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

Input:
Title: A NOVEL HISTOZOIC MYXOSPOREAN, ENTEROMYXUM CAESIO N. SP., INFECTING THE REDBELLY YELLOWTAIL FUSILIER, CAESIO CUNING, WITH THE CREATION OF THE ENTEROMYXIDAE N. FAM., TO FORMALLY ACCOMMODATE THIS COMMERCIALLY IMPORTANT GENUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study reports a novel Enteromyxum sp. in the stomach of the redbelly yellowtail fusilier in Malaysia as Enteromyxum caesio n. sp., and erects a new family, Enteromyxidae n. fam., for the genus Enteromyxum, currently containg four species. The manuscript is well constructed and logically described with clear figures. The reviewer has no indications or suggestions except for a few minor points as mentioned below:
Experimental design: No problem.
Validity of the findings: No problems.
Additional comments: The reviewer has no indications or suggestions except for a few minor points as mentioned below:

1.	L1 [Title]: ‘sp.’ after ‘Enteromyxum’ (?)
2.	L19: Is it necessary put ‘,’ after ‘mortalities’?
3.	L25, L30, L35, L77, L225: ‘Enteromyxidae n. fam.’
4.	L26, Figure 2 legend: Either ‘gastric’ or ‘of the stomach’ might be enough.
5.	L34: ‘spore valve’ or ‘shell valve’ (?)
6.	L35: ‘is’ (?)
7.	L44: Full description of scientific names at their first appearance might be welcome by the readers: ‘the genus Enteromyxum Palenzuela, Redondo et Alvarez-Pellitero, 2002’.
8.	L46, L50: The scientific name for the turbot and tiger puffer.
9.	L61, L235, L240: Two references of ‘Freeman & Kristmundsson, 2015’ are found in the Reference section.
10.	L68-69: Please place references chronologically.
11.	L71: ‘Caesio cuning (Bloch, 1791)’ (?)
12.	L83: What are ‘othres’?
13.	L87, L88, L95: ‘Dino-Eye’, ‘ImageJ’, and ‘Jeol JEM-1010’ are not familiar to the readers.
14.	L93: ‘PCR positive’ is too much simple expression for the readers.
15.	L96: ‘DNA lysis buffer’ (?).
16.	L83-L128: The reviewer recommend the authors to follow the basic manner of explaining some commercially available products as usual in the scientific articles.
17.	L184-L186: ‘Order Bivalvulida Shulman, 1959’ (?)  What is the reason for you ro omit it?
18.	L186-L188, L200-K202: If you use ‘et’ here, it is better to use ‘et’ for scientific names at any times. If you can use ‘& (and)’ for this purpose, use it thoroughly in this manuscript.
19.	L191: Does Enteromyxum caesio n. sp. prefer to parasitize only cardiac gastric glandular tissues only? How about other places of the stomach? Just for confirmation.
20.	L203: Full scientific name.
21.	L221: Full expression of Gastromydicae and Monomyxidae.
22.	L257: ‘Freeman & Kristmundsson, 2018’ not in the Reference section.
23.	L353: Finally, the reviewer understands that ‘2018’ must be placed here instead of ‘2015’. Please check citations of ‘Freeman & Kristmundsson, 2015’ and ‘Freeman & Kristmundsson, 2018’ thoroughly in the text.
24.	L389-390: ‘Scomphthalmus maximus’ in italic (?)
25.	L313: ‘Enteromyxum species.’
26.	Table 1: ‘Tin Tun et al. (2000)’ (?)
27.	Figure 4: Is it possible for the reviewer to see a high magnification TEM photographs of hyperinfected microsporidia just for review purpose? At low magnification, it seems a homogenous structure.
28.	Please check thoroughly the manuscript by themselves.

This manuscript is very interesting and valuable for us.


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

Input:
Title: A NOVEL HISTOZOIC MYXOSPOREAN, ENTEROMYXUM CAESIO N. SP., INFECTING THE REDBELLY YELLOWTAIL FUSILIER, CAESIO CUNING, WITH THE CREATION OF THE ENTEROMYXIDAE N. FAM., TO FORMALLY ACCOMMODATE THIS COMMERCIALLY IMPORTANT GENUS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article meets all the criteria.
Experimental design: The article meets all the criteria.
Validity of the findings: The article meets all the criteria.
Additional comments: The present paper describes a single myxosporean species assigned to the genus Enteromyxum. Authors provide morphological documentation of the parasite including also SEM and TEM ultrastructure details of the spores and trophozoites, respectively, as well as phylogenetic analysis of the studied myxosporean species and its relatives.
The main message of the manuscript is a species description of another species of important genus Enteromyxum, which member E. leei is a serious pathogen of cultured fish. In addition, authors erected a new family, Enteromyxidiidae, to accommodate four members of the genus Enteromyxum. Taxonomic changes within Myxozoa are challenging and have to be done very cautiously because of the presence of many paraphyletic and polyphyletic systematic groups. I agree with a new family Enteromyxidiidae - its establishment is another step to a better myxozoan taxonomy.
Specific comments:
The title of the manuscript should contain scientific Latin name of the fish host (English common names are not sufficient). I would also suggest adding higher classification for Enteromyxum (Myxozoa) in the title (not all parasitologists are aware of what group of parasite Enteromyxum is).
Similarly, in M&M, there should be a Latin name for fish host species provided. Please, provide also GPS coordinates for Kilim mangoves, Langkawi (is there a typo? “mangoves“  = “mangroves”).
Taxonomic summary, line 180-187: According to a book Myxozoan Evolution, Ecology and Development (Okamura et al. 2015), the taxonomic classification of Myxozoa is unranked subphylum, Myxosporea should be a class. There is an order Bivalvulida missing in the summary.
The histology section, as well as spore light microscopy, is of very poor quality in Fig.2. Do you have any better images?


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

Input:
Title: A NOVEL HISTOZOIC MYXOSPOREAN, ENTEROMYXUM CAESIO N. SP., INFECTING THE REDBELLY YELLOWTAIL FUSILIER, CAESIO CUNING, WITH THE CREATION OF THE ENTEROMYXIDAE N. FAM., TO FORMALLY ACCOMMODATE THIS COMMERCIALLY IMPORTANT GENUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article meets all the criteria.
Experimental design: The article meets all the criteria.
Validity of the findings: The article meets all the criteria.
Additional comments: The authors have improved the manuscript significantly, as well as clarified all issues.


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

Input:
Title: A NOVEL HISTOZOIC MYXOSPOREAN, ENTEROMYXUM CAESIO N. SP., INFECTING THE REDBELLY YELLOWTAIL FUSILIER, CAESIO CUNING, WITH THE CREATION OF THE ENTEROMYXIDAE N. FAM., TO FORMALLY ACCOMMODATE THIS COMMERCIALLY IMPORTANT GENUS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am satisfied with the author's responses and changes made in the manuscript.


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

Input:
Title: PREDICTIVE VALUES OF STOOL-BASED TESTS FOR MUCOSAL HEALING AMONG TAIWANESE PATIENTS WITH ULCERATIVE COLITIS: A RETROSPECTIVE COHORT ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The English language should be improved to ensure that an international audience can clearly understand your text.
2. The font size of the all figures should be identical.
3. Figure 2 was uploaded incompletely.
Experimental design: 1. In part of Materials & Methods, more details should be provided. I am confused that “the correlation of iFOBT and FC with the total Mayo score…” whether the total Mayo score was the baseline score. Additionally the evaluation points of the EMH and CMH were not provided in the study (main text and figure legends). This problem appears in many places of the manuscript. Please ensure that the points of all results including colonoscopy, stool samples and laboratory examinations should be marked clearly.
2. How many times did every patients enrolled in the study undergo colonoscopy in two years?
3. Please add the primary endpoints and second endpoints in Materials & Methods.
Validity of the findings: 1. In table 2 & 3, please add the results of multivariate analyses to find the independent factors which affect the outcomes of mucosal healing and show the results in part of Results.
2. Spearman rank correlation coefficient and P values especially less than 0.05 should be lucidly described.
3. In table 5, some information of the study by Naganuma et al was incomplete.
4. Were the best cutoff values of iFOBT and FC levels reported in others studies? Please discuss them in part of Discussion.
Additional comments: This study focused on the predictive role of stool-based tests, including fecal calprotectin (FC) and the immune fecal occult blood test (iFOBT) for mucosal healing among patients with UC in Taiwan. Multiple researches had demonstrated that the FC and iFOBT had similar roles in predicting (endoscopic mucosal healing) EMH among patients with UC. The present study was the first report from patients with UC in Taiwan that compared iFOBT and FC, which put forward a proposal that given the low cost and similar performance with FC, iFOBT may be advantageous as a first line monitoring tool for patients with UC in Taiwan. This study could guide the clinicians in Taiwan to choose the iFOBT, which is noninvasive and lower cost, as the prective of mucosal healing of UC in clinical practice and decision making.


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: PREDICTIVE VALUES OF STOOL-BASED TESTS FOR MUCOSAL HEALING AMONG TAIWANESE PATIENTS WITH ULCERATIVE COLITIS: A RETROSPECTIVE COHORT ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the language in the manuscript is clear, concise and acceptable. Introduction is well written and characterizes the context adequately. Minor edits are required on Line 68 (Introduction: add reference on colonoscopy complications.) and Line 96: (Materials & Methods: sentence structure in the inclusion criteria.)

