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: VARIATION OF 13C AND 15N ENRICHMENTS IN DIFFERENT PLANT COMPONENTS OF LABELED WINTER WHEAT (TRITICUM AESTIVUM L.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English should be checked before publication to polish some phrases, but otherwise is a well written manuscript.
Experimental design: All the suggested changes have been included so the experimental design is now acceptable.
Validity of the findings: THe novelty of the findings is not very high, but the results can be valuable for readers.
Additional comments: The manuscript has been considerably improved, but English grammar should be re-checked prior 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: WHOLE GENOME SEQUENCING OF A NOVEL, DICHLOROMETHANE-FERMENTING PEPTOCOCCACEAE FROM AN ENRICHMENT CULTURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Introduction seems very laconic to me. I suggest that you add a little bit more information about DCM degradation (line 46), and briefly describe Wood-Ljungdahl pathway or at least add the reference to the article where it is described in detail (line 52). I also suggest you to more heavily emphasize an importance of studying DCM-fermenting bacteria and its ecological impact.
Experimental design: Methods seem to be adequate and are described very thoroughly, with sufficient details to replicate. Though I think that some kind of basic visualization of the research pipeline would be useful and would make it easier to follow.
Validity of the findings: No comment
Additional comments: Statement about methylamine methyltransferases, which need a corrinoid cofactor to function, requires reference (line 310-311).
I would also suggest you to give more detailed focus to the metabolic abilities of the DCMF, explain how glycine/sarcosine/betaine reductases (line 301, 384) are connected to TMA utilization, how exactly methionine connects to the corrin ring formation (line 311-312) etc.


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

Input:
Title: WHOLE GENOME SEQUENCING OF A NOVEL, DICHLOROMETHANE-FERMENTING PEPTOCOCCACEAE FROM AN ENRICHMENT CULTURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is written in unambiguous, professional English. Although the information provided in introduction is not sufficient to understand the study. What is more, some of the information seems to be incorrect. Not all the necessary literature is mentioned in the introduction.
1. Lines 40-42: “Approximately 70% of 41 all DCM worldwide is of anthropogenic origin”. To confirm this fact author cite Marshall & Pottenger, 2016. There is no such statement in the mentioned article.
2. Lines 46-47: “To date, only two DCM-48 fermenting bacteria have been described” This claim seems suspicious, at least at Muller et al., 2011 (Research in Microbiology) at lot of such bacteria is mentioned.
In the same review (Muller et al., 2011) proposed mechanisms of DcmA action is described. It is improtant to mention in introduction that some details about DCM degradation are known.
3. Although in the introduction authors mention that methyl group have to be incorporated into Wood-Ljungdahl pathway, but do not mention that the presence of this pathway is not sufficient for DCM-degradation.
4. Also the important information for understanding the background is provided in the first paragraph of discussion section (lines 317-326). I suggest to move this to introduction.
5. The links to the raw Illumina and PacBio reads are provided. Reads are deposited in the SRA. Also authors provide link to the annotation files, that are surprisingly stored on their local server. In the Table 1 the accession to GenBank is also provided. But it seems it is annotated only with NCBI pipeline and does not include authors manual corrections. The GenBank Accession is crucial for genome availability for the scientific community, and though I think it will be important to include Accession in the main text and also possibly to update Genbank annotation with authors’ corrections.
6. The results provided are in general unclear. The part about methylamine methyltransferases seems unclear and unfinished.
Experimental design: The research is within Aims and Scope of the journal. The research question is not well defined, because only the fact that new bacteria is sequenced is provided, but no detailed metabolic reconstrction for the species is performed. I really appreciate here the part about genome assembly. The genome is accurately assembled from filtered reads, and the quality of assembly is accurately checked. Although some questions arise about manual gene corrections:
1. Why authors are so sure that the genes they corrected are indeed have pyrrolysines and selenocysteines, and not just pseudegenes? Especially, that most corrections were done in multi-copy genes.
2. As well it is not clear where the final annotation is stored, because on the website three independent annotations are provided.
3. The part about culture saturation is unclear. It is definitely part of previous experiment of archaea purification from the same region. The details for this part is hardly understandable for the general public. PeerJ considers articles in biological sciences in general, and that is why the process of obtaining the bacteria of interest have to be more clearly described, so it can be understood not only by specialists in the narrow field.
4. The selected method for taxonomy assignment is ok, but one expect more strait forward approach, such as comparison of 16S rRNA with SILVA database or RDP. It is unclear from methods what type of tree was build for 16S rRNA genes, and with what parameters.
5. The part about taxonomy assignment for individual proteins is unclear. I think the check for some some single-copy universal genes will be enough. Although those trees have to be ML ones with bootstraps.
6. In case of TMA methyltransferases it is unclear why not just to build multiple alignment and then phylogenetic tree. It will be easier to analyze clustered branches on such tree than from matrix provided by authors.
7. Why genomic DNA was extracted with the protocol developed for archaea DNA puification (line 97-98)?
Validity of the findings: Line 236:  Abbreviation BES is not explained in the text.
Line 248: The idea about “shift away from the Dehalobacter population” is unclear. It have to be described in more details.
Line 271: it is unclear how contaminant bacteria were identified.
My main concern about this paper is the lack of metabolic reconstruction. The absence of reductive dehalogenases is mentioned in Table 1. Still it is unclear where from the list of potential dehalogenases was obtained?
Authors discuss that pyrrolysine biosynthesis pathway is present, but there is nothing about selenocysteine, although in the methods it was stated that corrections were made for both aminoacids.
Why methylamine methyltransferases are so important? Some of them can be associated with pyrrolysine biosynthesis, but what about others?
With what genes are those  methyltransferases are surrounded? Such analysis may add some clues to the pathways behind DCM-utilization.
The more vivid metabolic reconstruction is important here because one of the main article claims is that the genome of the extracted bacteria have twice more genes than close relatives.
The discussion section is unstructured, although the description about genome assembly optimization is interesting.
Additional comments: The organism that you extracted seems to be interesting, but the article does not contain enough information to understand the importance of the finding.
First, the introduction does not provide enough information to understand the details and pathways behind DCM degradation. As well it will be useful to provide more explanations about Wood-Ljungdahl pathway.
Second, the sequenced genome itself is not that interesting. The more detailed analysis of this Peptococcaceae bacteria will add a lot to the text.
Third, some information have to be added in the text and not only mentioned in table 1, such as Genbank Accession and search for reductive dehalogenases.
Fourth, some methods used are not well-described. It is especially crucial for reproducing phylogeny reconstruction.
And last, there is no clear idea behind this text. The big part of manuscript is dedicated to optimization of the genome assembly, while less is discussed about sequenced bacteria properties. I think that the balance have to be shifted towards bacteria. That will add consistency to the text, and will be in better agreement with the abstract section.


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

Input:
Title: WHOLE GENOME SEQUENCING OF A NOVEL, DICHLOROMETHANE-FERMENTING PEPTOCOCCACEAE FROM AN ENRICHMENT CULTURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article is well written, unambiguous professional English is used, all necessary references, figures and tables are provided.
Authors have taken previous comments into consideration and added some more information about DCM metabolism and Wood-Ljungdahl pathway into the Introduction section. Though it still remains a little laconic and not easy to completely understand without reading cited articles. I would advise to introduce the metabolic sheme given in Figure 6 much earlier, not in the middle of Discussion section. Thus it will show the connections between DCM, TMA, glycine/betaine/sarcosine etc right from the beginning and will make the whole picture more clear and structured.
Experimental design: Methods are adequate and are described thoroughly, with sufficient details to replicate. Research question is quite important from the ecological point of view.
Validity of the findings: All underlying data is provided, conclusions are well stated.
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: WHOLE GENOME SEQUENCING OF A NOVEL, DICHLOROMETHANE-FERMENTING PEPTOCOCCACEAE FROM AN ENRICHMENT CULTURE
Review round: 2
Reviewer: 2

Output:
Basic reporting: I am satisfied with the changes authors made in this revision round.
I think that now this text has to be understandable by the general public.
Actually, the more detailed Discussion section now allows to better understand the unique properties of the isolated Bacteria.
Experimental design: The design is ok.
Now the methods are described in more details, so it can be reproduced.
Validity of the findings: The isolated DSMF bacteria is an interesting organism to study. Now when the obtained results are described and discussed in more details, I think that it will be evident to the general public, that this organism require more detailed biochemical analysis.
All the data is provided.
Additional comments: I really enjoyed reading the revised version of this manuscript, because now the findings are beautifully put into the wider scope and all the missing links are now explained.
I have only one small remark about Figure S2.
It seems like the font in this figure is somehow broken. And there is no explanation in this figure' legend about the black dots.


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: TRANS-ORAL ROBOTIC SURGERY VERSUS COBLATION TONGUE BASE REDUCTION FOR OBSTRUCTIVE SLEEP APNEA SYNDROME
Review round: 1
Reviewer: 1

Output:
Basic reporting: First, I’ll make a couple of general comments about the presentation of results:
* Whenever you use ± notation (e.g. Lines 31, 33, 147, 150, etc.), please indicate what the values before and after the ± symbol are, at least the first time you use this notation in each section. These could be assumed to be mean ± SD, but they could also be mean ± SE, mean ± 95% CI half width, etc.
* Given the number of surgeries (33 overall, 16 and 17 by group), there is no need for a decimal place in percentages (e.g. Line 35). Each person overall counts for around 3% themselves, and within groups, this is around 6% each, so the values after the decimal place are not meaningful.

