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 DECREASE IN REPORTS ON ROAD-KILLED ANIMALS BASED ON CITIZEN SCIENCE DURING COVID-19 LOCKDOWN
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors meet all basic reporting requirements of journal - see general comments for author for full review.
Experimental design: Authors meet all experimental requirements of journal - see general comments for author for full review.
Validity of the findings: Authors meet all validity requirements of journal - see general comments for author for full review.
Additional comments: General comments:

The authors have made major revisions to the manuscript in the introduction and added analyses and figures (though see comment on figures below). Overall, it is much improved and an interesting study.

I believe the authors are referring to the term “community-based science” rather than “community science.” It is true that the term “community-based science” usually refers to efforts led by the wider community and would not be fitting for a university-led project, but “community science” is used in lieu of “citizen science” as a more inclusive term for researcher/university/institution led volunteer data collection projects. This is not a major concern for the paper as it is on inclusive language rather than content, and I respect if the authors decide to continue to use “citizen science” instead as it is widely used. I very recently switched to using “community science” as a term myself.

After the paragraph describing other COVID roadkill studies in lines 86-96, there is an opportunity to discuss how some of these studies may not take possible bias of staying home vs actual decrease into consideration. This would be a good transition to describing project Roadkill and how this particular study is novel.

I appreciate the addition of taxon roadkill figures in Figure 1. Figures 2 and 3 seem to be very different than the figures from the original submission. If I’m reading the mosaic plot correctly, it looks like the proportions of more, same, and less reported roadkills are switched (not just the order). Please double check that this is the correct figure. Currently the figures read as most people reported more roadkills with reduced length and frequency of driving which seems incorrect compared to the results.

Check comma usage throughout. It is unnecessary in many places and missed in others. Some instances noted below.

Additionally, inconsistent usage of “Project/project Roadkill” – sometimes project is capitalized while other times it is not.

Specific comments:

59: comma unnecessary

70: “In some roadkill monitoring projects,” – sentence before is general so specifying is necessary

75: comma unnecessary

76: “provide the GPS coordinates of the roadkill” vs “locate the location of…”

80-82: comma usage and phrasing needs to be revised – “Through citizen science it is possible to sample a large geographical area (Theobald et al., 2015), to bring new expertise into the project, and last but not least, provide opportunities for topical education and science communication”

86: delete “this”

86: “the data collection process,”

87: “In the US,”

111: in () “e.g. when riding their bikes as a hobby or when commuting to work.”

117: “In previous years,”

117-119: revise sentence phrasing and add scientific names

121: “fewer” vs “less”

142: roadkill does not need to be capitalized unless referring to “project Roadkill”

219: “In Table 2,”

261: “Particularly,” vs “Especially”


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: MORPHOMETRY OF CELLULAR BEHAVIOR OF COELOMOCYTES FROM STARFISH ASTERIAS AMURENSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: This is one interesting study which revels that in contrast to the morphology of neurons, classical morphometry of which is well developed, the morphological description of fibroblast-like cells is less formalized. The authors make and specially emphasis about the conventional cytological parameters often provide an ambiguous picture, the development of an universal classification scheme requires a combination of more complex morphometry as well as genetic and biochemical methods of determining the cell specificity and cytodifferentiation data.

In this work the authors used 400 coelomocytes of the Asterias amurensis as example, in order to show the effectiveness of multiparametric analysis of the dynamics of the spreading of immune cells, including the identification of a number of characteristic morphotypes of cells and their transformation in the process of short-term cultivation.

Could be interesting mention the amazing size increases across the time.

These cells were adequate prepared for a qualitative light microscopy study, and morphometrically analyzed, for this study, photography’s were take using a 10-megapixel digital camera of a Zeiss Axiovert 200M Apotome microscope.

Also all the 39 measurements from all cellular parameters were made adequate as well as their statistical analyses. Thus, linear and fractal analyses were calculated with the software (Image J) and 12 linear parameters were exhaustive analyzed in 11 measurements and non-linear in 7.

They found lack of correlation for similar trends and vice versa as well as the difference in  correlation in different time groups. Thus, these founding’s generally indicate that this parameter in different cell groups describes different features of cell morphology.

In relation with the cell types, the parameters were used to generate a cluster model for each one, which clearly revealed three clusters corresponding to three cell morphotypes.

But, which one is the size of them?

They also, found that some parameters have sufficient discriminatory power for reliable identification of the most dendritic cells combined, and form variable correlations in different time groups. Also, included cluster and correlation analyses and an variance analysis using a variety of linear and nonlinear cell morphology parameters, enabled identification of cell characteristics, such as complexity of cell boundary microsculpture, overall asymmetry of the cell, sizes of the cell, bounding circle, and convex hull, and inhomogeneity of surrounding space filling by the cell and establishment of a relationship between these characteristics and differences in the dynamics of cell spreading at different culturing stages.

My principal comments are especially in the representation of the figures: Figures 1, 2, 4, why to put lettering below each graph and using point? (A. B. C. D. E.) also the codes will be better in the figure caption.

Figures 3, and 5. Although, the size of the type cells is clear, will be better to put an scale bar showing the micrometers.

In Table 1, it do not show the numbers of significant differences between groups.


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

Input:
Title: MORPHOMETRY OF CELLULAR BEHAVIOR OF COELOMOCYTES FROM STARFISH ASTERIAS AMURENSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This paper constitutes an original study about morphometry of Asterias amurensis immune cells after collection, using a wide range of linear and non-linear morphological parameters including size, asymmetry, complexity, form, area, circularity, diameter etc. Despite the interesting focus of the paper  I believe that the authors should make some changes and improvements of the text in order to make the paper suitable for the journal. I am most dubious about the parameters reported in the text and the presentation form choosen to report some data (see detailed comments). Also The English language should be improved to make comprehension less difficult and it is assumed a lot of sectoral knowledge for the reader and the readership. Considering this issue my suggestion to the authors is to revise and resubmit the paper in a new form.
Detailed comments:
Abstract:
-	Line 49: what does “cell boundary microsculpture” stand for? Please define it
-	Line 50: what does “convex hull of the cell” stand for? Please define it
Introduction
-	Line 85-91: This part is not well-written. Please revise
MATERIALS AND METHODS
-	Line 112: How many animals were used as donor?
-	Line 114-118: This part is not well-written. Please revise
-	Line 130-164: This part is not well-written and it assumes a lot of knowledge from the reader and the readership of the journal probably does not know what the major part of parameters selected are. This coud be helped with using the same style to explain each parameters: for example  Aspect ratio (AR):brief explanation; Roundness (Round): brief explanation; and so on…..
“the ratio of area parts of the cell that turned out to be in the two halves of the bounding circle, the diameter dividing cells was drawn in the direction that most unevenly divided the image cells and was used as a measure of cell asymmetry, (1/2half): this part is really confusing and impossible to understand. The same “The ratio of cell
 area in the outer half of the bounding circle to the area of the cells in the inner half of bounding circle”, “lacunarity calculated on the basis of differences in the number of
 pixels in each square of the grid for all orientations of the squares, with allowance for the
background pixels taken as zero (only pixels of the image of the object were taken into account in LF), (LE), etc.
The majority of the sentences are too long to be effective and hard to follow.
It could be a good idea to briefly introduce each parameter in this section and then develop each explanation in a new section as results, adding the real images of the cells in which the parameter is applied.

RESULTS
Visual description of cell morphology section: This section describes different type of cellular transformation that cannot be valued without any image that support this description.
Trends in parameter dynamics: when reading this section it is not clear the object of the discussion, the name of each parameter should be mentioned into the text.
Cell types: I would move this information at the beginning of the results.