Figures are not yet up to publication standards: Figure 2: legend makes reference to an A and B panel, but only a single panel is available in the document provided. Figure 2 Legend is formatted improperly. In Figures 2-4, no legend is given for the different depicted lines (CIs?).

Raw data is provided, but incompletely defined: Definition and calculation of Mayo score and subscores references four components, scored from 0-3, but partial Mayo scores are said to comprise only three variables; without mention of precisely which three components were selected (Line 103-106: Materials & Methods). Furthermore, raw data includes three mayo scores, without reference to which sub-components they denote; and total Mayo score is not explicitly given in the raw data.

Minor comment: Table 4: Please add asterisks denoting significance levels of the noted spearman correlations.
Experimental design: Yen et al provide a straightforward comparison of fecal calprotectin with the immune fecal occult blood test for the assessment of endoscopic healing. This research bears clinical importance and is of particular relevance in lower resource settings. A declaration with regards to the declaration of Helsinki is missing from the materials and methods.

Methods seem to be described in sufficient detail, though I defer to other reviewers for the quality of the description of the laboratory experiments. Statistical measures are explicitly described and accurately chosen, with the puzzling omission of the construction of a predictive model for the assessment of the predictive value. The authors have elected to impose seemingly arbitrary cutoffs on the predictors.
Validity of the findings: Overall, results are accurate and in-line with expectations. Basic statistical comparisons essentially tell the story and conclusions are in-line with these results. However, the authors stumble in the comparison of the relative predictive value of the FC and iFOBT predictor variables. Cutoff criteria for construction of the ROC are selected without rationale. No predictive model is used, two arbitrary cutoffs are selected (Line 155). This is a textbook example for the use of a logistic regression model. Figures pertaining to the ROC curves lack adequate descriptions and inadequately emphasize the difference between prediction of mucosal healing and complete mucosal healing.
Additional comments: This manuscript asks and answers a pertinent and clinically relevant question and the researchers should be commended for their work in this regard. A need for an alternative option to quantify endoscopic mucosal health exists, and avoiding the high costs associated with FC measurements is preferable. Results of basic statistical analysis support their conclusions, though the assessment and reporting of the relative predictive value of each candidate predictor is not yet up to rigorous scientific standards. If this is solved and figures are brought up to publication standards, I have no doubt this manuscript would be of interest to the scientific and clinical community.


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: PREDICTIVE VALUES OF STOOL-BASED TESTS FOR MUCOSAL HEALING AMONG TAIWANESE PATIENTS WITH ULCERATIVE COLITIS: A RETROSPECTIVE COHORT ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: Thanks for the authors carefulness and professional. I have no onther confusions and comments on the manuscript.


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

Input:
Title: BIOINFORMATICS ANALYSIS AND IDENTIFICATION OF CIRCULAR RNAS PROMOTING THE OSTEOGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS ON TITANIUM TREATED BY SURFACE MECHANICAL ATTRITION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled “Bioinformatics analysis and identiﬁcation of circle RNAs promoting the osteogenic diﬀerentiation of human bone marrow mesenchymal stem cells on titanium treated by surface mechanical attrition” by Shanshan Z et al, is appropriate for this journal audience. The authors identify discrete circRNAs that contribute to the osteogenic differentiation of hBMSCs on SMAT -related titanium surface. Overall, the authors have done a great work on analysis of cRNAs and used various in vitro experiments to apply this knowledge to future clinical use.
However, there remain many concerns that could be addressed to ensure appropriate interpretation and reproducibility of these results.
Experimental design: Major concerns
1. Figures - I would like to request the pictures of Alizarin red stained samples.  Also, please include the   SEM image of surface treated titanium sheets.
Line 240 – for the alizarin red staining, if the significant difference was found on the 14th day, there is no explanation on this at discussion part.
2. Line 149 – It looks like from the data, Bradfords (BCA) assay wasn’t used to normalize protein quantity before initiating ALP assay.  We cannot assure an equal amount of protein was present on all of 24 wells.  Looking at Figure 6, SMAT cells already had more cells than annealed group, which means ALP activity can be influenced by this leading to misinterpretation of the data shown in Figure 7.
Line 237 – for the detection of ALP activity, if the significant difference was found on days 3 and 5, there is no explanation on this. Please discuss.
3. Line 164 – why was qPCR only performed at 21 days?  Perhaps you can include more early timeframes to ensure a causal analyses.
4. Line 318 – why would you ‘predict’ something in the conclusion?  And why do you think this still needs further experimental investigation? I didn’t find that in discussion.
Validity of the findings: Minor concerns
1. At the abstract, why is has-circ-00326000 ‘most obvious’? This is not a good word to use.
2. Line 27 – What do you mean by ‘attractive’? I would suggest to be more specific on the words you choose.
3. Line 28 – ‘small’ doesn’t seem to be a proper word to be used here. Please change to more coherent word. ‘Better delay absorption of alveolar bone’ can be changed in a better phrase.
4. Line 38 – why is ‘high diffusion rate and high chemical reactivity’ an advantage?
5. Line 46 – what do you mean by ‘do not show immunological rejection’? Do you mean not at all or low rejection rate?
6. Line 113 – Typo error – 2 104 Hz.
7. Line 145 – specify what was osteogenic induction medium.
8. Line 266 – ‘They have many advantages’. This sentence seems very short and doesn’t seem to be coherent with the flow of the paragraph.
9. Line 321 – Typo error – ‘trelevant’
10. Overall, I would suggest the authors use a native-English speaker or service to review their paper so it is more professional.
Additional comments: This is a paper with a lot of significance if potential errors are corrected.


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

Input:
Title: BIOINFORMATICS ANALYSIS AND IDENTIFICATION OF CIRCULAR RNAS PROMOTING THE OSTEOGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS ON TITANIUM TREATED BY SURFACE MECHANICAL ATTRITION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript mainly wants to use biological information prediction to analyze the interactions between miRNAs, circRNAs and circRNA-miRNA-mRNA that interac with osteogenic mRNAs. In addition, the authors investigate the effects of surface mechanical attrition treatment (SMAT) on hBMSC osteogenic differentiation by affecting circRNA. The experimental objective is clear, while the English writing is poor, especially the Result Section. The literature references are sufficient and the sufficient background is provided. However, the results are not very clear and disordered. The number of Figures and Tables is too many to understand this manuscript.
Experimental design: The experimental design of this manuscript is relatively reasonable. The authors first used bioinformatics analysis and literature information to predict osteogenesis-related miRNAs, circRNAs and ceRNA networks of osteogenesis-related circRNAs by TargetScan, miRDB and miwalk3.0, and circRNA by circBase. Then, the authors used the SMAT titanium experimental material and the annealed titanium control material to detect the osteogenic differentiation ability of hBMSCs on the sample surfaces. However, the authors did not mention any information about the circRNAs that they detected. What’s the reason for choosing these circRNAs?
Validity of the findings: It is weak of evidence on the phenotype and functions of selected circRNAs. It also needs to add enough validation experiments about the effects of circRNAs on the osteogenic differentiation of hBMSCs by SAMT titanium.
Additional comments: It is a good idea to study the relationship between circRNA function and SMAT titanium material that promote hBMSCs osteogenic differentiation. In this study, the authors use a series of public dataset to support their research work by bioinformatics analysis, but it also needs more experimental evidence to prove the conclusions.

Major revisions:
  1, the authors need to give the details of circRNAs that choose in the ceRNA network, and directedly osteogenesis-related miRNA, mRNAs.
  2, the authors need to reorganized the orders of the figures. First is experimental results of SMAT titanium promoting osteogenic differentiation of hBMSCs, the second is bioinformatics prediction of osteoporosis-related circRNAs, and the third is the experimental validation of circRNAs function.
3, As presented, the English writing is not acceptable for the journal. The English writing of this manuscript must be improved carefully. Moreover, the introduction of Result section is too simple, not very logical and lack of summary. Therefore, this manuscript needs careful editing by someone with expertise in technical English editing paying attention to English grammar, spelling, and sentence structure.


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: BIOINFORMATICS ANALYSIS AND IDENTIFICATION OF CIRCULAR RNAS PROMOTING THE OSTEOGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS ON TITANIUM TREATED BY SURFACE MECHANICAL ATTRITION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: On Figure 3, please pick one of the sample from the each group that shows most dramatic changes between alizarin red staining.
Validity of the findings: In line 27, change 'attractive' to 'esthetic'.
Additional comments: Thank you for all your corrections.


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

Input:
Title: EVOLUTIONARY HISTORY OF A SCOTTISH HARBOUR SEAL POPULATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: no comment
Additional comments: This is a scientifically sound manuscript that evaluates the genetic variability and describes the demographic history of harbor seals in Moray Firth, North East Scotland using a good number of nuclear markers (17 microsatellite loci). The data are properly analyzed employing statistical software adequate to the nature of the markers. The study relies on standard techniques for quantification of genetic variation such as observed and expected heterozygosity, Bayesian analysis, estimation of F-statistics, assignment tests, and factorial correspondence analysis. The demographic history evaluation was tackled using two approaches to estimate present and historical effective population size (MSVAR and VAREFF), which are appropriate for this kind of data. The results presented in this article can be of importance for conservation of this species. I only have one minor concern that could be easily solved in the discussion. The samples used in this study were collected almost 30 years ago. Even though, this does not affect the historical population events detected here, the “actual” effective population size is in fact not that recent, therefore, it should be discussed. The recommendation of “accept, with minor revision” refers mostly to minor revisions and some desired clarifications, which are enumerated in the paragraph-itemized comments below.