I will suggest some additional information be added to the abstract for the reader who might not read the entire manuscript. Specifically:
* The reader of the abstract will probably want to know when the follow-up measurements were taken (Lines 29–30, for both the follow-up AHI and ESS, and the period during which complicated were recorded). The first of these is given as 3 months+ (what was the minimum–maximum?) on Line 78, but the other time periods are not explicitly described in the manuscript (Lines 85 and 100).
* I would suggest starting the abstract results with a sentence about these patients (Line 31), including noting any potentially important differences between the surgery groups or the lack of such.
* The study aim is addressed by between-group comparisons (i.e. between TORS and coblation assisted tongue base reduction surgery) rather than within-group comparisons. While providing the pre and post values in the abstract on Lines 31–33 is potentially helpful, rather than or as well as showing within-group p-values here, please present these crucial between-group p-values for all outcomes in the abstract (currently AHI, success, and complications), rather than only for the second of these. Why is ESS not included here also?

The introduction could be improved with some additional references. Any fact or claim made here should be supported by the literature. In particular, I think references are needed for Lines 45 (symptoms), 47–48 (tolerance and surgery seeking), 49 (variable responses), 53 (increasing use of multilevel surgery), 58 (reason for failure), and 65 (hesitance to use TORS).

When reporting the results (Lines 165–186), I think it would be easier for the reader if you interleave the within- and between-group analyses so that each variable is covered in turn, rather than presenting within-group analyses for all variables and then doing the same for the between-group analyses. The between-group analyses are the ones that address your research aim and so I feel that these need to be emphasised more, with the within-group results there to support the efficacy of the surgeries more generally.

A reasonable amount of the Discussion (Lines 198–237, possibly Lines 239–250, and perhaps also Lines 253–255) could be removed or moved (in whole or in part) to the Introduction. None of this material discussed your study’s findings, the relationship of these findings with the literature, or its implications for practice or research. Note that it is tradition to begin the Discussion with a description of the main or overall pattern of findings from the results before comparing these to the literature, leading into these implications. Some, but not all, of the material on Lines 281–311 could be moved to the start of the Discussion to summarise your findings. Results from the literature that are not going to be compared to your study results in some way or another should, I think, be moved to the Introduction or deleted entirely.

Some minor specific comments about the writing are listed below:
Lines 39–40: Perhaps either “…is a successful approach for the management of OSAS.” or “…is successful for managing OSAS.” A similar comment would apply to Line 198.
Line 57: Do you mean “…as the most important…” or “…as an important…” here?
Line 58: Do mean “procedures” here?
Line 90: 50% would appear to be in both grade II and grade III here.
Line 108: I suggest just “literature” here.
Lines 117–118: The sentence here (“The curved frame of the retractor could fit with the shape patient’s face and the extended framework could make the movement of the robotic arm easily.”) needs rewriting.
Line 129: “held” rather than “hold” here?

A data dictionary would be a very useful complement to the data provided. This could be an additional worksheet within the workbook or as an external document. In either case, this would list all variables and give the meaning of the variable and the interpretation of each value for binary, ordinal, and categorical variables. For example, at the moment it is not clear whether sex=0 or sex=1 is male. I think the variable named “posai” should be named “postai”.
Experimental design: How many patients did not have follow-up data (Lines 77–78)? What were the reasons for this and did these patients differ from those with follow-up data? How many were also excluded for having had previous surgery (Line 79)? This would appear to tighten the research question to being about differences in the procedures amongst those who have not had previous surgery and, if so, this should be made clear back on Lines 24–25 and 71. You might find a patient flow diagram (like a CONSORT diagram despite the lack of randomisation here) to be helpful here in showing the missing data both overall and by surgery group.

Note that all statistical tests make some form of assumptions and these need to be explained in the statistical methods (Lines 139–142). This includes assumptions about the residuals for independent sample t-tests (normality and homoscedasticity) and paired sample t-tests (normality of delta values, note also that this is the usual nomenclature rather than “dependent t-test” on Lines 141 and 165), and most statisticians require expected cell counts to exceed 5 in at least 80% of cells for a Chi-squared test. For the t-tests, based on the data shown later in the tables, these assumptions will not be met here in all cases. Better than independent sample t-tests would be an ANCOVA comparing follow-up values between groups and including baseline values as a covariate. This increases power if changes are correlated with baseline values, which is often the case (the patients who are worst off might have the potential for greater improvements in absolute terms—this might be the case with CT90 in Table 3 for example). Again, if you use this approach, you will still need to discuss model checks, now including linearity of residuals with respect to baseline values. You might find natural logarithmic transformations to be helpful, or you may need to look at semi- or non-parametric alternatives.

Another advantage from using a regression-based approach such as ANCOVA is that you could adjust for potentially important variables that differ between surgery groups. While you have not at this stage identified any statistically significant differences between groups (Line 163), you might still feel that some values are numerically different enough to be worth adjusting for.

Note that means and SDs are not useful for most readers in most cases for non-normally distributed data and the values for AHI, AI, ESS, Min-SpO2, and CT90 in Table 3 are all skewed to a greater or lesser extent. You might find it more useful to present medians and IQRs in this case, or you could investigate transformations of these variables. The changes for these variables in Table 4 should also be investigated for normality.
Validity of the findings: If you wish to make statements such as “comparable surgical outcomes” (Line 37), this would require an equivalence approach to comparing the groups rather than a superiority one. You would need to define equivalence/comparable (in units for each variable) and then focus on the 95% CIs rather than the point estimates and p-values for this interpretation. Given the higher cost of TORS, I think an equivalence or a non-inferiority design would better address what I see as the primary question (can coblation assisted tongue base reduction surgery be used in place of TORS?)

Table 2 presents means and SDs for some ordinal items (stages and grades) which I would not consider valid, nor would I expect the t-tests using these to be valid due to the discreteness of the distributions. Note that the tests used should be apparent in the table itself as well as explained in the text. This last point also applies to Tables 3 and 4. Note also that using the standard approach to Chi-squared tests (80% of expected cell counts exceeding 5), Fisher’s exact test could be preferred for comparing sex between groups.

Table 3 needs to present within-group changes and 95% CIs to help make it clear what values are consistent with the data. Similarly, Table 4 needs to present between-group differences and 95% CIs for all outcomes (this will particularly help when you present and discuss the LoS result on Lines 190 and 305–306). I suggest moving the between-group comparisons for AHI reduction, AI reduction, ESS reduction, Min-SpO2 improvement, and CT90 reduction from Table 3 to Table 4, leaving the post-surgery outcomes in Table 4. Given the small sample size, if the between-group intervals are wide, this would mean that no conclusions about the outcomes from the surgeries being comparable could be made and further research is needed. On the other hand, if these intervals did not include meaningful differences, you would be able to make slightly stronger claims about the lack of differences between the surgeries, subject to selection effects for each.

An important aspect not covered here is the cost of the procedures (aside from a mention in the Introduction around Line 64–67, and Discussion around Line 310). While a full cost-effectiveness analysis might be beyond the scope of the present work, some readers will be interested in the relative cost of the two surgeries. Are you able to provide any data on this (e.g., operating theatre time alongside LoS data, ideally with standard costs used for each?)
Additional comments: While this is an interesting and important topic, there is work needed, particularly for the statistical analyses and in improving the flow of the Discussion.


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: TRANS-ORAL ROBOTIC SURGERY VERSUS COBLATION TONGUE BASE REDUCTION FOR OBSTRUCTIVE SLEEP APNEA SYNDROME
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 103 Are there differences in the way of doing UPPP in those patients? Are there reposition pharyngoplasty or expansion sphincter pharyngoplasty done to these patient? Because in these multilevel surgeries, different pharyngoplasty chosen may also affect the outcome.

148 To report percentage of male number and percentage is enough, as well as in the table.

156 Please report the range of AHI values for both groups, so we could know better the patient selection of the authors in the very beginning.(Seems the authors chose the severe OSA group as their surgery target?)
Graphs for individual AHI decrease may also be helpful, because AHI values for some unsuccessful patients may increase after surgery.

165 t test may not be appropriate in this small patient volume. Non-parametric test like Mann-Whitney and Wilcoxon test are more appropriate if these values failed  normal distribution test, and median values with quartiles should be reported instead of mean and standard deviation.

198 Using more effective than UPPP only rather than just successful would be more appropriate.

295 Hwan-->Hwang

326 Conclusion of abstract and main article: successful treatment is an exaggeration. 50 plus success rate is far from success. Although Sher criteria is too strict for severe OSA group, 20 plus residual AHI for about half patients does not mean success either. Better statement would be: Multilevel surgery using either TORS or coblation tongue base reduction combined with uvulopalatoplasty are effective in reducing disease severity in some severe OSA cases.

