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: EFFECTS OF GRAZING INTENSITY ON FORAGE NUTRITIVE VALUE OF DOMINANT GRASS SPECIES IN BORANA RANGELANDS OF SOUTHERN ETHIOPIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1) Figures and tables:

Some figures and tables are presenting the same data. For example, Figure 7 and Table 3. It is necessary to review all figures and tables to decide which are necessary.

Figure 5: What is GI in axes “X”? It was not described in material and methods.

2) I couldn't found the raw data.

3) Hypotheses.
Is not clear to me why the authors building these hypotheses? In line 246 is showed some references (Fanselow et al., 2011; Haiyan et al., 2016; Schiborra et al., 2009), confirming that GI increases the forage nutritive value. I think that need a clear introduction to postulate the current hypothesis.
Experimental design: Is not clear what kind of experimental design was used. That needs to be much clearer.

Figure 1: What experimental design is that? How the repetition was randomly sorted?

Line 118 and Figure 3: What means 5 main plots? Is not clear.

Line 144: General Linear Model (GLM) is not the appropriate way to analyze the data. There were two seasons (dry and rain) and I understood that was used the same plot to collect the samples (repeated measurements), for that are necessary to use MIXED model.

Line 143: Linear regression model. To that, the quantitative factor describes in line 110 needs to be more specific. Non-grazing (NG), moderate grazing (MG) and overgrazing (OG) are more like a qualitative factor in line 110. It needs to appropriately describe, to be clear that regression analysis can be done.
Validity of the findings: 1) The results are valid, but the manuscript needs to be adjusted to archive the quality necessary. But the big concern that I have is about the short experimental period (6 months). To understand all effects on grassland is necessary to investigate, at last, one year, but ideally two years.

2) Conclusion: (Line 346) With the data shown is not possible to make the conclusion, that MG should be recommended. To do that is necessary to look to forage mass and animal performance.
Additional comments: A. Introduction:

•	Line 86: Hypotheses. Is not clear to me why the authors building these hypotheses? In line 246 is showed some references (Fanselow et al., 2011; Haiyan et al., 2016; Schiborra et al., 2009), confirming that GI increases the forage nutritive value. I think that need a clear introduction to postulate the current hypothesis.

B. Material and methods:

•	Need much more details.

•	How the carrying-capacity was kept across the seasons (dry and rainy)? What kind of animal was used? How the carrying-capacity was adjusted and which frequency?

•	Line 101: The soil chemical analyses were done?

•	Figure 2: If possible, add the historical data about the weather condition. That is important to understanding if the experimental period was in a normal condition for the location.

•	Line 104: I think that information is not so important to the manuscript.

•	Line 110: non- grazing (NG) (~ 0 ha AU-1Y-1), moderate grazing (MG) (6 ha AU-1 Y-111 1), and overgrazing (OG) (12 ha AU-1 Y-1 and above), is it correct? In this way, for example, I understand that OG has more area by the animal than in MG. I think the units should be wrong.

•	Line 113: How many samples, by treatment, were collected by season (dry and rainy)? When? The samples collected need to be better explained.

•	Line 118: Main plots. What was that?

•	Line 123: How the dominant grass species were determined? How the relative abundances (≥ 40%) was done? Was it a subjective observation?

•	Line 128: Was the sample directly dried at 105oC? That normally is done after a previous dried at 55-60oC. Dry directly at 105oC may change the qualitative characteristic of the grass, and that is the key point of the manuscript.

•	Line 133 and Table 1: Dry matter intake (DMI). How is it was done? There is no detail about it.

C. Statistical analyses:
•	In my point of view, the analyses need to be redone. In experimental design, I make my suggestion.

D. Results:

•	To review if all 7 figures and 6 tables are necessary.
•	As carrying-capacity was calculated I think the authors could present the data of forage mass too, it can be helpful to the manuscript.
•	Lines 169 and 177: The authors are making discussing in the results session.
•	Line 191: Biomass production. There is this information (data)?

E. Discussion:
•	As a suggestion, the discussion should be done without subtopics. The use of subtopics makes the discussion less fluent. Many times the authors are telling the same discussion.
•	The use of the term “palatable” (Lines 47 and 271) is not appropriated. Palatable is applied to us (humans), for the animal we can not know. The appropriate term, in this case, is acceptable (dominant grasses are acceptable by the animals).

F. Conclusion:
•	Line 346: With the data shown is not possible to make that conclusion, that MG should be recommended. To do that is necessary to look to forage mass and animal performance.

G. Figures and tables:

•	Some figures and tables are presenting the same data. For example, Figure 7 and Table 3. It is necessary to review all figures and tables to decide which are necessary.
•	Figure 5: What is GI in axes “X”? It was not described in material and methods.


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: EFFECTS OF GRAZING INTENSITY ON FORAGE NUTRITIVE VALUE OF DOMINANT GRASS SPECIES IN BORANA RANGELANDS OF SOUTHERN ETHIOPIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made the suggested corrections so that the paper is now accepted for publication.
Experimental design: The authors have made the suggested corrections by the reviewers on the experimental desgin and methods for analyses, so that the paper is now accepted for publication.
Validity of the findings: The findings are valid, and with the corrections, the paper has been substantially improved. It is now accepted for publications.
Additional comments: The paper is now accepted for publication.


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

Input:
Title: EFFECTS OF GRAZING INTENSITY ON FORAGE NUTRITIVE VALUE OF DOMINANT GRASS SPECIES IN BORANA RANGELANDS OF SOUTHERN ETHIOPIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Point 1: Description of treatments - The way how the authors are describing the grazing intensity (GI) treatments is confusing, in the first revision both reviewers had the same difficulty to understanding. After reading the authors' answers, I still had difficulty understanding, but I guess I could understand. In my point of view, that will confuse to the readers too. What I understood was that the author did not apply the treatments, they select areas that were no-grazed (NG) or had moderated (MG) and overgrazed (OG) and calculated the carrying capacity of those areas (treatments). To clarify that, in the manuscript, will be helpful to better understanding.

Point 2: Conclusion (Line 381) – Looks like that the authors made a discussion in the conclusion section, the discussion is good but should be added in the discussion section and the conclusion should be more concise.

Point 3: Line 45 - The use of the term “palatable”  is not appropriated, for an animal in the pasture is better to use “acceptable”.  I made that comment in the first review.

Point 4: Line 127 – Two or three treatments?

Point 5: Line 174  - The dry matter intake (DMI) was not evaluated, was done an estimate of the potential of dry matter intake. I guess should be used as “potential dry matter intake” to avoid misunderstanding.

Point 6: Line 173 – There are no units to variables forage nutritive value as, for example, crude protein.
Experimental design: Ok
Validity of the findings: Ok
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: DEVELOPMENT OF A NOVEL EMBRYONIC GERMLINE GENE-RELATED PROGNOSTIC MODEL OF LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the submitted manuscript Linjun Liu et al have done a commendable job performing integrated bioinformatic analysis on embryonic germline genes in relation to lung adenocarcinoma (LUAD) development. The authors pose a logical and relevant question at the introduction and designed rigorous experiments to develop a risk score, by putting appropriate weightage on differentially expressed germline genes that can be used as a prognostic biomarker for LUAD development.
Experimental design: The authors performed rigorous experiments to address a relevant query. The methodology is substantially detailed. Although, I would strongly encourage the authors to describe in detail the methodology of developing the risk score. In the method section, it is not fully clear how the risk score was constructed.
Validity of the findings: Though a similar bioinformatic approach is being taken to develop biomarkers for different cancer progression models, the development of risk-score showed novelty from the research group’s point.

Some minor comments to be addressed by the authors –

Figure 1: Legend – ‘FDR’ full form – False Discovery Rate. As much as I understand that the False positive rate conveys a similar meaning, please correct the abbreviation.

Material and Methods: Line 132 -  “The performance of the nomogram was evaluate…….” Please change it to “ was evaluated…”

Please consider rearranging figures 4, 5, and 6 to have a better flow with the text.

Please rearrange figure 5 to have similar pathological status distributions in the same row for better readability. Please provide the distributions of risk scores to all the pathological statuses in the training sets to show parallel comparisons to the distribution of representative ERGS genes’ expressions.
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: DEVELOPMENT OF A NOVEL EMBRYONIC GERMLINE GENE-RELATED PROGNOSTIC MODEL OF LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors outline methodologies to identify genes, specifically embryonic germline related genes, which can be used as prognostic predictors of lung adenocarcinoma. The article is fairly comprehensible with the article sections, figures and raw data in line with the journal standards. There are few minor grammatical corrections which have been marked in the manuscript, which should be addressed by the authors.
Experimental design: The authors have used several lines/methods of investigations in order to arrive at the conclusions.
The study is supported by relevant analysis, explanation of the techniques used and their relevance.
Validity of the findings: Overall, the potential implications of the study would be of interest in the field of LUAD and it prognostic significance. However, there are a few concerns regarding the results, which the authors must look into addressing (detailed in the comments section).
Additional comments: The study results are promising in terms of potential future studies related to the prognostic indicators for lung adenocarcinoma. The authors are suggested to address the following in order improve the overall impact of the study.
Minor suggestions:
The authors are urged to rectify certain grammatical errors and revise few sentences in the following lines of the manuscript (the specific edits are highlighted in the attached manuscript file)
Lines 59, 69, 151, 225, 251, 255 and 279.