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

Input:
Title: MORPHOMETRY OF CELLULAR BEHAVIOR OF COELOMOCYTES FROM STARFISH ASTERIAS AMURENSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors analyzed using a range of linear and non- linear morphological parameters in three stages of cell behavior of immune cells of the marine invertebrate Asterias.
In each stage they described chronological differences in cell complexity, such as size and asymmetry among other. The main statistical found is due to the characterization in several linear and nonlinear cell parameters of the external morphology for all cell types.
Experimental design: This is interesting since of the dynamics of cellular behavior shows a number of transitional forms, which dynamics can also reflect the species-specific features of cellular behavior, the state of the animal's immune status or reaction to the experimental effect.
Validity of the findings: In this context and due to the difficulty of this morphological cellular changes is necessary the use of multiparametric cellular analysis in vitro and in short-term.
The statistical design and correlations are correct supporting the results.
Additional comments: My comment is about the conclusions when a comparative issue es in classical neuronal morphometry which is well developed, in contrast with the fibroblast-like cells, is important to mentioned the morphometrical analysis in microglial cells, in which skeleton was well as fractal analysis is also well studied, I recommend to include these biblio:
Young, K., Morrison, H. Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ. J. Vis. Exp. (136), e57648, doi:10.3791/57648 (2018).
Morrison Y, et al., Quantitative microglial analyses revels….Scientific Reports | 7: 13211 | DOI:10.1038/s41598-017-13581-z


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: LONG-TERM FOLLOW-UP OF APPLYING AUTOLOGOUS BONE GRAFTS FOR RECONSTRUCTING TYMPANOMASTOID DEFECTS IN FUNCTIONAL CHOLESTEATOMA SURGERY
Review round: 1
Reviewer: 1

Output:
Basic reporting: First of all, I would like to congratulate the authors for assembling such an interesting article. The article is generally well-written and structurally adequate. The authors have successfully built the rationale behind their study, providing solid grounds to demonstrate why their study would be important to the literature and practicing ENT physicians.
I have minor general concerns at this point of the review:
- Although I commend the authors for performing such a detailed literature review, the manuscript is too long. Some of the informations contained in the introduction and discussion are of general knowledge of physicians who are more likely to read the manuscript. Therefore, I recommend shortening the manuscript to include only information that is critical to the understanding of the article.
- The english language is generally acceptable. However, some sentences are wordy, with minor syntax issues. I recommend the article should be proof-read by an expert.
- There are some sentences in the introduction and discussion that are lacking references.
- Another surgical technique proposed in the past that is similar to yours was also described by Prof. Paparella, with the name "flexible endaural approach". I think this is worth mentioning in the introduction.
- The images are excellent; however, they are very small and condensed in a single picture. I strongly suggest the authors to separate the figures in at least 3 different pictures so they can be expanded and seen in more detail. Additionally, it would be interesting to include more pictures of how the bone chips were harvested.
Experimental design: The experimental design was described in detail, and seems adequate to answer the proposed objectives.  I particularly enjoyed the strictness of your inclusion/exclusion criteria, and the detailed description of the surgical technique. However, I have major concerns regarding the experimental design used:
- There is no control group to compare the findings.
- Were all patients operated by the same surgeon? If the surgeries were performed by more than 1 surgeon, it would be interesting to describe if the surgical technique changed among surgeons and if there were a significant heterogeneity in the results obtained by each one of them.
- The authors performed hearing tests at the 3-year timeline following the surgeries. Although this is very interesting, the lack of a control group does not allow comparison regarding the success of this specific reconstruction (using bone chips) with other approaches. As the auditory results are largely dependent of the degree of bone erosion/ossicular chain abnormalities, it seems that this information - although interesting - does not add significantly to your results of conclusions.
- In the same line of thought, it is not possible to compare the success regarding lack of recurrence as there is no control group to compare the findings.
- I was particularly confused by the use of the term "bone chips". Bone chips usually refer to particles of bone that accidentally separate from the drilled area. As the "methodology" only briefly cites how the "bone chips" were harvested and there are no pictures to illustrate this, I thought that the authors have collected bone pate and performed a mastoid occlusion with this material before I saw the surgical image later on. I would personally recommend the use of other terminology to better illustrate the technique used - such as "autologous bone grafts", or "bone graft palisade".
- There are plenty of articles that performed a bone reconstruction of the posterior canal wall using different techniques that were not cited or included in the discussion.
Validity of the findings: The results are meaningful and interesting. The data yields interesting information that could potentially improve the practice of ENT physicians that are involved with cholesteatoma surgery. Although the article could be a significant contribution to the literature, the lack of a control group critically undermines the validity of your results.
Additional comments: No additional comments


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

Input:
Title: LONG-TERM FOLLOW-UP OF APPLYING AUTOLOGOUS BONE GRAFTS FOR RECONSTRUCTING TYMPANOMASTOID DEFECTS IN FUNCTIONAL CHOLESTEATOMA SURGERY
Review round: 1
Reviewer: 2

Output:
Basic reporting: I would like to congratulate the authors, as this is a very well-written study, and although it is a retrospective study, methodologically, inclusion and exclusion criteria were carefully used, making the sample very homogeneous, making the results comparable with other studies. However, only 66 ears in total are a small sample as some subgroups were left with an extremely limited number of patients (less than 10 individuals) and some variables that are not very prevalent, such as the performance of Type III-M, Type IV tympanoplasty and absence of the stapes suprastructure, were not very representative and therefore are difficult to be statistically evaluated.
I must emphasize that both the figures and the tables are very clear and informative.
I suggest checking references number 20 and 27 as they are outside the formation suggested by PeerJ.
Experimental design: This manuscript presents a follow-up of at least 3 years with functional results within expectations both from the point of view of relapse, recurrence and hearing recovery, although in cases of cholesteatoma there is the possibility of late recurrences with a 5 year evolution. In relation to the surgical aspects used for the removal of the cholesteatoma, the material used for the reconstruction does not present any novelty and the impact can be evaluated only in relation to the follow-up time and in relation to the preservation of the auditory thresholds as the recovery rate observed despite the statistically significant was only 10dB, which may or may not be clinically relevant depending on the previous aero-bone gap of everyone.
Validity of the findings: Studies on cholesteatoma and its treatments have been carried out since the twentieth century and unfortunately, the surgical exeresis of cholesteatoma, regardless of its origin, is still the only therapy capable of controlling the evolution of this disease and preventing its complications in both the auditory aspect and its fatal risk. Several techniques are described and currently the most used are those that remove the disease trying to preserve the anatomical structures responsible for the auditory function, a technique used in the study and called functional surgery for cholesteatoma. The reconstructive phase of this surgery has also been widely explored and several materials have been reported with the individual's own cortical bone but also several other synthetic materials for the reconstruction of both the posterior wall and the compromised ossicular chain. Therefore, neither the surgical technique used, nor the material used for reconstruction are innovative, on the contrary, it has been widely used because economically it does not generate additional costs with heterologous prostheses, and when performed by well-trained surgeons, they achieve reasonable functional results. For this reason, there are already several studies that examine the results of the follow-up of the application of autograft bone to reconstruct tympanomastoid defects in surgery for functional cholesteatoma.
The conclusions are based on the results; however, it does not add any additional data to those already provided by the current literature.
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: LONG-TERM FOLLOW-UP OF APPLYING AUTOLOGOUS BONE GRAFTS FOR RECONSTRUCTING TYMPANOMASTOID DEFECTS IN FUNCTIONAL CHOLESTEATOMA SURGERY
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
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: LONG-TERM FOLLOW-UP OF APPLYING AUTOLOGOUS BONE GRAFTS FOR RECONSTRUCTING TYMPANOMASTOID DEFECTS IN FUNCTIONAL CHOLESTEATOMA SURGERY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No issues found
Experimental design: No issues found
Validity of the findings: Although the study replicates methodology from other past studies, the findings are valid.
Additional comments: Thank you for addressing the comments made by the reviewers. The manuscript has increased a lot in quality. I have no further comments.


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

Input:
Title: LONG-TERM FOLLOW-UP OF APPLYING AUTOLOGOUS BONE GRAFTS FOR RECONSTRUCTING TYMPANOMASTOID DEFECTS IN FUNCTIONAL CHOLESTEATOMA SURGERY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thank you for addressing the comments in reviewing this manuscript. The authors provided additional information and after had been proof-read by an expert and adopting specific terminology, the manuscript improved overall. The findings are interesting and remain relevant to the journal's readers.
Experimental design: Although it was not possible to expand the sample size in different subgroups, the results indicate an adequate functional result using autologous bone grafts for reconstruction external ear canal defect and mastoid obliteration without additional materials can improve operational efficiency, which greatly reduces surgical costs.
Validity of the findings: The findings are interesting and remain relevant to the journal's readers.
Additional comments: The separation of the figures really facilitated the interpretation of the data however in figure 1, the word "Cholesteatoma" has a typo and must be corrected. I also suggest putting an acronym for CMO, CWD, EAC.


