for use globally). And moreover, the data in this current manuscript sheds light onto skeletal health across the lifespan.

•	Line 100-101. A bit more expansion is needed here – in particular, a description of the demographic transition here. The assumption is that Chile is characterized by an expanding aging population and a shrinking subadult population but this needs to be stated.

METHODS
•	Line 115: Is 41,200 the number of people in the province or the study population from which a randomized sample was pulled? Figure 2 states that this number is the “total population of the province” but in this line, it’s not stated as such. Please clarify.

•	Line 117-118: It would be helpful to give age ranges for the categories of participants. For example, what age range are participants considered “children”, “adolescents” etc. And moreover, what does “youth” include and how is that different from “children and adolescents”? This term youth is not specific enough and likely redundant.

•	Lines 128-132: I don’t think it’s necessary to include Chile’s HDI here. If it must be included, provide a reference for understanding the index of 0.847. Is this high? Is this low?

o	The education system is described as being divided into four phases yet three are listed, unless a comma is missing: early childhood, primary media (is the comma missing here?), and higher education? Although ultimately, I don’t think this information is entirely necessary in the manuscript.

•	Line 135-136. How was nationality determined? By passport? Did participants self identify? Were all participants born in Chile? Please elaborate briefly on this.

•	Line 138-139: Delete the zero before the number of children and adults excluded from the study (i.e., 4 children, 6 adults).

•	Line 166: Provide ages for what qualifies as pediatric populations (i.e, < 18 or < 21 years old).

•	Line 175: What is a test-re-test? I think these two sentences can be combined and rephrased as: “To ensure quality control for the variables collected, 10% of the total number of participants were evaluated twice for both anthropometry and DXA scans.”

•	Line 187: This should be reworded as “Differences between sexes” and not genders.

•	Line 193: Regarding R2, the 2 should be a superscript: R2

RESULTS
•	Line 208-209: The authors can simply state from 13 to 80 years old (and not 13 to 70-80).
o	Also, consider revising the wording here to simplify. Suggestion “…females had lower BMC compared to males. No significant differences in BMI between sexes were identified.”

•	Line 212-214. The comparison between Chilean females and US females in this sentence is not clearly described. A few words are missing (i.e., the mean values for BMD were relatively similar compared to US females?)
o	Also, there should be a statistical test included in the methods that compares the US and Chilean means across the life course.

•	Line 221-223. I just want to confirm that the age at peak bone mass achievement is the same age for both BMD and BMC in males and females. So males achieve peak bone mineral density AND content at age 30 and females at age 28 for both variables?

•	Line 225-227: Consider revising these sentences for clarity: Suggestion “…followed by a plateau with no significant gains or losses until 40 years old. After age 40, BMD and BMC undergo declines that continue through to advanced age.”

•	Line 229-232: These sentences can be streamlined. Suggestion: “The values for BMD in both sexes were similar from age 5 to 13 after which males have a higher median compared to females. This difference persists until advanced age. However, males have higher BMC values compared to females across all ages with the greatest difference manifesting at 10 years old and persisting until 80 years old.”

DISCUSSION:
•	Line 237-239. Consider revising for clarity and succinctness. Suggestion: “The results from this study demonstrate that Chilean males from Maule have marginally higher BMC and BMD values compared to US populations, based on NHANES data while Chilean females display similar values to that of US females.”
o	In addition to revising wording for clarity, it is important to repeat that no statistical test of these differences were presented in the results and this should be remedied.

•	Line 242-249: The entire first paragraph of the discussion compares BMC and BMD values from this study to other studies with no mention of a) the methodology of these studies (i.e., were these all DXA based studies?) And b) no statistical analysis performed to confirm these comparisons. While I don’t think statistical comparisons are needed for all of the references mentioned, at the minimum, some statistical comparison between this study population and the US NHANES is warranted.

•	Line 250- 258. These two sentences can be combined and streamlined. Suggestion: “These findings are consistent with other research that have reported population differences in BMD and BMC across the life course.”

o	This sentence can be followed up with this suggested revision “Differences in skeletal health values across diverse geographical and ethnic populations may be due to various factors including diet, nutritional status….etc.”
o	Also, do the authors mean “timing of” sexual maturation? What do they mean by “sexual maturation” as a factor related to skeletal health?

•	Line 265: As mentioned earlier, MBM or preferably PBM should be introduced as an abbreviation in the beginning of the manuscript.

•	Line 265: Here, the authors state that MBM is determined from maximum BMD. However, this needs to be stated earlier on in the manuscript, in the methodology, or in the introduction. Currently MBM is defined in lines 71-74 as a broad term encompassing mass, density, micro-architecture etc. but then gets distilled as maximum BMD in this paper. So a justification needs to be made for this, however, brief, that here, maximum bone mass is really maximum bone density.

•	Line 270-272. Instead of just stating that MBM in this study was similar to US, Mexican, and Singaporean populations, providing those ages identified in these studies would be helpful (as they do in the next sentence).

o	Instead of beginning the sentence with “Although, evidently, other research studies have” consider revising to: “Other research studies have reported younger age at peak bone mass achievement as young as 18-20 years old (Nakavachara et al., 2014, Yi et al., 2014, van Der Sluis et al., 2002)”
o	Also, please clarify if the above studies used DXA scans.

•	Lines 275-278: Can the authors state why a specific age at MBM achievement is important? Line 277-278 begins to highlight the importance but falls short. Why are these values important for informing preventative approaches for bone loss and osteoporosis? This needs to be made more clear throughout.

•	Line 279-282: The abbreviation PMO is introduced without any explanation or previous use in the manuscript.

o	This sentence is redundant and needs to be streamlined altogether.