Abstract
Line 20: “microsatellites markers” should be “microsatellite markers”
Line 22: “North eastern” should be “Northeastern”

Methods
Line 133-134: It is not clear how the markers were tested first and then the primers defined. If the microsatellites were already described (lines 130-133), then the primers were designed too, or where they redesigned and then tested again? Please, explain.
Line 141: Delete space in “MgCl2 ,”
Figure 2: X-axis legend says 1+2. It should be 1-2

Results
Line 395-397: It seems the ancestral effective size estimated with MSVAR is much larger (128,000-340,000) than with VAREFF (65,000), however it says is same order of magnitude. Please clarify.
Line 401: Reference is missing.
Line 433-434: Figure letters should be capitalized (e.g. Figure 3A). Please check throughout the document.
Line 437: The decline starting at around the las 100 generations is clear; however, it is hard to see if the population remained constant after that in figure 4. Please, explain in more detail why it is assumed that the population remained constant.

Figure 4B: the x-axis is confusing. The legend indicates from calendar year 1995 to 500 years ago (where 500 years ago means 1520), however, it seems to be from year 500 to 1995, correct?

Discussion
Line 564: A comma is needed after “However”
Line 611: U.K. should be UK. It has to be consistent throughout the document.


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: EVOLUTIONARY HISTORY OF A SCOTTISH HARBOUR SEAL POPULATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This study characterizes the evolutionary history of harbor seals in North-East Scotland. Specifically, the authors assess changes in effective population sizes at one Special Area of Conservation over time using measures of current, neutral genetic differentiation and population structure. The data and study are novel and, in my opinion, of great importance given that they may be used directly for conservation of a species whose populations have undergone drastic declines. For that reason, the study is highly deserving of publication. The authors have done excellent and thorough work, especially with their methods. They give plenty of detail on how the data were collected, how microsatellites were chosen and scored, and how analyses were carried out. The methods are very thorough, using multiple software programs to verify estimates of genetic differentiation and robust models for inferring historic effective population size. They also explain why their chosen method for assessing coalescence (MSVAR) is more favorable than other, similar methods.

My only overall concern is some apparent inconsistencies between the data presented in the tables/figures and the interpretations of those data.

For example: lines 323 – 325 list the loci that significantly deviated from HWE. Table 1 appears to show the P values from the exact tests for each loci. But, that column is described as the “probability of deviations from HWE” in the table caption. I would then read the values in the table as the probability that they deviate from HWE, rather than as p-values indicating significant deviation. I would suggest describing that column simply as “HWE p values” or something like that rather than as “probability of deviations from HWE”.

Additionally, on line 340, the p value for HWE for the whole seal population is given as P = 0.519, but I do not see this value in Table 1. Please clarify this.

Lines 409 – 421: I found the results from VAREFF on effective population sizes to be confusing and inconsistent at times. For example, lines 415 – 416 indicate that the different measures of effective population size through time (arithmetic mean, harmonic mean, mode, median) were “similar”. This is not consistent with what is shown in Figure 3. In Figure 3A, estimates of mode (in blue) and median (in black) look similar, but the arithmetic mean and harmonic mean look drastically different in effective population size over time. The word “similar” should be clarified and explained in further detail here.

Other concerns:

For analyses in STRUCTURE (in the Clustering analysis section of the methods, lines 216 – 225), why were both STRUCTURE HARVESTER AND Structure Selector used for structure inference? I would suggest only using one of these programs to reduce confusion and wordiness. Additionally, which structure inference methods were used to estimate the best K. Both Maximum Likelihood (Evanno et al 2005) and Puechmaille (2016) methods are mentioned, but it is not currently clear which method was used (or if both were used).

Additionally, lines 363 – 366 describe STRUCTURE and DAPC results, but no data from these analyses are shown, either in tables or in figure format. For greater accessibility to the reader, I would suggest showing at least the Delta K (or Puechmaille) results in a supplementary table and the DAPC results in a figure. I think that would give greater clarity to the purpose behind those analyses.

Lines 525 – 527 give a definition of effective population size according to Frankham 1995, but this is a highly simplified definition, since we know that effective population size is directly related to genetic drift. Since the paper is focused largely on estimating effective population size, I would like to see a more detailed and thorough explanation of the genetic consequences of low effective population size.

I would like to see one final paragraph in the conclusion discussing the potential consequences of continually decreasing effective population size in this seal population. In addition, I would like to see a bit more discussion of how these results, if at all or if possible, will contribute to conservation efforts for these seals or for other species.

Minor considerations:

Line 20: “microsatellites” should be made singular to “microsatellite”

Line 46: omit the word “the” before “abundance”

Line 72: “is” should be made past tense to be consistent with the rest of the methods

Line 108: Omit the word “have” and change “an” to “a” before “SAC”

Line 155: “seal” should be plural as “seals”

Line 228: “Performed” should be lower case

Line 236: There should be an “a” before “Mantel Test”

Line 288: Should “Time to the Most Recent Common Allele” be “Time to the Most Recent Common Acestor” instead?

Line 317: The number of decimal places given for heterozygosity here in the text is two (0.38), but in Table 1, this value is given as 0.384, if I am reading correctly. The number of decimal places should be kept consistent between the text and tables.

Line 359: at the end of this line, I believe “no” should be “not”

Line 363: at the end of this line, I believe the word “support” should be “supported”

Line 366: at the beginning of this line, I believe “is” should be “was”

Line 509: “Association” should be “associated”

Line 522: At the end of the line, the word “thus” should be moved before the word “does”

Line 621: The word “leading” should be “led”

The figures do not appear to have all been created using the same software, so they are not formatted uniformly. Please consider using a single medium, ie. R or Excel, for all figures.

Figure 2: the label for the second tick mark on the x-axis should be 1-2

Figure 3 B,C: Please increase font size on y-axis.

Figure 5: Please increase font size


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: EVOLUTIONARY HISTORY OF A SCOTTISH HARBOUR SEAL POPULATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The majority of the manuscript reads very well with clear, professional and technical english. There are a handful of places where I have made some edits and corrected spelling and grammatical typos. Mostly, the manuscript is well supported by the relevant literature. There are some papers I have suggested throughout to include as they may support some of the authors statements. There are also some noted referencing errors in the text: ommisions of “et al.” in a handful of locations, incorrect years (eg. Hanson et al. should be 2017 and not 2015) and in-text citations that do not appear in the reference list (eg. Hall et al. In press and Thompson et al. In press). There is an absence of the most up-to-date counts and trends in harbour seals (which are available in Thompson et al. 2019), with references pertaining to the time period when the samples were collected (30+ years ago). Although these references are appropriate for this manuscript, I also think it is important to include the most updated data.
The manuscript is well laid out with professional structure, figures and tables. Some notes have been made in the figures/tables to help clarify them for the readers. This manuscript is a self-contained study with a direct connection between the hypotheses, methods and findings. Appropriate scope is provided from outside studies.
Experimental design: Although an extension to other harbour seal studies in the UK, this study has unique aims that fall within the scope of this journal. Both biological and environmental studies are covered in this study. The authors clearly state the research question of the sutdy in final paragraph of the introduction. They are also clear about the current state of knowledge regarding harbour seal biology as well as population structure. It is made clear that the study is acknowledging the knowledge gap regarding historic estimates of population demography and changes. The sample size seems adequate considering the population size of the region and the difficulties of obtaining samples from seals. Although a slighly older technology, microsatellites are still perfectly suited for the type of analyses and questions approached in this study. Ethical considerations are clearly stated with appropriate permits. There is insufficient detail in the lab portion of the methods section to replicate the study. I have left comments in the text with regards to the DNA extraction and PCR steps where some extra detail should be included.
Validity of the findings: The study design, results and their interpretation are valid and robust. The findings contribute nicely to the growing field of conservation genetics and fill a knowledge gap referring to the demographic hisotory of a protected population of harbour seals. With the suggested additions to the laboratory section, the methods should be reproducible for other researchers to apply in other wild populations. By providing the sample metadata and microsatellite genotypes, the data can be re-analysed following the computer-based methods.
Additional comments: a.	Due to the nature of working with genetics and marine mammals specifically, I understand that sometimes studies are conducted because the data is available and not always as a result of a driving question. Although impact and novely are not assessed in this review, the manuscript would benefit from more information as to what it is about the Moray Firth specifically (compared to the other UK Harbour seal populations) that warrants a more in-depth analysis than the structure and diversity results that have been found in previous studies (such as Oslen et al. 2017) with added microsatellite loci? Was this location more stable during the two PDV outbreaks? What is the current population trend (since this paper focuses exclusively on the population approximately 30 years ago when the samples were obtained)?
b.	Estimating historic abundance, effective population sizes and bottlenecks are very interesting and important for understanding the current status of a population. Although the manuscript mentions that harbour seals are protected, it would be helpful for the reader to understand just how impacted UK harbour seal populations have been in recent decades (using total declines or percentages, see Thomspon et al. 2019: “The status of harbour seals (Phoca vitulina) in the UK”, Aquatic Conservation: Marine and Freshwater Ecosystems, 29:S1).
c.	The Moray Firth harbour seals (Loch Fleet) are one of the best studied populations of harbour seals providing the only estimates of vital rates (birth rates, survival rates, fecundity, etc.) in the UK. I imagine this was a factor in choosing this location to investigate further and if so, I recommend highlighting this invaluable dataset in the manuscript.
d.	As one of the justifications for this study seems to be a lack of data for conservation measures, how can your new findings about the historic state of this population be incorporated into contemporary management?
e.	The most recent estimates of harbour seal abundance in the UK are now available in Thompson et al. 2019. It was reported that mean counts decreased by ~40% between the 1995 and 2005-2016 surveys down to about 900 animals. Although your data come from animals sampled before these more recent declines, it would be advisable to incorporate the more recent into your conclusions and justificaitons for understanding the historical abundances.