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: RISK FACTORS ASSOCIATED WITH PROLONGED VIRAL CLEARANCE IN PATIENTS WITH A REFRACTORY COURSE OF COVID-19: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: Dear authors and editors,
Thank you for the opportunity to review this paper. In this study, the authors reported the viral clearance in COVID-19 patients with refractory course and the risk factors. Since reinfection, relapsed infection and recurrence of positive (re-positive) nucleic acid detection are not rare in clinical practice and might have clinical and epidemiological implications for treatment and infection control measures. Thus, this topic is important, and relevant research to understand the underling mechanism and the risk factors are urgently needed. However, there are several significant weaknesses in this paper. My comments regarding this manuscript are provider below:
Major points:
1. According to the description of the manuscript, it seems that the study population was COVID-19 patients with refractory course rather than those with readmission. To the best of my knowledge, the reason that patients were admitted to the Huoshenshan hospital was not necessary to be readmission. Some of them were did not discharge from another hospital previously and were directly transferred to the hospital due to the absence of ward beds on elsewhere. Thus, the term “patients with more than one COVID-19 related hospitalization” means nothing, and the title is not accordance with the contents of the manuscript. I suggest the authors to revise the title correspondingly.
2. Given the tremendous body of literature demonstrating the viral clearance in COVID-19 patients with refractory course and the risk factors, it is not fully clear what the authors wish to add to the literature in the manuscript. The novelty of findings from this cohort seems to be limited.
3. The authors intended to explore the risk factors associated with the duration of viral clearance. However, the definition of the duration of viral clearance was the period from the date of illness onset (as stated in the history of present illness) to the date of discharge from Huoshenshan hospital. This definition was less accurate. Some patients might still be still hospitalization due to the complications from other organs (e.g. bacterial pneumonia, urinary tract infection, heart failure, etc.), even the negative results of SARS-Cov-2 RNA were received. This situation is more common in severe or critical patients. Thus, the research question is more about the duration of prolonged hospitalization instead of viral clearance. Please reflect on this.
4. It is noted that there were missing data in the antibodies of SARS-Cov-2 and PCT. However, no statement on the degree to which their variables had missing data and what was done in the cases of missing data or inconclusive data. In particular, how the incomplete data of antibodies were included into the COX model with other complete data? Are there any statistical methods performed? (e.g. imputation?)
5. The writing of this manuscript is poor. I suggest revising the introduction to make it succinct, so that that the rationale is clearer. Moreover, the discussion is very higgledy-piggledy, and the key points are difficult to grasp. Particularly, the authors did not show how their results and interpretations agree (or contrast) with previously published work.
6. The English language should be improved to ensure that an international audience can clearly understand your text. The current phrasing makes comprehension difficult. I suggest you have a colleague who is proficient in English and familiar with the subject matter review your manuscript, or read a textbook on how to write an academic article (e.g. Robert A. Day. How to write and published a scientific paper) before rewriting.
Minor points:
1.	Line 43: “has” rather than “have”
2.	Line 64: This paper did not focus on the underlying mechanism, and therefore the statement should be revised.
3.	Line 126: The symptoms were presented from the illness onset or on this admission?
4.	Line 132: 60+9=68?
5.	Line 73: There was no definition of critical case, whereas critical cases were presented in the results.
6.	Line 165: To the best of my knowledge, this is not the first study that reported the viral clearance in COVID-19 patients with refractory course and the risk factors.
7.	The limitation of this study was not fully presented.


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: RISK FACTORS ASSOCIATED WITH PROLONGED VIRAL CLEARANCE IN PATIENTS WITH A REFRACTORY COURSE OF COVID-19: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors of the MS titled with “Risk factors associated with longer viral clearance in patients with more than one COVID-19-related hospitalizations”, firstly investigated the viral clearance in patients with a refractory course of COVID-19. They performed the time-dependent cox regression and found that fever at illness onset, serum level of aspartate aminotransferase > 21.7 IU/L, and titer of SARS-CoV-2 IgG > 141.69 were the three independent risk factors associated with delayed viral clearance. The study is novelty, had clinical importance, used comprehensive analyses, and the findings are of important value in management of COVID-19 patients.

Comments:
1. How was the sample size determined? Please add statistical analyses details of how the sample size was determined.
2. Whether the discharge criteria were same or not between the first and second COVID-19-related hospitalization?
3. Authors should further discuss the underlying mechanisms or some related factors for the associations among  fever at illness onset, serum level of aspartate aminotransferase, titer of SARS-CoV-2 IgG and the delayed viral clearance.
4. The MS was well written.


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: RISK FACTORS ASSOCIATED WITH PROLONGED VIRAL CLEARANCE IN PATIENTS WITH A REFRACTORY COURSE OF COVID-19: A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: Dear editors and authors
Thank you for addressing the majority of my concerns. The quality of the manuscript has been improved significantly. However, there are still some issues that should be further addressed.
#1. Commonly, readmission is the act of taking somebody into a hospital again after they had been allowed to leave. As 78% of the COVID-19 patients were admitted to the Huoshengshan Hospital due to the persistent symptoms, the term “readmission” is not suitable for the study population. Please revise it across the manuscript. In addition, is the presentation “more than one COVID-19 related hospitalization” suitable? Please reflect on this.
#2. In the revised manuscript, the title as well as relevant presentation was revised to be “patients with a refractory course of COVID-19”. What is the exact definition of refractory course? No definition was presented in the manuscript. I have noticed that there were COVID-19 patients who had the duration of viral clearance with only 11 days. Did these patients meet the definition of refractory course? Please consider carefully how to describe the study population exactly.
#3 As the authors stated, there were 2 patients who were still hospitalization due to the complications from other organs, even the negative results of SARS-Cov-2 RNA were received. The definition of the duration of viral clearance the authors defined is not rigorous in case that the two patients were excluded. Whether the inclusion of the two patients would influence the results is still unknown (underestimate, overestimate, or change the result?). This problem can not be resolved by presenting it as a limitation. In this case, it is better to exclude the two patients, or only their durations from illness onset to viral shedding could be analyzed in statistical analysis. Or else, the research question is more about the duration of prolonged hospitalization instead of viral clearance.
#4. To deal with the missing data, the authors delete all the cases that had missing values. The sensitive analysis is nice. However, as cases with missing data were excluded from the COX model, there were only 36 cases in the model. Since the sample size may not be sufficient, the goodness-of-fit of the COX model should be reported. Additionally, the results should be interpreted carefully.
#5. The presentation of the conclusion in the abstract is not suitable. Commonly, this section is limited to the major findings based on the current study rather than the perspectives from the findings.
#6. Additionally, some minor work needed is grammatical errors throughout. Some examples include:
Line 197: …as the viral load was found to (be) associated with more severe COVID-19 and (a) longer virus-shedding period…
Please check the grammatical issues carefully across 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: RISK FACTORS ASSOCIATED WITH PROLONGED VIRAL CLEARANCE IN PATIENTS WITH A REFRACTORY COURSE OF COVID-19: A RETROSPECTIVE STUDY
Review round: 3
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: OK
Validity of the findings: OK
Additional comments: Dear authors,
Thank you for addressing all of my concerns. The quality of the manuscript has been improved more significantly. As a scientific article, a clear, unambiguous, professional presentation is needed throughout. However, there are still several minor issues to be corrected, majority of which are regarding professional presentation.
1. Professional presentations regarding the medical nomenclature
For example:
#line 35: The presentation “mildly impaired liver function” is not correct. As the upper limit range of AST Is 60 IU/L, a level of AST> 21.8 IU/L does not mean mild impaired liver function.
#line 37: What does the “COVID-19 diseases” mean? COVID-19 means Coronavirus disease 2019.
#line 43:The presentation “ COVID-19 infection” is inaccurate. Maybe “SARS-Cov-2 infection” is more precise.
#line 141: As I have point out previously, the presentation “readmitted” is not correct.
#Table 2: “20-30 days”. Maybe “21-30 days” will be more precise because that the duration of 20 days was excluded.
2. There are also several ambiguous presentations across the manuscript. As an academic writer with English as a second language, I strongly recommend the authors to read a textbook regarding the scientific writing named “English Exposed-Common Mistakes Made by Chinese Speakers” (Steve Hart, Hongkong University Press).
For example:
#line 36 What is the exact mean of “positive treatment”? Is the presentation “positive” suitable here?
#line 60: What is the exact mean of “diseases”? Are the transmission of any other diseases was also involved?
#Line 141:”hospitals”. Did the included patients be admitted in other hospitals?
#Line 172, 177, 208, 355……: The exact titer of SARS-Cov-2 IgG should be 142.09 AU/ml. The unit should not be missed. Please re-check it across the manuscript.
#line 184 “virus”. Which virus do you mention in this sentence?
#Line 199: was was…..
3. Others
#line 217: Commonly, the exact statistical values in the result are not recommended to be presented in the discussion repeatedly.


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: RISK FACTORS ASSOCIATED WITH PROLONGED VIRAL CLEARANCE IN PATIENTS WITH A REFRACTORY COURSE OF COVID-19: A RETROSPECTIVE STUDY
Review round: 4
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK
Additional comments: The authors have addressed all my concerns. Good luck!


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: APPLICATIONS OF THE INDOLE-ALKALOID GRAMINE MODULATE THE ASSEMBLY OF INDIVIDUAL MEMBERS OF THE BARLEY RHIZOSPHERE MICROBIOTA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English could be simplified in a few points (see additional comments section) in order to improve the text flow... but overall the English is of high quality.

Also there are discussion points in the results (see additional comments section) which need to be rephrased/removed.