Major concerns:
(1)	Although, the authors provide a rationale for studying LUAD, it is recommended that the authors show the relevance of the 11 genes they have identified to be of prognostic value, in other tumor types as well. The studies by Bruggeman et al have already identified several germ-line associated genes in various cancer cell lines. The authors should at the very least look into the expression profiles of the 11 genes in the cancer cell line databases to identify the impact and relevance of their findings. Can the findings be applicable to any other tumor types in addition to lung cancer? Since the genes were identified by taking into account the embryonic germline databases, the authors are especially encouraged to state their thoughts and potential impact of their findings in cancers specifically associated with the reproductive organs (ovarian, endometrium, prostrate cancers to name a few)
(2)	The authors have constructed a prognostic model related to the embryonic germline genes (Fig. 4). Can the authors comment on the number of relapse-free events in the patient sets (training set)?
(3)	Can the authors clarify how many patients were considered for the nomogram depicted in Fig. 7? The authors state that the expression of 11 genes were calculated from an individual patient. If this is indeed the case, the authors must construct nomograms for a statistically significant number of patients in order to make any claims regarding prediction of survival rates of LUAD patients.
(4)	 The authors must show protein expression profiles for the 11 genes (or atleast few of the genes) they have identified, either through in vitro experiments measuring protein levels in tumor samples or cancer cell lines or through protein databases. There are several studies showing poor correlation between gene expression data and the corresponding protein expression patterns (such as the study by Vogel, C. et al., Nat. Rev. Genet. 2012, 13, 227–232). The relative protein expressions levels will be one way to provide proof of the functional relevance of the expression of the identified genes to the  progression of the disease.


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

Input:
Title: DEVELOPMENT OF A NOVEL EMBRYONIC GERMLINE GENE-RELATED PROGNOSTIC MODEL OF LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: In the revised manuscript the authors Linjun Liu et al have established a risk score, considering appropriate weightage on differentially expressed germline genes to use as a potential biomarker for Lung Adenocarcinoma development.
Experimental design: No comments
Validity of the findings: No Comments
Additional comments: The revised manuscript is stronger with the suggested 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: SEXUAL AND PSYCHOLOGICAL HEALTH OF COUPLES WITH AZOOSPERMIA IN THE CONTEXT OF THE COVID-19 PANDEMIC
Review round: 1
Reviewer: 1

Output:
Basic reporting: • This manuscript might benefit from a more extensive report of background literature. Specifically, I felt as though the authors could have developed a stronger argument for the study’s focus on azoospermic couples, as opposed to couples who suffer from other kinds of fertility issues. Why might couples suffering from azoospermia be particularly vulnerable to the effects of the COVID-19 pandemic?

• The authors might also strengthen their rationale for focusing on azoospermic couples by discussing the prevalence of this type of fertility problem. What percentage of couples suffer from azoospermia? Are certain demographics affected more than others?

• The information presented in the introduction might also benefit from some slight reorganization. The last paragraph of the introduction discusses the impact of COVID-19 on sexual health, in general. It might make more sense to first talk about the impact of COVID-19 on sexual health, generally, and to then discuss the impact of COVID-19 on the sexual health of infertile couples, specifically.

• The authors provide a definition of azoospermia in the methods section of this manuscript, which may be helpful to readers who are unfamiliar with this condition. However, providing this definition earlier (i.e. in the introduction) might increase the readability of this manuscript.
Experimental design: • In the Participant Selection section of the paper, the authors note that “all patients [were] informed that the study… did not involve any privacy”. Is this a typo or grammatical error? If not, the authors should elaborate on this point.

• The authors state that some couples were removed from analyses due to providing inconsistent answers. The authors should explain the method by which they determined inconsistent responses.
Validity of the findings: • Under the subheading “Comparison of sexual health and sexuality between azoospermic and normozoospermic couples” the authors make several statements regarding group differences on several measures, including the IIEF-15, PEDT and FSFI. Were these the results from independent samples t-tests? If so, why are p values the only statistic reported? Means and standard deviations are reported in Table 2, but these (along with t values) should appear in-text as well.

• Similarly, the standard errors and 95% confidence intervals should be reported in-text when the authors discuss the results of their SEM.

• The authors note in the limitations section that they are unable to compare the effects of azoospermia on mental health, sexual health, and relationship satisfaction before and after the pandemic. Although it is good that the authors address this limitation, I don’t know that it is given the appropriate amount of consideration. One of the key themes of this paper is the negative effect of the pandemic on various psychological and behavioral outcomes, yet this claim was not empirically tested and the authors have no way of determining how/whether the pandemic impacted the results of this study.

• In the discussion section, the authors seem fairly confident in their interpretation of the results of their SEM; namely, that increased frequency of masturbation (which they speculate is the result of the global pandemic) leads to decreased relationship satisfaction, which in turn leads to decreased sexual health. I think it would be extremely helpful for readers if this pattern of results (assuming it was hypothesized by the authors beforehand) be laid out in the introduction section of the paper. This also relates to a broader criticism I have of this manuscript, which is that the overall narrative could be greatly clarified and strengthened in both the introduction and discussion sections of this paper. Specifically, in the introduction, the authors should clearly define each of the relevant psychological constructs, lay out the expected relationships between these constructs, and give a justification as to why they predict this pattern of relationships to emerge. Then, in the discussion, these predictions should be contrasted with the results of the statistical analyses. To summarize my point: I think that it is, at times, unclear what message the authors most wish to convey in this paper, and this message could be made clearer by revising both the introduction and discussion.

• That said, I also think the authors should take some time to consider alternate interpretations of their results. The authors did not implement an experimental design (which might also be listed a limitation). Thus, the authors should recognize that the pattern of variables laid out in their SEM is not the only possible interpretation. Sexual health was the outcome variable in the present study, but other research has suggested that a deterioration of sexual health might lead to a decrease in relationship satisfaction (e.g., Young et al., 1998), while others have proposed that, at least in men, sexual disfunction may underlie psychological distress, such as depression and anxiety (e.g., Althof, 2002).
Additional comments: I think that this manuscript presents a number of interesting findings that are absolutely worth being presented publicly. However, I have some concerns with the authors’ framing of these data, as well as some recommendations for more technical aspects of this paper in the methodology and results (noted in my comments above). I believe that these concerns should be addressed before this article is accepted for publication.


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

Input:
Title: SEXUAL AND PSYCHOLOGICAL HEALTH OF COUPLES WITH AZOOSPERMIA IN THE CONTEXT OF THE COVID-19 PANDEMIC
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are some instances throughout the article in which incorrect English is used with some grammar issues/ambiguous descriptions of study details being an issue. A thorough copyediting of the manuscript to check for such instances is recommended.
Experimental design: No comment for this area.
Validity of the findings: This is the area that requires the most improvement before publication is recommended. Most notably, details of many analyses (e.g. t-tests and ANOVAs) have been omitted from the results, and methods for scoring and coding some of the variables is questionable without a reasonable accompanying rationale. There is no mention of attempts to control for Type I error despite the use of several comparisons. These issues need to be addressed, and more detailed suggestions for revision are provided in the general comments section.
Additional comments: This article investigates an interesting and novel topic: how the COVID-19 pandemic affects azoospermic couples in terms of various relationship dynamics (e.g. sexual satisfaction, psychological distress, relationship satisfaction). Additionally, the study directly compares the relationship dynamics of azoospermic couples with normozoospermic couples. The novelty of the study and the population investigated are certainly strengths of the study. However, there are numerous methodological and statistical weaknesses that need to be addressed before publication is recommended. Most notably, details of many analyses (e.g. t-tests and ANOVAs) have been omitted from the results, and methods for scoring and coding some of the variables is questionable without a reasonable accompanying rationale. See below for more detailed suggestions for revision:

In the Introduction, line 51, it should read as “It has had an unprecedented impact ON individual psychological and relationship health.”

In the Participants section, in line 92, it should read as “Before participating, all patients WERE informed that the study was voluntary….”. Also, in that same sentence, the phrasing “did not involve any privacy” does not make sense. Are the authors trying to say that it did not involve any threats to privacy? This requires clarification.

In the Self-reported Questionnaires section, in lines 111 – 112, the item the authors set up: “How satisfied are you with intimacy and sexual activity with your wife (husband)?” is double-barreled, which makes it more difficult to interpret the meaning of the responses. After all, intimacy is a construct that is distinct from sexual activity. This should at least be mentioned as a limitation. Similarly, in lines 133 - 134, the authors created another double-barreled item which would be associated with the interpretability issues: “Do you have difficulty with or pain during sexual intercourse (dyspareunia)?” Once again, “difficulty” might not always involve pain, so the exact construct being measured here is ambiguous.

In the Statistical Analysis section, in lines 144 – 145, be sure to mention that the t-test is an independent-samples t-test.

In the Results section, it would be easier for the reader to follow along if the authors state which analysis was done for each of the comparisons being made (t-test, chi-square, or ANOVA).

There are many comparisons being referenced in the Results section and no mention of attempts to control for Type I error. Were any procedures put in place to reduce Type I error? If so, please describe those. If not, explain the rationale for not implementing them.

Also, in the Results section, some type of effect size measure for the various comparison analyses (such as Cohen’s d and/or partial eta squared) would also help with interpretation of the results, especially in the case that Type I error was not controlled for.