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: ANALYSIS OF FACTORS CONTRIBUTING TO POSTOPERATIVE BODY WEIGHT CHANGE IN PATIENTS WITH GASTRIC CANCER: BASED ON GENERALIZED ESTIMATION EQUATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are many basic reporting problems with this article:

1) The abbreviation TNM is used extensively but never explained.

2) In the abstract, the P values of body weight at T1 and T2 have an unusual symbol with Greek letters instead of <.

3) The abstract is clumsily written. The sentences "The result of generalized estimation equation showed that female, Buddhism religion, and lower body mass index significantly influenced postoperative body weight changes independently (all P < 0.05). Specifically, compared with male patients, female patients experienced more weight loss. Compared with Buddhism religion, the non-region were protective factors for postoperative body weight change. Taking obesity patients as reference, underweight patients experienced the largest body weight loss, followed by normal weight patients, and overweight patients" are largely redundant and should be summarised in one sentence i.e. "being female, of Buddhism religion, and having lower body mass index were independent predictors of greater postoperative weight loss."

4) English language writing is poor throughout and requires significant revision by a native English speaker. Particularly problematic terms include "dig out", "zoomed in on", "dug out that", "come to", "comes to"- none of these are acceptable in formal scientific writing.

5) Furthermore, the phrase "no less than 10%" is confusing and not used conventionally in scientific writing. Why not just use the phrase "10% or greater?"

6) Similarly, the phrase "surgical complication grade no less than two" is confusing and not used conventionally in scientific writing. Why not just use the phrase "surgical complication grade of 2 or greater?" Also, in this citation, surgical complication grade is not explained- which validated scale is being used exactly to determine surgical complications?

7) The sentence "After surgical treatment, appetite and food intake in GC patients changed, resulting in the recovery of nutritional status last up to one year(Ryu & Kim 2010), where body weight loss usually accompanied with" is particularly poorly written and requires revision.

8) In the first 2 sentences of the "Results" paragraph, there is inconsistent use of Roman numerals and words for numbers. "six" should be "6" and "one" should be "1"

9) The sentence "More than half of them were female (55.10%), married (91.80%), without religious brief (85.7%), and with a stage III cancer (57.1%)" is poorly phrased. It implies that more than half the patients met all the above criteria.

10) The sentences "specifically, the body weight continued to reduce 1.16 kg from T1 to T2, and 0.31kg from T2 to T3" are redundant.

11) The three sentences "Lower preoperative BMI was a risk factor for body weight loss during three months after gastrectomy in GC patients. The lower the preoperative BMI was, the higher the body weight loss was. Patients with underweight normal weight, and overweight experienced significantly body weight loss compared with obese patients" are all saying the same thing. Two of these sentences should be deleted for redundancy.

12) Table 4 has missing information in the legend, including explaining T1/T2/T3 and II/III/IV, and also the meaning of "B" and "SE".

13) Table 3 has no legend, including explaining T1/T2/T3 and also the meaning of "B" and "SE".
Experimental design: METHODOLOGICAL PROBLEMS
1) The abstract states that covariates were entered into generalized estimation equation after univariate analysis. However in the main body of the text, univariate analysis is never described. Instead, the authors state, confusingly, that generalized estimation equations are used for multivariate analysis.

2) The sentence "And time variable on postoperative body weight change should be considered in the model" is poorly written as the authors have not referred to any particular mathematical models by this point in the manuscript. Furthermore, the authors actually fail to discuss any further time variables on postoperative body weight change in the results or the conclusion, even though Table 4 shows that a lower weight at T1 is an independent predictor of greater weight loss at T3.

3) It is unclear why, according to the statistical analysis, data is presented as median combined with minimum and maximum. This is not standard practice in scientific writing: why do the authors not use median, quartiles and interquartile range?

4) Throughout the manuscript and tables/figures, body weight is referred to as "body weight", "average body weight", etc. It should always be consistently referred to as mean body weight.

5) The authors should not use mean absolute changes in body weight or BMI as variable or outcomes, as this may be dependent on patient baseline weight. They should use mean percentage changes in body weight instead, just like most of the previous studies that they cite (e.g. Climent et al, Aoyama et al.

6) The choice of generalized estimation equations as a statistical method is extremely difficult to comprehend in this study, and poorly explained.

The sentence "QIC value in independent form was smaller than that in the other four forms in this study" is confusing- what are the "other four forms" that the authors refer to?

The entire explanation of the GEEs in the paragraph entitled "Factors contributed to body weight change during three months after gastrectomy" is confusing. Firstly the authors state that the results of GEEs showed that sex, religion, TNM, CCI significantly influenced body weight changes. However then these variables are entered into GEEs for mutlivariable analysis- this is confusing- were two sets of GEEs involved? Furthermore, if only sex, religion, TNM, CCI were entered into GEE to do multivariate analysis, why does the analysis then include other variables such as preoperative BMI, ONS, nutrition knowledge/attitude/practice?

Furthermore, the copula function of GEE is not explained at all. Is this a mathematical model to predict absolute weight at 3 months? Or absolute BMI at 3 months? At what time point are the BMI in the copula function measured? And what does the variable "1 month after gastrectomy" in the copula function mean? In what practical way can clinicians use this copula function?

Why was the crucial variable of partial versus complete gastrectomy not analysed in the multivariate analysis?

I propose that for such a small, simple study, with less than 50 patients and only three time points, the authors abandon such a complex statistical methodology which introduces significant confusion. Instead, they should simply perform univariate linear regression with patient variables (including baseline variables, as well as weight change at T1 and T2) and a continuous outcome of weight loss at T3. After this, multivariate linear regression of variables where P <0.1 should then be performed to identify independent predictors of weight loss at T3.
Validity of the findings: DISCUSSION/CONCLUSION PROBLEMS
1) The authors do not provide an adequate explanation of why Buddhist religion may lead to greater weight loss. The statement "Buddhist refuses or limits the meat intake...aggravating nutritional status and body weight loss" is an unproven generalisation, not supported by cited studies or data in this study actually assessing meat intake. It is therefore speculation and should be deleted.

2) The sentence "Actually, the compliance of ONS is poor in cancer patients in clinical practice, which may explain why ONS in this study did not showed significant effect on reducing body weight loss" is unproven, not supported by cited studies, and of course not supported by data in this study as the authors acknowledge their failure to record ONS intake quantity. It is therefore speculation and should be deleted.

3) The discussion of limitations is poor. The authors fail to acknowledge in this section that they did not collect data about patient nutritional intake, let alone ONS intake, post surgery. The statement "this was a single center study, most of patients were with advanced GC admitted to our hospital, which may come to patient bias" is poorly explained. What "patient bias" are they referring to? Is it selection bias- and if so, does it affect the internal validity or external validity of the study, or both?

Finally, the statement "we did not analyze the relationship between body weight change and adverse event, as we just followed three months" is misleading. The authors failed to collect any information of adverse events after the surgery during the hospital recovery period, which would have been easily available from patient records and could have been an important variable predicting post-operative weight loss, even confounding all of their conclusions (as they themselves acknowledge in the previous sentence quoting Zhao et al. 2018).
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: ANALYSIS OF FACTORS CONTRIBUTING TO POSTOPERATIVE BODY WEIGHT CHANGE IN PATIENTS WITH GASTRIC CANCER: BASED ON GENERALIZED ESTIMATION EQUATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1) Some errors in editing and grammar were found. I mentioned these in the "General comments for the author".
2) References well support the plot.
3) Well structured and raw data were also shared.
4) Relevant results.
Experimental design: 1) Within aims and scope of the journal
2) Relevant and meaningful results.
3) Proper investigations for clinical questions.
4) There is room for improvement in M & M section and I mentioned in the "General comments for the author".
Validity of the findings: Relevant.
Additional comments: Overall, this is an interesting study describing factors associated with postoperative weight loss in gastric cancer patients. Although the conclusion of the study should be validated in a larger number of cohorts in the future, this manuscript is suitable for publication. There are several minor suggestions to improve the quality of the paper.

Materials and methods
1) There is no specific description of how the body weight was measured (e.g. self-report and so on). Please make up for it. If weight measurements were not performed uniformly, please indicate in the discussion section as a limitation of the study.

Result
1) Are there any cases of relapse? Since recurrence of cancer can contribute to weight loss, please reanalyze the effects of recurrence on weight loss if any cases exist. If no recurrence was observed during follow-up period, please indicate this fact.
2) The first section of the Result has no title. Please add the title such as “baseline characteristics of the study population”.
3) Mark the expansion for PTX if this word was first used in the manuscript.
4) In the 2nd section of Result, there should be a gap between the number and the unit (e.g. kg).
5) In Table 1, upper case should be used for ‘f’emale and so on.
6) In Table 1, what is the ‘type of C’?
7) In Table 3 and 4, please indicate the expansion for ‘B’, ‘SE’, and ‘CI’?