Table 1 and discussion :Are there differences of success rate or AHI reduction between patients with partial or complete retroglossal collapse in DISE? i.e. Are preoperative DISE findings of tongue affect surgery outcome?

Table 3 is a little confusing. TORS , Coblation and their related variables should be at the left vertical panel; preoperative, postoperative comparison and p values should be reported at the horizontal panel.

Table 4 To emphasize mean SD in the left panel of the table is not necessary. Just mention that in the note would be fine.


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: TRANS-ORAL ROBOTIC SURGERY VERSUS COBLATION TONGUE BASE REDUCTION FOR OBSTRUCTIVE SLEEP APNEA SYNDROME
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for addressing the ± notation in the abstract. Can you also do this in the results around Line 162 when you first use this? Some readers will of course skip the abstract and read only the manuscript body.

My requests about the data (“A data dictionary would be a very useful complement to the data provided. This could be an additional worksheet within the workbook or as an external document. In either case, this would list all variables and give the meaning of the variable and the interpretation of each value for binary, ordinal, and categorical variables. For example, at the moment it is not clear whether sex=0 or sex=1 is male. I think the variable named “posai” should be named “postai”.) do not appear to have resulted in any changes despite the positive remark in the rebuttal (“Answer: Thank you, and we supply it.”). Was the updated spreadsheet uploaded along with the revised manuscript (the last edited date on Raw_data_.xlsx is 30-April-2019 which suggests that this is the old data spreadsheet)? Can you please also check that all variables presented in the manuscript are included in the data, I couldn’t find the Friedman stage or ESS, as examples, there (my apologies if I’m overlooking them, although this is also where a data dictionary is helpful).

The statistical test used to produce each and every p-value should be evident from the table through notes in that table.

Some minor comments about the writing:

Line 62: Perhaps “THE Friedman tonsil grading…”

Line 64: Perhaps “THE Friedman tongue position…”

Line 66: Perhaps “THE Friedman staging system…”

Line 85: Perhaps “…in the field of tongue base reduction…” (deleting the second “the”).

Line 88: Suggest deleting “make” and “of” here (already use “makes” earlier in the sentence), leaving “…makes operators and patients hesitant to use it.”

Line 119: “…AND cumulative…” for the last list item.

Lines 156–157: Please explain which descriptive statistics were used here.

Line 157: Note that there are two Wilcoxon tests, the between- and within-group tests. If this is referring to the within-group test, “signed Wilcoxon test” would make this clear, even more so if “between and within groups respectively” was added at the end of the clause (just before the comma on Line 158).

Line 158: “…Fisher’s EXACT test…”

Line 164: I suggest “of the” for both instances of “in the” here. Perhaps also start the sentence (Line 163) slightly differently, perhaps “Males comprised…”.

Line 176: “Demographics AND baseline PSG data…”

Line 182: The tilde (“~”) is normally used for “approximately” rather than to indicate ranges. I suggest a hyphen (“-“) or, better, an en-dash (“–“) here instead. Also Line 187 and elsewhere.

Line 194: “…STATISTICALLY SIGNIFICANT differences…”

Lines 195–196: “…had a GREATER reduction IN…

Line 199: “mean” rather than “average”. See also Lines 224 and 277.

For the results on Lines 199–201, it would be much easier to appreciate these, especially the different in length of stay, with descriptive statistics included here. Also on Line 277–280 where values will help you indicate potential clinical significance as well as the results of a hypothesis test.

Line 206: An integer percentage would be more appropriate given the sample size. Same point on Line 268, 281, and possibly elsewhere.

Line 209: I’d avoid such a definite term as “demonstrate” here as there is room for the patients to vary between the two treatments.

Line 222: “THE coblation lingual tonsil removal technique…”

Lines 242–243: You can delete “the comparison of” as you’ve described this as a “comparative study”.

Line 251: I’m not sure what you mean by “a lack of fine and matched studies”. Do you mean “finely matched” or “high quality and matched”?

Lines 273–274: These values could be misread as min–max BMI rather than the two means (this is not stated on Line 273 as you do for other outcomes on Lines 274 and 275, for example). As there are only two means, you could list them instead here and for other outcomes.
Experimental design: As noted previously, there is substantial skew for many variables (e.g. Lines 172–173, 175, 176, many of the variables in Table 2 and 3). This makes reporting means and SDs questionable at best. It is not usual to report means and SDs, which are generally treated as parametric descriptives in that they assume a normal distribution, at least without another context being provided, and then to use non-parametric analyses. When non-parametric analyses are used, this generally indicates that means are not normally distributed, leading to medians (and the IQRs or 25th and 75th percentiles for variability).

There appear to be some rounding errors in Table 2, e.g. mean BMI for TORS is 28.15625 which rounds to 28.2 and not 28.1 as reported. Similarly, mean BMI for the coblation group should be 27.3 not 27.3 and the SD 5.6 not 5.5. I get quite different values for AI (means of 33.3 versus 34.3 and 31.3 versus 28.2). This all assumes that the uploaded data is correct of course. I have not checked for all other possible discrepancies, so I suggest that all values in Table 2 are also checked.

The statistical methods refer to Mann-Whitney U, (signed) Wilcoxon, and Fisher’s exact tests (“The Mann-Whitney and Wilcoxon test were used for comparing numerical variables, and Fisher’s test was used for categorical variables” on Lines 157–158), but the handful of p-values for non-categorical variables that I looked at in the Tables seemed to be from t-tests instead or did not match either this or the listed non-parametric test. As also noted previously, t-tests are not appropriate for discrete data, such as tonsil grades. I am unable to replicate some of the p-values using either t-tests or Mann-Whitney U tests so I am unsure what was done here.

While I can replicate some descriptive statistics and p-values in Tables 3 and 4, some of the values I get from the data are quite different. It required some trial and error for me to realise that some outcomes not indicated as percentages in Table 4 were being analysed as percentages, e.g. AHI, which was also changed to 0 in the case of worsening (this should be made clear somewhere if it is not already). Even after being able to replicate the means for AHI % change, the SD for the TORS group still differed for me (SD 38.0 not 38.9). I wonder if the uploaded data are what was used to produce the results in the manuscript.

Related to the above, the values presented on Lines 185–186 say they are events per hour, but they appear to be percentage reductions instead. Note that for the coblation group, the reduction of 45.3 “events/h” (Line 186, Table 3) is greater than the pre-surgery mean of 44.8 (Line 184, Table 2).
Validity of the findings: An important limitation that follows from using non-parametric approaches is that you are unable to adjust for potential confounders. Given the data set size, this adjustment would always have been limited in terms of the number of variables that could be accommodated. Possibly related to this, can you be more precise about the “selection bias” on Line 286 and, if possible, consider the expected direction of this bias for your results. The other major limitation of non-parametric approaches is the lack of resulting confidence intervals, which makes interpreting results, including non-statistically significant results, difficult. Even with this lack, however, I feel that more could be made of the results by adding clinical interpretation of differences. Is the 1.1 (not 1.2 as suggested in Table 4 if the uploaded data is correct) day difference in hospital stays clinically relevant or not in your view?
Additional comments: The manuscript is much improved but after noticing some confusing results (at least to me), I looked at the data and found what appears to me to be a number of issues. Some of these are minor rounding discrepancies, others are different values obtained from the uploaded data compared to the manuscript. Some variables were not clear in their meaning (e.g. the reduction in AHI events/h appears to be a percentage) and other variables seemed to be lacking in the data set and could not be examined. Some of the p-values could not be replicated using the statistical methods described, but could using alternative tests, and some could not be replicated even after investigating alternatives. As far as I can tell, none of these affects the overall findings, but I would strongly suggest the authors carefully check each and every value using the uploaded data file to ensure that everything is correct. If any of this is due to my misunderstandings, I hope that this is determined quickly and I apologise in advance.


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: TRANS-ORAL ROBOTIC SURGERY VERSUS COBLATION TONGUE BASE REDUCTION FOR OBSTRUCTIVE SLEEP APNEA SYNDROME
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: One of your inclusion criteria is AHI > 20. However, there are 4 patients in your raw data did not meet this criteria(see below). Please explain, change your inclusion criteria or reselect your patients.

		       preAHI
1050527		14.4
1020702		16.9
1051210		17.8
1050902		19.5

[Graphs for individual AHI decrease may also be helpful, because AHI values for some unsuccessful patients may increase after surgery.]

For this question, I didn’t mean whisker bar graph. Can you provide graph like figure 1B of this paper?
 DOI:10.1038/s41598-018-22710-1
It would be much easier for us to follow.

[Table 3 is a little confusing. TORS , Coblation and their related variables should be at the left vertical panel; preoperative, postoperative comparison and p values should be reported at the horizontal panel.
Answer: Yes, we had revised them.
Table 4 To emphasize mean SD in the left panel of the table is not necessary. Just mention that in the note would be fine.
Answer: Yes, we had revised them.]

For these questions, I suggest you authors should show the original version and changed version in the response letter at the same time, because it is hard for me to follow the difference in the manuscript. Actually, I would love to see you authors answer all my questions in this way.