•	Line 283-287: These sentences are very confusing and come across as rambling. Please clarify – eliminating some words and stating simply that finding ways to maximize bone mass by 10% could benefit older adults and could reduce the risk of fractures and heightened health care costs substantially.

•	Line 288: Remove the words “In this context” as it doesn’t make sense following the last paragraph. Simply beginning with “Recent studies have suggested…” would suffice.

•	Line 294: The statement “Therefore, it is necessary to improve lifestyles” is a vague, broad, and not really helpful conclusion. I would delete this altogether as specific modifiable factors have already been presented. Also, it would be useful to emphasize here that ameliorating bone health across the life course is a multi-factorial endeavor, with no single factor driving bone loss and gain.

•	Line 296-299: This paragraph begins by discussion the percentiles determined in the study and then switches focus to environmental factors and ends with “these need to be permanently monitored”. None of these statements make sense here – more consideration of the percentiles presented is warranted (for some reason lines 305-309 elaborate on the percentiles but this section can be moved up here). Lines 297-299 can be deleted altogether as they don’t contribute anything new to the discussion.

•	Line 303-304: Osteopenia should be defined briefly here (i.e., low bone mass) as well as osteoporosis.

•	Line 310-314: Again, this section is rambling and needs some focus. Consider revising some of the wording here. Suggestion: “The pattern observed in this study is consistent with other research suggesting the importance in monitoring BMD and BMC across the life course, particularly during early growth and development when bone mass is rapidly being accrued”.

•	Line 315-318: This should be reframed as “DXA-based population references for BMD and BMC are scare not only for Chile, but South America in general” as I know of a few QUS based references for South American populations.

•	Line 323-325: Please qualify the statement “This is the first cross-sectional study to be carried out in Chile as well as South America”. First, do the authors mean “DXA-based skeletal health study”? Keep in mind that I am aware of a number of non-DXA based bone health studies from South America, as well as a few DXA studies. So, this statement needs to be expanded upon.

•	Line 326: Why use the words “It is possible that these results may be used for Chilean populations”. If I understand this correctly the authors mean that these data can be useful for Chilean populations more broadly – I’d eliminate the words “it is possible” in this statement because clearly local references are more applicable than international references.

•	Line 330-332: I’m not clear about what the website is presenting based on the manuscript description here. Is this website for participants to see their results? Or for people interested in seeing where their values fall in relation to the study participants? Please clarify the utility of this website (which has value!) in the manuscript.

•	Line 333 – 339: This is an awkward beginning to a sentence. The authors simply need to state that ideally, bone mass across the life course would be studied longitudinally.

o	Include the sample size for males between 30-80 vs. females if it is going to be mentioned as a limitation to this study.
o	The sentence beginning with “As well, it was not possible to evaluate some variables…” should be moved up where right after “…identify causal relationships”.

FIGURES
•	In figure 2, the total number of excluded participants is indicated as 104 although the numbers added up equals 103.

TABLES
•	The tables have a lot of data in them and are visually hard to track – however one thing that can be easily addressed is emphasizing MALE and FEMALE in the table by bolding the words or finding some other way to emphasize these sections.

ABSTRACT
•	In the first sentence of the background, “fractures” risk should be “fracture” risk.
•	In the results and discussion section, instead of “Discrepancies” consider revising to: “Differences between Chilean and US male mean values were identified for BMD and BMC.” Or something similar to streamline the sentence.
Experimental design: As indicated in my response to 1) most of the important revisions that I suggest are particular to the framing, results and discussion. This includes but is not limited to:  1) Framing the importance of this study more directly (i.e., emphasizing why local population-specific references are needed); 2) Performing a statistical analysis that directly compares US and Chilean mean values for BMC and BMD; 3) Clearly defining BMC and BMD in the manuscript; and 4) Streamlining the discussion to more clearly reflect the results of this study.
Validity of the findings: The findings are sound but a more clear, streamlined discussion of the results is warranted. Additionally, a statistical analysis of the US and Chilean mean values for BMC and BMD should be included rather than simply stating "they look similar".
Additional comments: see other sections for specific details.


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: REFERENCE VALUES FOR BONE DENSITY AND BONE MINERAL CONTENT FROM 5 TO 80 YEARS OLD IN A PROVINCE OF CHILE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The aim of the study is clear
The design is adequate
Population is large enough and well stratified
Experimental design: It is well designed and population stratified
Validity of the findings: Validity of the findings are right
I have only two observations
1 . Individuals between 5 and 25 years of age should be correlated with Tanner score of pubertal development rather than with chronological age
2 BMD and peak bone mass should be correlated with calcium intake and nutritional data .
Additional comments: It is a good study
My observations are
1 BMD should be correlated with Tanner score of pubertal development in individuals between 5 and 25 years of age
2 BMD should be correlated with calcium intake and nutritional data


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

Input:
Title: A FLEXIBLE AND ACCURATE METHOD FOR ELECTROENCEPHALOGRAPHY RHYTHMS EXTRACTION BASED ON CIRCULANT SINGULAR SPECTRUM ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Comments included in the attached file.
Experimental design: Comments included in the attached file.
Validity of the findings: Comments included in the attached file.
Additional comments: Comments included in the attached file.


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

Input:
Title: A FLEXIBLE AND ACCURATE METHOD FOR ELECTROENCEPHALOGRAPHY RHYTHMS EXTRACTION BASED ON CIRCULANT SINGULAR SPECTRUM ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study, authors have applied circulant singular spectrum analysis method to decompose an electroencephalography (EEG) signal in to a set of orthogonal reconstructed components, then the reconstructed components are then grouped flexibly to extract the desired EEG rhythms based on the known frequencies.