What was the rationale for the high rate of categorization of variables that often are treated as continuous? For instance, the QMI is typically measured in a continuous fashion, with higher scores indicating higher satisfaction. However, the authors categorize couples into “fine, general, or deteriorate.” How are these categorizations determined? Furthermore, why use such categorization? Typically, creating arbitrary cutoff points in continuous scales is associated with decreased quality of analyses.

In the SEM analyses, the coding for frequency of masturbation is not intuitive, with increased masturbation coded as 0 and 3 coded as none. This makes it confusing for the reader to interpret the figures depicting these analyses with frequency of masturbation negatively predicting psychological distress and positively predicting relationship satisfaction. Consider recoding for improved interpretability.

In the Discussion, in line 334 “healthing” is not a word. Please revise.

In the Discussion, line 347, “…compare the situation…” should be changed to “…comparing the situation…”

In the Discussion, in lines 350 – 351. The privacy concern is reasonable when speaking of reasons for not including measures such as serum hormone levels and azoospermia classification. However, couldn’t there be some type of code number assigned to participants’ clinical data to retain privacy?


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: SEXUAL AND PSYCHOLOGICAL HEALTH OF COUPLES WITH AZOOSPERMIA IN THE CONTEXT OF THE COVID-19 PANDEMIC
Review round: 2
Reviewer: 1

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


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: SEXUAL AND PSYCHOLOGICAL HEALTH OF COUPLES WITH AZOOSPERMIA IN THE CONTEXT OF THE COVID-19 PANDEMIC
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed the the reviewers' comments and concerns regarding the manuscript.
Experimental design: No comment here.
Validity of the findings: No comment here.
Additional comments: No additional comments


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

Input:
Title: DEVELOPMENT OF A GENE DOPING DETECTION METHOD TO DETECT OVEREXPRESSED HUMAN FOLLISTATIN USING AN ADENOVIRUS VECTOR IN MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: - some essential literature is missing, referencing needs thorough review and upate (details given in the general reviewer comments section)

- the Figure quality / resolution needs to be improved (details given in the general reviewer comments section)
Experimental design: - the methods' description should be expanded to enable reproducing the experiments (details given in the general reviewer comments section)
Validity of the findings: no comments
Additional comments: Line 17-18: “Gene doping is a gene-editing technique to improve athletic performance without a target.”
	This introductory sentence should be phrased more precisely. Possible gene doping techniques comprise genome-editing techniques as well a gene therapeutic techniques without the potential of chromosomal integration. All are targeted techniques with the intention to manipulate specific genes or gene functions, respectively.

Line 48-50: “Gene doping is defined as "the non-therapeutic use of cells, genes, genetic elements or regulation of gene expression that have the ability to enhance athletic performance" (WADA, 2008).”
	Instead of citing the definition of the WADA prohibited list 2008, the reviewer suggests to cite the current version (WADA, 2021) as the definition has been updated and specified over the years (e.g. 2013, 2018).

Line 51-53: WADA has added gene doping to the prohibited list in 2003 and established a committee in 2004 to investigate the latest advances in the field of gene therapy and methods of detecting gene doping (WADA, 2016).

	Please provide the URL. Is it this one (from 2015)? http://antidopinglearninghub.org/sites/default/files/supporting-material/Anti-Doping%20Textbook%20-%202015%20Code.pdf

Line 54-55: However, standard methods to detect gene doping have not been established yet, and there is an urgent need to develop detection techniques.

	Several groups have already published successful detection approaches for potential gene doping targets via nested PCR, qPCR or ddPCR (e.g. Baoutina et. al. 2010; Beiter et. al., 2010; Ni et. al., 2011; Moser et. al., 2014, Neuberger et. al. 2016). In January 2021, WADA has published laboratory guidelines for gene doping detection based on polymerase chain reaction (PCR) indicating the establishment of standardization methods is on its way. The reviewer suggests that the authors elaborate/cite the current literature in more detail.

Line 56-60: In gene therapy, gene carriers called vectors are used. Data on vectors in Table 1 were
obtained from the Gene Therapy Clinical Trials Worldwide (Gene Therapy Clinical Trials
 (2021)) website. Viral vectors are widely used, with recombinant adenovirus (rAdV) vectors
being the most common. They are also used in gene therapy (Liang, 2018; Xia et al., 2018; Zhang et al., 2018).

	“in gene therapy …viral verctors are used…they are also used in gene therapy…” The last sentence reiterates the two sentences before, please rephrase.

Line 86-88: The following plasmids were used in this study: Gen EZTM ORF clone hFST in pcDNA3.1(+) (GenScript, NJ, USA), pENTR4 (Thermo Fisher Scientific, MA, USA), and pAd/CMV/V5-DEST (Thermo Fisher Scientific).

	Please indicate if “Gen EZTM ORF clone hFST in pcDNA3.1(+)” is intronless.

Line 91-92 PCR product was inserted into the pENTR4 plasmid using T4 ligase (Promega software,
92 Madison, WI, USA).

	Do the authors mean „promega software“ or the enzyme from Promega? Please correct if so. Has the correct insertion of the hFST sequence confirmed by Sanger Sequencing? Please state here if so.

Line 97 ff: “2.1. Cell Culture…”

	Please add supplier information of cell culture media and antibiotics. Please add more information for the reader about the kind (and reason) of cell lines used in the experiments.

Line 178: Total DNA was extracted using a phenol/chloroform/isoamyl alcohol solution.

	Please add supplier information.

Line 218: The sequences of primers and TaqMan probes are listed in Table S2.

	Please provide sequence accession numbers used for primer designs and indicate primers used for SYBR Green assays or Taq Man Assays. Please provide information about the detection of hFST isoforms as well as on possible mice or human gDNA detection via the assays in the text document. Please give a description of primer location (e.g. exon 4-exon 5 spanning). Please indicate in which assay / figure mFST primer assay has been used.

Line 287-289 In addition, because the phosphorylation of Akt and p70S6K significantly increased, it is expected that muscle synthesis would have been activated to induce muscle hypertrophy.

	Phosphorylation of Akt and p70S6K has been shown in the mouse cell line modell (C2C12) but not in the human cell line (HuH7). Assumptions concerning functional effects via induction of muscle synthesis should be discussed more carefully and differentiated between species.

Line 325-327 These results indicate that the DNA extraction method, primer and TaqMan probe design, thermal cycling conditions, and reagents for TaqMan-qPCR in this study were accurate and optimal.

	Results show specific amplification of the intended target. Please provide further information about assay efficiency and LOD to substantiate “optimal” set up of the assay and cycling conditions.

Line 343 -344 In this study, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping using rAdV<hFST>.

	Multiple transgene fragments? just hFST under CMVp?

Figure Legends

	In general, figure legends (e.g. “Figure 1 (A) hFST gene expression”) should provide more information for the reader about the experimental set up and shown results.

Figure 1 A) and B)
The reviewer recommends to provide SYBR Green melting curve profiles of qPCR assays in the supplements as well as agarose gel separation profiles to underline specific target amplification of the assays and proof of amplicon sizes.

Figure 5 A) and B)

	Please provide amplification plots in higher resolution and indicate NTCs.


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

Input:
Title: DEVELOPMENT OF A GENE DOPING DETECTION METHOD TO DETECT OVEREXPRESSED HUMAN FOLLISTATIN USING AN ADENOVIRUS VECTOR IN MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
==>
Before final submitting, the manuscript should be edited by natives or the editorial office.

Literature references, sufficient field background/context provided.
==>
Yes

Professional article structure, figures, tables. Raw data shared.
==>
Yes

Self-contained with relevant results to hypotheses.
==>
Yes
Experimental design: 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: Impact and novelty not assessed. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
==>
No problem.

All underlying data have been provided; they are robust, statistically sound, & 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.
==>
No problem.
Additional comments: Recently, WADA published PCR-based detection guidelines. Does the detection method developed in this study follow this guideline?

https://www.wada-ama.org/sites/default/files/resources/files/wada_guidelines_for_gene_doping_pcr_test_v1_jan_2021_eng.pdf

Although the authors studied using a mouse model, the authors may need to follow the guideline to develop gene doping tests in sports. If there is a reason not to follow, the authors should consider the reason in the texts.

Primer, probe, sequencing
In this study, hFST was administered into mice and detected by PCR. After administering hFST to humans, can this be specifically detected the hFST with the primers and probes designed?
Sequencing may be difficult because of amplifying human genome .

Minor:
Line 17-18: "without a target". What does this mean?
Line 17-18: The authors mentioned "Gene doping is a gene-editing technique to improve athletic performance", but does "adenoviral (rAdV) vectors containing human follistatin (hFST) genes" correspond to gene editing?
Line 48-53: WADA defines gene doping as "The use of nucleic acids or nucleic acid analogues that may alter genome sequences and/ or alter gene expression by any mechanism. This includes but is not limited to gene editing, gene silencing and gene transfer technologies."
Line 54-55: The authors should cite the WADA guideline here.