Example(please do this in tables):
Page	Line	   Original	            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: STRUCTURAL ANALYSIS OF URINARY LIGHT CHAINS AND PROTEOMIC ANALYSIS OF HYALINE TUBULAR CASTS IN LIGHT CHAIN ASSOCIATED KIDNEY DISORDERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is clear and well written. The language used is technically appropriate. Both the background and the objectives are clearly described in the introduction as in the discussion. The authors demonstrate to deeply know the previous literature in the specific field of investigation.
The table and figures are clear, well detailed and labeled. Legends are exhaustive. Although the absence of uromodulin and the consequent deductions on the formation of the casts in the considered patient appear relevant (lines 233-246 in the text), the Figure 3C should be removed and the result of the related experiment should be reported just in the text.
For the data shown in figure 4, it would be advisable to be reported as a table, maybe using the format proposed by the software used to analyze (and appropriately filter) the large amount of data got by proteomic experiments.
Experimental design: The research questions are well defined and discussed throughout the text. The experiments are rigorous, taking into account the technological approaches used today for such these studies.
Although this is a preliminary study, to move to clinical trials the statistics of some data and the strengthness of the conclusions should be improved increasing the total number of patients involved.  In particular, data relating to a single patient, or to a few patients, should be verified on similar population of patients, even considering, as was correctly done in this work, the pharmacological treatments to which they have been subjected.
The technical aspects of proteomic nano LC-MS analysis are partial and, although they refer to previous studies, they cannot fail to report some essential technical information to allow these experiments to be reproduced by other researchers. In particular, the proteases used to obtain the peptides, subsequently analyzed and characterized, as well as the digestion procedure (in solution, in column; why not in gel?, etc.), should be described in the detail.
Validity of the findings: The results are novel and partly confirm what has been shown in previous studies. This study undoubtedly contains interesting data for the advancement of knowledge in the study and treatment of patients with light chain disease. Although circumscribed to a limited and, in some respects, heterogeneous number patients  the obtained data can be meaningful to think of developments that lead to an optimized, single patient, dialysis treatment.
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: STRUCTURAL ANALYSIS OF URINARY LIGHT CHAINS AND PROTEOMIC ANALYSIS OF HYALINE TUBULAR CASTS IN LIGHT CHAIN ASSOCIATED KIDNEY DISORDERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors studied urinary casts composition in 4 patients with light chain associated kidney disorders. The method seems robust. However, detailed renal biopsy findings in these patients are lacking. The second part of this study focus on urine light chain examined by immunoblotting in 17 patients with monoclonal gammopathy.
1. Would consider revising the title. Perhaps the word “architecture” could be deleted and maybe “light chain associated kidney disorders” instead of "renal light chain disease”.
2. The term “light chain nephropathy” (for example line 53) is not appropriate and may be confusing. The authors should prefer “light chain associated kidney disorders”.
3. Since multiple myeloma is a plasma cell dyscrasia, the distinction used (for example line 52) is not appropriate. The authors should classified patients with symptomatic multiple myeloma or monoclonal gammopathy of renal significance (MGRS) since this distinction is more relevant in the context of paraprotein associated kidney disorders.
4. Your introduction should better highlight that the spectrum of renal complications associated with paraproteins is heterogeneous with two main categories according to the characteristics of the underlying B-cell clone. The first group is always associated with high tumor mass and light chain cast nephropathy is the more frequent complication in this context. The second group include all patients with renal symptoms related to a nephrotoxic monoclonal immunoglobulin by any mechanism other than the tumor burden and is now defined as MGRS (ref Leung N et al. Blood 2012).
5. The reference Leung N et al. Nature reviews Nephrology. 2019 (https://doi.org/10.1038/s41581-018-0077-4) needs to be added to the introduction section.
6. Line 64 : free light chain measurement in the urine is not recommended in the management of patients with paraprotein associated kidney disorders. Please discuss this point.
7. Line 79 : please replace “renal deposition disease” by “monoclonal immunoglobulin deposition disease”
Experimental design: No comment
Validity of the findings: Renal biopsy was not performed in all patients. This point is an important limitation of the study. How many patients had a renal biopsy? What means “0” in column renal pathology in Table 1?
Additional comments: The authors should better described renal pathological findings and hematological characteristics of the patients included in this study. You should distinguish in Table 1 patients with symptomatic multiple myeloma or MGRS.


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: STRUCTURAL ANALYSIS OF URINARY LIGHT CHAINS AND PROTEOMIC ANALYSIS OF HYALINE TUBULAR CASTS IN LIGHT CHAIN ASSOCIATED KIDNEY DISORDERS
Review round: 1
Reviewer: 3

Output:
Basic reporting:
Experimental design:
Validity of the findings:
Additional comments: "Structural analysis of urinary light chain architecture and proteomic analysis of hyaline tubular casts in renal light chain disease"

The work is well conducted, the main topic is clear and the description of the procedure is really well done. The laboratory part is very accurate and all the conclusions are strictly clear and give messages that can be really useful for clinicians and for researchers.

Overall is a very good article

The only remark is about clinical aspects:

* authors describes only architectural and structural composition of casts, basing the whole work on the morphological carachteristic of chains. Nevertheless, they don't describe number of free chains, creatinine at the time of diagnosis, previous clinical treatment patients underwent (a number of patients has a long history of mieloma) making difficult to evaluate the patient in all his complexity. Lots of datas about the kind of free chains and casts could depend on the patients previous conditions and it could be useful to describe.
* giving the limited number of patients seems difficult to divide into subgroups but the age of patients and the age of illness could play an important role in histones precipitation and reaggregation.
* at the beginning authors describes only three forms of renal involvment during multiple myeloma while there are a few more (Immunotactoid, crioglobulinemic nephrotic syndrome, leukaemia invasion, nephropaties with organised Immunoglobulin deposits). The description of all the different kind of lesions is very important in the context of electronic microscopy.


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: STRUCTURAL ANALYSIS OF URINARY LIGHT CHAINS AND PROTEOMIC ANALYSIS OF HYALINE TUBULAR CASTS IN LIGHT CHAIN ASSOCIATED KIDNEY DISORDERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Since the authors have implemented all the suggested changes , in my opinion the manuscript can be accepted for publication
Experimental design: Since the authors have implemented all the suggested changes , in my opinion the manuscript can be accepted for publication
Validity of the findings: Since the authors have implemented all the suggested changes , in my opinion the manuscript can be accepted for publication
Additional comments: No additional comments


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

Input:
Title: STRUCTURAL ANALYSIS OF URINARY LIGHT CHAINS AND PROTEOMIC ANALYSIS OF HYALINE TUBULAR CASTS IN LIGHT CHAIN ASSOCIATED KIDNEY DISORDERS
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: CLINICAL IMPLICATIONS OF ABERRANT NEUROVASCULAR STRUCTURES COURSING THROUGH THE SUBMANDIBULAR GLAND
Review round: 1
Reviewer: 1

Output:
Basic reporting: While it is acknowledged in the head and neck clinical literature that the facial artery sometimes passes through the SMG, I was surprised to find that there were no percentages of this variant reported in the literature. This manuscript will be a valuable contribution to any clinical study interested in reporting anatomic variations in the submandibular triangle.

I did find two papers that discuss variations in the submandibular triangle that are not cited in this manuscript. Please consider including these papers in their revision:

Vadgaonkar, Rajanigandha, et al. "Variant facial artery in the submandibular region." Journal of Craniofacial Surgery 23.4 (2012): e355-e357.

Venugopal, S. V., et al. "RELATIONSHIP BETWEEN THE FACIAL ARTERY AND SUB MANDIBULAR SALIVARY GLAND." Int J Anat Res 2.3 (2014): 597-600.

The data are clear and unambiguous

The manuscript is well written. There are a couple minor typos that the authors should consider fixing
ln 35 "for numerous health conditions and procedures to be occur"
Edit: Wording
Ln 44 "for neoplastic origin, is Fine Needle Aspiration"
Edit: comma
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have done an excellent job with the discussion. It was thorough and highly relevant. This manuscript will be a great resource.


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: CLINICAL IMPLICATIONS OF ABERRANT NEUROVASCULAR STRUCTURES COURSING THROUGH THE SUBMANDIBULAR GLAND
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language of the manuscript was in clear and professional English. Proper background and context was provided and citations were appropriate. The figures and tables were very well done and offered clear detail. There were a few instances where the flow of the text wandered off course or was redundant (e.g., line 48, “Thus…”), but his was not really detrimental to the text.
Experimental design: The research question was well defined and meaningful. The relevance of the investigation is, to me, not of critical importance as surgical variations are well known and surgeons and anatomists have been working in this region for generations. The important aspect of this manuscript is the frequency data of each structure found within the substance of the submandibular gland. The research was conducted on body donors from a medical school anatomy lab (with documentation of IRB waiver), which introduced some limitations to the study, which was clearly addressed in the text.
Validity of the findings: My only concern is the possibility of some bias due to the inclusion of donors with a minimum of one gland. If a donor with a damaged gland that may or may not have had a variation is compared with both grands from another donor, some bias might creep into the dataset and influence the overall results. Also, using a chi-square test that is robust to the effect of small sample size is likely appropriate. Other than those thoughts, the data were collected and documented well, appropriately defined, and support the overall conclusions of the manuscript.
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: DETECTION AND IDENTIFICATION OF XANTHOMONAS PATHOTYPES ASSOCIATED WITH CITRUS DISEASES USING COMPARATIVE GENOMICS AND MULTIPLEX PCR
Review round: 1
Reviewer: 1

Output:
Basic reporting: This text of this manuscript is generally in clear, unambiguous and professional English. However, there are several exceptions where the meaning is unclear or ambiguous. There are a few minor lapses of grammar.