Simulated EEG data and real EEG data have been used to test the performance of this addressed method, which is better than basic singular spectrum analysis method, the wavelet decomposition method, and the infinite impulse response filtering method.
Experimental design: The simulated EEG and real EEG data with the closed and open eys states have been tested.
Validity of the findings: The comparison with other methods has been used to valid the performance of this proposed method.
Additional comments: No comments.


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

Input:
Title: A FLEXIBLE AND ACCURATE METHOD FOR ELECTROENCEPHALOGRAPHY RHYTHMS EXTRACTION BASED ON CIRCULANT SINGULAR SPECTRUM ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an interesting manuscript on using time-delay embedding to isolate spectral components in a signal. This is not the first application of decomposing time-delay embedded matrices for neural signal processing, but it does provide some new information that could be useful. Below are my comments.

The authors repeatedly claim that the extracted rhythms do not include mixing from other frequencies or artifacts. I don't really understand the significance of the first point -- band-pass filtering also excludes other frequencies (within the range of the passband frequencies and filter transition zones, etc). Furthermore, how does the CSSA method exclude artifacts? Broadband artifacts still have energy in, e.g., the alpha band, and some artifacts such a blinks are concentrated in low frequencies such as delta/theta. All of the methods I'm aware of that isolate narrowband activity while suppressing artifacts are spatial multivariate methods that leverage the linearly dissociable spatial distribution of artifacts vs. signal. That does not seem to be the case here.

Please add some information about how to set L. Setting it arbitrarily to 40 seems like a bad idea, because it depends entirely on the data sampling rate and the desired bandwidth. I can imagine two approaches: (1) Use eq 9 to get L = Fs/Fb. So then users can specify their desired bandwidth to obtain L. (2) Define L based on the number of cycles at the desired center frequency. For example, the center of alpha is 10 Hz, and having, e.g., two cycles at 10 Hz would give a decent spectral estimation (if the SNR is sufficient), so then L should be at least 200 for a sampling rate of 1 kHz. Thinking about it this way, L=40 with Fs=1000 would lead to only .4 of one cycle of alpha, which isn't enough to get any reliable results.

I don't think that the comparison shown in Figure 5 is fair. The bandwidth of CSSA was narrower than the bandwidths of the IIR and wavelet filters. So it's trivial that the filters will pass through energy at a wider frequency range, which here leads to mixing. A narrower filter (and probably a better one, e.g., FIR) would give (I guess) comparable performance to CSSA. By the same token, if the authors had simulated the data to have a wider alpha spectral peak but used the same L parameter, then IIR would have given more accurate results because the CSSA bandwidth would have been too narrow.
And I don't understand what's happening with the wavelet in panel B. The wavelet appears to pass through frequencies from 4-10 Hz, so obviously it will miss the alpha. It's an inappropriate filter, so what's the point of showing it here?

Same comment for Figure 9: The wavelet is passing through a strange collection of frequencies. This is not the appropriate way to do wavelet convolution in electrophysiology data; a useful wavelet would have narrowband characteristics, e.g., a Morlet or Gabor wavelet.

I have to admit, I was more enthusiastic about the study from the intro/methods than when seeing the results. This results appear to show that CSSA is effectively a narrowband filter. I don't think this should preclude publication, but I think the authors need to include a more detailed description of the implications, possible uses, and advantages of CSSA over a carefully designed narrowband filter, which is more commonly done in neuroscience, and which is also computationally more efficient.
Experimental design: .
Validity of the findings: .
Additional comments: No additional comments


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

Input:
Title: A FLEXIBLE AND ACCURATE METHOD FOR ELECTROENCEPHALOGRAPHY RHYTHMS EXTRACTION BASED ON CIRCULANT SINGULAR SPECTRUM ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Comments included in the attached file.
Experimental design: Comments included in the attached file.
Validity of the findings: Comments included in the attached file.
Additional comments: Comments included in the attached file.


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

Input:
Title: A FLEXIBLE AND ACCURATE METHOD FOR ELECTROENCEPHALOGRAPHY RHYTHMS EXTRACTION BASED ON CIRCULANT SINGULAR SPECTRUM ANALYSIS
Review round: 2
Reviewer: 2

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: MULTILINGUAL VALIDATION OF THE SHORT FORM OF THE UNESP-BOTUCATU FELINE PAIN SCALE (UFEPS-SF)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The report by Luna and colleagues is clear and thorough and provides a wealth of references to the appropriate literature.
Experimental design: The methods are well-defined and rigorous. There is a lack of statistical details in the results and one is forced to read through the figure captions for this information. Some key details should be placed into the Results section. The breadth of the analyses is impressive, but most can be placed into the Supplemental section. Eight figures and twelve tables is too many for a journal article and dilutes the findings.
Validity of the findings: The findings are valid and can add to the assessment of pain-like behavior in cats across cultures and languages. The work can be quite impactful and has been rigorously tested.
Additional comments: 1. There is an error in the Abstract: "...were randomly and evaluated.." seems to be missing a word.
2. The addition of the names and affiliation for the committee organized for content validation seems unnecessary and irrelevant. It can simply be stated that experts in the field from multiple institutions provided feedback.
3. In the Video Analysis section, lines 156 and 165 both state that the wound area was covered at all time points to avoid bias. Only one mention is needed.
4. As a more general comment, there is considerable disagreement as to the use of the term "pain". The most updated definition from IASP includes a note that verbal communication is not necessary, thus including non-human animals as experiencing pain. However, if in the M1 period, a cat is scored as showing pain when there is no reason to expect that pain is being experienced, it may be worth considering a statement that the scale assesses pain-related behaviors. In other non-human animal scales, it is understood that the closing of eyes or lack of body movement is a normal behavior that can be exacerbated by pain, but is not, in itself, a definitive sign of pain.