Discussion
1) The effect of religion on postoperative weight loss cannot be generalized easily. Please include this as a limitation of the study.


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

Input:
Title: ANALYSIS OF FACTORS CONTRIBUTING TO POSTOPERATIVE BODY WEIGHT CHANGE IN PATIENTS WITH GASTRIC CANCER: BASED ON GENERALIZED ESTIMATION EQUATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: Not clear description of this study's design: prospective cohort or retrospective electric medical record review.

Small number of patients - not enough to conclude and sub-analyze

Very short follow-up period
Validity of the findings: No novelty - the fact of weight loss after gastrectomy is well known
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: ANALYSIS OF FACTORS CONTRIBUTING TO POSTOPERATIVE BODY WEIGHT CHANGE IN PATIENTS WITH GASTRIC CANCER: BASED ON GENERALIZED ESTIMATION EQUATION
Review round: 1
Reviewer: 4

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors investigated factors contributing to postoperative body weight change in gastric cancer patients, and showed female, Buddhism religion, and lower preoperative BMI were risk factors to post-op body weight loss by using generalized estimating equation analysis. As they mentioned in the introduction section, it has been well known that body weight loss after surgery was associated with an unfavorable long-term outcome in gastric cancer patients. That's why it would be useful if surgeons predicted such patients, and commenced exercise and nutrition intervention preoperatively.  At a glance, this is a well-written paper that presents interesting data, however, I have the following concerns.
1) In clinical practice, after TOTAL GASTRECTOMY, the severe body weight loss is commonly observed. But, the authors didn’t present the data and their interpretation about patients undergoing total gastrectomy. I suggest that the authors should touch on the issue and details in the manuscript.
2) In the discussion section, they interpreted the risk factor to weight loss of Buddhism religion as the limitation of meal intake. Their interpretation of that is not plausible. It would be useful if the authors gave Buddhist patients’ eating habits or nutritional knowledge, attitude and practice.


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: ANALYSIS OF FACTORS CONTRIBUTING TO POSTOPERATIVE BODY WEIGHT CHANGE IN PATIENTS WITH GASTRIC CANCER: BASED ON GENERALIZED ESTIMATION EQUATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Generally improved since last review. Small errors in English grammar will likely be cleaned up in the editing process.

Some important vocabulary changes needed in the discussion though:

1) line 268- "considered to" should be "expected to"

2) line 277- "Coherent" should be "consistent"

3) line 284- "suitable for" should be "generalizable to".

4) in the tables "stand error" should be "standard error"
Experimental design: Methodology has been better explained now.

1) However in my previous review, I wrote that "The authors should not use mean absolute changes in body weight or BMI as variable or outcomes, as this may be dependent on patient baseline weight. They should use mean percentage changes in body weight instead, just like most of the previous studies that they cite (e.g. Climent et al, Aoyama et al.) This has not yet been addressed by the authors.

Studies such as Climent et al, Aoyama et al, Davis et al, Kubo et al all express weight change for each given patient as a % weight change compared to baseline.

This is a much more meaningful measurement than absolute weight change in kilograms, which is what the authors of the current study use. It puts the absolute weight change in the context of the patient's baseline weight. For example, a 100kg person losing 5kg is clinically of far less concern than than a 40kg person losing 5kg, even it is seems that their absolute weight loss is identical. The first person has lost 5% of their body weight. The second person has lost 12.5% of their body weight. I understand that there is not so dramatic a range of weights in the patients in the current study. However, the mathematical principle remains the same: using % weight change is a more appropriate scientific measurement, and has extensive precedent in the worldwide literature quoted by the authors. It is unclear why the authors have chosen to persist with expressing measurements with absolute weight change in kilograms.

The authors do find that a lower baseline weight is independently associated with greater absolute weight loss in kg. I argue that performing their analysis according to % body weight loss would actually accentuate and statistically strengthen their findings! It would show that the % weight loss in the underweight group is far higher than the % weight loss in the normal weight or overweight group, and the difference is of much greater magnitude than when measuring absolute weight loss in kg. It would make this an even more convincing and powerful article. If the authors choose not to take up my suggestion, they should at least include this as a major methodological limitation.

2) The explanation of GEEs in univariate analysis from line 173 to 180 needs to be in the Statistics section of Materials and Methods, not in the Results.  Furthermore, it needs to include explicit mention of each study variable that was studied in univariate analysis. Furthermore, it needs to mention that multivariate analysis was then performed, which variables from the univariate analysis were chosen to undergo multivariate analysis, and on what basis they were chosen. That is scientific convention in the statistics section of an article.

3) These sentences from line 180 are confusing "The results of the GEEs for univariate analysis showed that sex, religion, TNM, CCI significantly influenced body weight changes (all P < 0.05), and education level, operation type (partial vs completely gastrectomy), chemotherapy type, cancer therapy type, and perioperative complication showed no statistically significance with the body weight change. Therefore, those variables (sex, religion, TNM, CCI) were entered into GEE to do multivariable analysis, and the results of GEE multivariable analysis indicated that sex, religion, and preoperative BMI were contributing factors of body weight change (P < 0.05), while other variables, such as TNM stage,ONS, CCI, patients' nutrition knowledge, nutrition attitude, and nutrition practice were not relevant." It suggests that many variables were included in the multivariable analysis that were not included in the preceding univariate analysis such as preoperative BMI, ONS, patients' nutrition knowledge, nutrition attitude, and nutrition practice. In fact, the authors confirm this in the rebuttal letter to me. This is not, to my knowledge, the correct way to perform univariate and multivariate analysis. Univariate analysis should start by including all the possible study variables. Then, only those variables that meet a pre-specified level of statistical significance on univariate analysis go on to be included in multivariate analysis. Why did the authors not do this?

4) Continuing from point 3 above, there are demographic variables that were collected but not included in analysis such as marriage status, smoking, and drinking. Firstly, how exactly was 'smoking' and 'drinking' defined? Does 'drinking' mean alcohol? Secondly, what was the point of collecting or presenting these variables if they were not included in the analysis? I believe they should also be included in the univariate +/- multivariate analysis (as long as 'smoking' and 'drinking' can be adequately defined).

5) Continuing from point 3 above, table 4 is incomplete and confusing. It states p values but does not state whether this is on univariate or multivariate analysis. Despite it being called "Parameters associated with body weight change", it presents many variables that actually were not associated with body weight change as they did not reach statistical significance. If, indeed, the authors instead wanted to provide a table that included all variables, they should change the title, and also include many variables that were omitted including education level, operation type (partial vs completely gastrectomy), chemotherapy type, cancer therapy type, and perioperative complication.

I suggest the authors update table 4 so it includes every single study variable, one column for p values of univariate analysis, and one column for p values of multivariate analysis. This could be titled "Association of study variables with body weight change three months after gastrectomy". It should be arranged intuitively: with variables that reached significance on multivariate analysis clustered together in rows at the top of the table, and variables that did not reach significance clustered together in rows separately at the bottom of the table.
Validity of the findings: Nothing to add here
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: ANALYSIS OF FACTORS CONTRIBUTING TO POSTOPERATIVE BODY WEIGHT CHANGE IN PATIENTS WITH GASTRIC CANCER: BASED ON GENERALIZED ESTIMATION EQUATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1) Some errors in editing and grammar were found. I mentioned these in the "General comments for the author".
2) References well support the plot.
3) Well structured and raw data were also shared.
4) Relevant results.
Experimental design: 1) Within aims and scope of the journal
2) Relevant and meaningful results.
3) Proper investigations for clinical questions.
4) There is room for improvement in M & M section and I mentioned in the "General comments for the author".
Validity of the findings: Relevant.
Additional comments: All issues raised by me have been satisfactory addressed.


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: ANALYSIS OF FACTORS CONTRIBUTING TO POSTOPERATIVE BODY WEIGHT CHANGE IN PATIENTS WITH GASTRIC CANCER: BASED ON GENERALIZED ESTIMATION EQUATION
Review round: 2
Reviewer: 3

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: ANALYSIS OF FACTORS CONTRIBUTING TO POSTOPERATIVE BODY WEIGHT CHANGE IN PATIENTS WITH GASTRIC CANCER: BASED ON GENERALIZED ESTIMATION EQUATION
Review round: 3
Reviewer: 1

Output:
Basic reporting: There are confusing contradictory statements in the discussion: line 238, "there was no significant difference in weight loss between total and subtotal gastrectomy in this research. The result was similar to previous studies, where total gastrectomy was a risk factor for postoperative weight loss." These two sentences are directly contradictory. Also the first sentence is wrong. There was a significant difference in weight loss between total and subtotal gastrectomy in this research, as reported all throughout the manuscript.
Experimental design: My previous comments asked "how exactly was 'smoking' and 'drinking' defined?"

The answer from the authors has been inadequate. I quote: "Smoking was defined as with the habits of smoking. Drinking was defined as with the habit of alcohol drinking."

This is inadequate. What does 'with the habit of smoking mean'? Does it mean the patients are current smokers? Or current or former smokers? Did the authors apply a quantitative number of pack-years of smoking? If the authors cannot show they have made any attempt to quantify the amount of smoking exposure, then smoking should be removed from the manuscript altogether.