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

Input:
Title: DEVELOPMENT OF A GENE DOPING DETECTION METHOD TO DETECT OVEREXPRESSED HUMAN FOLLISTATIN USING AN ADENOVIRUS VECTOR IN MICE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscritp is clear and the English language editing was done. There is one point that could be re-evaluated in the tile that is the term "adenovirus vector", because the focus in the title is gene doping method instead the method for overexpression. I believe that the result section should be more explored and the figure legend must be more detailed. Moreover, the conclusion section repets results and aim that must be avoided.
Experimental design: The authors did not say the number of independent experiments to validate the results. I believe that only one independent experiment was done based on raw data file.
The major concern is the capacity of adenovirus system to reach muscle cells or liver after infusion promoting the overexpression of FST over there, the current data do not bring this information.
The detection of hFst by PCR in the peripheral blood had as template the compounds of adenovirus available systemicaly, then how do the authors detect the gene doping after long time when adenovirus is gone and the target cells were modified. Also, how do the authors address the gene doping using integrated DNA or infusion of RNA that will not be readly available in the blood.
Validity of the findings: Some informations already said in the topic 2.
Additional comments: Dear auhtors,
The manuscript is interesting and the overexpression of FST was clearly demonstrated. I feel that the authors worked hard in the overexpression to simulate a gene doping, whereas the detection of gene doping is not a new method. I know that the protocol for detection of gene in the peripheral blood is an advantage developed by authors, but qPCR is already previously proposed to detect gene editing. So far, I believe that authors should demonstrate that the overexpression of FST really occured, in vivo, in the target cells.


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

Input:
Title: DEVELOPMENT OF A GENE DOPING DETECTION METHOD TO DETECT OVEREXPRESSED HUMAN FOLLISTATIN USING AN ADENOVIRUS VECTOR IN MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors suggest a methodology to detect gene doping using adenovirus vector and the gene used to demonstrate the strategy evidenced the application of the proposal. The authors answered my comments that will be evaluated properly in the future studies.
Experimental design: This point was evaluated previously.
Validity of the findings: This point was evaluated previously.
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: COMPARATIVE TRANSCRIPTOME ANALYSES OF DIFFERENT SALVIA MILTIORRHIZA VARIETIES DURING THE ACCUMULATION OF TANSHINONES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The basic reporting of this manuscript has to be improved intensively:
1.	The English or writing of the manuscript has to be improved. For example:
•	Line 63: “…. in the family Labiatae, its dry roots and rhizomes…” should be separated into two sentences
•	Lines 76-77: change “…Zhang (Zhang et al. 2005), Xu (Xu et al. 2016)… carried out…” to “Zhang et al. (2005), Xu et al. (2016)…carried out…”
•	Line 128: “….contents were originated? collected? from Sichuan…”
•	Line 293: “Totally 1420 transcription…”
•	Line 294: “..675 differentially expression TFs..”
•	Other grammatical changes: suggest to be reviewed by a native English speaker
2.	The quality and resolution of some of the figures have to be improved (e.g. Fig. 4). However, I am not sure if this is due to the reduced resolution in the reviewed copy of the manuscript or due to the original image submitted.
3.	The literature review of the study of secondary metabolite biosynthetic mechanism is too long (i.e. lines 93-106) and covering different types of plants. If possible, it should be focusing on research related to the Salvia genus or its family/order.
4.	The introduction is not being well written to give the readers sufficient background information about this research and methodology. For example:
•	As the research was done on S. miltiorrhiza sampled at early and late stages of tanshinones accumulation, the general life stages of the plant and its secondary metabolite accumulation (i.e. tanshinones) should be briefly reviewed. This should be linked to the rationale of selecting the early and late stages for this study.
•	This research was conducted on S. miltiorrhiza with different varieties, ecological differences and genetic diversity. However, the background information S. miltiorrhiza variety, genetics and ecology was not being described, with only general information of “The strong ecological …. formed rich genetic diversity.” from lines 67-70. This should be linked to the rationale of selecting the specific 4 varieties for this study.
•	The authors should also elaborate further on tanshinones, such as its components, chemistry, properties or biosynthesis process.
5.	Fig. 2A: The values for S1 (5730) and S2 (12001) are the sums for all 4 varieties? Please revise the figure caption to make it clearer for readers
6.	Fig 4: black bars and red lines?
Experimental design: The research question is well-defined but a clearer methodology must be presented:
1.	The rationale of selecting the 4 varieties and 2 different stages, as mentioned in point 4 in Basic Reporting, should be enhanced.
2.	Some additional information is required for methodology. For example,
•	Line 130: how long is the cultivation being done?
•	Line 131: which part of the plants was sampled? Roots?
•	Line 154: provide information on reverse transcription, enrichment method and library preparation, i.e. kits and manufacturers
•	Line 156: provide information on RNA sequencing e.g. paired-end sequencing? Read length? Throughout?
•	Line 162: what is the QC stringency used for Q30? Additionally, the use of Q30 (e.g. >90%) in clean reads should be mentioned prior to the Trinity assembly.
•	Line 167: how to define “best aligned”?
•	Line 171: what is the method for transcription factor prediction?
•	Line 175: elaborate “Gene expression was calculated.”. How is it being done, e.g. mapping method and software, criteria, against de novo Trinity transcriptome?
•	Line 187: “thresholds above 0.70” = P<0.30?
•	Line 190: change “The experiments were performed…” to “The validation was performed…”
•	Line 191: why only samples for stage S2 were included in the validation?
•	Line 192: 400 ng of total RNA or mRNA?
•	Line 193: remove “First-strand cDNA…Real-time PCR”
•	Line 194: what is the starting material amount for reverse transcripted template?
•	Line 196: any reference(s) of using actin as a housekeeping gene in S. miltiorrhiza as a number of S. miltiorrhiza transcriptome studies have been reported. This is to support the “suitability” of using actin (as traditional housekeeping might have variable expression as well) and a single housekeeping gene (as most studies use 2-3 control genes) for normalisation
3.	The authors have mentioned that the whole genomes of S. miltiorrhiza are available (3 different studies). Therefore, the authors should provide the rationale for using de novo transcriptome assembly instead of genome-based reference transcriptome mapping.
4.	The methodology and comparison group for RNA-Seq results should be indicated in Materials and Methods, e.g. read mapping and comparison group for 4 different variety versus 2 stages, how it is being done?
5.	The authors should elaborate on selection criteria for DEGs for real-time PCR validation and what are the criteria for primer design. How about reference or housekeeping genes?
Validity of the findings: Data is sufficiently provided but the presentation and result description has to be improved:
1.	Line 206: cite Fig 1 in the first sentence
2.	Fig. 1: the differences between stage 1 and stage 2 should be tested with a suitable statistical test such as t-test and defined as significant at P<0,05 or P<0.01 level. Additional statistical test such as two-way ANOVA may provide statistical information into effect of stages, variety and interaction between stages and variety on tanshinones accumulation
3.	Line 210: the phrase “…increased to some extent” is not professional and scientific. In general, it should be described as “increased by XX-XX fold for all XXX except for XXXXX”, or “increased by XX-XX % for all XXX except for XXXXX”.
4.	Lines 211-213: the description of “did not change”, “increased greatly” and “increased significantly” is weak without support from the statistical test
5.	Line 214: are DTN, TNIIB and MTN being identified (or being proposed?) as the intermediates based on cited studies? The authors have to be careful about the use of “maybe” if it is already a fact or proposed fact by others. Again, this link to the needs for a brief description of tanshinones biosynthesis in the Introduction section
6.	There is no comparison on tanshinones profiling among the four varieties, which should be the key focus of this study. How different are the tanshinones content and components among the four varieties? This is not being answered and well presented in Figure 1, although the data is already there. This is important as the comparison is essential to be correlated with the RNA-Seq data for interpretation.
7.	Line 225: change “analysed by Trinity” to “assembled by Trinity”
8.	Line 228: 19,4454 unigenes?
9.	Line 240: do not start a sentence with a number
10.	Line 271: any examples of the 10 DEGs common to all 4 comparison groups? At least some interesting candidates to be listed in the result description?
11.	Lines 272-273: “the expression of many DEGs …. than at stage 1” indicates an “up-regulation”? Please make it clearer by improving the sentence
12.	Lines 294-298: how many TFs being differentially expressed? 675 or 124?
13.	Line 308: what is the degree of consistency (quantitative) between RNA-Seq and real-time PCR?
14.	Lines 419-422: the statement is too sceptical and not conclusive if it is just based on the current results. It should be indicated as a speculation
Additional comments: The present manuscript is interesting as it targets to investigate the transcriptional and phenotypic differences among 4 different Salvia miltiorrhiza varieties with medicinal properties at 2 different metabolite accumulation timepoint. The current manuscript is a very beginning draft where various improvement has to be made before it can be evaluated further and reconsidered for publication.


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

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSES OF DIFFERENT SALVIA MILTIORRHIZA VARIETIES DURING THE ACCUMULATION OF TANSHINONES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	Figure 1
It would be more clear understanding if showing the concentrations of thashinones at two stages in 4 varieties of S. militiorrhiza in one histogram.
2.	The text within Figure 4 is not clear enough.
3.	It’s necessary to show the relationship between the expression levels of related genes and the concentrations of thashinones. It would be helpful for understanding the contribution of different transcriptional factors and functional genes in tanshinone accumulation.
4.	Line 98
“Lycium Barbaru and L. Ruthenicum” should be “Lycium barbaru and L. ruthenicum”.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors showed comparative transcriptome analysis of four different varieties of S. miltiorrhizaduring tanshinones accumulation. That’s of interest but the manuscript could be improved.