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: MULTILINGUAL VALIDATION OF THE SHORT FORM OF THE UNESP-BOTUCATU FELINE PAIN SCALE (UFEPS-SF)
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, Luna et al. present data demonstrating the validity of a recently developed feline acute pain scale in multiple languages. It represents a valuable addition to the literature as the recognition of feline pain is arguably substandard worldwide.
The article is well written and I have no real concerns in terms of the quality of the writing, the narrative or the article structure. I would urge the authors to consider reducing the numbers of figures and tables somewhat but including some the data as supplemental instead. The sheer number of figures and tables is overwhelming and perhaps distracting. I agree the data should be presented but most of the figures and some of the tables could be supplemental and simply referred to in the text.
Experimental design: The design of this study uses well accepted statistical techniques for validation of behavioural pain scales. The methods are well described and all relevant data is presented.
I would like to see clarification that all evaluators were viewing the videos for the first time as part of this study. If they had viewed these videos specifically beforehand, or perhaps used them extensively for education purposes then this would invalidate the results due to lack of blinding. Can this be clarified in the text?
Validity of the findings: The conclusions are supported by the findings and overall the scale performs admirably across different languages. Major potential issues with the study design are discussed well (e.g. the rationale for the use of video recordings)
The authors admit themselves in the discussion that they have only used 1 evaluator per language rather than the recommended 3. In addition all these evaluators are highly experienced. The validity of the scale may not be as good when used by a broader population of veterinarians who may well be less experienced and 'pain-savy'. While the authors already make this clear, I would like to see a mention of the validity being confined to experienced observers in the abstract and conclusions as I feel this represents an important limitation on the validity of the results.
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: RAPIDHRV: AN OPEN-SOURCE TOOLBOX FOR EXTRACTING HEART RATE AND HEART RATE VARIABILITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: -	The introduction should go directly to the point (HRV analysis and physiological signal processing, without reviewing the HRV literature in itself).
-	The authors should mention the neuroKit2 toolbox and probably cite the following paper, as it is directly related to this area:
Pham, T., Lau, Z. J., Chen, S. H. A., & Makowski, D. (2021). Heart Rate Variability in Psychology: A Review of HRV Indices and an Analysis Tutorial. Sensors, 21(12), 3998. https://doi.org/10.3390/s21123998

-	Describe how does the k-mean clustering could help with P and T wave contamination.
-	What do the parameters “liberal”, “moderate” and “conservative” refer to? What are the parameters affected?
Experimental design: This paper does not report experimental results but uses open datasets to test the performances of the new methods that were introduced. I think this is nicely done, but I have concerns regarding the way it is tested.

- It is not clear to me that the different tests detailed in the paper demonstrate the accuracy of the package. The authors often state that they found “excellent” or “good-to-excellent” agreements, but these statements are not grounded on model comparison. Measuring the error of the estimate (eg. RMSE) and comparing this with other toolboxes, or ground truth might be more appropriate.
- One of the originalities of this toolbox is a new peaks detection algorithm based on clustering. The performance of this algorithm should be directly compared to the state-of-the-art algorithms for the same task (see e.g. https://github.com/berndporr/py-ecg-detectors for ECG). As other tools are already provided in the Python ecosystem, it would be relevant here to compare the PPG performances with NeuroKit2 and/or Systole, as it is not clear for now that this pipeline performs better, we just see that it is not performing badly.
Validity of the findings: This paper introduces a new open-sourced Python pipeline to perform heart rate variability analyses in an easy and reproducible way. Heart rate variability is widely used in psychology, physiology, and neuroscience in general and, as Python itself is gaining popularity in these fields, this contribution will certainly find a large range of potential users. There are some alternatives already available in the same area. This toolbox introduces original components but it might be too limited to be considered as a package in itself. The paper also details several analyses that are interesting from a method point of view, but the pipeline proposed is debatable and not really tested against other algorithms.
I think the following points that concern the novelty of this work, and the way the package is tested should be addressed before this paper can be considered for publication.

- This paper introduces rapidHRV as a heart rate variability toolbox, however, the range of metrics this package can compute is extremely limited. I understand that the HRV literature already incorporates hundreds of mathematical derivations of variability, and including all of them in every package might not be necessary. But some gold standards are critically missing (e.g. frequency domain and non-linear domain). The authors should point to other packages that can be used to extract such features, as only extracting the RMSSD from a given recording might appear extremely limiting. It also questions the name of the package itself, as rapidHRV would suggest something oriented toward heart rate variability in general, this module could be renamed rapidRMSSD? I do not think that the package should be renamed, I think its scope should be enlarged.
Additional comments: Some of my comments concern the codebase in itself (as it can be found in https://github.com/peterakirk/RapidHRV):
-	I am a bit concern that this package does not include any test suite (as far as I can see). I think this is the main problem, for now. The code should be tested, ideally, this should be performed automatically using Github action or something similar (TravisCI …). The code coverage should be estimated from this test suite and should be maximized.
-	Providing example data set that can be automatically downloaded using some function would make it easier to test the code and run tutorials (right now, running the code snippet from the Readme returns an error as the variable `mydata` is not defined).
-	It would be convenient to have tutorials provided as notebooks, making it run on Google Colab can also increase readability and help users test the package before installing it.
-	The code should follow clear formatting rules (flake8, black…). Providing type hints and static checks can be a plus.
-	The install requires the exact version of packages, which is a bit brutal for the user and probably not necessary, I would recommend version >= something instead wherever possible.
-	The API could be simplified (e.g. rapidhrv.preprocess.preprocess could be shortened into rapidhrv.preprocess for import)