(1) In the Abstract, the authors first mention "three species" and then subsequently mention "four species". This is confusing for the reader. The confusion arises because there are three distinct pathogens and these collectively fall within three taxonomic species. The authors should carefully re-word the text to make this clear. See also point number (21) below.

(2) The authors write: "Although these species exhibit different levels of
virulence and aggressiveness, only limited alternatives are currently available for proper
and early detection of these diseases in the fields." I don't really follow the logic here. Why would the exhibition of different levels of virulence and aggression mean that there would be not be limited alternatives? In other words, (a) why use the word "although"? and (b) surely the alternatives will inevitably be limited, even if there are many. Please re-word this to make the intended meaning more clear.

(3) In the Abstract, the authors write: " It is freely available and can be utilized for any other model organism." Why specifically 'model' organism? I imagine that the most frequent scenario will be that a researcher wants to apply detection to non-model organisms rather than model organisms. For a defintion of 'model organism', see for example: https://doi.org/10.1016/j.endeavour.2013.06.001

(4) Line 62. "Gram-negative", not "gram-negative". This is named after Hans Christian Gram.

(5) The authors write "Xanthomonas is a genus of phytopathogenic gram-negative bacteria that cause a variety of diseases in several Citrus trees [1]." This is potentially misleading, since only a small minority of Xanthomonas bacteria cause disease in Citrus trees.

(6) Lines 65 and 66. The authors should not use italics for "sp." as it is not part of the taxonomic name. Furthermore, the authors need to consider whether they really mean "sp." (singular) or "spp." (plural) when they write "... for instance, X. oryzae in Oryza sp. (rice), X. campestris in Brassica sp. 66 (cabbage), X. vesicatoria in Solanum sp. (potato), and X. citri in Citrus sp. (orange) [2–6].".  See also "Citrus sp." at lines 100- 101.

(7) Delete "that" from line 67.

(8) Line 70. Sometimes the authors write "Citrus" and sometimes "citrus". What is their rationale for distinguishing between these two terms? Would it be more helpful to the reader to be consistent?

(9) Line 73. I don't understand the expression "directly reflects on". What does this actually mean? Please use more precise language.

(10) Line 78. The authors write "Although they are genetically close to type A, these variants are related to a smaller set of compatible hosts (Table 1) [9,13].". It is confusing to say "related to" here because (a) the first part of the sentence concerns genetic relatedness, and (b) I don't really understand what related means here; does it mean something like 'associated with'? Or causal?

(11) Line 89. The authors write "... bacteria are classified among the main phytosanitary problems ...". this is confusing. The choice of the word 'classify' suggests taxonomic classification classification; but this is not what the authors mean. Do they simply mean that these bacteria present one/some of the most important phytosanitary problems?

(12) The authors write (line 296): "there are no reports available in the literature of other species of Xanthomonas not associated with citrus surviving in contact with this host, which would massively reduce the possibility of amplification of false-positives".   That statement is true but it is not useful and the problem arises from the use of the word 'species'. For example, X. fuscans subsp. fuscans could be considered to be a member of a species associated with Citrus because the species X. fuscans contains the pathogen X. fuscans subsp. aurantifolii. Please carefully re-word this section to make the meaning clear and precise.

(13) Line 110 and Table 2. When refering to genome sequences obtained from public databases, it would be much more useful to provide the GenBank or RefSeq accession numbers of the actual genome sequence rather than the BioSample accession number for the biological sample from which those sequences were derived.

(14) Lines 113 and 214. "MAUVE" versus "Mauve"; please be consistent.

(15) Line 122. "set ... were" should be "sets ... were".

(16) Line 132. "compared against all the species deposited in the NCBI". Species are no deposited in NCBI; sequence data from species are deposited in various sequence databases available via the NCBI (and indeed other portals such as that of the EBI).

(17) What is meant by a "citrus dependent Xanthomonas isolate" (line 143)? In what sense are they 'dependent'? Perhaps the authors mean "Citrus-pathogenic"?

(18) The term "Electronic PCR" is confusing (line 208). Perhaps the authors really mean "in-silico PCR"?

(19) Line 228. "However, it does not occur upon using broad-coverage primers". What does not occur? Is it amplification that does not occur? Currently, the way it is written suggests that it is Stenotrophomonas which does not occur. Also, what is meant exactly by "broad-coverage primers"? Which primers, specifically, does this refer to?

(20) Line 261. "citrus canker is not the central pathology that currently affects citrus". What is meant by "central"? Does it mean something like "most prevalent"? Or "most economically important"? Or something else?

(21) Line 245. The authors write "In contrast, Xacmm amplified a weak fragment for five of the investigated species (X. axonopodis pv. vesicatoria 1689, X. axonopodis pv. passiflorae M89, X. axonopodis pv. manihotis IBSBF1954, X. axonopodis pv. axonopodis 1696, and X. axonopodis pv. glycines 1694) ...". They say "five ... species" but actually this is only one species, i.e. X. axonopodis.

Actually, it more complicated still, since X. axonopodis pv. manihotis has now been transferred into X. phaseoli (https://doi.org/10.1111/ppa.12461)!

(22) Line 305. "the diagnosis". I am not entirely sure that "diagnosis" is the correct word here. The purpose of the tool is detection, or perhaps identification, rather than diagnosis.

(23) There are some problems with formatting of  references. See for example inappropriate use of capital letters in author names in reference number 6.

(24) Figure 1. "Primer Design Tool" should have capital initial letters as in line number 123, where the authors write "using Primer Designing tool [33]." Actually, if we look at the title of reference number 33, it suggests that the tool is properly called "Primer-BLAST"? Please use the correct name in both the text and the figure and be consistent in use of capital letters.
Experimental design: (1) The authors propose PCR-based tools.They could also consider discussing the possibility of adapting their approach to isothermal amplification methods such as LAMP, which might be more convenient in the field. Or at least they could add a sentence or two that justifies their choice of approach.

(2) In the Abstract, the authors say "The bioinformatics pipeline developed in this study to design specific genomic regions is capable of generating specific primers. It is freely available and can be utilized for any other model organism.". From where can the reader download this pipeline? In what format or programming language is it implemented?

(3) The authors, sensibly, check their primer sequences against available genome sequences to assess selectivity. However, it is important that the primers do not make false positives against other bacteria in the (normal/healthy) Citrus microbiome. The authors should consider searching the sequences of available microbiome metagenomic data such as from this study: DOI: 10.1128/AEM.00210-17.
Validity of the findings: (1) In the Introduction, lines 96-98, the authors write "the use of comparative
97 genomics as a tool for molecular diagnosis is still at a nascent stage. This is even more evident in the case of bacteria of the genus Xanthomonas".

This is quite a bold claim and the authors do not cite any references or present any other evidence to support this. Arr the authors aware of the numerous examples of genomics-informed molecular detection and diagnostics in Xanthomonas?  Here are nine examples that I am aware of; there may be more:

https://doi.org/10.1111/j.1365-3059.2012.02654.x

and

https://doi.org/10.1111/ppa.12289

and

1: Larrea-Sarmiento A, Dhakal U, Boluk G, Fatdal L, Alvarez A, Strayer-Scherer A,
Paret M, Jones J, Jenkins D, Arif M. Development of a genome-informed
loop-mediated isothermal amplification assay for rapid and specific detection of
Xanthomonas euvesicatoria. Sci Rep. 2018 Sep 24;8(1):14298. doi:
10.1038/s41598-018-32295-4. PubMed PMID: 30250161; PubMed Central PMCID:
PMC6155141.

2: Lang JM, Hamilton JP, Diaz MGQ, Van Sluys MA, Burgos MRG, Vera Cruz CM, Buell
CR, Tisserat NA, Leach JE. Genomics-Based Diagnostic Marker Development for
Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. Plant Dis. 2010
Mar;94(3):311-319. doi: 10.1094/PDIS-94-3-0311. PubMed PMID: 30754246.

3: Palacio-Bielsa A, López-Soriano P, Bühlmann A, van Doorn J, Pham K, Cambra MA,
Berruete IM, Pothier JF, Duffy B, Olmos A, López MM. Evaluation of a real-time
PCR and a loop-mediated isothermal amplification for detection of Xanthomonas
arboricola pv. pruni in plant tissue samples. J Microbiol Methods. 2015
May;112:36-9. doi: 10.1016/j.mimet.2015.03.005. Epub 2015 Mar 11. PubMed PMID:
25769438.

4: Wang H, Turechek WW. A Loop-Mediated Isothermal Amplification Assay and Sample
Preparation Procedure for Sensitive Detection of Xanthomonas fragariae in
Strawberry. PLoS One. 2016 Jan 14;11(1):e0147122. doi:
10.1371/journal.pone.0147122. eCollection 2016. PubMed PMID: 26766068; PubMed
Central PMCID: PMC4713083.