Otherwise, the manuscript meets the journal standards.
Experimental design: The authors have not included any gramine producing wild-type and its gramine deficient mutant in their study… an addition as such would have greatly enhanced their discussion. I would suggest to discuss this event in the least.
Validity of the findings: There are some results that are somewhat overstated, but this could be addressed through text changes. See the "additional comments" section for more details.
Additional comments: Herewith, I provide my views on the manuscript entitled “Applications of the indole-alkaloid gramine shape the prokaryotic microbiota thriving at the barley root-soil interface” by Maver et al describing the effects of the allelopathetic gramine on the prokaryotic microbial community. The authors have used a bulk soil vs two gramine deprived varieties, and endeavoured to understand the effects of gramine by contrasting three different gramine application levels (0, 24 and 48 µmol L-1) of applied gramine.

General comments:

I believe that there is merit in this study particularly for the portion of the research community interested in the applied aspects of alkaloids. I also think that the authors did a meticulous work in several parts of the experimental design although they could have improved their setup as I will explain below. Finally, The English is of high level, although sometimes difficult to follow due to the use of very literate language (I like it in most cases).

Having mentioned the strong points of this work, I would like to mention some weaknesses (which are less important but could be easily addressed by toning down the discussion or acknowledging the associated pitfalls).

E.g. one weakness is the fact that the authors have not included any gramine producing wild-type and its gramine deficient mutant in their study… an addition as such would have greatly their enhanced discussion. I would suggest to discuss this event in the least.

Another weakness is that the authors seem to consider gramine solely (or in the most manuscript) as a toxicant against prokaryotes (although gramine has been studied mostly for its untifungal properties). They do not consider the fact that gramine could also be C/N source for prokaryotes, leading to the manipulation of the microbial composition due to changes in the nutrient balance… or gramine could be toxic against fungi and, hence, affect their degradation. There are some comments in the end of the report about indirect effects but most of the document passes the feeling to the reader that graine is toxic against all tested prokaryotic members.

I see highly meticulous attitude in some aspects of the experimental design (e.g. negative controls for all indexes used in multiplex sequencing, several controls in other design aspects etc…) and quite relaxed attitude in some other parameters (e.g. 2x150 in >300bp amplicons)

Finally (less important point… but needs to be addressed), there are some discussion points made in the results section… I would suggest changing or removing these.

Specific comments:

Introduction

L51: “support” instead of “supports”

L58: I am not sure the authors want to use the word “checkpoints”… maybe “milestones”… either way the sentence needs rephrasing.

L66-87: Here the authors apparently want to state that domestication is related to the production of allelopathetic metabolites (like gramine) which may contribute to the manipulation of the rhizosphere microbial communities. I would suggest adding such an opening sentence in the beginning of this part that will immediately make clear to the reader what the two paragraphs below are about. Besides, these paragraphs are the most important for introducing the aims.

Material and methods

L130: media? Only agar? Else, what was the composition? A reference could support as well.

L138-139: what was the basis of the choice of the gramine concentration? Is it close to the naturally occurring concentration?

L207-210: This strategy barely leaves any chance for amplicon reconstruction (the amplicon plus the barcode is more than 300bp long while the 2x150 cover just 300 allowing no reliable overlap for amplicons to be reconstructed). Hence, no second strand verification is provided (at least at the terminal, most error prone, parts of the sequence reads) by this strategy and the quality filtering relies solely on estimated errors by the base-calling software rather than physical evidence. This approach has been published but does affect the confidence by which these data should be considered.

L219: please provide a link or a reference for the pipeline

L222: high quality? Please provide a brief description of the criteria… I remind that since no overlap exists there is no second strand verification of the read-end error prone sequence parts. I do accept the fact that this approach was published in the past, but I would like the authors to acknowledge its weaknesses.

L227: I might be missing something, but, read merging is not possible for the vast majority of the amplicons obtained with this primer set (~300bp amplicons with some length variance) and given the sequencing-approach (2x150 = 300bp per amplicon, hence no overlapping bases between the reads)… the authors mast mean that they used the parameter allowing concatenation of the reads in case no overlap is obtained… is that right? Please describe it clearly.

L232: The authors mean “screened” or “ASVs of non-target taxa were filtered out” instead of pruned. The authors seem to be literate but at some points their expressions are somewhat odd.

L237: I agree with this approach (i.e. probably the authors wanted to retain sequences that were clearly associated with the treatments)… however, it has to be justified. Please provide the reasoning.

Results

L277: the title looks like a conclusion… since this belongs in the results section, I would suggest that the subsection titles be more descriptive

L314-317:  This sentence looks to be more of a discussion statement than a results description.

L322: look at my comment for the previous subsection title.

L331: “respond” seems to be a strong statement… I would suggest using “coincide” or similar

L334: same as above for “responsive”

L356-361: This belongs in the discussion not the results. I would also suggest toning down statements like “otherwise suppressed by gramine application, appeared rescued”. The authors did not provide or cite any mechanistic information about possible toxicity of gramine on microbial cells, let alone particular taxa. E.g. gramine itself may act as a nutrient and therefore selection means as do root excretes of nutritional value. Without a gramine producing wild type control (which I understand is difficult to find in a way that it will have a similar genotype with the tested plant varieties), these statements seem a bit far-fetched.

Discussion

L378: Or may act as nutrient that causes outperformance of responsive microorganisms over others that lower their relative abundance which the authors may assume to be suppressed by gramine.

L381: a 0.5 pH difference seems marginal given between lab differences in experimental performances… I would rather avoid discussing it as an important difference between experiments. What about the organic matter (which is also a major microbial diversity driver – actually it is the most important one)?
L393: from a study… or … studies

L394: there the authors used benzoxazinoid impaired mutants along with the wild type… something that could help enhancing the impact of this study as well (i.e. the use of wild-type and gramine impaired wild-types next to the tested varieties).


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: APPLICATIONS OF THE INDOLE-ALKALOID GRAMINE MODULATE THE ASSEMBLY OF INDIVIDUAL MEMBERS OF THE BARLEY RHIZOSPHERE MICROBIOTA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Maver and co-authors analyzed the changes in barley rhizosphere and bulk soil bacterial communities after addition of the secondary metabolite gramine to soil in a pot experiment. Gramine is present in root exudates of wild genotypes, but absent in some modern varieties, such as Morex and Bark analyzed in the present study. This topic is important, since understanding the exudate molecules that shape the rhizosphere microbiome is essential to guide plant breeding and management strategies aiming to explore the beneficial plant-microbe interactions for more sustainable agricultural systems. The manuscript is relatively well written and organized, but requires grammar corrections in many sentences. The literature is in general well referenced, needing just a few inclusions (e.g. acknowledge other factors that affect the rhizosphere microbiome selection in addition to exudates - Intro; mention other secondary metabolites that shape the rhizobiome such as coumarins, glucosinolates and camalexin - Discussion). The figures are in high quality and the legends are clear.
Experimental design: The hypothesis of the study is clear and methods are standard in the field. The gramine adsorption assay was particularly interesting to make sure this compound is available in the soil solution to influence the soil bacteria. However, the experimental design would be more appropriate to test their hypothesis if the modern barley genotypes absent in gramine exudation were compared to the wild genotypes able to exude gramine. More genotypes would be needed for a stronger statistical power, avoiding bias associated with specific genotype background. The methods are properly described for replicability, but sometimes too much detail is provided, please summarize (e.g. sequence analysis). Moreover, the Methods should not be organized with checkpoints, as it is in some parts (Lines 185-189, 215-252). Removing the ASVs with <20 reads in at least 11% of samples (Lines 237-239) is too stringent, a lot of taxa was possibly lost in this process. Where did authors find this approach? Please add reference.
Validity of the findings: Although the authors claimed in the title and throughout the manuscript that the microbiota was affected by gramine addition, the results showed the opposite. Permanova results indicated that gramine concentration did not affect the bacterial community structure (Table 1) and the CAP showed a clear separation of samples according to gramine concentration only for the bulk soil (Figure 2). Observing specific differences in the relative abundance of a few bacterial genera does not mean the prokaryotic microbiota was shaped by gramine concentration. The greater effect in the bulk soil than in the rhizosphere beta diversity found here does not support the idea that gramine is a selecting factor for the barley rhizosphere microbiome, despite a previous study showed a significant effect (Schütz et al., 2021). The authors should change the focus of the manuscript to better reflect their findings.
Additional comments: No additional comments


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

Input:
Title: APPLICATIONS OF THE INDOLE-ALKALOID GRAMINE MODULATE THE ASSEMBLY OF INDIVIDUAL MEMBERS OF THE BARLEY RHIZOSPHERE MICROBIOTA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Maver et al. tested the effect of exogenous application of gramine (two doses) on bacterial communities of two modern barley cultivars and compared to unplanted soil (bulk soil). Their conclusion is that gramine can impact the bacterial communities in the rhizosphere of barley, with some enriched taxa.