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: RAPIDHRV: AN OPEN-SOURCE TOOLBOX FOR EXTRACTING HEART RATE AND HEART RATE VARIABILITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript by Kirk and colleagues presents and describes RapidHRV as an open-source Python toolbox for extracting heart rate and time-domain heart rate variability measures. It does so by introducing the background and the motivation of the package. The authors also present different testing scenarios, using simulated and real datasets against different signal-to-noise ratios and different sampling rates, and with signals of different modalities (ECG, PPG, and pulse oximetry).
I think this toolbox is within the scope of PeerJ and will be of interest to the readers even though some issues would need to be addressed before publication. I recommend "major revision" to have the possibility to read the modified version. As part of this review, I have looked at the source code of the package in the GitHub repository, tried to install and run the example scripts. You'll find below my suggestions and questions. Note that authors are free to not take them into an account provided they explain why.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: General comments:

•	I was able to install the package and run the example found in the GitHub repository. I commend the authors for putting together the documentation for the functions and the arguments in the docstrings which greatly help with the readability of the code. However, I have a few comments about the styling of the code and the documentation which can be found below.

•	While the authors did briefly mention other toolboxes for HRV in the introduction (line 51), they have missed out on some toolboxes that are also open-source and have been developed to rigorously work with noisy data such as:
o	pyHRV: https://github.com/PGomes92/pyhrv
o	NeuroKit2: https://github.com/neuropsychology/NeuroKit

I recommend the authors further elaborate on the advantages of RapidHRV compared to these existing packages (heartPy, pyHRV, NeuroKit2, hrv) and how the proposed pipeline in RapidHRV would work more rigorously with data from wearable devices. I understand that doing a benchmarking study against these packages would be a lot of work and might be out of scope for this manuscript. However, it would be good to acknowledge the existing packages, how are they different/similar to RapidHRV, and perhaps discuss a few features that are uniquely RapidHRV (e.g., clustering of QRS waves).

Major issues:
•	K-mean clustering to discern different QRS features seems to be a unique feature of RapidHRV. I would recommend the authors to illustrate with an example when and how it can be useful. If there is literature to support this step, do include it too. And if adding an example would disrupt the flow of the current manuscript, the authors can do so in the GitHub repository and refer to the example accordingly.

•	As the toolbox specifically focuses on the time-domain features, I believe that having only 3 measures (BPM, RMSSD, and SDNN) would significantly limit the usage of the package. Perhaps the authors can discuss a future plan to include more of them? If you are looking for a review of different HRV indices, you can consider our recent work: https://www.mdpi.com/1424-8220/21/12/3998, which includes an exhaustive list of time-domain measures. Nevertheless, please feel free to refer to any other suitable works.

•	“Time from the first peak to the last peak does not recede 50% of the fixed window width.  This is to ensure the user that the actual length of time for extracting BPM/RMSSD is not less than half of that which is specified in the window width argument.” (line 152). The relevance and usefulness of this statistical constraint are not as clear as the others. Could the authors elaborate a little more?

•	Comments about the software:
o	Unit-test to maintain package: currently, the functions are not being tested (using pytest or Travis). Having the functions tested and benchmarked helps to maintain the stability of the package (e.g., functions can break due to updates in other dependencies). Thus, the authors are recommended to add basic tests, at least for the three main functions, to ensure the quality of the code.
o	Plot legends in visualization: authors should add matplotlib legends to specify to users what do the “X”, the black, grey, and purple dashed lines represent in the plot. We were able to read Figure 3, thanks to its legend. However, for your future users, plot legends should be added so that the information in the plot can be conveyed clearly.

Minor issues:

•	While the normalization steps in the manuscript are described as
“Normalization is performed at the level of a sliding window during the analysis stage (‘extract_heart’ module), wherein the mean signal is subtracted from each data point in a given window, then multiplied by 100. Finally, this is divided by the range of values (maximum - minimum data point in the window). “,
the source code in the GitHub repository for normalization is:
“(data - np.min(data)) * 100 / (np.max(data) - np.min(data))”.
There seems to be a discrepancy here. It seems like the authors would like to perform the min-max normalization (as described in the source code) and had made a typo in the manuscript.

•	Since normalization is a step performed in the analysis stage, maybe the authors could consider describing them in the later paragraph, instead of having them together with the pre-processing steps (line 79).

•	The caption of Table 2 should specify what is the value inside and outside of the brackets.

•	Code styling and readability:
o	For code readability, authors should consider following the PEP guidelines (https://www.python.org/dev/peps/pep-0008/). For instance, underscores can be used in the argument names to separate the words (e.g., `input_data` instead of `inputdata`). Nevertheless, this is a minor comment about styling and needs not to be addressed by the authors for the manuscript to be considered for publication.
o	Authors should also follow the standard recommendations for readable docstring syntax. The common ones are google docstrings (https://google.github.io/styleguide/pyguide.html#38-comments-and-docstrings) or numpydoc (https://numpydoc.readthedocs.io/en/latest/format.html). Again, this comment about styling is to help the package to have better readability and thus should not be critical for the decision 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: RAPIDHRV: AN OPEN-SOURCE TOOLBOX FOR EXTRACTING HEART RATE AND HEART RATE VARIABILITY
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 made all the changes that were requested in the first review and I do not have additional comments to make on the current version of the paper.

I would only recommend checking the citations one last time before publishing (e.g. the Pandas citation is not the one recommended on the website).

Congratulation to the authors for their work and their efforts in the review process.