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: COMPARATIVE TRANSCRIPTOME ANALYSES OF DIFFERENT SALVIA MILTIORRHIZA VARIETIES DURING THE ACCUMULATION OF TANSHINONES
Review round: 2
Reviewer: 1

Output:
Basic reporting: See General Comments to Author
Experimental design: See General Comments to Author
Validity of the findings: See General Comments to Author
Additional comments: Overall, the manuscript has been improved significantly and is ready to be considered for acceptance with minor revision based on the following comments:

Major comments:
1. Provide some sequencing and assembly statistics of the RNA-Seq data, such as how many throughput (Gb of data) per sample, total length (bp) of the assembly, GC content and N50 of the assembly, average unigene length etc. It could be summarised in one or two sentences in the Results section.
2. The grammar has to double check another round as some minor mistakes still exist although the language for the overall manuscript has been improved.
3. The author should add one or two sentences in Conclusion on the significance of the present research, i.e. how the results are useful for future work or applications

Minor comments:
1. Line 40: formatting error, "among these varieties, The tanshinones content"
2. Line 75: Perhaps can make " S. miltiorrhiza is widely distributed and cultivated in China...." into a new paragraph
3. Line 157: Indicate the cDNA kit used (Brand, Country)
4. Line 169: "were co-assembled." into ?? contig? unigene?
5. Lines 191-199: these sentences can be in the same paragraph
6. Line 219: are designed > were designed
7. Line 216: are used > were used
8. Line 223: "0.8 μL of the primer, and 1 μL of cDNA", do not use μL as it is meaningless, use μM/mM or primers and μg or ng for cDNA template
9. Line 239: DTN or DTNI?
10. Lines 237-240: the increment mentioned here is referring to the comparison between S1 and S2?
11. Line 243: "had significant effects", indicate the P value, P<0.05? P<0.01?
12. Line 282: " 73 ~ 136 KEGG", ~ or -?
13. Line 336: "accumulation(Table S1)" - spacing and full stop
14. Figure 4: what is mean by y-axis and x-axis? fold change? log2 fold change? Most of the "DEGs" are below the value 1 which is not optimal, the validation should cover a wide range of dynamic value
15. Line 406: change to 23,369 (44.75%). Same for Lines 421-422
16. Line 433: change to "we found that"
17. Figure 2: " two stages (S1 and S2)", suggest to change to early stage (S1) and late stage (S2) of tanshinones accumulation
18. Line 161: the information of 150 bp read is not mentioned
19. Line 172: "cDNA was synthesized"
20. Line 165: "comprised of"
21. Line 183: "...data were de novo..."
22. Line 182: high quality or high throughput? choose only 1. There is typo for throughput
23. Line 265: degrees ...... previous research results
24: Line 282: one database


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: COMPARATIVE TRANSCRIPTOME ANALYSES OF DIFFERENT SALVIA MILTIORRHIZA VARIETIES DURING THE ACCUMULATION OF TANSHINONES
Review round: 2
Reviewer: 2

Output:
Basic reporting: See general comments
Experimental design: Well done.
Validity of the findings: Well done.
Additional comments: 1.	The author wrote in the manuscript: "(SmMAPK1, SmMAPK3) might play a role in tanshinones biosynthesis (Xie et al. 355 2020). Among the DEGs associated with tanshinones accumulation obtained in this study, there were six MAPK-related genes s, and these genes had higher levels of expression in YN samples 357 with high tanshinones content (Table S10)". It is stated that there are 6 SmMAPKs highly expressed in YN samples. According to Xie et al. (2020) study, we know that SmMAPK1 and SmMAPK3 are associated with tanshinone synthesis. So are there SmMAPK1 and SmMAPK3 among the 6 MAPKs stated by the authors and which one is it respectively.

2.	The authors quantified the expression of the SmMYC. We know that there are two SmMYC2 in Salvia, SmMYC2a and SmMYC2b, which act as positive regulators of the synthesis of tanshinones and phenolic acids, so which one is the authors' SmMYC2? This needs to be clearly stated.

3.	The authors quantified the expression of SmMYB36. Is this gene SmMYB36 as reported by Ding et al (2017)? According to the report, we know that this gene is a positive regulator of tanshinone synthesis and a negative factor for phenolic acids. If it is SmMYB36, the authors need to explicitly specify. However, we checked the primers (Forward primer: AAGAGCTGCAGGTTGAGATG; Reverse primer: GTAACTGAGATGCTATGACCGAC) for the quantitative PCR of the SmMYB36 gene, and this primer did not match the sequence reported by Ding et al (2017). Please explain why this primer does not match the sequence reported by Ding et al (2017).
>SmMYB36 Ding et al. 2017 KF059390
ATGGCGAGTGATGCATCTCTGAACAAAGGGGCGTGGAGTGTGGATGAAGATTCGACTCTGGCCCAATACGTCGCCCTTCACGGCCCCAAGAGGTGGAAATCTGTGGCCATCAAATCAGGCCTCAACAGGTGCGGTAAGAGTTGCAGGTTGAGATGGCTCAACTATCTTAGTCCTGATATCAAAAGAGGCAACTTCTCCGATGCTGAAGAGGACTTGATTCTTAGGTTACATAGGCTCCTAGGAAATAAGTGGTCGTTGATCGCCAAGAGAATCCCTGGGCGGACTGACAACGAGATCAAGAACTACTGGAATGCTCACTTGAGGAAGAAAGCCATGTTGATGGACAAGTTACCCGCAATCTCAACCGCAGCAATGAAGCAAAACATTTGGAATGAAACCGGAGGCGACGATGAATCCTTGGACGTCTCTGCCTCTGGGTTGGATTGGGTTAAAAGTTTTCTTGAACTCGACGAGGATGAATGA


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: COMPARATIVE TRANSCRIPTOME ANALYSES OF DIFFERENT SALVIA MILTIORRHIZA VARIETIES DURING THE ACCUMULATION OF TANSHINONES
Review round: 3
Reviewer: 1

Output:
Basic reporting: See Additional Comments.
Experimental design: See Additional Comments.
Validity of the findings: See Additional Comments.
Additional comments: Minor comments:
1. The previous comment about providing the cDNA synthesis kit name has not been addressed. This should be different from the PCR purification kit where the author highlighted in their rebuttal.
2. Line 251: Are you sure such a high amount of cDNA (80 micrograms) was used in a single 20u PCR reaction?
3. Line 43: ....that are responsible for the differences...
4. Line 67: dried roots?
5. Line 125: metabolome
6. Line 259: ...were low.
7. Line 409: responding


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: COMPARATIVE TRANSCRIPTOME ANALYSES OF DIFFERENT SALVIA MILTIORRHIZA VARIETIES DURING THE ACCUMULATION OF TANSHINONES
Review round: 3
Reviewer: 2

Output:
Basic reporting: The manuscript could be accepted.
Experimental design: The manuscript could be accepted.
Validity of the findings: The manuscript could be accepted.
Additional comments: The manuscript could be accepted.


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: DNA BARCODE REFERENCE LIBRARY CONSTRUCTION AND GENETIC DIVERSITY AND STRUCTURE ANALYSIS OF AMOMUM VILLOSUM LOUR. (ZINGIBERACEAE) POPULATIONS IN GUANGDONG PROVINCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study, the authors analyzed the genetic diversity and genetic structures of A. villosum populations and they used DNA barcoding and Inter-Simple Sequence Repeat (ISSR) markers for investigation of the population genetics of A. villosum.
Experimental design: Your experimental needs more detail. I suggest that you improve the description at lines 81- 92 to provide more justification for your study.
-The methods of collection and references is needed.
-For example the voucher number of each population is needed.
-The quality of image is very low and needs to improve.
Validity of the findings: However the genetic diversity is very important in medicinal plant but the phytochemical and morphological characterization is also need. For this reason I highly suggested that this detail must be added to this paper.
Additional comments: Your experimental needs more detail. I suggest that you improve the description at lines 81- 92 to provide more justification for your study.
-The methods of collection and references is needed.
-For example the voucher number of each population is needed.
-The quality of image is very low and needs to improve.
However the genetic diversity is very important in medicinal plant but the phytochemical and morphological characterization is also need. For this reason I highly suggested that this detail must be added to this 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: DNA BARCODE REFERENCE LIBRARY CONSTRUCTION AND GENETIC DIVERSITY AND STRUCTURE ANALYSIS OF AMOMUM VILLOSUM LOUR. (ZINGIBERACEAE) POPULATIONS IN GUANGDONG PROVINCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript entitled "DNA barcode reference library construction and genetic diversity and structure analysis of Amomum villosum Lour. populations in Guangdong Province" reports genetic diversity and populations genetic structures of collected A. villosum from Guangdong. The data, in the complex, contribute to increase the knowledge about the genomic structure and give correct information about the dimension of the gene pool useful to define the core collection to preserve genetic diversity.The manuscript needs to be re-read and there are a few questions and errors that should be answered and corrected by the authors:
Abstract
1.	Background: Line 23: Please replace "Ammomum villosum Lour" by "Amomum villosum Lour".
2.	Results: Line 43-44: "…56 bands, 84.85% for all the seven A. villosum populations, were amplified polymorphic". In this sentence, it is better to delete the word "amplified".
Materials and Methods
Plant Material Sampling: Line 81-82: "…Research Department of The Second Clinical College". Word "The" in this sentence should be written in non-capital.
Plant Material Sampling: For some populations, ten individuals and for another population 33 individuals have been selected. It is better to explain on what basis the number of samples are different?