The submitted paper conforms to professional standards as regarding to the use of English. The references are sufficient and provide a good overview of the topic. The provided figures are relevant to the understanding of the paper. Raw data is available at ENA and I liked the way they made the scripts available at Github, so their results can be reproduced.
Experimental design: The research is original and methods are also in accordance with what people are currently using (DADA2, ASVs).
Methods are briefly described and the full information can be seen in Terrazas et al. (2019). However, it's a biorxiv paper. I would recommend to add the full details in the present paper if there is more information to be added.
I could not see the script using phyloseq, vegan and deseq2 in R, regarding the removal of mitochondria, chloroplasts, filtering low abundance of ASVs, agglomeration of ASVs at genus level, alpha diversity analyses, PCoA, CAP, Permanova and deseq2 analysis. Please add it to your Github repository.
Validity of the findings: I have one question, related to the choice of cultivars. Why did you not compare the application of gramine in both modern and wild barley cultivars? It would have been interesting to see the differential bacterial recruitment between elite x wild. Perhaps include somewhere in the text why this comparison was not made.
Additional comments: Minor comments:

Line 64 – remove the comma before metabolites

Lines 124-125 - Missing reference – International Barley Genome Sequencing Consortium, 2012)

Line 240 – how were ASVs agglomerated at Genus level? Was it done manually? Please provide additional information regarding the way it was done.

Line 332 – In the text you say 18 genera, but looking at figure 4 it’s taxa mostly at the “order” level. Correct that in the text. Is there any phyla there?

Line 420 – “17 prokaryotic orders”. Weren’t there 18?

Table 1 – I would suggest you don’t need that table as you provided the same information in the text (lines 310, 312, 314).

Figure 3 – I liked the idea of the upsetR plot, however, it requires further explanation. When you show the number of enriched genera (comparison between bulk soil and Morex – A) and (bulk soil and Barke – B), do the numbers belong to enriched genera in the rhizosphere or in the bulk soil? E. g. By looking at the plot you can say that when you apply G46 you have 4 enriched genera. But in which niche? Are they enriched in both bulk soil and rhizosphere of Morex? If that is the case, would it be possible to show which ones are enriched in bulk soil and rhizosphere, separately? In that way we could see the rhizosphere effect.

Figure 5 – In the legend, what is the taxa correspondent to gray?


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: APPLICATIONS OF THE INDOLE-ALKALOID GRAMINE MODULATE THE ASSEMBLY OF INDIVIDUAL MEMBERS OF THE BARLEY RHIZOSPHERE MICROBIOTA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all my previous comments clearly. All field associated parameters comply with the journal standards.
Experimental design: All field associated parameters comply with the journal standards.
Validity of the findings: All field associated parameters comply with the journal standards.
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: ATYPICAL FUNCTIONING OF FEMALE GENITALIA EXPLAINS MONANDRY IN A BUTTERFLY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study is focused on an unusual condition (lacking muscularis on corpus bursae) of the female genitalia of a butterfly, related to monandry. The text is well written, concise and clear. All relevant literature sources are cited. The figures demonstrate well the results and are of very good quality.
Following the tips, I can confirm:
Clear and unambiguous, professional English used throughout. YES
Literature references, sufficient field background/context provided. YES
Professional article structure, figures, tables. YES
Self-contained with relevant results to hypotheses. YES
Experimental design: The research is original and is focused on an important but rarely studied field of the sexual selection. The monandry is a rare phenomenon among Lepidoptera and the present study reveal the reasons for its existence in a model species. All ethical standards applicable to the studied group of organisms are followed. The methods used in the study are appropriate and are described in detail in the Material and methods section, ensuring reproducibility of the results.

I confirm thefollowing:
Original primary research within Aims and Scope of the journal.
Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.YES
Rigorous investigation performed to a high technical & ethical standard. YES
Methods described with sufficient detail & information to replicate. YES
Validity of the findings: The results are clearly explained. They are commented in the Discussion without any excessive speculations.

All underlying data have been provided; they are robust and controlled. YES
Conclusions are well stated, linked to original research question & limited to supporting results. YES
Speculation is welcome, but should be identified as such. Speculations without supporting results are not formulated in the paper.
Additional comments: Though undesirable, I would like to say that the study is of particular interest for me. The problems of the sexual selection and related phenomena are of fundamental importance for understanding the evolution in general. I believe that the paper will be of interest for most biologists, not only entomologists. I made some minor suggestions in the manuscript (mostly editorial), as well as a comment, using the editing mode of Word. I recommend publication of the manuscript after minor corrections, without any extensive changes.


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: ATYPICAL FUNCTIONING OF FEMALE GENITALIA EXPLAINS MONANDRY IN A BUTTERFLY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Because this manuscript is very short and does not really involve "experiments" or "data analysis", I have simply put all of my detailed comments here in the "Basic reporting" section. I hope that is OK.

This short manuscript describes some interesting (and possibly unique) anatomical features of a (mostly) monandrous butterfly that might help explain the mating behavior of the species.

I very much enjoyed this manuscript. The paper is very clearly written and concise, but at the same time appropriately detailed. The opening arguments (lines 34-124) very clearly make a compelling case for why the findings to come later are of interest. Although the paper does not make the point explicitly, it is a very good example of what can come of the appropriate overlap of basic natural history, anatomy and physiology, and evolutionary biology. In addition to being interesting and well-written, the figures are very nice.

I have really no substantive criticisms of the paper and only a few suggestions.

1)	First, if photos, with scale-bars, of all of the dissected spermatophores exist would it be possible to provide some basic summary statistics of the sizes of the spermatophores? If the photographs are not sufficiently standardized then maybe this wouldn’t be appropriate. However, in addition to strengthening the case for their hypothesis some summary statistics could be useful for future work, whether in this species or others, that might wish to conduct similar analyses.

2)	Second, could the authors propose some future experimental manipulations that might further support their hypothesis? I have on mind a couple of ideas although I admit I have not thought these through in detail, nor do I know if they are actually possible. First, do methods exist that would allow one to inject the bursa of females with something that would gradually “digest”? If so, you might predict that this simulation of a digesting spermatophore would result in the initial loss of, and eventual resumption of, female receptivity. This could show that monandry is indeed a function of activities inside the bursa and related to the physical size of the spermatophore and not some other cause. Second, could females be mated to virgin males and recently mated males to show that smaller spermatophores do indeed fail to induce monandry? I should add that I am not suggesting that this paper needs to do these experiments; I am just wondering if the authors have thoughts, beyond the phylogenetic analyses they already have proposed, for further work that could investigate the ideas in the future.

3)	I have also included some very minor suggestions about wording changes that would make some sentences more consistent with conventional English. In particular, there are several places where the article “the” should probably be inserted. Those, and some other instances, are reported below by line number.

L97: “More generally…” would be more appropriate than “More general…”

L105: “mentions the following: ‘The bursa…” is more appropriate, since a colon should be preceded by a complete clause.

L34-124: This opening argument is very well written. It is clear and sets up the purpose of the study nicely.

L170: Add “the”, as in “known as the ostium”

L173: I think “associated with” instead of “associated to” is a bit more conventional in English.

L175: “injected with Karnovsky’s fixative”

L187: “allowed to continue laying eggs”

L212: “called the collum”

L261: “is a result of”

L283: “signa appear” (instead of appears, since signa is the plural)

L300: “where it at least receives irrigation”
Experimental design: Please see my comments in the "basic reporting" section. The research approach is appropriate, as I explained, with only some minor suggestions.
Validity of the findings: Please see my comments in the "basic reporting" section. The authors do a very good job arguing for the validity and novelty of their findings.
Additional comments: No additional comments


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

Input:
Title: ATYPICAL FUNCTIONING OF FEMALE GENITALIA EXPLAINS MONANDRY IN A BUTTERFLY
Review round: 1
Reviewer: 3

Output:
Basic reporting: “Atypical functioning of female genitalia explains monandry in a butterfly” by Xochipiltecatl et al documents the physiological abnormalities on the corpus bursae of Leptophobia aripa. This study demonstrates that the CB does not puncture and digest the spermatophore as is common in polyandrous species, and that the CB itself appears devoid of muscles.

Previous studies have shown that the presence/absence of the signa correlates with the propensity for a female to remate, but this study is the first to my knowledge that observes a lack of muscle on the CB which could potentially lead to the inability of females to digest the spermatophore. Such a finding is exciting and has the potential to shed light on the sexual antagonistic coevolution that is common across the Pieridae clade.