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

Input:
Title: NOVEL DECORATING BEHAVIOUR OF SILK RETREATS IN A CHALLENGING HABITAT
Review round: 1
Reviewer: 1

Output:
Basic reporting: I really enjoyed reading this manuscript. It was clearly written, well organized, and well referenced. The only place I thought might need more references was lines 70-73 to support those statements.
Experimental design: This is an observational study to learn more about the retreat building and foraging behavior of a newly discovered species of jumping spider. The extensive observations and measurements were appropriate to achieve this goal.
Validity of the findings: I loved learning more about these spiders and how they survive on smooth tree bark. Most of the conclusions reached in the Discussion section are consistent with the data presented. The only conclusion that I was less convinced of was "spiders were not transient inhabitants" (line 303). While I agree that the data is supportive of the conclusion that the spiders live and forage on exposed trunk surfaces, it still seems possible that they could move among trees. Even jumping spiders could balloon, or could crawl on the ground to a nearby tree.
Additional comments: Fun read!


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

Input:
Title: NOVEL DECORATING BEHAVIOUR OF SILK RETREATS IN A CHALLENGING HABITAT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript “Novel decorating behaviour of silk retreats in a challenging habitat” describes the retreat building of the jumping spider A. mullion and provides details of their life history based on observational data. In general, the manuscript can benefit from some re-writing (see bellow and line comments).

The abstract could benefit from a reduction in the amount of background information and the addition of more detail regarding the work and main findings. I suggest you condense the first 3-4 sentences (lines 10-19), state your hypothesis, and add the main findings.

Introduction lacks focus and the research question is not well defined. The concept of habitat structural complexity should be mentioned in the second paragraph, as it stands there is an abrupt change of topics with no linkage. I suggest rewording the examples of orb-web spiders that build retreats in terms of habitat complexity. Are these webs in a highly complex environment and thus building a retreat increases survival or allows for expansion into more complex habitats. More detail on the species used in the study will be a good addition in the introduction and the section of the methods can be shortened. Why was this species chosen? Distribution? Other than it resides on a particular tree? Is it the only species of jumping spiders on the tree? Such description could be added in line 79.

The Discussion can benefit from more cited work as well as more emphasis on why the current work is significant to the reader. There should also be a discussion regarding the role of trunk scrapping in retreat decoration.

The conclusion section reads like the last paragraph of the discussion instead of a conclusion. The conclusion should summarize the work, major findings and how they support your hypothesis, etc.
Experimental design: While the findings are novel and interesting to the scientific community, the research question is not well defined. As mentioned elsewhere, the introduction and the abstract need to be reworded to clearly state the hypothesis and how the data is/or not supporting it.
Validity of the findings: See other comments.
Additional comments: After the first use of the species name, abbreviate the genus name. Check the manuscript for consistency. To aid with clarity, please use commas to separate three or more items. Figure 1: Species names should be in italics

Video S1: If possible, I would suggest that the video is presented in full instead of cropped to see the complete sequence in retreat construction. You can change the video speed to match the desired length.

Line Comments:
Line 53: If the common name for spiders in the genus Seothyra is known please include it (e.g. sand-dwelling/spoor).

Line 61: comma after flexibility

Line 61-64: this sentence is confusing as written. Please revise and rewrite. Maybe something like: “Considering the functionality, flexibility, and broad benefits of web building, it is surprising that some groups have abandoned capture webs and instead hunt by vision and ambush (e.g. jumping spiders).” I suggest the edit because technically speaking active hunting spiders have not lost the ability to build webs, they still have the machinery (silk-producing glands) to do so.

Line 65-66: replace parenthesis with commas

Line 68: replace “Because” with “Given the astounding acute vision of jumping spiders,”

Line 71: what other types of active outbound journeys are known for spiders? If none, consider explicitly stating searching for prey and mates.

Line 71: Can you provide a reference for this statement as well as more detail? Which jumping spiders? Or generalize the statement to say that there are jumping spiders that do build silk retreats.

Line 79: add (Zabka, 2002) after the species name

Line 125: change “was” for “were”

Line 132: add “R” after conducted. Alternatively, the sentence can be written as: “All statistical analyses, including log transformations to meet statistical assumptions, were conducted in R (R Core Team, 2021).”

Line 136: The species of spiders should be included. It is unclear what type of spiders were used for the comparison.

Line 149: why were the measurements restricted to 300 to 700 nm?

Line 157: comma after “decorate”

Line 181: add “(Video S1)” to the end of the paragraph.

Line 190: Is there a time frame for retreat construction? An average time should be added here.

Line 201: Remain the reader of the importance of the observations by including that this is a species of jumping spiders. Something like: “The jumping spider A. mullion spend their entire life on the surface of tree trunks. Unlike other salticids, A. mullion spiders stayed in their silken retreat and were rarely … trunks.”

Line 210: use instead of “run”

Lines 233-236: these sentences are confusing to me, and I think they would benefit from a rewrite. You recorded 94 retreats in 2018 with 1 new retreat added in the second survey? Add the date for clarity. Being more explicit about the dates could help clear this confusion. For example: The variation in retreat persistence was 47-82%, but for which dates? All surveys? Half? You could combine the decline of retreat persistence (8.75% from XX) to coinciding with tree shedding (Lines 235-238).

Lines 254- 260: Spectral reflectance section. As per my previous comment, the reason why the wavelengths of 300 and 700 nm were chosen needs to be included. Is this change in mean reflectance significant?

Line 268: Would it be appropriate to label each retreat entrance to note differences in usage?

Line 293: The discussion would benefit from a few introductory sentences reminding the reader what the paper is about and why the work is significant. I would suggest adding details such as the fact that A. mullion is a jumping spider, part of one of the largest families of spiders. Jumping spiders usually have a cursorial lifestyle and are active predators instead of a sit and wait, salticids are known for jumping at prey/or as defense, hence the name. They are known for their large eyes and great vision which makes them very good active diurnal hunters. Some of these details should also be included in the introduction.