ISSR-PCR amplification system Line 113: Please specify the template DNA concentration.
Discussion
In the discussion section, you could refer to other genetic diversity work done on this plant or nearby species.
Figure:
The populations in Figure 1 should be completely separated and be clearly marked on the map.
Table 4: Please explain the abbreviations (Na, Ne, H and …) below the table.

The whole text is not justify and in in somewhere the space between words and lines is not suitable.


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: TRANSCRIPTOMIC ANALYSIS REVEALS CANDIDATE GENES FOR MALE STERILITY IN PRUNUS SIBIRICA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Interesting transcriptomic study related to male sterility in Prunus sibirica.

However, some important defects must be revised before publication.
Experimental design: The assyed P. sibirica clone must be described or refernced.

In figure 9 and in the methodology authors must incorporated the correlation coefficient for the statistical evakluation of correlation between RNA-Seq and qPCR analysis.

Tables 3 and 4 are not of great inmteerst. Should be incorporated as supplementary.
Validity of the findings: Conclusions must be improved including main implications of the obtained results from a breeding and production point of view.
Additional comments: Introduction section shopuld be completed with some key refernces about prunus male sterility and Prunus RNA-Seq estudies.

Please clarify objectives of the work in a separated parragrapgh.

Figures 2 5 and 8 should be aded as supplementary material.

Descritpion of Results is very poor. Authors must clarify results showed in the Figures and Tables.

Discussion section should be improved including key refernces about prunus male sterility and Prunus RNA-Seq estudies.


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: TRANSCRIPTOMIC ANALYSIS REVEALS CANDIDATE GENES FOR MALE STERILITY IN PRUNUS SIBIRICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Recommendation: Major Revision

In the manuscript “Transcriptomic analysis reveals candidate genes for male sterility in Prunus sibirica”, authors documented morphological characteristics of male sterile flower buds and male fertile flower bus in Prunus sibirica and conducted a RNA-seq analysis on these materials. Male sterility (MS) is an important trait that has been used successfully and widely for plant breeding. This paper addresses an important biology question and authors identified candidate genes responsible for MS in Prunus sibirica. RNA-seq data generated in this study are successfully validated by using qRT-PCR. However, the writing is problematic and improvement is needed. It’s very difficult to see the significance of this research partly due to this reason. What authors present here are just a collection of general descriptive dataset without thoughtful elaboration and meaningful interpretation.
There are major aspects within this manuscript that need to be emended or addressed, authors need to conduct a thorough self-examination according to scientific publication standard.
Specific comments are as following.
1.Introduction
1.1 Authors should give a little information about pollen development and its relationship with the degeneration of the tapetum and middle layer in introduction, so that readers can understand their suggestion that “the reason of male sterility in Prunus sibirica was that tapetum and middle layer cells failed to degrade sufficiently”.
1.2 Relevant references should be cited properly. For instance, L51-55, “the low and unstable yield...affects the yield of Prunus sibirica”, refer to relevant references here. This is true throughout the entire manuscript.
1.3 L61-64, Please update the references. It is not appropriate to use the references published in 2016 or even earlier to represent the work “in recent years”.
1.4 L65-66, “there are few reports on the application of transcriptome sequencing technology to study male sterility”, this is clearly not the case.
1.5 L71, biological functions were not investigated in this work. Authors only annotated DEGs in 7 database. Please rephrase.
2.Materials & Methods
2.1 I am concerned about the genetic backgrounds of clone No. 1 and No. 60, because I consider that there is no scientific meaning in comparing two materials with different genetic backgrounds.
2.2 L91-93, what do the authors mean with “MSFB” and “MFFB”? Are they used to represent male sterile clone No. 1 and male fertile clone No. 60, respectively? Please explain what do they mean.
3. Results
3.1 Fig. 1 are very low-resolution and it is very hard to read. The color of stained anthers in Fig. 1C was not uniform with other panels. It would be much easier to read Fig. 1 if the length of scale bars is uniform in the two columns when showing male sterile flower buds and male fertile flower bud. It is very tedious to check the differences between Fig 1C and G at microspore stage, Fig 1D and H at anther maturity, particularly when the magnification of panels in the two columns is different. This makes it even more difficult to interpret the figure.
The legend of Fig. 1 is unclear. “A, E: sporogenous cell stage; B, F: tetrad stage; C, G: microspore stage; D, H: anther maturity stage.... A, D: sporogenous cell stage; B, F: tapetum stage.” please correct.
Fig. 1D is similar with Fig. 1G. How do you judge that the pollen in Fig. 1D is at anther maturity stage rather than microspore stage as showed in Fig. 1G.
3.2 Instead of showing morphological characterization of male fertile and male fertile plants using paraffin section, it would be clearly more informative to provide evidence using semi-thin sections (1 μm) stained with toluidine blue.
3.3 L170-177, “In male fertile flower bud...(Fig. 1C, D). In male sterile flower buds...(Fig. 1G, H)”. However, in Fig. 1, A-D showed the microstructure of male sterile flower buds and E-H represented male fertile flower bud. Please refer to the correct subgraphs.
3.4 L178-179, “...was one of the causes of male sterility...”, do you mean there are other causes? What are they?
3.5 L189, please explain the critical stage. Isn’t this the “microspore stage”?
3.6 L200, describe the specific databases, not simply mention as “7 databases”.
3.7 L209-210, “Gene Ontology (GO) functional...GO functional classification”, please correct.
3.8 L229-230, “Among them, a total of 297 related genes were enriched.” I don't see any result supports this claim. Please provide more details, either by figure or table.
3.9 L237, please define “4 categories”.
3.10 L246-247, authors mentioned that “these pathways played a key role in regulating the formation of male sterility”. KEGG pathway enrichment analysis alone is not enough to draw such a conclusion. A biological explanation is needed for such a significant result, I don’t see that in the current manuscript.
3.11 Sequencing data should be deposited into public databases, such as NCBI GEO/SRA and include the accession IDs in the manuscript.
4 Discussion
4.1 L275-276, “Male sterile flowers appeared withered and atrophic in full bloom, and pollen sacs can’t crack normally”. I don’t see the description in the Results section of the current manuscript.
4.2 L284-286, the authors stated that “We speculated that part of the proteins regulating anther development were degraded, and the related cellular metabolism process was blocked, thus leading to male sterility”. That statement comes out of the blue without any connection to the data presented.
5 Table
5.1 Table 1
In general, male sterile plants show a reduction in size compared with its male fertile line. However, clone No. 1 shows an increase in tree height, trunk diameter and crown width. This result makes us very curious. Please indicate the genetic backgrounds of clone No. 1 and No. 60.
5.2 Table 2-6 should be included as a supplemental material.
6. The quality of language in the manuscript needs some extensively edited.


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: TRANSCRIPTOMIC ANALYSIS REVEALS CANDIDATE GENES FOR MALE STERILITY IN PRUNUS SIBIRICA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English language should be improved to facilitate understanding of the text. I suggest you have a colleague who is proficient in English and familiar with the subject matter review your manuscript, or contact a professional editing service.
The Introduction contextualizes properly the knowledge gap to be filled. However, the last paragraph needs to be improved with a more realistic perspective regarding the findings of this manuscript.
The literature data are not accessible, since there are too many articles that are not available in the databases or are available only in Chinese, which prevents the access of the international audience. Thus, the references need to be improved by changing the articles unavailable or in Chinese to articles available in English.
The figures and labels need to be revised since some do not properly report the individual names or disagree with the Results.
Experimental design: The research is original, relevant, and extremely meaningful. However, the anatomical analysis conducted was not the most appropriate and prevented that the aims of this study were achieved. Besides that, the anatomical method was based on an article in Chinese and was not sufficiently detailed, which does not permit be replicated. Therefore, a more accurate anatomical analysis needs to be conducted, as suggested in the Material and Methods.
Validity of the findings: The manuscript was revised in order to enrich the quality of the article, and all considerations were made throughout the text in the PDF file.
Unfortunately, the structural analysis conducted precluded an accurate structural characterization of the pollen grain at the different developmental stages and the identification of the tissues that compose the anther, as this manuscript aims. Therefore, the inferences of the tissues and the pollen grain developmental stage related to the male sterility cannot be realized. Besides that, as the transcriptome analysis was based on the results obtained by the structural analysis, both structural and transcriptome data need to be reviewed.
Additional comments: I encourage the authors to resubmit the manuscript at another time. I believe that with new structural analysis, as suggested in the Material and Methods, the pollen grain developmental stage and the tissues involved in the male sterility will be determined and the combination of the ontogenesis data with the transcriptome data will provide important references for the study of the reproductive development and the mechanism of male sterility in this species.


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

Input:
Title: EFFECT OF SARS-COV-2 INFECTION ON HOST COMPETING ENDOGENOUS RNA AND MIRNA NETWORK
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have reported an important finding on the competing endogenous RNAs (ceRNA). Overall, the manuscript is well written. However, I have some comments:

Abstract: It is well written and structured. But, please mention the gaps in the previous study in line and the strengths of the new finding briefly.