5: Wang XQ, Allen TW, Wang H, Peterson DG, Nichols RL, Liu A, Li XD, Deng P, Jia
D, Lu SE. Development of a qPCR Protocol to Detect the Cotton Bacterial Blight
Pathogen, Xanthomonas citri pv. malvacearum, from Cotton Leaves and Seeds. Plant
Dis. 2019 Mar;103(3):422-429. doi: 10.1094/PDIS-07-18-1150-RE. Epub 2019 Jan 11.
PubMed PMID: 30632895.

6: Nakato GV, Wicker E, Coutinho TA, Mahuku G, Studholme DJ. A highly specific
tool for identification of Xanthomonas vasicola pv. musacearum based on five
Xvm-specific coding sequences. Heliyon. 2018 Dec 27;4(12):e01080. doi:
10.1016/j.heliyon.2018.e01080. eCollection 2018 Dec. PubMed PMID: 30603713;
PubMed Central PMCID: PMC6307341.

7: Hodgetts J, Hall J, Karamura G, Grant M, Studholme DJ, Boonham N, Karamura E,
Smith JJ. Rapid, specific, simple, in-field detection of Xanthomonas campestris
pathovar musacearum by loop-mediated isothermal amplification. J Appl Microbiol.
2015 Dec;119(6):1651-8. doi: 10.1111/jam.12959. PubMed PMID: 26425811.

(2) The authors write at line 101 "These sequences are intended to be used
only for detecting isolates derived from Citrus sp.; these sequences do not distinguish citrus-associated Xanthomonas from any other bacteria."

I find this slightly puzzling. In order for the tool to successfully detect isolates from Citrus spp., it must be able to distinguish these from other bacteria. If it cannot distinguish the target organism from other bacteria, then there will be false positive results. For example, the microbiome of the Citrus tree must surely contain "other bacteria" and thus are a source of potential false positives. Furthermore, I don't really understand why the authors are being so cautious in this claim, given that they have experimentally demonstrated good specificity of their primers in silico, in vitro and in vivo?

I appreciate that in line 289, the authors write "It is important to emphasize that the method presented here is not a general method for identifying the Xanthomonas strains discussed here with respect to other bacterial isolates. The application setting for which the diagnostic tool was designed is for strains isolated from citrus trees only."

(3) Line 115. The "custom Perl scripts" must be made available, e.g. as supplementary information or submitted to a repository such as GitHub. See PeerJ Data and Materials Sharing policy.

(4) Line 214. "Mauve found a total of 5,217 genomic islands.". What is the definition of a 'genomic island'? One definition is "Genomic islands are clusters of genes within a bacterial genome that appear to have been acquired by horizontal gene transfer." (J.P. Ramsay, ... C.W. Ronson, in Reference Module in Life Sciences, 2017). Does Mauve actually check for evidence of horizontal transfer? Or does it simply look for sequences that are private to one genome or set of genomes?

(5) The authors present a new pipeline for detecting pathogen-specific sequence signatures. It would be beneficial to the reader if the authors could include some discussion about how their pipeline compares with previous approaches (why re-invent the wheel?)  to solving this problem for example, but not limited to, these two previous publications:

1: Phillippy AM, Ayanbule K, Edwards NJ, Salzberg SL. Insignia: a DNA signature
search web server for diagnostic assay development. Nucleic Acids Res. 2009
Jul;37(Web Server issue):W229-34. doi: 10.1093/nar/gkp286. Epub 2009 May 5.
PubMed PMID: 19417071; PubMed Central PMCID: PMC2703920.

2: Phillippy AM, Mason JA, Ayanbule K, Sommer DD, Taviani E, Huq A, Colwell RR,
Knight IT, Salzberg SL. Comprehensive DNA signature discovery and validation.
PLoS Comput Biol. 2007 May;3(5):e98. Epub 2007 Apr 20. PubMed PMID: 17511514;
PubMed Central PMCID: PMC1868776.
Additional comments: This paper presents a method or pipeline for the design of pathogen-specific PCR primers and successfully applies it to the case of distinguishing among several important Xanthomonas pathogens of Citrus crops. It makes a useful contribution and is essentially scientifically sound, notwithstanding the specific issues that I raised above.


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

Input:
Title: DETECTION AND IDENTIFICATION OF XANTHOMONAS PATHOTYPES ASSOCIATED WITH CITRUS DISEASES USING COMPARATIVE GENOMICS AND MULTIPLEX PCR
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the paper is well presented and researched. The authors have conducted a very through literature review and provided good context.

Suggest that the introduction provides more detail or makes it clearer on how their work is novel compared to previous work on citrus canker diagnostics. For example, is this the first genome informed analysis to design PCR primers for Xanthomonas pathogens on citrus, some of the current published PCR tests are over 14 years  old (e.g. Cubero et al. 2005). Could consider describing some of the false-positive and test specificity issues with the current diagnostics e.g. Delcourt et al (2013) (cited reference 52).

The concluding sentences in the introduction (lines 100 -102) are a bit ambigous and needs to be rewritten to clarify what the developed assay targets. For e.g. on one hand the paper states that their target sequences are specific to bacteria on citrus but then say they do not distinguish citrus associated Xanthomonas from any other bacteria. From their results there assay is specific and targets the 4 Citrus pathotypes.

The paper has 7 figures that could be condensed. The figures only need to present results that are representative. For e.g. Figure 5 is a photo of a blank gel with negative results (should at least provide a positive control on the gel).

The discussion could be revised to make some of the sentences clear.
Experimental design: The experimental design is robust. The papers describes the key components of assay design e.g. primer design, specificity, and sensitivity.

Specificity - a good in silico evaluation was good conducted. Lab testing of the target strains were reasonably well covered. More lab testing of the expected background microflora on citrus plants would have been useful to further support their findings. For example, testing of environmental and saprophytic strains - Pantoea agglomerans, Enterobacer sp, Stenotrophomonas sp etc.

Line 123 - when you refer to Primer Designing tool - is this Primer-Blast on NCBI?\

Line 140 - receipe for nutrient agar - "meat extract" should be beef extract.

Line 208 - useful to have a sentence to describe what eletronic PCR does? Note: NCBI replaced electronic PCR with Primer BLAST.
Validity of the findings: The results for testing assay specificity and sensitivity are robust and well supported.

Would be useful to describe how you assessed repeatability and robustness of the assay. For example, can you use different reagents and DNA extraction methods and get the same result, are results consistent when you test other citrus varieties, how were samples replicated.

Do you get a competition effect amongst the different primers when using multiplex PCR on all 4 targets at the same time. Figure 6 - suggest to might as the amplicon at 160 bp is much fainter than the other targets.

The discussion suggests that TaqMan could be used or developed with these primers to enable better test sensitivity. TaqMan is only likely to work with the smaller amplicon targets (<300 bp).

No information is provided to what region of the genome the primers amplify. For e.g. are the amplicons derived from known genes or genetic regions, what do the amplicon sequences match when you blast them?
Additional comments: No additional comments