Line 297: add “and” before 3)

Lines 203- 317: Are there trade-offs that could account for A. mullion strategy such as size, prey abundance, predation, clutch size, survival rate, etc.? Are they the only species of spiders to use this tactic? Only salticid? Why is living on the tree trunks and relying on silk retreats significant to A. mullion?

Line 378: Should Stephanopsis by Stephanopis? Also, make sure to use italics font.

Line 421: this statement needs a citation and maybe an example.


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: MUTATION BREEDING OF ASPERGILLUS NIGER BY ATMOSPHERIC ROOM TEMPERATURE PLASMA TO ENHANCE PHOSPHORUS SOLUBILIZATION ABILITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is readable and technically correct except for the following errors which should be corrected.

L332-333 there is no the data of xj180–22. And “xj190–32”? Is it xj90–32?

L343-344 What is the difference compared with others? Please point it out.
Experimental design: This manuscript is suitable for publication for its research questions are well defined and meaningful. However the following questions should be answered before publication.

L77-79 Silva U et al. (PLoS ONE, 2014. doi.org/10.1371/journal.pone.0110246) have showed UV mutagenesis can also improve the P-solubilizing of Aspergillus niger. Please explain why you choose ARTP not UV.

L110 Please write down the ARTP parameters, for instance temperature, helium flow rate and irradiation distance. It is very important for this study.

L119-120 What are the standards for the preliminary screening? The author did not describe it clearly. The size of halo zone of dissolving phosphate? Please describe it in detail.

L226-227 Generally, we think the lethality rate of 50~60% is better and we can get more positive strains. However, the results showed that the positive mutation rate was the highest when the lethal rate was 37%. Please explain the reason for this phenomenon.

L319-320 P-solubilizing abilities of xj180–22 is better than xj120–12 under different influencing factors. Why is xj120–12 better for the biomass of peanut plant than xj180–22?
Validity of the findings: It is meaningful for this manuscript to find a fungi xj120–12 to be used as a dominant fungi with the potential of P solubilization and growth promotion and provide the basis for the production of P solubilization fungi fertilizer.
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: MUTATION BREEDING OF ASPERGILLUS NIGER BY ATMOSPHERIC ROOM TEMPERATURE PLASMA TO ENHANCE PHOSPHORUS SOLUBILIZATION ABILITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.This article text is quite professional English language used throughout. Please check Line 388 : fungi fertilizer should be fungal fertilizer?
Structure conforms to PeerJ standards, discipline norm, or improved for clarity.
Figures are relevant, high quality, well labelled & described.
2. Your introduction needs more detail. I suggest that you improve at lines 49 by adding ref Kuna et al. 2021, Frontiers in Microbiology about the Aspergillus section Nigre for mineral solubilizing activities to provide more information in general properties.

3.Reference This part is very weak need to improve much more for consistency of writing pattern according to Journal rule.
1.	Scientific name need to be in italic  ex L396, L472, L486, L488, L490,L517,  L535
2.	Journal name should be in full and not in italic. Check how to write journal name.
3.	Title of article : check constancy of writing. Only the beginning of sentence that first letter is capital the other word is in small letter
Experimental design: Original primary research within Scope of the journal.
Research question well defined, relevant& meaningful. It is stated how the research fills an identified knowledge gap. Rigorous investigation performed to ahigh technical & ethical standard.
Methods described with sufficient detail & information to replicate.
Question 1. Author wrote in methodology L 183 add Ca3(PO4)2 1:1000  this is by weight or by volume ratio?
Question 2: Line 184   author use inoculum at 106 CFU why not use 1x108 CFUg-1  as state before?
Validity of the findings: Impact and novelty are provided.
All underlying data have been provided; they are robust, statistically sound, & controlled.
Conclusions are well stated, linked to original research question & limited tosupporting results
Additional comments: The manuscript is clearly written in professional, unambiguous language. If there is a weakness, it is in the writing of  References pattern (as I have noted above) which should be improved upon before Acceptance


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: E-VOLVE: UNDERSTANDING THE IMPACT OF MUTATIONS IN SARS-COV-2 VARIANTS SPIKE PROTEIN ON ANTIBODIES AND ACE2 AFFINITY THROUGH PATTERNS OF CHEMICAL INTERACTIONS AT PROTEIN INTERFACES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English language in the manuscript should be improved to ensure that your research is clearly understood and appreciated by the international audience. I have listed specific details in my comments below but overall, the introduction, result, and conclusion sections need major rewriting due to the use of ambiguous English language. I suggest the author contact a professional editing service to assist them. This will add great value to this research manuscript.

Detailed comments:
Authors should provide an Abstract which is unified and concisely reports the research problem, methods, key results, and conclusion. Currently, authors have segmented each of these sections as a substitute for a more succinct and clearer abstract.

Please reframe the paragraph starting from lines 27-32 Page 7 it's not clear and difficult to follow for the reader.

Line 35-36 Please clarify what do you mean by electrical alterations? Please re-frame.

Line 40 page 7  "this effect by changes at the mutant’s dynamics equilibrium" authors should re-write this paragraph as it's unclear what the author meant by “mutant’s dynamics equilibrium”.

The use of the word "empirically" by the authors doesn’t seem relevant. It is often used for citing other researchers' work in the manuscript. Authors should use “as observed” or provide more details as to why they think other cited studies are empirical in nature. One such example is Line 376, page 16 where authors are citing work by Wang et al. 2021.
I thank the authors for providing relevant detailed figures however in the case of the delta variant protein-protein interaction authors should provide a figure similar to Figure 4 as supplementary material.