The article was presented in a straightforward manner with a clear narrative that was simple to understand and follow. There were, however, a few places in the manuscript in which sentence structure and grammar could be revisited for improved clarity:
•	Line 174 “introduced in a freezer” might be better phrased as “placed in a freezer” or alternatively “frozen at”
•	Line 181 “during 20 minutes” could be “for 20 minutes”
•	Line 187-188 “continuing laying eggs” instead might be “to continue laying eggs”
•	Line 205-207 this is a very long sentence, I recommend breaking it apart for increased clarity
•	Line 300 “where at least receives irrigation”. I’m unclear what this statement is supposed to mean, please consider rephrasing

As far as literature references and background go, this manuscript provides a robust reference base for the system and previous knowledge (though would benefit from citing literature on the European Corn Borer conducted by Al-Wathiqui et al). However, the discussion does make note of enzymatic digestion of the spermatophore in combination with mechanical digestion (line 250) yet the introduction does not provide background on alternative digestion of the spermatophore except for mechanical. I would suggest including some background on the various mechanisms used to digest the spermatophore in order to be able to more completely discuss the lack of digestion observed.
Experimental design: My one concern about this manuscript is the general lack of data and quantification for the phenotypes observed. For example, visually it appears that the spermatophores are not being digested, but are there any quantifiable measures for this? For example the surface area or mass of the spermatophores across numerous individuals in order to show that no digestion is occurring?

Second, while it seems like there are no muscles on the CB, are there any ways to verify this? I would personally recommend staining for muscle in order to show lack of staining on the CB in contrast to likely strong staining on the ductus bursa.
Validity of the findings: I will echo my comments above that there are no quantifiable data provided, which would greatly increase the strength of the manuscript.

As far as the conclusions for the manuscript, I appreciated the proposed hypotheses that the lack of muscle is a female adaptation to monandry. I find it strange that there was no comparison to or contrast to the systems in which signa have been lost, and further still that in a system where females no longer digest the spermatophore, that males would continue to produce such a costly gift. Perhaps this could be addressed in the discussion?
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: ATYPICAL FUNCTIONING OF FEMALE GENITALIA EXPLAINS MONANDRY IN A BUTTERFLY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Since I reviwed the manuscript at the first review round, I am not going to repeat my review but I write on some tips that I supopse should be mentioned, generally following the instructions:

Clear and unambiguous, professional English used throughout. YES
Literature references, sufficient field background/context provided. YES
Professional article structure, figures, tables. YES; the authors added a figure of a bursa with developed muscularis and now even non-specialists can realise what is it;
Self-contained with relevant results to hypotheses. YES
Experimental design: Study designed and performed excellently.

Original primary research within Aims and Scope of the journal. YES
Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.YES
Rigorous investigation performed to a high technical & ethical standard. YES
Methods described with sufficient detail & information to replicate. YES
Validity of the findings: The results are clearly explained. They are commented in the Discussion without any excessive speculations.
All underlying data have been provided; they are robust and controlled. YES
Conclusions are well stated, linked to original research question & limited to supporting results. YES
Additional comments: The authors addressed all comments of the reviewers, and extended some paragraphs accordingly. I would be happy to see this work published.


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: ATYPICAL FUNCTIONING OF FEMALE GENITALIA EXPLAINS MONANDRY IN A BUTTERFLY
Review round: 2
Reviewer: 2

Output:
Basic reporting: As I said in my first review, my most substantive comment was a request for some quantitative data and the authors have now provided that, so I find this version of the manuscript satisfactory. The other changes made in response to my comments and those of the other reviewers also improved the manuscript, in my opinion.
Experimental design: See comments in "Basic reporting" section.
Validity of the findings: See comments in "Basic reporting" section.
Additional comments: See comments in "Basic reporting" section.


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

Input:
Title: ATYPICAL FUNCTIONING OF FEMALE GENITALIA EXPLAINS MONANDRY IN A BUTTERFLY
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript, after undergoing revisions, has been updated to address all concerns I had previously. I have no further comments or suggestions for improvement of 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: ASSOCIATION OF ENDOTHELIAL DYSFUNCTION WITH SARCOPENIA AND MUSCLE FUNCTION IN A RELATIVELY YOUNG COHORT OF KIDNEY TRANSPLANT RECIPIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written throughout, with a clear background provided in the introduction that allows the reader to understand the context to which the work is relevant.

specific points:

Title
The authors might consider amending the title of the paper to acknowledge the relatively young cohort of patients included in the study. Furthermore, I think it would be beneficial to refer to ‘muscle function’ in the title, to better characterise the work.

Abstract
‘Kg’ should be ‘kg’

Introduction
Line 64: it would be useful to include a citation to back up this statement, there a plenty of studies on the potential role of the muscle microcirculation/endothelial dysfunction (see Larsson et al 2019 Phys Rev).

Line 66-68: ‘cardiovascular events’ implies pathological cardiac incidents/performance rather than endothelial dysfunction, per se.

Table 1
Add in a line on the patient data from men.

Inclusion of brackets and ‘±’ is used interchangeably throughout; if this is deliberate then the authors should make clear the significance of this distinction.

Figure 1
Rather than noting the percentage values above each bar, error bars should be included to enable the variation in each sample to be taken into account.

Figure 2
I think that this figure would be better presented in a table similar to Table 3.
Experimental design: Methods
Make clear why the unadjusted and adjusted multivariate approaches are presented in the results.
Validity of the findings: Results
It would be interesting to explore the interaction of BMI, VRI and sarcopenia, because it would seem that these sarcopenic patients have consistently low BMI. If you consider variation in the multivariate model after removal of BMI (which accounts for the greatest proportion according to p-values in Table 3), I wonder if any of the other covariates become statistically significant? It strikes me that BMI will covary closely with SMI due to the muscle loss that drives decreases in both indices, so BMI is perhaps not an independent predictor but rather a derived measure of sarcopenia, in which case it might be obscuring other (currently non-significant) factors in the multivariate model.

Line 204: the univariate models indicated that these factors were not significantly related to the development of sarcopenia, so cannot strictly be cited as ‘confounding’ factors; no evidence of any test on interactions between dependent variables is presented in the multivariate model, so it is difficult to envisage why they would account for more of the variation (in the independent variable) after inclusion of more covariates.

Line 208: I appreciate that the P-value for handgrip strength is close to the 5% significance level, but by the statistical definition provided in the manuscript, this P-value remains non-significant, marginal or not.

Discussion

Line 213: same as line 208, the result was non-significant; I interpret this from a physiological point of view – the handgrip protocol will elicit rapid muscle force production that does not depend upon enhanced local blood flow whereas the gait score protocol likely does, and so endothelial reactivity is potentially more important for this sustained exercise.
Additional comments: I think that the authors have produced an interesting study on the incidence of endothelial dysfunction in the development of, and/or coincidence with, sarcopenia. Overall this paper is well-written with a clearly defined objective. I think the authors should consider my comments to improve the focus and clarity of 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: ASSOCIATION OF ENDOTHELIAL DYSFUNCTION WITH SARCOPENIA AND MUSCLE FUNCTION IN A RELATIVELY YOUNG COHORT OF KIDNEY TRANSPLANT RECIPIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: It was a well written article, with adequate english, literature references and background.
The article structure, figures and tables are well done. And, the raw data shared is organized, well described.
I appreciated the hypotheses and results.
However, as I have no experience with the terms, methods and clinic of the cardiovascular area, I believe that a second reviewer with a background in cardiovascular disease could enrich the review.
Experimental design: Introduction, well adressed the content.

About methods I have only one question. There is no sample size calculation, therefore the authors are not authorized to say this is either a small or a large sample, as it was written in the last paragraph of discussion section.
In fact, this is the basic essential tenet of any research question, and when not estimated or reached, it is a huge limitation of the conclusions. Sample size insufficiency threatens the validity and generalizability of studies’ results. The size of a sample influences two statistical properties: 1) the precision of estimates and 2) the power of the study to draw conclusions.
So, having said so, none of the statistics can support the conclusions, unless the authors provide this fundamental, basic methodological scientific requirement. Otherwise, they must discuss why they have not carried it out and discuss as a huge limitation of the study...
Validity of the findings: About discussion section:
- Line 213: According to the results, VRI appear to predict only GS. HGS and SMI had a p value >0.05, so, VRI do not predicted both.
Limitation: add as limitation of the study the limitations of body composition evaluated by BIA.
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: ASSOCIATION OF ENDOTHELIAL DYSFUNCTION WITH SARCOPENIA AND MUSCLE FUNCTION IN A RELATIVELY YOUNG COHORT OF KIDNEY TRANSPLANT RECIPIENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have carefully revised the manuscript, and I think that it is improved as a consequence. Therefore I have no further comments on the paper.


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

Input:
Title: CLIMATE CHANGE FAVOURS CONNECTIVITY BETWEEN VIRUS-BEARING PEST AND RICE CULTIVATIONS IN SUB-SAHARAN AFRICA, DEPRESSING LOCAL ECONOMIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is very interesting but the presentation requires minor revision because of the following.
1. Semi-colon is overused. In most of the use cases, the authors can just replace them with a period (.) and create two simple sentences instead. As a result, there are many long sentences separated by semi-colons (;). This makes it hard to read.
2. There are several editorial revisions I have highlighted (with suggestions) in the annotated PDF.
Experimental design: The paper is great in its experimental design. However, I would like to seek some clarifications from the authors.