What does "with the habit of alcohol drinking mean'? Did the authors apply a quantitative number of alcohol intake e.g in mean grams of ethanol per day? Or in mean standard drinks per day? If the authors cannot show they have made any attempt to quantify alcohol intake, then alcohol should be removed from the manuscript altogether.
Validity of the findings: The reporting of the variables associated with weight loss in univariate and multivariate analysis in lines 180 to 190 needs to be clearer in terms of the direction of effect.

The authors mention operation type, preoperative BMI, postoperative complication and nutrition practice score as influencing body weight change. For each variable, what was the direction of effect? Did total gastrectomy or partial gastrectomy associate with greater weight loss? Did higher pre-operative BMI or lower pre-operative BMI associate with greater weight loss? Did more or less complications associate with greater weight loss? Did higher or lower nutrition practice score associate with greater weight loss?

Similarly, in the multivariate analysis, total gastrectomy and preoperative BMI were contributing factors of body weight change. What was the direction of effect? Did total gastrectomy or partial gastrectomy associate with greater weight loss? Did higher pre-operative BMI or lower pre-operative BMI associate with greater weight loss?
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: ANALYSIS OF FACTORS CONTRIBUTING TO POSTOPERATIVE BODY WEIGHT CHANGE IN PATIENTS WITH GASTRIC CANCER: BASED ON GENERALIZED ESTIMATION EQUATION
Review round: 4
Reviewer: 1

Output:
Basic reporting: No further issues.
Experimental design: No further issues. All my criticisms answered
Validity of the findings: No further issues. All my criticisms answered
Additional comments: No additional comments


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

Input:
Title: EFFECT OF SODIUM (S)-2-HYDROXYGLUTARATE IN MALE, AND SUCCINIC ACID IN FEMALE WISTAR RATS AGAINST RENAL ISCHEMIA-REPERFUSION INJURY, SUGGESTING A ROLE OF THE HIF-1 PATHWAY
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: 1. The best and standard model of renal IR injury must be C57mouse. Why did the authors choose Wister rats?
2. In the reference 32 by Wei and Dong, a mouse model was used, and they chose the back incision, but in the present study, the author chose the midline incision. Was this reference proper?
3. Why did you choose 15 hours reperfusion but not 24 hours, please provide a reasonable explanation?
Validity of the findings: 1. The elevation of HIF may be critical for the effectiveness and mechanism of the two drugs. So, the only detection of HIF using ELISA in tissue homogenates. The author should supplement the results of Western Blot.
2. The statements in the Result sections may be improper,  such as "There were no
significant differences between  A  and B."  A more explicit statement must like "A is higher than B, or 2-HG decreased the level of creatinine."
3. The nephroprotective effects must be most important, which should be firstly described in the Result section
4. If possible, other interventions should be used as a positive control, such as local IPC or remote IPC.
Additional comments: No comments


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

Input:
Title: EFFECT OF SODIUM (S)-2-HYDROXYGLUTARATE IN MALE, AND SUCCINIC ACID IN FEMALE WISTAR RATS AGAINST RENAL ISCHEMIA-REPERFUSION INJURY, SUGGESTING A ROLE OF THE HIF-1 PATHWAY
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: Please have a look at Major concerns: 1 to 6.
Validity of the findings: No comment.
Additional comments: Kidney transplantation is the definitive treatment for patients with end-stage chronic kidney disease. However, the shortage of available organs for transplantation is a formidable challenge. Ischemia–reperfusion (IR) injury is the main cause of delayed graft function in solid organ transplantation. It is inspiriting and beneficial to analyze the activities and potential pharmacological properties of preconditioning drugs (hypoxia mimetics) on preventing or limitting IR injury. In this manuscript, Dr. Eduardo Cienfuegos-Pecina and colleagues looked at the nephroprotective effect of the egg-laying defective nine homolog (EGLN) inhibitors, (S)-2-hydroxyglutarate [(S)-2HG] and succinic acid (SA) in IR injury rat. The study’s conclusion showed that only (S)-2HG showed a dose-dependent nephroprotective effect at the evaluated doses, which involved the promotion of HIF-1alpha accumulation. Although the current study is well and interesting, there are some major concerns that need to be addressed before consideration of publication.

Major concerns:
1. “Materials & Methods – Animals” Line 158-165. “Wistar rats, weighing 250–300 g, were used: 32 male rats to evaluate (S)-2HG activity and 42 females to evaluate SA activity.” Why study the activity of (S)-2HG in male rats and SA in female rats?
2. “Materials & Methods – Experimental design” Line 168-202. “Rats were treated with (S)-2HG at a dose of 12.5 or 25 mg/kg (12.5Tox and 25Tox, respectively) in double-distilled water administered p.o. twice per day for 2 days.” Please give clear reasons and references for the above concentration and administration time.
3. “Materials & Methods – Experimental design” Line 168-202. “Rats were treated with SA at a dose of 12.5, 25, or 50 mg/kg (12.5Tox, 25Tox, and 50Tox, respectively) in double-distilled water under the same conditions as the SH group.” Please give clear reasons and references for the above concentration and administration time.
4. “Table 2 & Table 4”. The Scores of Tubular Necrosis and Acidophilic Casts in IR group were 4.00 (4.00-4.00) and 4.00 (3.00-4.00) respectively in Table 2. However, the Scores of Tubular Necrosis and Acidophilic Casts in IR group were 3.50 (3.00-4.00) and 2.00 (2.00-3.00) in Table 4. It shows that the Score of Acidophilic Casts was different between the two IR groups. Why is there a difference? Combined with the first major question, is it a gender difference?
5. “Figure 2 & Figure 4”. The serum concentrations of creatinine can reflects kidney function. It seem that the creatinine of IR group was about 3.1 mg/dL in Figure 2. However, the creatinine of IR group was about 2.5 mg/dL in Figure 4. It shows that the creatinine was different between the two IR groups. Why is there a difference? Combined with the major question 1 and 4, is it a gender difference?
6. “Results – Evaluation of the effect of sodium (S)-2HG treatment on the tissue HIF-1α concentration” Line 382-389 and Figure 6. Although the results of the manuscript showed that there was not remarkable nephroprotective effect of SA against IR injury in rats, the control group was not stable in the experiment of studying the activity of SA. Thus, I could not judge whether SA was indeed inactive. I suggest the author to add experiments to detect whether the SA has an effect on the concentration of HIF-1alpha, to reflect the characteristics of SA from this side.

Minor comments:
1. “Figure 3 and 5”. Missing the scale in the pathological images.
2. “Abstract”. Missing the “Conclusion”.


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 SODIUM (S)-2-HYDROXYGLUTARATE IN MALE, AND SUCCINIC ACID IN FEMALE WISTAR RATS AGAINST RENAL ISCHEMIA-REPERFUSION INJURY, SUGGESTING A ROLE OF THE HIF-1 PATHWAY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Eduardo Cienfuegos-Pecina et al. ‘Sodium (S)-2-hydroxyglutarate, but not succinic acid, has a nephroprotective eﬀect against ischemia-reperfusion injury in Wistar rats’, suggests a role of the HIF-1 pathway’, dealing with the ameliorative effect of (S)-2-hydroxyglutarate in renal IR injury in Wistar rats.
The study objectives are clear, and experiments are nicely done.
Author should add some latest research/references. Which are lacking in text. Like discuss some basic signaling related to HIF pathways which may be effected by S)-2-hydroxyglutarate and succinic acid.
Experimental design: Authors performed many experiments and assessed all the basic parameters with good stats.
Validity of the findings: no comments
Additional comments: Authors need to justify following comments and some of which should be included in the discussion of the manuscript so that readers can easily understand.
Comments:
1.	Author should justify why male rats were used for assessing (S)-2-hydroxyglutarate while female for succinic acid?
2.	Authors should justify why they use rat IR model? Whether rats are good for IR models than mice? What are the metabolic changes induced by HIF 1 accumulation in these models? (S)-2-hydroxyglutarate metabolism in rat vs mice system? Which model is more relevant to humans?

3.	How would authors justify the doses of (S)-2-hydroxyglutarate which ameliorates the effects of renal IR injury but what about its other potential molecular effects like activation of oncogene or inactivation of tumor suppressor genes in the long term? Such effects are already established to be induced by high concentration of (S)-2-hydroxyglutarate.

4.	As authors previously published same type of study on plant extract ‘Nephroprotective Effect of Sonchus oleraceus Extract against Kidney Injury Induced by Ischemia-Reperfusion in Wistar Rats’, I hope they are wellaware of using different compounds in the protection renal IR injury as well as the longterm effects of those compounds. Please justify my point.

5.	Moreover, Authors only assessed kidney tissue but what about the effects of (S)-2-hydroxyglutarate and succinic acid in other organs? We should be cautious when giving non targeted compounds by orally.

6.	What is the conversion rate of (S)-2-hydroxyglutarate to alpha ketoglutarate?

7.	Is it possible that (S)-2-hydroxyglutarate treatment may change the epigenetic pattern of DNA viz; hypermethylation of DNA and cause tumor in long term effect? Because in this study all rats were sacrificed within 24 hrs only following start of treatment schedule.

8.	Is it possible that tramadol induces some immunomodulatory effects or neutralizes the immunomodulatory effects of IR / (S)-2-hydroxyglutarate?

9.	In Abstract methods section line 37 … (S)-2HG and SA were independently evaluated in rats after renal IR. It should be ‘renal IR injury’.