Introduction: It is well written but very briefly. The authors should focus on details of the biology, such as what are mRNAs and miRNAs? Classify RNAs and their relations to SARS-CoV2 viral replication. Please state the previous study limitations and how this one will overcome those, and clearly write the objective of the study. You should not cite other papers in the last paragraph of any introduction. The authors have missed a couple of recent vital pieces of literature, such as this very interesting and high-impact one: https://doi.org/10.1016/j.gene.2020.145057.

DIscussion: Please add a brief hypothesis and findings in the first paragraph. Mention the strengths and limitations of the study.
Experimental design: No comments.
Validity of the findings: The findings are valid and logical to the writing flow.
Additional comments: No additional comments


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

Input:
Title: EFFECT OF SARS-COV-2 INFECTION ON HOST COMPETING ENDOGENOUS RNA AND MIRNA NETWORK
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript aims to investigate effects of SARS-CoV-2 infection on host competing endogeneous RNA and miRNA network through application of their previously developed R package.
Although it is a well written manuscript, there are various points needed to be improved:

1.	According to Salmena et. al. 2011 paper, the ceRNA hypothesis which is also mentioned in the title of the submission, “a unifying hypothesis about how messenger RNAs, transcribed pseudogenes, and long noncoding RNAs “talk” to each other using microRNA response elements (MREs) as letters of a new language”. In addition to this, circular RNAs also have miRNA binding sites. This study should provide information/results about the other players of ceRNA rather than looking only at host mRNA and viral RNA targets of host miRNAs.

2.	In the introduction section current knowledge about miRNAs in SARS-CoV-2 infection are explained in detail with many citations. The results of those cited papers should be compared and discussed with original findings of the study.
Experimental design: 3.	A major concern about the experimental design is the authors do not perform any predictions about miRNAs and their targets. It is understandable to obtain experimentally validated miRNA – mRNA targets from mirtarbase but human miRNAs targeting of SARS-CoV-1 and/or SARS-CoV-2 were extracted from a previous study. If existing datasets would be used, data from more sources should be considered. There are various publications for SARS-CoV-2 and human miRNAs, some are also available in the references list.

4.	In line with the 3rd point, there are reports of SARS-CoV-2 encoded miRNAs, their viral and human targets. These should also be included in a ceRNA based work.

5. 6 genes and 16 miRNAs "which vary in competitive behavior specifically in presence of SARS-CoV-2 RNA" should be experimentally validated to show that the results are due to competition and not any other possible mechanism.
Validity of the findings: In the abstract, it is mentioned that “These transcripts have associations with COVID-19-related symptoms suggesting that our perturbation ability perspective has the potential to reveal the molecular basis of SARS-CoV-2 induced systemic dysfunctions.” This should be further explained in discussion. What type of molecular basis of SARS-CoV-2? Would they be helpful for development of new therapies or drugs? Impact and novelty of the study is not clearly stated.
Additional comments: No additional comments


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

Input:
Title: EFFECT OF SARS-COV-2 INFECTION ON HOST COMPETING ENDOGENOUS RNA AND MIRNA NETWORK
Review round: 1
Reviewer: 3

Output:
Basic reporting: This research uses Single-cell RNA-Seq and bulk miRNA-Seq data of Severe acute respiratory syndrome-related coronavirus(SARS-CoV-1) and Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infected human cell lines to generate  ceRNA network simulations using the ceRNAnetsim R package.  miRNA:target interaction networks are constructed using miRTarBase (version 8) and Human miRNAs targeting SARS-CoV-1 and/or SARS-CoV-2 genome are extracted from a previous report (Khan et al., 2020). This study tried to examine the regulatory role of miRNAs and viral RNA in SARS-CoV-2 infections and identified 6 genes and 16 miRNAs that vary in competitive behaviour, specifically in presence of SARS-CoV-2 RNA.
The experimental conditions of the two study datasets are not explained in details. Why only two datasets were selected? How relevant are they to support the study hypothesis?
There should have been a flowchart to show the study pipeline to make general readers understand and follow the study easily.
How did Khan et al. arrived at the list of miRNAs potentially targeting the SARS genomes should be explained.
The paragraph starting at line 38 looks orphan and out of context and the statement is made without any reference too.
The introduction lacks review of related studies already reported in the literature.
The manuscript requires language corrections. for example, Figure 1 legend "p value < 0,05)" should be "p-value < 0.05", and many others.
Experimental design: The interaction of host miRNAs and viral RNAs has been predicted by several groups.
Sufficient attempt has not been made to connect the findings with previous reports.
It is not clear from the results how the findings fill an identified knowledge gap.
Functional annotation of miRNAs is not thorough and sounds incomplete and vague. For example, miRNAs have been reported to be associated with different disorders (liver, etc.), without giving specific details and pathways.
Figure 2- It is not clear why carcinoma cases were excluded during calculations. Please explain.
Validity of the findings: The study lacks any experimental validation and sounds rather too descriptive. It could have been rigourously linked to the already conducted computional and experimental studies, as a validation.
The impact and novelty of the study are not highlighted properly.
The study limitations are not mentioned or discussed.
Additional comments: No additional comments


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

Input:
Title: IN SILICO EVALUATION OF MOLECULAR VIRUS–VIRUS INTERACTIONS TAKING PLACE BETWEEN COTTON LEAF CURL KOKHRAN VIRUS- BUREWALA STRAIN AND TOMATO LEAF CURL NEW DELHI VIRUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The written English language is OK but could be improved. The word "the" is frequently used and misused throughout the manuscript. Additionally, the authors used non-scientific terms (possibly to avoid plagiarism), which should be corrected. Need to be succinct and specific.

At several places, irrelevant references are cited (see the comments).

Figures 1 and 2 – Authors failed to produce the exact genome organization of begomoviruses, esp the C4 and TrAP (C2) ORFs. The C2 is truncated (~35 aa) in CLCuKoV-Bur only but not in ToLCNDV, but the authors illustrated the same for both the begomoviruses. Similarly, the size of C4 ORF is usually variable, on some viruses it completely localized within the Rep and sometimes does not. So, the sizes of the proteins depict the presence of anomalies. If authors used some particular sequence of a bipartite begomovirus then they should mention the accession number here (or refer to a suitable table). In addition, the sizes of DNA A and DNA B are not the same. These anomalies raised serious concerns over the sizes of the used proteins, their structures, and protein-protein interaction presented in the study.
Experimental design: The designed study is within the aims and scope. But the authors superficially discussed their results (see the comments).

The demonstrated In silico analyses are performed to a substantially good level. However, these findings should be supported with some wet lab work (see the comments).
Validity of the findings: The performed in silico work is good enough to decipher the molecular interactions between the subjected proteins.

The authors concluded “the antagonistic behavior between the investigated viruses”. Although, this is far from the conclusion – as the opposite is also possible. However, this notion should be addressed by co-inoculating these two viruses, in all possible combinations, in host/model plants (the same is mentioned in comments too)
Additional comments: 1.	It is a well-established fact that viruses interact (in a synergistic or antagonistic manner) during co-infections. The results obtained here indicate that the majority, but not all, of the proteins interact with the same protein from another virus, such as ToLCNDV-Rep and CLCuKoV-Bur-Rep. Again, it is well established that the majority of these proteins form oligomers as a result of their interacting residues – thus, these interactions are simply a result of those specific domains.
2.	The produced title is not a well fit. The word in silico must be included.
3.	Overall, the introduction section lacks a sequential presentation and did not provide vital information. Based on the results - authors should demonstrate “how Rep performs it function, esp the hypotheses demonstrated by Nawaz ul Rahman et al., 2009. Additionally, the study demonstrated by Akbar et al., 2016 could be discussed.
4.	Scope exists to improve the ‘Discussion’ section. Specifically, the work presented by Zaidi et al., 2017 and Iqbal et al., 2017 may be beneficial in discussing the region's current situation in light of the findings of the presented study.
5.	The authors concluded “the antagonistic behavior between the investigated viruses”. Although, this is far from the conclusion – as the opposite is also possible. However, this notion should be addressed by co-inoculating these two viruses, in all possible combinations, in host/model plants.

Minor Comments
L 80: italicize the genus name.
L 81: not the exact reference.
L87: not an exact reference.
L91: As authors are using a bipartite begomovirus, they may use (A)V2 rather than just V2. Do the same for other ORFs too, wherever is applicable.
L94: replace “anti virion sense strand” with a “complementary sense strand.”
L95: supplemented? Not a precise term. Need substantial English improvement.
L96: authors should mention the family and genus of beta- and alphasatellites.
L100-102: ambiguous sentence – rewrite it.
L140 & 145: authors must learn to write the name of begomoviruses according to the recent ICTV taxonomy.
L153: cotton samples?
L178-179: mention accession numbers.
L239: Reword the sentence to “Protein-protein Interactions (PPIs) of CLCuKoV-Bur and ToLCNDV encoded proteins…..”
Table 1: Mention the size of the proteins. In the first column of the table mention Virus/satellite
L315, 319, 322, 327, & 333: This looks somewhat more similar to the M&M section rather than the result section. Better omit it or describe it as a result section.
L323: replace the word “depicted” with either revealed or showed.
L323: percentage identity is a meaningless term. Better change it to “Percentage sequence identity”.
L375: replace “associative” with “associated”.
L447: Sentence needs a full stop.
L444 & 452: results of these parameters are not mentioned.
L473: looks quite odd using the term “interacted” here.
L481: italicize “in vivo”
L482: formatting needed.
L510: Authors failed to accredit the exact reference here. Zaidi et al. 2016 is not an exact reference.
L514: Should discuss the work performed by Akbar et al., 2016 here.
L517: Self-isolation? Please elaborate this phenomenon.
L533: omit parenthesis.
L541: Substitute navigation with a precise scientific word.