Preamble: The fusion of the 2010 C3S land cover map and the SPAM’s 2016 map is acceptable only when data is unavailable. If you refer to this link (http://2016africalandcover20m.esrin.esa.int/) you will find the 2016 land cover map at 20-m spatial resolution produce from Sentinel-2 by the European Space Agency.

Question: What was the reason for fusing the 2010 C3S land cover map with the SPAM’s 2016 map? Which year then became the baseline for the studies, 2010 or 2016?
Validity of the findings: no comment
Additional comments: General comment: This paper is very interesting and has policy relevance in SSA in respect of food security. Congratulations to the authors for the great job done.

Suggestions for improvement:
1. The authors should justify in the paper why they fused data from the years 2010 and 2016 and explain the reason, for example, to choose 2010 as a baseline instead of 2016?
2. The interpretation of Figure 5b appears slightly different from what the figure shows (L366). Authors are to check and make changes if required.
3. Please provide further information about the nineteen bioclimatic variables so that others verify results in future studies. What exactly are the variables?
4. Refer to the annotated PDF for more specific revision suggestions.


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: CLIMATE CHANGE FAVOURS CONNECTIVITY BETWEEN VIRUS-BEARING PEST AND RICE CULTIVATIONS IN SUB-SAHARAN AFRICA, DEPRESSING LOCAL ECONOMIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors are specialists of the biology of the flea beetles Chaetocnema pulla species group (pulla group), a harmful rice pest vector of rice yellow mottle virus, an important pathogen of rice in Africa. In this study, they implement a large range of statistical techniques, not necessarily familiar to the biologists, to assess (i) the impact of ongoing climate and land-use changes for both the pulla group and rice in two climatic/socioeconomic future scenarios through ecological niche modelling, (ii) the connectivity potential of the pulla group populations towards rice cultivations for both current and future predictions, through a circuit theory-based approach, (iii) the rice production and GPD loss per country through the spatial index named “Pest Aggression Index”, based on the inferred connectivity magnitude.

These are quite important objectives, not attainable through direct biological experimentation. Additional information on the datasets used in this study on Chaetocnema pulla, on rice in Africa, and on the impact of the virus on rice would be useful. How the degree of precision and the biases of the different datasets impact the conclusions should be discussed. Otherwise, some conclusions (especially those of figure 5) may be questionable. These queries are detailed below.

Data used in figures 2, 3 and 4.
Chaetocnema pulla. The authors are specialists of Chaetocnema pulla. Does it mean that the insect is not present in the areas where it is not reported on figure 2? For instance, in the Niger Inner Delta (in Mali) which is the region of diversification of the virus and where damaging epidemics often occur? If so (i.e., the insect is not present), this would weaken the putative link between Chaetocnema pulla and rice yellow mottle (see remark below). If not (the insect is present but has not been reported), how this sample bias affect the conclusions?

Rice. There are several maps available on rice cultivation in Africa. That used in Dellicour et al 2018 to assess the link between rice yellow mottle spread and rice cultivation was generated from You et al 2009 (reference below). It displayed rice distribution patterns sometimes different from that of the study. A justification of the map used in this study (vs that used in Dellicour et al for instance) would be useful. Would different conclusions on the relationships between Chaetocnema pulla and rice be reached if the You et al map had been used?

Data used in figure 5.
Recent studies showed that beetles are not necessarily the main vector of rice yellow mottle (see Traoré et al 2009 for a review), and underlined the role of transmission through cultural practices. The means of transmission are quite variable between regions and years. Morevoer, several transmission means are involved simultaneously. These results are at odds with the sentence “ the presence of pulla group mostly determines the infection” (line 404). Are these observations on the complexity and the variability of the ecology of the disease taken into account in the study? How is the associated uncertainty incorporated in the analyses?

The link between rice yellow mottle infection and rice loss is highly dependent on the cultivar used, the date of infection, the climatic conditions… Are these observations on the complexity of the link between the disease and the crop loss, as well as the scarcity of information available on these relationships taken into account in the study? How is the associated uncertainty incorporated in the analyses?

Methodology. There are several sources of uncertainties and biases in the datasets used (see above). Were negative controls - such as those used in Dellicour et al  2018 – implemented to test the significance of the relationships obtained in the study? Would it be possible to calculate a a confidence interval for the quantitative conclusions?

References
The list of references on rice yellow mottle epidemiology should be enlarged.

References quoted in the review
Dellicour et al. (2018) ‘On the Importance of Negative Controls in Viral Landscape Phylogeography’, Virus Evolution, 4: 23.
HarvestedChoice (2011) Rice Area Harvested (ha) (2000). Washington, DC: International Food Policy Research Institute and St. Paul, MN: University of Minnesota. http://harvest choice.org/node/4799
Traore´, O. et al. (2009) ‘A Reassessment of the Epidemiology of Rice Yellow Mottle Virus following Recent Advances in Field and Molecular Studies’, Virus Research, 141: 258–67.
You, L., Wood, S., and Wood-Sichra, U. (2009) ‘Generating Plausible Crop Distribution Maps for Sub-Saharan Africa Using a Spatially Disaggregated Data Fusion and Optimization Approach’, Agricultural Systems, 99: 126–40.
Experimental design: not applicable
Validity of the findings: not applicable
Additional comments: not applicable


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: CLIMATE CHANGE FAVOURS CONNECTIVITY BETWEEN VIRUS-BEARING PEST AND RICE CULTIVATIONS IN SUB-SAHARAN AFRICA, DEPRESSING LOCAL ECONOMIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I'm happy about the changes made in the revised manuscript. Congratulations to the authors.


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: CLIMATE CHANGE FAVOURS CONNECTIVITY BETWEEN VIRUS-BEARING PEST AND RICE CULTIVATIONS IN SUB-SAHARAN AFRICA, DEPRESSING LOCAL ECONOMIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: In the rebuttal letter, the authors replied satisfactorily to the queries I raised. They made the necessary modifications in the manuscript.
Experimental design: OK
Validity of the findings: OK
Additional comments: none


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: COMMERCIAL UTILITIES AND FUTURE PERSPECTIVE OF NANOMEDICINES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment.
Experimental design: In the present study, the authors tried to collect material on the importance of the field of nanomedicine. Although the topic is very broad, in general, materials have been collected in some formats such as the application of nanomedicine, economic aspects, as well as challenges and future perspectives.
Validity of the findings: This study has many problems in terms of writing. My advice is that if it is to be published, it must first be greatly modified in writing. The next thing that worries me is the generality of the content. Although the topic has been selected as a whole, looking at each section, we see that no specific information is provided to the reader. For example, the part that talks about Taiwan's strategy is very unnecessary and vague. On the other hand, the Standards and Guidelines section does not contain detailed information. Content generally does not add anything new to the reader. As another example, in the Clinical Trials section, it is not stated how long these phases last for drugs derived from nanoparticle technology.
Another concern that cannot be ignored is the inconsistency of references with the content. For example, there is no connection between references “Blanco E, Hen H, Ferrari M. 2018” and “Brigger I, Dubernet C, and Couvreur P. 2002” with the referenced material.
This manuscript also requires tables that contain useful and up-to-date information such as nanoparticle classification (based on application or world usage or …), number of FDA-approved nano-drugs, and diseases that are currently treatable with nanoparticles.
Here are some grammatical errors.
Line 37, “was” must change with “were”.
Line 43, “precise” must change with “precision”.
Line 50, “offers” should be replaced with “offer”.
Line 51, “in case” must change with “in the case”.
Line 97, “has” must change with “have”.
Lines 108-112, the sentence (The progress discussed for…) is very long and confusing. Please amend it.
Line 116, “that” should be deleted.
Line 151, “or identifying them from”.
Line 199, “to increased” should be replaced with “to increase”.
Line 151, “steps”.
Line 392, “results”.
Line 539, “phenomena”
Additional comments: Providing more detailed data, eliminating some unnecessary parts such as Taiwan strategy, providing a rich table of contents, and synchronizing references with the content provided are important operations that should be considered.