Please place the references in order as they appear in the manuscript. Example: Line 49 page 8 reference (Hu et al. 2021) is mentioned but can't be found in the reference list.
Please provide a reference for Line 26 page 7 written in the Abstract
Page 8 Line 56-59 Please provide relevant references for this statement.
Line 91 page 8 Authors should use the full form of ACE2 (Angiotensin-Converting Enzyme 2) at the earlier occurrence of ACE2 in the manuscript (introduction section).

Line 413 page 17 special characters "฀?1'/?฀2’loop" are not displayed properly in the text. Please correct this across the entire manuscript

Line 109 page 9 Supplementary Table S1 provided by the authors doesn’t provide any descriptions for the list of PDB's, please provide a description for these PDBs.

Authors should choose one nomenclature for the mutants like alpha, beta, delta and consistently use that in the manuscripts rather than switching between nomenclatures B1.1.X vs Greek names of the variants.
Please provide high-quality 300 DPI images to be included in the manuscript. Example: The set of images in Figure 1 are hard to read, and gets blurred when zoomed in.
Experimental design: Line 116 page 9 please provide a reference for the MODELLER methodology or describe it briefly in the manuscript’s Material and Method section.

Line 274-280 Page 13 Authors should elaborate on the TM-score and describe the methodology they have employed to generate the heat maps. For example, it was not clear how a “previously generated database” (constituents of this database are not detailed) is employed to emphasize the impact of mutations on protein-protein interactions?
Validity of the findings: Line 333 Page 15 "Y501/Y41 bond, hydrophobic interaction in the spike-ACE2 complex" contradicts with Figure 3B where Y501/Y14 interaction is shown. Authors should clarify which interaction is correct Y501/Y14 or Y501/Y41 and make changes either in the text or figure.

In Figure 5A mobility around the 417 position doesn’t seem to be significant compared to nearby positions, authors should clarify or provide more evidence to support their statement made regarding the mobility around 417 positions (Line 421 Page 17)

I would like the authors to comment on the higher mobility observed around the 438-450 residues for the P1 (gamma mutant) compared to wildtype (Figure 5B) and its implication on the conformational state of the protein.

I would recommend that the authors report only the observed results in the Result section of the paper and elaborate their findings in more detail in the discussion section. Currently, it’s not the case making it difficult for the readers to focus on the key findings.
Additional comments: In this manuscript the authors have demonstrated a web-based tool named "E-Volve", to systematically analyze interchain interactions given a PDB identifier, list of mutations, chain pairs as inputs. The tool evaluates protein-protein interaction using a contact comparison strategy. Authors have used E-Volve to analyze variants of SARS-CoV-2 spike proteins and their impact on their ability to interact with ACE2 and antibodies. I commend the authors for their extensive work on the SARS-COV-2 spike's protein-protein interactions. I appreciate the use of molecular dynamics simulations for the gamma variant studies that corroborate the findings from their proposed E-Volve tool. However, the manuscript needs to be re-written in an unambiguous language before 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: E-VOLVE: UNDERSTANDING THE IMPACT OF MUTATIONS IN SARS-COV-2 VARIANTS SPIKE PROTEIN ON ANTIBODIES AND ACE2 AFFINITY THROUGH PATTERNS OF CHEMICAL INTERACTIONS AT PROTEIN INTERFACES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see my additional comments.
Experimental design: Please see my additional comments.
Validity of the findings: Please see my additional comments.
Additional comments: The manuscript entitled “The E-Volve: understanding the impact of mutations in SARS-CoV-2 variants spike protein on antibodies and ACE2 affinity through patterns of chemical interactions at protein interfaces” by
Vitor Pimentel Dos Santos et al. investigated the mechanisms that mutations in the viral spike protein of SARS-CoV-2 have decreased the activity of neutralizing antibodies, and increased the affinity to ACE2 receptor using E-Volve. Authors have demonstrated highly frequent interface changes mediated by mutations (N501Y and E484K). The spike-ACE2 complex induced new hydrophobic bonds. Remarkable electrical alterations that breached important sites in the spike-Antibody complexes were observed. This study is of clinical significance, providing a web tool for real-time tracking of the virus mutant lineages. It will potentially help doctors adjust treatments according to virus mutations. However, some issues need solving.
Major issue:
1.	This study manuscript were largely dependent on E-Volve algorithm. How do authors validate their findings experimentally?
Minor issues:
2.	Why was Figure 1C referred before Figure 1B?
3.	In text line 412-421, fonts are not consistent and there are some typos.


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

Input:
Title: E-VOLVE: UNDERSTANDING THE IMPACT OF MUTATIONS IN SARS-COV-2 VARIANTS SPIKE PROTEIN ON ANTIBODIES AND ACE2 AFFINITY THROUGH PATTERNS OF CHEMICAL INTERACTIONS AT PROTEIN INTERFACES
Review round: 1
Reviewer: 3

Output:
Basic reporting: OK.
Experimental design: Ok.
Validity of the findings: OK.
Additional comments: The manuscript needs English proofreading.


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: E-VOLVE: UNDERSTANDING THE IMPACT OF MUTATIONS IN SARS-COV-2 VARIANTS SPIKE PROTEIN ON ANTIBODIES AND ACE2 AFFINITY THROUGH PATTERNS OF CHEMICAL INTERACTIONS AT PROTEIN INTERFACES
Review round: 1
Reviewer: 4

Output:
Basic reporting: no comment
Experimental design: Various methods needs to be used to validate the models.
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: E-VOLVE: UNDERSTANDING THE IMPACT OF MUTATIONS IN SARS-COV-2 VARIANTS SPIKE PROTEIN ON ANTIBODIES AND ACE2 AFFINITY THROUGH PATTERNS OF CHEMICAL INTERACTIONS AT PROTEIN INTERFACES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have successfully responded to my comments.
Experimental design: None.
Validity of the findings: None.
Additional comments: None.