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

Input:
Title: IN SILICO EVALUATION OF MOLECULAR VIRUS–VIRUS INTERACTIONS TAKING PLACE BETWEEN COTTON LEAF CURL KOKHRAN VIRUS- BUREWALA STRAIN AND TOMATO LEAF CURL NEW DELHI VIRUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general the article is well written with professional English used. Further editing to ensure minor typos are fixed and correct words are used would be beneficial, in particular spaces between numbers and symbols (e.g 3.0 kb not 3.0kb) and conversely not consequently. Some sections could be combined to reduce wordiness (e.g RMSD and RMSF lines 444 to 459). There are too many figures and tables in the article. These could be reduced to only show differences, for example, figures 8-11. Alternatively some could be put in supplementary data.

The study is very interesting and the choice to predict protein interactions using models is worthy of investigation. The conclusions drawn, however, are not supported by the evidence presented in the article. The reference to ToLCNDV being similar to CLCuKoV-Bur demonstrates a lacking in virology input to the article. They are both begomovirus species, but one is a bipartite and the other a monopartite which are quite different. The explanation of results in relation to virus epidemiology are not clear. Further detail is provided below.
Experimental design: The use of in silico modelling of protein interactions is very interesting and a useful technique to investigate virus-virus interactions.

The meaningfulness of the results presented would be improved if data was presented on homologous protein interactions. For example, what is the strength of the interaction of the CLCuKoV-Bur Rep with its own MP, NSP and TrAP and similarly for the ToLCNDV proteins with themselves. This would allow comparison of the strength of the interactions between proteins of the heterologous viruses to their own predicted interactions. Having this will improve inferences made on the likelihood the interactions would affect virus replication or movement etc. Without this its difficult to put the results in perspective. As such the experimental design is considered flawed in relation to linking the results with functional evidence of virus replication and/or in planta movement.

Additionally, a measure of functionality to support the in silico predictions would be beneficial. This could be through mutational knockouts of infectious virus clones, particularly using the identified amino acid residues predicted to be involved in the interaction. The infectious clones could also be used to evaluate whether the interactions are antagonistic through measure of virus titre in active infections by qPCR.
Validity of the findings: The conclusions made about protein interactions are well presented in relation to the likely strength of the interaction using multiple complementary methods. The strength of the interaction is further supported by detailed analyses of amino acid residues involved in the interaction. This analyses would benefit from the addition of the homologous virus protein interactions suggested above. It provides an internal control for the models used to measure protein interactions and puts the results of heterologous virus protein interactions into perspective.

The speculation on the interactions affecting virus epidemiology is logical. The conclusion, however, doesn't fit the data provided. As there is no functional evidence to show if a dual virus infection enhances or reduces virus replication or spread of one of the viruses, the speculation must cover all contingencies. Instead the article is indicating the interactions was antagonistic due to the spread and virulence of one virus species being reduced. There is no evidence in the article that supports that conclusion.
Additional comments: The study is very interesting and the use of in silico predictions for protein interactions is highly valuable as a first step in virus-virus interaction studies. Further data analyses is suggested, however, before the article is progressed for publication. This is to ensure the overall conclusions are valid in relation to predictions on virus epidemiology. For stronger investigation of the virus-virus interactions, functional experimental work is needed.


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

Input:
Title: IN SILICO EVALUATION OF MOLECULAR VIRUS–VIRUS INTERACTIONS TAKING PLACE BETWEEN COTTON LEAF CURL KOKHRAN VIRUS- BUREWALA STRAIN AND TOMATO LEAF CURL NEW DELHI VIRUS
Review round: 1
Reviewer: 3

Output:
Basic reporting: I have evaluated the manuscript. manuscript is interesting and written clearly. Figures, tables and their captions are self-explanatory. But there are some short coming especially in introduction and discussion section with reference to literature. Although historic data is very important, but authors should use recent literature to present the current state of problem. Moreover, results must be discussed with reference to both old and new literature
Experimental design: Experimental design and statistics are presented in a very good manner and in sufficient details and meet the journal standards

Authors should present, when and where this study was conducted? i.e. lab, years  etc.
Validity of the findings: results are presented in a sufficient details, but authors are suggested to add some quantified values in the results section.

Although results are discussed and justified, but authors are suggested to use recent literature to justify the results to present current state of knowledge

In conclusion section, authors must present scope of the study
Additional comments: Please address the above stated suggestions to improve 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: IN SILICO EVALUATION OF MOLECULAR VIRUS–VIRUS INTERACTIONS TAKING PLACE BETWEEN COTTON LEAF CURL KOKHRAN VIRUS- BUREWALA STRAIN AND TOMATO LEAF CURL NEW DELHI VIRUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Further editing of the manuscript is needed, particularly in the results and discussion sections. Comments are provided on the manuscript. Essentially some results are presented in the discussion section without appropriate description in the results section.
Experimental design: Authors addressed previous comments very well. No further comments for this section.
Validity of the findings: As mentioned in point 1 - further editing of the results and discussion section is needed to clearly identify and describe the findings of the study. Comments are on the manuscript.
Additional comments: Congratulations on an interesting study, I look forward to reading the next manuscript describing the biological data further investigating this virus interaction.


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: VISUAL ASSESSMENT OF DYNAMIC KNEE JOINT ALIGNMENT IN PATIENTS WITH PATELLOFEMORAL PAIN: AN AGREEMENT STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Table 1. Describe the training experience of participants.
Line 132. Indicate the frames per second of the video records.
A hot map figure (with individual data) can be helpful to understanding the dispersion of your data.
Experimental design: My principal concern about the methodology used in the present study is that the dynamic knee joint alignment scoring was not contrasted with a gold standard (e.g., kinematic). The agreement to the visual evaluation of dynamic knee joint alignment was made based on an assessment by two experts, limiting the extrapolation of results (e.g., professionals < 5 yr experience).
Line 182. Can you indicate if this scoring used is validated, or has it been used in a previous study?.
Line 146 and 154. Did the author controlled/registered de velocity of the tests?. Did the participant practiced the tests before to applied the measures?.
Line. 184. Explain how the sample size was chosen. State the determined number of raters, subjects/objects, and replicate observations.
Line 246. The limitations must be expanded.
Validity of the findings: 'no comment'
Additional comments: This paper is well written and is an interesting topic worthy of further investigation. Importantly, however, the results must be more clearly presented. More information about the protocol is needed.


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: VISUAL ASSESSMENT OF DYNAMIC KNEE JOINT ALIGNMENT IN PATIENTS WITH PATELLOFEMORAL PAIN: AN AGREEMENT STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Page 9, line 180, missing a quotation mark after the word malalignment

There are also a few things that need to be clarified about the methology:

The participants performed the exercises without practicing it, however, there may have been some among the participants who were familiar with the gesture, which could have been a determining factor in their quality of execution due to prior motor learning. Were the study subjects sedentary or physically active?

Were the exercises performed at a speed selected by the participant? How was the influence of acceleration on the knee excursion controlled? This factor could influence the knee valgus.

What criteria did the authors use to categorize as severe, moderate, mild and doubtful varus / valgus? Does this represent an adaptation of the system used by Nae et al (2017)? It is necessary to clarify this to allow the reproducibility of your system, considering that subjects with doubtful varus / valgus were finally categorized as "No definite evidence ...". The authors could give an example or brief description of this categorization, otherwise it is too dependent on the judgment of the evaluator, independent of their clinical experience.
Validity of the findings: Page 11, line 242. The authors state "Forward lunge is therefore a promising clinical tool for evaluating knee alignment". Since this study deals with intra- and inter-rater reliability, this statement cannot be attributed to the results. The authors did not measure the sensitivity or specificity of the test. I understand that you meant that forward lunge is a reliable test to be used in clinic enviroment, as you say later.

An element to note is that the video analysis carried out did not include slow motion. This makes the validity results more representative of clinical practice than other studies.
Additional comments: It is a good article, clear and consistent between its objective and methodology. The authors' interest in bringing research closer to daily clinical aspects is appreciated.

There are methodological appraisals that must be reviewed, mainly considering the reproducibility of the classification system they propose.


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: VISUAL ASSESSMENT OF DYNAMIC KNEE JOINT ALIGNMENT IN PATIENTS WITH PATELLOFEMORAL PAIN: AN AGREEMENT STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: I believe the authors have satisfactorily addressed my comments and I recommend that the article be accepted for publication.
Experimental design: I have no comments
Validity of the findings: I have no comments
Additional comments: I have 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: VISUAL ASSESSMENT OF DYNAMIC KNEE JOINT ALIGNMENT IN PATIENTS WITH PATELLOFEMORAL PAIN: AN AGREEMENT STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors made any necessary modifications in consideration of the comments of the previous review. The article is better understood from a methodological perspective