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: COMMERCIAL UTILITIES AND FUTURE PERSPECTIVE OF NANOMEDICINES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a review on nanomedicine commercialization, but it seemed unfocused and the presented information appeared arbitrary. Few commercialization details are provided. The first part focused on regulatory frameworks for nanomedicine, but didn't include much concrete information. The next part discussed basics of nanomedicine, but again without depth and seemingly arbitrary manner. The selection of table 1 which focuses on Taiwanese nanomedicine and table 2 which lists seemingly random patent literature also makes this work unfit for publication.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


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: COMMERCIAL UTILITIES AND FUTURE PERSPECTIVE OF NANOMEDICINES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you to the authors for providing all corrections.
Experimental design: No comment
Validity of the findings: As far as my knowledge, the manuscript was well written and had no grammar or spelling errors. However, if the Editor finds this necessary, it would be very great if the authors provide a list of abbreviations used in the body of the texts.
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 CORE GENES FOR COLORECTAL CANCER PROGNOSIS BASED ON IMMUNE AND STROMAL SCORES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The title, introduction, methods, results and discussion are appropriate for the content of the text. Furthermore, the article is well constructed, the experiments are well conducted, and analysis is well performed. The figures are relevant, high quality, well labelled and described. The abstract needs more editing with more details and summary level sentences.
Experimental design: The experimental design is original and the research is within the scope of the journal. Research question is well defined, relevant and meaningful. The methods are highly technical, ethical and logistical. Statistical methods are chosen correctly.
Validity of the findings: All underlying data have been provided in detail. The findings are meaningful. The conclusions are well stated and relevant to the research questions.
Additional comments: This paper investigates the function of expression of  tumor micro-environment-related genes in overall survival(OS) and prognosis of colorectal cancer(CRC) by analyzing TCGA datasets. The authors identified avlist of tumor micro-environment-related genes that strongly correlate with the prognosis of CRC patients by a series of bioinformatic analysis. Moreover, the authors validate their findings in a GEO dataset. Furthermore, the authors explore the related pathways through enrichment analysis. In short, this study identified several tumor micro-environment-related genes as potential predictors for the prognosis of CRC.

Editorial Criteria
BASIC REPORTING
The title, introduction, methods, results and discussion are appropriate for the content of the text. Furthermore, the article is well constructed, the experiments are well conducted, and analysis is well performed. The figures are relevant, high quality, well labelled and described. The abstract needs more editing with more details and summary level sentences.
EXPERIMENTAL DESIGN
The experimental design is original and the research is within the scope of the journal. Research question is well defined, relevant and meaningful. The methods are highly technical, ethical and logistical. Statistical methods are chosen correctly.
VALIDITY OF THE FINDINGS
All underlying data have been provided in detail. The findings are meaningful. The conclusions are well stated and relevant to the research questions.

Overall, I think this paper is novel and will be of interest to the community of lung cancer genetics, especially CRC research. The statistical part is valid and makes sense. The authors make it comprehensive by integrating analysis of multiple sources including GEO and TCGA. The main strengths of this paper is that it addresses an interesting and unexplored question, finds a novel discovery based on a carefully selected set of bioinformatic procedures. As such this article represents an excellent and elegant bioinformatics study which will almost certainly influence our thinking about the function of ferroptosis-related genes in CRC. Some of the weaknesses are the lack of in vitro or in vivo validation experiments. In general, the work is convincing except some major and minor comments below:

Major Comments:

The Methods of the Abstract are too simple, I would recommend expanding it and adding details like what datasets were used, what the aims are for PPI, GO, KEGG, Pivot analysis, etc, what the discovery dataset is, what the validation dataset is.

The Conclusions of the Abstract are too simple. Please add one more sentence about the importance of this study.

Line 85 to 90: Please add sample size and sequencing platforms for both TCGA and GEO datasets.

I do see multiple normalization methods for COAD (Colon adenocarcinoma) samples in GDC, which includes HTSeq - Counts (sample size: 514), HTSeq - FPKM(sample size: 514) and HTSeq - FPKM-UQ(sample size: 514). Please explain why the sample size in the manuscript is 362 rather than 514. Please also explain which normalization method was chosen for this analysis and reason.
 (https://portal.gdc.cancer.gov/repository?facetTab=files&filters=%7B%22op%22%3A%22and%22%2C%22content%22%3A%5B%7B%22op%22%3A%22in%22%2C%22content%22%3A%7B%22field%22%3A%22cases.primary_site%22%2C%22value%22%3A%5B%22colon%22%5D%7D%7D%2C%7B%22op%22%3A%22in%22%2C%22content%22%3A%7B%22field%22%3A%22cases.project.program.name%22%2C%22value%22%3A%5B%22TCGA%22%5D%7D%7D%2C%7B%22op%22%3A%22in%22%2C%22content%22%3A%7B%22field%22%3A%22cases.project.project_id%22%2C%22value%22%3A%5B%22TCGA-COAD%22%5D%7D%7D%2C%7B%22op%22%3A%22in%22%2C%22content%22%3A%7B%22field%22%3A%22files.data_category%22%2C%22value%22%3A%5B%22transcriptome%20profiling%22%5D%7D%7D%2C%7B%22op%22%3A%22in%22%2C%22content%22%3A%7B%22field%22%3A%22files.experimental_strategy%22%2C%22value%22%3A%5B%22RNA-Seq%22%5D%7D%7D%5D%7D)

I’m wondering if there are any ongoing clinical trials focusing on micro-environment associated genes identified in this study in CRC? It will be very strong evidence for the significance of the current study if so.

Minor Comments:
Line 32: please add “(CRC)” after “Colorectal cancer”.

Line 38, 39: please add the full name of GO and TF.

Please make sure all the gene names should be italic.

Line 86 to 87: please replace “Gene expression profile data and clinical information of colorectal cancer patients were obtained from TCGA database” with “Gene expression profile data and clinical information of colorectal cancer patients from TCGA was obtained from NCI Genomic Data Commons (https://portal.gdc.cancer.gov)”. Since GDC is the official data host for TCGA data. Please also add this paper as reference for GDC: https://www.nature.com/articles/s41588-021-00791-5 .

Line 166 to 167: I would recommend adding more details about the top hits for the results of the enrichment analysis.

It would be great if there is a session of abbreviations to list all the abbreviations for the database names. I would also recommend including abbreviations like CRC, TCGA, GEO, DEG, GO, KEGG etc in that list.


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 CORE GENES FOR COLORECTAL CANCER PROGNOSIS BASED ON IMMUNE AND STROMAL SCORES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Missing words in sentences at various paragraphs. For example: "In 2011, scientist began to various stages (TNMI - IV) of patients with". Please revise the manuscript to check for consistency in grammar.
Experimental design: The tools and software used in the manuscript is in accordance with the aim of experiment.
Validity of the findings: Please see the below comments

1) Please try GSEA on the expressed genes. It may further output interesting pathways and gene sets
2) How many genes were used for GO analysis?
A small number of genes (22 prognostic genes) used for the GO analysis are too few. Please explain the results.
Additional comments: Overall the authors have done a good work with experimental design and providing evidence. Please revise the manuscript with recommendation as stated in the previous sections.


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 CORE GENES FOR COLORECTAL CANCER PROGNOSIS BASED ON IMMUNE AND STROMAL SCORES
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.Poor English expression and lack of technical terminology
2.Many relevant articles not cited
Experimental design: Lack of innovation in experimental design, unconvincing results and lack of validation
Validity of the findings: Lack of innovation in experimental design, unconvincing results and lack of validation
Additional comments: The authors identified prognostic genes in colorectal cancer. There are several important flaws in the article that I believe cannot be published in PeerJ
1.	This paper is a purely bioinformatic analysis, and in recent years there have been so many similar studies that this paper cannot provide additional value.
2.	The title of the article is significantly flawed. How do we know that these genes are genes of the tumor microenvironment? Tumour cells do not express these genes?
3.	A single algorithm is used in this paper, and it is recommended that 2 similar algorithms be added to verify the conclusions of this paper.
4.	The lack of experimental validation in this paper is an important flaw.
5.	In this paper, GSE102479 is used as a validation. It is recommended that all colorectal cancer-related data from the GEO database be included for validation, and to my knowledge, there should be >10 similar datasets.
6.	The pictures in this article are not aesthetically pleasing and reflect a lack of technical skills as a bioinformatics analyst.


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 CORE GENES FOR COLORECTAL CANCER PROGNOSIS BASED ON IMMUNE AND STROMAL SCORES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The languages still need to be polished a little bit.
Experimental design: The experimental design is original and the research is within the scope of the journal. Research question is well defined, relevant and meaningful. The methods are highly technical, ethical and logistical. Statistical methods are chosen correctly.
Validity of the findings: All underlying data have been provided in detail. The findings are meaningful. The conclusions are well stated and relevant to the research questions.
Additional comments: The revised version of the manuscript has addressed all the comments I raised.


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 CORE GENES FOR COLORECTAL CANCER PROGNOSIS BASED ON IMMUNE AND STROMAL SCORES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Professional English used.
Experimental design: The bioinformatics tools applied are in accordance with the design of experiment.
Validity of the findings: Some parts of analysis still needs clarification. For example:
1) The statement "The 22 core prognosis genes obtained were validated in the GEO data set, and 22 genes were expressed.
183 Further analysis of survival in this dataset identified only one gene consistent with TCGA results (P<0.05).
184 The results of gene survival analysis are shown in Figure 4B. "

How 22 core prognosis genes were validated in GEO dataset. Did you see the expression of these genes? If yes, please upload the expression/violin plots

2) For GSEA, you would want to perform the analysis on whole gene list as GSEA ranks the gene list and performs the pathway enrichment analysis.
Additional comments: The manuscript needs some more clarification as stated above.