10.	In the results, the authors report that 12.5 tox and IR groups show HIF 1 alpha accumulation but not in 12.5+IR and 25+IR groups. Can authors clarify in the manuscript whether these are negative results as per the hypothesis in terms of HIF1 alpha accumulation.

11.	At some places authors write inhibition of EGLN and at others as inhibition of prolyl-4-hydroxylases. They should either write EGLN family member prolyl-4-hydroxylases or only inhibition of prolyl-4-hydroxylases at all the places. I suggest it would be best if they focus on what has been analyzed in their experiments.

12.	Please write H1F1 alpha or H1F1 α at all places throughout text to maintain uniformity.

Line 32 in abstract please write renal IR injury.

Line 48 result section please write SA did not ‘show’ instead of ‘have a’ nephroprotective effect

Line 415 Discussion…(S)-2HG caused no toxic effects at the hepatic or renal level, as shown by the normal values for the biochemical markers…..should be… as shown by the normal values ‘of’ biochemical markers.

Line 455-456 Discussion…. (S)-2HG caused a significant accumulation of HIF-1α in the kidney tissue in the nontoxicity groups of rats. Please write….(S)-2HG caused a significant accumulation of HIF-1α in the kidney tissue of rats in nontoxicity groups.

Line 464 concentration in the 12.5+IR and 25+IR groups in the late phase in our study Please write ………. in the late/reperfusion phase of this study.


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

Input:
Title: EFFECT OF SODIUM (S)-2-HYDROXYGLUTARATE IN MALE, AND SUCCINIC ACID IN FEMALE WISTAR RATS AGAINST RENAL ISCHEMIA-REPERFUSION INJURY, SUGGESTING A ROLE OF THE HIF-1 PATHWAY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Kidney transplantation is the definitive treatment for patients with end-stage chronic kidney disease. However, the shortage of available organs for transplantation is a formidable challenge. Ischemia–reperfusion (IR) injury is the main cause of delayed graft function in solid organ transplantation. It is inspiriting and beneficial to analyze the activities and potential pharmacological properties of preconditioning drugs (hypoxia mimetics) on preventing or limitting IR injury. In this manuscript, Dr. Eduardo Cienfuegos-Pecina and colleagues looked at the nephroprotective effect of the egg-laying defective nine homolog (EGLN) inhibitors, (S)-2-hydroxyglutarate [(S)-2HG] and succinic acid (SA) in IR injury rat. The study’s conclusion showed that only (S)-2HG showed a dose-dependent nephroprotective effect at the evaluated doses, which involved the promotion of HIF-1alpha accumulation. The paper is improved and most concerned raised by the reviewer have been addressed. I think it is might suitable for publication at this version of revised manuscript.


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

Input:
Title: CELLULAR HETEROGENEITY MAP OF DIVERSE IMMUNE AND STROMAL PHENOTYPES WITHIN BREAST TUMOR MICROENVIRONMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.1 Discussion should be added to existing relevant reports.
Eg.
[1] Deng L, Lu D, Bai Y, et al. Immune Profiles of Tumor Microenvironment and Clinical Prognosis among Women with Triple-Negative Breast Cancer [J]. Cancer Epidemiol Biomarkers Prev, 2019, 28(12): 1977-1985. DOI: 10.1158/1055-9965.EPI-19-0469.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Breast cancer is one of the predominant types of tumors in women worldwide. Despite great progress in the early diagnosis and treatment recently, drug resistance and distal metastasis remain major causes of cancer-related mortality. Tumor microenvironment (TME) cells are important elements in tumor tissue. There is increasing evidence that they have important clinical pathological significance in predicting tumor clinical outcomes and therapeutic effects. It is inspiriting and beneficial to analyze the global view of tumor-infiltrating immune and stromal cells in tumor tissues and adjacent normal tissues, and investigate the correlation of TME cells’ signature with breast cancer prognosis. In this manuscript, Dr. Li and colleagues looked at the role of the “64 types of immune and stroma cells’ signature” in diagnosis and prognosis of breast cancer. The study’s conclusion showed that "immune and stroma cells’ signature" was expected to be used to predict the prognostic survival of breast cancer. Although the current study is well and interesting, there are some major concerns that need to be addressed before consideration of publication.

Major concerns:
1. In the newly published article [1], Dr. Deng and colleagues also analyzed BRCA dataset from TCGA using xCell, so it is necessary to discuss the similarities and differences between the two studies.
[1] Deng L, Lu D, Bai Y, et al. Immune Profiles of Tumor Microenvironment and Clinical Prognosis among Women with Triple-Negative Breast Cancer [J]. Cancer Epidemiol Biomarkers Prev, 2019, 28(12): 1977-1985. DOI: 10.1158/1055-9965.EPI-19-0469.
2. “Abstract – Methods”. The method should be described more detailed.
3. “Abstract – Results”. The results should be briefly descripted.
4. “Results – Breast tumor tissues had higher fractions of immunes cells than normal tissues” Line 128-142, Figure 1D to J. Paired samples (e.g. 112 cases) are recommended for comparison, in order to reduce the error caused by differential number of samples analyzed between groups.
5. “Results – Inhibitory receptors were co-expressed on certain subtypes of T cells” Line 150-154, Figure 2A and B. In order to show similarities and differences between tumor samples and normal tissues, the Tumor (F2A) and normal tissues (F2B) should be combined on a heat map, and t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis should be preform.
6. “Results – Prognostic model was built with survival associated cell types” Line 211-214, What is the meaning of “…prediction model…”? The method should be described more detailed, including software, risk factor calculation formula, and references.
7. “Results – Subtypes of breast cancer had diverse phenotypes of cellular heterogeneity” Line 229. What is the meaning of “…ImmuneScore and StromalScore…”? The method should be described more detailed.

Minor comments:
1. “Figure 1B”. The information of the axis and unit should be added to depict the “blue to red” color.
2. “Figure 1D to J”. Missing the y-axis label.
3. “Figure 2A to D”. The information of the axis and unit should be added to depict the “blue to red” color.
4. “Figure 2E to H”. The scatter plot should show P-values and R-values.
5. “Figure 3B to F”. Missing the y-axis label.
6. “Figure 4 figure legend”. “ESCC”? Please examine your manuscript carefully.
7. “Figure 5 B to N”. The values of cutoff (can be noted “best”), Hazard Ratio (HR), and 95% Confidence Interval (CI) of ratio should be shown in survival curve, and the unit of survival time should use months or years.
8. “Results – Subtypes of breast cancer had diverse phenotypes of cellular heterogeneity” Line 231-232. “…t-SNE cluster analysis…” What data analyzed? Is RNA profile or the fractions for 64 cell type? Please state clearly.
9. “Results – Subtypes of breast cancer had diverse phenotypes of cellular heterogeneity” Line 231-232. Did “…distinguish these five subtypes (Figure 6B-C)…” should be “ (Figure 6B)”; and “…among these five subtypes (Figure 6D)…” should be “(Figure 6C-D)”?
10. “Figure 6D to F”. The text in charts is too small to read, so charts need to be rearranged.
11. “Figure 7”. The text in charts should not be distorted and should be easily recognizable to the reader.


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: CELLULAR HETEROGENEITY MAP OF DIVERSE IMMUNE AND STROMAL PHENOTYPES WITHIN BREAST TUMOR MICROENVIRONMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the current study, the authors aimed to provide a unique cellular heterogeneity map of diverse immune and stromal phenotypes in breast cancer microenvironment and uncover potential therapeutic targets and biomarkers. xCell was used to portrayed the cellular heterogeneity map of 64 tumor microenvironment -associated cell types in RNA-seq dataset of breast cancer and normal tissues downloaded from TCGA. They found that specific immune and stromal cells, such as CD8+ T cells, keratinocytes, NKT and Class switched memory B cells in the tumor microenvironment, may serve as independent prognostic factors in the subsequent progression of breast cancer. Furthermore, they analyzed a ImmuneScore and StromalScore system to distinguish the five subtypes of breast cancer, and they owned diverse phenotypes of cellular heterogeneity. The research is interesting. I have some major concerns as the following:
1. The method used in this study should be described in more details, and a flowchart of study design and patient’s information should be added.
2. How did the authors calculate the ImmuneScore and StromalScore from 64 cell types? And the scores of each immune and stromal cell should be demonstrated in table.
3. What did the prognostic prediction model exactly mean in Figure 5? The evalution criteria of significant prognosis factors used to predict the survival should be provided.
4. It would be easier to understand if the illustration of figure legend was presented in more specifically way.
5. As shown in Figure 2E-H, the immune inhibitory receptors were significantly correlated with certain subsets of T cells. The correlation coefficient and P value should be provided in scatter plot.
6. What is the y-axis supposed to be in Figure 1D-J, Figure 4B-F and Figure 6E-F?
7. The figure legend title in Figure 4 “Involvement of cellular heterogeneity in clinic-pathology of ESCC” was miswritten, it should be “breast cancer”.
8. The number of samples of each group should be provided in figure.
9. Writing of this article may need further modification.


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: CELLULAR HETEROGENEITY MAP OF DIVERSE IMMUNE AND STROMAL PHENOTYPES WITHIN BREAST TUMOR MICROENVIRONMENT
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is generally well written, but there are some issues with English usage that will need to be addressed. This might be accomplished by a native English speaker upon revision by the authors, or by the journal's scientific editor and production staff.

The literature cited is appropriate and complete.