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

Input:
Title: CD36 IDENTIFIED BY WEIGHTED GENE CO-EXPRESSION NETWORK ANALYSIS AS A HUB CANDIDATE GENE IN LUPUS NEPHRITIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Yang et al report the results of a gene co-expression network analysis in the context of a manifestation of systemic lupus erythematosus, lupus nephritis, to identify hub genes that could represent new biomarkers of disease and/or potential therapeutic targets. The authors analyzed previously published gene expression microarray data and identified CD36 as hub gene associated to the condition and then try to validate the results using g additional gene expression data from four microarray datasets.
The English language is globally understandable, despite the use of a few terms seems unusual (i.e. excavation instead of Identification at line 204, “to distinguish the biological themes of gene” line 170).
The introduction provides a clear context for the study. References should be added at line 69, when authors refer that WGCNA has been rarely used for studying LN. The structure of the paper is clear. Some figures should be improved for clarity. Figure 3A showed the dendrogram with the results of two different clustering methods. The authors should specify in the text that they chose the merged dynamic method and justify their choice. Figure 3B show a different heatmap dendrogram compared to the modelled gene network: this can lead to confusion. Eigengene adjacency map provided in Figure 3 should be improved by adding at least the labels indicating module color. The authors should consider to provide also a clustering representation together with the heatmap to improve the interpretation of the figure.
Finally the rawdata are accessible by GEO, so that readers can identify original data.
Experimental design: The research question is well defined and relevant and the work has the potential to make the field moving forward.
Concerning the overall design of the study, a major question is whether the results are linked to the true pathogenesis of the disease or to the fact that in LN the tissues are infiltrated with immune cells, and the different gene expression profiles between control tissues and LN are simply due to cell type composition. The authors should comment on this point, eventually providing additional information on the pathology of the studied tissues.
Concerning the co-expression analysis, I wonder why the authors restricted their discovery analysis using WGCNA to only one gene expression dataset, when three out of the four datasets used for the validation (Figure 9) are based on the same or very similar microarray platform (all of them being Affymetrix arrays). The same analysis performed on one microarray should be performed on the pooled data and validated by the fourth dataset based on Nanostring technology. This approach should be tested to provide results that are expected to be statistically more robust and biologically more meaningful. Finally, the authors should state why they discarded other modules still significantly associated to LN in their subsequent analysis (lightyellow and darkgrey).
Validity of the findings: While the authors provide some evidence that CD36 could be an important gene in LN, the limits of the design do not allow a conclusion on the validity of the results.
Additional comments: No other major 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: CD36 IDENTIFIED BY WEIGHTED GENE CO-EXPRESSION NETWORK ANALYSIS AS A HUB CANDIDATE GENE IN LUPUS NEPHRITIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: General comment
Reusing publicly available genomic data for different bioinformatics approaches can help reveal novel candidate genes responsible for disease. The present work is interesting and the authors have managed to find a differentially expressed gene that could be relevant to Lupus nephritis. However, important details are missing from the manuscript and the relevance of the proposed hub gene has not been confirmed in any way. I think that the manuscript should be thoroughly revised to be suitable for publication.
Experimental design: Specific comments:
1. In Material and Methods – Expression profile data collection: I checked the GEO accession number provided by the authors and I was not able to figure out which samples were included in the study. Please give more details on the study group including preferably a supplementary table with the GEO IDs and associated information. Also, provide more details on the type of controls you are using (what’s normal and why did they undergo a biopsy?).
2. In Material and Methods – Weighted co-expression network construction and module division: Please specify whether any variance-related filtering was performed. Also, were lowly variable/non-expressed genes taken into consideration in the WGCNA analysis? If so, you are at a high risk of including genes in your study with weak biological significance. Please detail this part.
3.  In Material and Methods – GO and KEGG pathway enrichment analysis: You point out that a nominal p value<0.01 and a BH-corrected p value<0.05 are used for GO and KEGG pathway enrichment analyses (respectively, I guess). These thresholds seem to be arbitrary and one cannot understand why you use a corrected p value in a case and a nominal p value in the other. Please explain your choice or be more consistent and restrictive.
4. In Material and Methods – Differentially expressed gene analysis: Same comment as before. You perform a genome-wide analysis but do not correct for multiple comparison. Additionally, this results in an extremely large heat map that is difficult to read and interpret. I would be more restrictive, at least for data visualization.
5. In Material and Methods – Identification and validation of hub gene: You mention different GEO datasets that you used for the validation of the hub gene. However you do not provide any information (not even accession numbers) nor describe the sample size and characteristics. I propose to provide this information in the manuscript and to complete the abovementioned supplementary table with IDs/associated info corresponding to these confirmatory datasets as well.
Validity of the findings: Specific comments:
6. Results – Data processing: You mention a clustering for sample filtering. Can you provide it as a supplementary figure or at least make it clear which are the final samples from GEO included in the analysis?
7. Results – Identification of clinically significant modules (exclude DEGs from the tittle in this section, you speak about them in the following section and not in this one): I don’t think the authors interpret correctly Figure 3c. In fact, it represents adjacency between eigengenes from different modules and therefore, do not provide any information about the accuracy in the division of the modules (as stated in the last sentence of this section), but the relationship among them. Moreover, you don’t interpret further such a relationship and I do not think you should mention in Results anything that is not going to be discussed. Please revise both the figure legend and the main text. Please specify what dynamic tree cut and merged dynamic mean in your dendogram (Figure 3A).
8. Results – Identification of clinically significant modules: At the end of the section you mention that your results reveal that the darkgreen/lightgreen modules are suitable for further investigation. I think the most important finding here is the strong positive correlation you find between module membership and trait significance, suggesting that the highest the membership the highest the association with LN or normality. I think this should be highlighted, probably in both Results and Discussion. Additionally, you should rename your main modules (colors do not have any biological meaning; I would replace colors by something like top LN and top non-LN modules). Finally, I think you should remind the readers about the number of genes contained in the further investigated modules.
9. Results – Enrichment analysis and DEG analysis of trait-related modules: The last paragraph of this section is highly speculative and should be rewritten and moved to Discussion.
10. Discussion: I miss more references to previously performed trascriptomic analyses of LN.
11. Discussion: I think there are parts in the Discussion that are too speculative/ overoptimistic in the interpretation of the Results.
12. Discussion: There are a number of genes appearing for the first time in the Discussion. I think being more restrictive in the DEG analysis would give as a result a shorter candidate gene list and a more comprehensive heat map (please revise Figure 7). I think the text would flow better if you mentioned at Results anything you would like to discuss later.
Additional comments: 13. I miss a survival analysis that proves the importance of CD36 in LN. From my point of view, overexpression is not a strong enough evidence to prove the centrality of CD36. Could you please try to perform such an analysis by using for example TCGA data (if available)? I think a positive result would substantially increase the impact of this work.
14. Please mention abbreviations at first appearance and revise English.


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: CD36 IDENTIFIED BY WEIGHTED GENE CO-EXPRESSION NETWORK ANALYSIS AS A HUB CANDIDATE GENE IN LUPUS NEPHRITIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I believe the authors have been addressed the major criticisms I have raised.
Experimental design: Clarifications have been made on the design and it is now clear the choice to include only one dataset for the co-expression analysis and to use the others for further validation.
Validity of the findings: The new analysis provided by the authors seems to validate the previous 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: CD36 IDENTIFIED BY WEIGHTED GENE CO-EXPRESSION NETWORK ANALYSIS AS A HUB CANDIDATE GENE IN LUPUS NEPHRITIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made substantial changes to the manuscript including the reanalysis of the data after a variation-based filtering and a differential expression analysis according to the LN class in an independent database. Additionally, they have detailed the Methods section as required by the Reviewers. In general, I think that the manuscript has considerably improved and I congratulate the authors for they thorough revision.
Experimental design: The Methods section is more accurate now. A supplemmentary table and document have been added to provide details and IDs on the specific samples used in the analysis.
Validity of the findings: Some of the Figures have been redone, new analyses have been conducted. Discussion has been rewritten to reduce the speculative considerations.
Additional comments: Minor comments:

-I would mention Supplementary Table 1 in Methods for the first time.

-Sorry for suggesting TCGA (I was thinking about a similar database on Lupus but obviously TCGA is not the one). I think Nephroseq was a good choice and certainly, your new analysis strenghtens your previous results. However, you do not comment about this in the Discussion. Moreover, you plot class II and III vs IV separately (Figure 10), and group IV looks different in the two plots although it is supposed to be expression data of the same samples, right? Please, pay attention to this Figure and accordingly discuss in the appropriate section.


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

Input:
Title: EFFECT OF BIRDSONGS AND TRAFFIC NOISE ON PEDESTRIAN WALKING SPEED DURING DIFFERENT SEASONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article uses clear and technically correct text. On the introduction, some paragraphs could be rearranged to improve the text flow. See my comments on the uploaded file.

The article includes sufficient background to demonstrate how the work fits into the broader field of knowledge. Nevertheless, some results of previous studies were presented as showing the expected effect even if they were not statistically significant. This could lead to misunderstandings.

The structure of the article is conforming to an acceptable format. Figures are relevant to the content of the article, but Figure 1 do not has a reference from where it was taken from.

Please see my comments about tables’ labels on the upload file.

The raw data have been made available. I suggest to provide information about time of walking for each section (1– 8) and for total route in the raw data.

The authors claimed that the work is self-contained justifying it in an appropriate manner.
Experimental design: The research question is well defined, relevant and meaningful. The authors intend to fill the knowledge gap regarding the evaluation of direct behavioral responses (walking speed) to noise in specific environments.

The authors affirm that there were no known risks for participants in this study. Nevertheless, any potential risks of walking around the route and the discomfort it may entail should be considered for ethical purposes.

The soundtrack link of the relaxation sound condition was not correctly typed. The stimuli were not described with sufficient detail to replicate and there is no credit attribution for the authors of the soundtracks.

The authors have presented the effect size value for the analysis. The sample size of each of the three conditions was different and it was not specified in the text for the analysis.
Validity of the findings: The authors did not state that the data were made available in an acceptable discipline-specific repository but they uploaded them as Supplemental files.

The data was not fully controlled because the research was conducted in an outdoor environment. Nonetheless, according to the authors they have tried to reduce the effects of the external variables by balancing the experimental conditions during the days on which the experiment was performed.

The results were more robust for the interactions between the effects of the specific environment of the route and walking speed than for the interaction between the presented sounds and walking speed.

In some parts of the manuscript, the authors attribute that there was a difference between two conditions even if this difference was not statistically significant. This should be considered very carefully.

The conclusions are appropriately stated but in some cases are not supported by the results, or are overestimated. Some of the hypotheses presented in the introduction were not taken up during the discussion of the results.
Additional comments: Please see my comments in the uploaded 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: EFFECT OF BIRDSONGS AND TRAFFIC NOISE ON PEDESTRIAN WALKING SPEED DURING DIFFERENT SEASONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Reporting is good throughout. some additional references are suggested below.
Experimental design: Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap.
Methods described with sufficient detail & information to replicate.
Validity of the findings: Impact and novelty could be assessed more deeply (see suggestions below on implications and relevance).
Additional comments: The paper explores the effects of relaxation nature sounds (birdsong) compared to traffic noise on walking speed in a real outdoor urban environment. This is an interesting and novel topic that has important implications for policy and practice. The methodology is robust and the reporting is very precise and high quality. I suggest below some changes that would help to make the article stronger.
First of all, while the title refers to relaxation nature sounds, I recommend to change this into birdsongs and make explicit reference from the title and abstract that the sound considered was that of birdsongs. This can help readers to identify the topic of the study more clearly.