The manuscript is nicely prepared. The figures look good. Table 1 describes the cell types evaluated in this study and provides abbreviations when applicable. This table is important for its content, but the content might be better delivered in the text rather than a table.

**It is not at all clear that the raw data is available for other investigators. This study is based upon 1092 patients from the TCGA. There is no description of how patients were selected and so whereas the TCGA data is available, it is not possible to reconstruct this cohort.
Experimental design: This manuscript describes a study of 1092 breast cancers from TCGA where the gene expression signatures were used to identify 64 cell types including immune cells, stromal cells, and other cell types. Given that the cellular composition of a cancer can impact on its biology and by extension its treatability and prognosis, this is considered a significant study. Overall, the authors used sound methods and applied good statistical analysis.

1. The authors curated gene expression data from TCGA for 1092 breast cancers. No selection criteria are provided and so it is not possible for a reader to recreate this data set. It is not clear that the raw data is shared in the form it was used.
2. The authors don't provide summary statistics for the breast cancers examined, such as age of patients, ER/PR/HER2 status, etc. This information is contained in the results, but really belongs in the Methods. In fact, the cohort included 12 make breast cancers and this was not revealed until late in the results.
3. Some of the text included in Results (like the first paragraph on cell types analyzed) could be included in the Methods.
Validity of the findings: This manuscript describes an interesting study. The authors used good methods and statistics. The figures are nicely prepared. The text of the results and discussion is well done. Overall, the findings are very interesting and presented in a fairly straightforward manner. In general, the approach taken by these investigators provides a nice discovery study. What it lacks is a validation study where a smaller subset of breast cancers is analyzed in a different manner (immunostaining for instance) to verify the gene expression mining results.

1. The results shown in Figure 1 include 112 normal breast tissues in comparison to 1092 breast cancers. The authors have chosen to show results for all cancers compared to the normals instead of using the paired tissues in a much more powerful manner.
2. Figure 4 includes "ESCC" in the figure title. Please define this abbreviation. I would not expect this abbreviation in a breast cancer manuscript.
Additional comments: Overall, this is a very well prepared manuscript that describes an interesting study. The manuscript can be improved through minor edits, and perhaps consideration of additional analysis (paired samples) and/or a validation study (with immunostaining of tissues anlyzed by gene expression).


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: CELLULAR HETEROGENEITY MAP OF DIVERSE IMMUNE AND STROMAL PHENOTYPES WITHIN BREAST TUMOR MICROENVIRONMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Breast cancer is one of the predominant types of tumors in women worldwide. Despite great progress in the early diagnosis and treatment recently, drug resistance and distal metastasis remain major causes of cancer-related mortality. Tumor microenvironment (TME) cells are important elements in tumor tissue. There is increasing evidence that they have important clinical pathological significance in predicting tumor clinical outcomes and therapeutic effects. It is inspiriting and beneficial to analyze the global view of tumor-infiltrating immune and stromal cells in tumor tissues and adjacent normal tissues, and investigate the correlation of TME cells’ signature with breast cancer prognosis. In this manuscript, Dr. Li and colleagues looked at the role of the “64 types of immune and stroma cells’ signature” in diagnosis and prognosis of breast cancer. The study’s conclusion showed that "immune and stroma cells’ signature" was expected to be used to predict the prognostic survival of breast cancer. The paper is improved and most concerned raised by the reviewer have been addressed. I think it is might suitable for publication at this version of revised manuscript.

Minor comments:
1. Figure 4 B and C. The two side-by-side charts are not the same size. To maximize the quality of the article, set the same type of chart to the same magnification as possible.
2. Figure 4 K to O. Pictures in the same row, be sure to align horizontally. Authors should check all the pictures one last time.


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: CELLULAR HETEROGENEITY MAP OF DIVERSE IMMUNE AND STROMAL PHENOTYPES WITHIN BREAST TUMOR MICROENVIRONMENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In the revised manuscript, the authors provided a unique cellular heterogeneity map of diverse immune and stromal phenotypes in breast cancer microenvironment using xCell method, which might uncover potential therapeutic targets and biomarkers. They found that specific immune and stromal cells in the tumor microenvironment may serve as independent prognostic factors in the subsequent progression of breast cancer. The authors have revised the paper carefully according to the concerns raised by reviewers in the current version. I think it is suitable for publication at this point for this version of revised manuscript.


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

Input:
Title: METHYLOBACTERIUM, A MAJOR COMPONENT OF THE CULTURABLE BACTERIAL ENDOPHYTE COMMUNITY OF WILD BRASSICA SEED
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article was written in clear English. However, the paper has some problems in punctuation marks and font style. For example, lines 38 and 51 of the abstract.

There are some misspellings (lines 188, 192, 197, 199, 202, 212and 214).

In the results of the “Plant growth promotion” section (line: 254), you report the increase of the growth in dry weight. However, on the axis of the figure 2 was noted like “fresh weight”. Also, check the raw data table.

References: you must correct the molecular nitrogen annotation (lines 484 and 562).

All these observations were noted in the original text. This document will be attached in PDF format.
Experimental design: In materials and methods, in the section “Identification of seed-associated bacterial endophytes” (line: 156), the authors used partial 16S rDNA sequences in agreement to Weisburg et al. (1991). However, it is important that you analyze the work of Kato et al. 2005 (J. Gen. Appl. Microbiol., 51, 287–299). Because, the HV-region of 16S rDNA used in this study produced a better grouping of Methylobacterium species. In addition, this region has been used in other genres and produces the same results (for example: Bacillus, Goto et al. 2002). Maybe you could adjust the length of their sequences and produce a new tree.
Validity of the findings: You should add images of the “Dual culture test” (line: 261). I believe that these results are important to confirm the importance of Methylobacterium like antifungal.
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: METHYLOBACTERIUM, A MAJOR COMPONENT OF THE CULTURABLE BACTERIAL ENDOPHYTE COMMUNITY OF WILD BRASSICA SEED
Review round: 1
Reviewer: 2

Output:
Basic reporting: Endophytes are important plant inhabitants that f.e. provide tolerance against biotic and abiotic stress. They were discovered and defined in the 80th; methods of microbiome research (multi-omics, advanced microscopy) boost our knowledge about endophytes and the seed microbiome during the last ten years. However, this study based on a traditional cultivation approach of endophytes of Brassica seeds.Such approach is of value for applied research, e.g. looking for novel plant growth promoting or biocontrol bacteria. For basic research, methods are not state of the art.
Experimental design: Authors studied seeds of 83 wild and landrace Brassica accessions composed of 14 different species with a worldwide distribution for seed-associated bacterial endophytes.
However, I see problems with experimental design in the following points:
•	The methods applied are very traditional, and based on cultivation of microbes only. A combined approach e.g. with 16S rDNA amplicon sequencing would be of value and allow a much better assessment of the cultivated strains.
•	The design of the seed samples is not well explained. There is an impact of seed age and origin, which is not considered.
•	29 accessions were excluded due to exhibited epiphytic fungal growth of Alternaria sp.
Validity of the findings: Due to the problems with the experimental design I don’t think that the conclusions are really representative.

Other comments:
Abstract: Microbial species cannot be parasitic. Half of the abstract is introduction only, on the other side half of the experiments is not yet mentioned in the abstract (PGPR by Methylobacteria).
"Seed-associated" is not correct because the authors studied the phyllosphere and rhizosphere of plants - "seed-originating" would be better. Despite the differentiation of isolates according to their habitat, this is not well considered in the results and discussion section but would be interesting.
Abbreviations of the genera are not correct.
Missing important references:
Hardoim PR, van Overbeek LS, Berg G, Pirttilä AM, Compant S, Campisano A, Döring M, Sessitsch A. 2015. The Hidden World within Plants: Ecological and Evolutionary Considerations for Defining Functioning of Microbial Endophytes. Microbiol Mol Biol Rev. 79(3):293-320
Wassermann B, Rybakova D, Müller C, Berg G. 2017. Harnessing the microbiomes of Brassica vegetables for health issues. Sci Rep. 7(1):17649.
Additional comments: Best wishes Gabriele


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

Input:
Title: IN OR OUT: RESPONSE SLOWING ACROSS HOUSING CONDITIONS AS A MEASURE OF AFFECT IN THREE WESTERN LOWLAND GORILLAS (GORILLA GORILLA GORILLA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an interesting and clearly written study about gorilla affective states.  The background literature is well understood and covered.

The word "states" is missing from line 1 of the Abstract.

"these individuals spend a relatively high proportion of time
vying for dominance and it may be that they experience a tradeoff concerning these two sleep locations"
please provide data to support this suggestion.
Experimental design: The experimental design appears robust and is clearly described.

My first main concern is the sample size of 3.  I appreciate that great ape studies are typically limited in terms of the possible sample sizes available, but given that the study overall finds no consistent effects, it may just be a sample size issue. This has not been discussed.
Validity of the findings: My other main overall concerns is that the effects of one previous night on the current mood of the gorillas might not be strong enough to detect an effect. It seems that the night time conditions/enclosures for the three gorillas were changing quite often. Details of how often changes occurred and where the gorillas spent their nights (husbandry routine of the zoo) should be given early in the Methods. There is some information currently between lines 165 and 171. I could not find Table 1 among all the submitted files (see line 171).

Given that the gorillas seem to have been moved often between preferred and less-preferred night enclosures, this might explain the lack of consistent effect.  This possibility has not been discussed.
Additional comments: n/a