Abstract

Suggest to remove “Although there is a large body of studies based on subjective evaluations of noise annoyance or environmental preference” and focus on what the study is about.
Since one of the main points of the study was to test congruence, I suggest to make this clearer in the abstract earlier on.
Please add: The participants (n=x), young university students, walked along a 1.8-km urban route in the city of xx.
Please remove because the outdoor environment was, in general, quite noisy. – the word because is misleading as it is not possible to assess causality. Same suggestion for: because of the congruence between the sounds and the visual environment. – please rephrase avoiding word because.

Introduction

The section gives context, but the start doesn’t do a great job in identifying what is the core topic of the article. Since the article is on sounds, I suggest starting it with background information on the role of sound in restorative experiences, especially on birdsongs.

Also, since the study is about stress and walking in cities, I suggest to add background information on the relevance of these topics. For example, some references to urbanisation trends, or to the need to encourage walking and improving psychological wellbeing of city residents. These additions would reinforce the relevance and significance of the study.

Line 179
Regarding the negative health consequences of the fast pace of walking, I suggest to add further references, including some more recent research. This would reinforce the claim that high walking speed is detrimental for health.

Methods

Please add info on the city where the studies were performed.
Were Incentives/rewards offered to students?

I suggest to add one table with dates and weather conditions of each day. This would make reading easier.

Discussion

The start of the section needs more detail. I suggest that you improve the description at lines 543-547 to provide more detail on the main findings.

Regarding the congruence (lines 569 and following), I suggest to deepen the discussion on the practical implications of this matter. For example, one practical implication could be related to the use of relaxing artificial natural sounds in urban roads such as birdsongs: the findings seem to suggest that such artificial sounds should match weather and seasonal conditions to aid relaxation.

Line 574: it would be useful to re-state the finding on speed and season.

Lines 597-601 are unclear, I suggest to rephrase and reorganise.

Line 614: I suggest to change to The types of sounds experienced might have also influenced the ratings of visual environments. This is because it is hard to assess causality between the two, also due to external factors that were not controlled for.

It would be worth including some reflections and related literature on why people might walk faster in presence of traffic sound. This could be due to environmental stressors (Evans 2003) and safety concerns for example (Bornioli et al., 2019).

Limitations:
it seems that the order of walks was not randomised. If this is the case, I suggest to discuss the limitations and implications of this choice.
I also suggest to expand lines 632-636 and reflect more deeply on the strengths of a real-world environment. Is there any study that has compared relaxation effects between field and lab environments? See for example Conniff and Craig, 2016; Shaw et al., 2015 on relaxation and sensorial experience.

I suggest to add recommendations for future research, for example on:
-	Research on benefits of nature and the role of seasons;
-	Congruence between visual and audio stimuli and effects on behaviours and wellbeing;
-	Personal characteristics and effects on walking speed and relaxation.

Conclusions are well stated, linked to original research question & limited to supporting results. I suggest to add recommendations for policy (for example see above on the use of artificial birdsong sounds in cities).

Minor changes:

Throughout text: When using the word significantly, I suggest to specify statistical significance.
Line 45 and 179
The research? Is it previous research or the current research?
Lines 45-57
Please add references to support your statements.
Line 544
There is something missing: First… (there is no “second”?)
Line 559
Suspicious? Not sure that’s the right word?
Table 2: title seems not correct as the table reports rating on walk not on speed?
For tables on ratings on walking experience, I suggest to add histograms that would also show statistical differences between ratings.


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

Input:
Title: EFFECT OF BIRDSONGS AND TRAFFIC NOISE ON PEDESTRIAN WALKING SPEED DURING DIFFERENT SEASONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study reports the results of an experiment involving the effect of visual and auditory stimuli to relaxation in walking behaviour. The author looks at relaxation by means of direct simple individual reports (questionnaires) and walking speed across different urban environment settings. The study acknowledges that it is part of a sequence of studies related to walking, sound and urban environment.

As a study that is part of a larger initiative, the conceptual proposal of the experiments seems coherent and previous findings support part of the assumptions used by the authors. The experimental setup is not complex but let many possible interfering variables take part actual results. Most of the hypotheses and questions seem to reproduce other studies and the majority of the results reported are not conclusive, regarding the initial hypotheses. My general opinion is that the text could only be accepted if the authors promote large changes in the content, review of the literature and report of results.

Thought not-significant results should be reported and should be taken as an important part of the scientific development, the text and statistical reporting fail in providing a creative exploratory view of the data and information. Distributions, variability and possible cross-correlations between tendencies in the data are not clearly reported. Tendencies are hidden and the authors seem to avoid discussing null-hypotheses. This bleak report of non-significant data just reinforces some experimental design problems and hide insights that could help the reader.

 I find problematic the definition (or lack of definition ) of the concept of relaxation behind the text. Although relaxation and stress have been studied for a long time, the authors do not discuss it from the literature, alternative definitions, or cultural variability. The choice for the stimuli seems to represent a culturally specific notion of relaxing sound. There is no problem in choosing an urban, Western, 2020's concept of relaxing sound.  The problem here is that the study does not acknowledges the cultural, social, historical and psychological limitations of it, while makes culturally-centric choices. Since the concept of relax is very important for the study, my opinion is that it is not possible to understand the work without a clear review of the definition.

Following the problems of definition of relaxation and stress, the stimuli used as traffic noise seem to be very problematic. My guess is that the track includes an abnormal density of traffic noise. In other words, I don't think the surroundings of the experimental location exhibit, at anytime or context, the level of noise exhibited in the stimuli. It might impose a kind of cultural mismatch since, most probably,  the subjects never experienced a similar context. It was recorded in a totally different cultural and urban environment and also includes an unreported/not-discussed concept of noise. It includes voices from different languages which, create another layer of psychological aspects of "noise" (in this case, traffic noise and speech).
Experimental design: The overall experimental design is feasible but the control of variables and consequent definition of limitations are weak. It does not mean that researchers must control all the aspects of the experiment (real experiments) but it seems that the authors and the statistical procedures ignored limitations coming from cultural assumptions, variability and uncontrolled variables that have an effect on the results. If the authors want to use ANOVA as a test for significant differences I strongly recommend discussing if the Anova's assumptions are violated. Another option is just use anova or other descriptive methods, as data visualization. Given the current state of the variability of the data that are reported and not reported,  no test of significance can be easily applied. First, because there is no need for it (significance is useless in such a heavily influenced cultural behaviour), and second, because it is almost impossible to accept the assumptions of these methods.

Regarding the selection of participants, please, report if the participated in the previous studies and some better descriptions of their cultural background.

line 203: Since the previous study raises important questions tackled by the present one, the authors should be more specific and less simplistic concerning the overall design and results. For example:
- 203: " the sound variable was fully controlled": it is very difficult o control sound in the open environment. Please, specify the method
- 2017: "The participants listening to traffic noise walked
208 significantly faster on the route than those listening to forest birdsong sounds". Please, report what means significantly fast.
218: " the walking route was located in green areas with a relatively
219 small amount of traffic." I find "green areas and small amount of traffic" a very subjective definition that should be specified

237: "On the other hand, it is almost impossible to have a perfect control condition in a  real outdoor environment.". I would also indicate that it is almost impossible to have similar treatment conditions in a real outdoor environment. How this impact in the assumptions required in the statistical methods? (e.g. how can you assume that deviations come from random processes?)
Validity of the findings: See discussion above in item 2

638: "Furthermore, it was not considered individual differences in noise annoyance or attitudes toward particular types of noise." I understand that the authors envisaged this problem. But it deserves a clear discussion using proper literature as base.

657 "In conclusion, the study demonstrated the interaction between auditory and visual stimuli in the perception of an environment and showed consequent behavioral reactions, specifically changes in walking speed." I think this conclusion is not supported by evidence. Authors should avoid typical terminology used in hard sciences and acknowledge uncertainty. I think the data explores, illustrates or suggests some relationships but the results (from a traditional statistical perspective) are not conclusive.
Additional comments: The content first paragraph is somewhat loose and seems to be repeated in the following paragraphs.

Lines 134, 137: "e.g." abbreviations should be placed inside the parentheses.

179: I cannot fully understand what means "pace of life" since life is very difficult to define or measure. Apart from the vagueness of the expression, it seems that there are cultural biases that seem to present an alternative slow pace of life as a generalizable solution. This may only reflect the viewpoints of subjects living in urban regions. Individuals from rural areas might have a different opinion or different problems with walking pace, the speed of changes, events and responses from the environments. This bias was also reproduced in the selection of subjects, who were probably selected from a group living in an urbanized area.  Please, clarify or acknowledge the viewpoint.
244: Typo in "with to "..