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: POPULATION GENETICS OF SWAMP EEL IN THE YANGTZE RIVER: COMPARATIVE ANALYSES BETWEEN MITOCHONDRIAL AND MICROSATELLITE DATA PROVIDE NOVEL INSIGHTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: First of all, I salute authors for conducting this high level genetic investigation of Monopterus albus. It is not so common to see such investigations carried out by researchers of one laboratory. I could go on by saying that this work was interesting and novatoric but unfortunately the policy of the journal is that such criteria are not evaluated. Therefore, I tried to prepare my review based on objective criteria. Despite the fact that I wrote many comments and suggestions how to improve your manuscript I can say that you need to prepare your manuscript only for minor-major changes.

Basic Reporting
In general, article text is written quite good but English of the manuscript could and should be improved due to high requirements of the journal. Introduction, and especially Discussion, could be improved too because not always sufficient information provided (see General comments section). There are no noticeable problems concerning the structure of the article. However, I was not able to see submitted sequences in GenBank using presented accession numbers and there is room for improvement for some figures and tables and raw data. Due to the fact that submission should include all results relevant to the hypothesis original MJ haplotype network files, at least rdf, should be also presented.
Experimental design: Authors presented original primary research within aims and scope of the journal. Research question (How population genetic structure of Monopterus albus is affected by this fish biology, i.e. sex reversal?) is relevant and meaningful but could be defined better. I think that after suggested changes in the manuscript (see General comments section) there will be no problems concerning research question, identification of the knowledge gap and statements how research fills this gap. In general, this investigation performed to a high technical and ethical standard. However, not all methods and procedures are described with sufficient information. It is really necessary to clarify why mtDNA COI and Cyt b markers were chosen.
Validity of the findings: As mentioned before, original data, at least rdf file, of MJ haplotype network should be provided. This would help to remove all doubts whether truly 47 mutation steps were detected between black dot (mv?) and H-67 haplotype. Ideally, one homologous sequence of species related to M. albus should be used for the same MJ haplotype network. This would reveal how many mutation steps should be expected between different related species from Monopterus genus. In general, major changes are not required in conclusions (see General comments section).
Additional comments: Submission
There are no problems concerning Section, Subjects, Keywords, Manuscript type, DNA data deposition statement, field study permits. However, I could not access the deposited data in GenBank. Experiments were necessary and ethical. Dear authors, despite the fact that it is only formality make sure that not some of You but all of You approved final draft of the manuscript. “( Monopterus albus )” I advise to change to “(Monopterus albus)”.

Abstract and Introduction
From Abstract it is not clear what these mainstream populations are but given in mind that Abstract have words limitation and this is later explained in the Introduction I think that it is not necessary to make big changes in Abstract text. In the Introduction text authors should explain why the population genetic research of the wild eel become a hot topic due to the development of the eel farming. It is not clear what Li et al (2013) determined after their research. Similarly, it is not clear how Liang et al (2016) investigated these six populations. That was natural populations? Finally, in Line 62 it is really necessary to clarify what mtDNA markers were chosen and why.

Minor changes:
Line 42: “nutritional” I advise to change to “nutritional value”.
Line 45 “eel” I advise to change to “swamp eel”. Otherwise some readers could be confused because “eel farming” is commonly used term for Anguillidae species.
Lines 47-48 “microsatellites” I advise to change to “DNA microsatellites”. Abbreviation ISSR should be explained.
Line 51 “researches” I advise to change to “investigations” or “studies”. It is not clear enough why the results of these investigations are important for swamp eel breeding.
Line 55 “normal” I advise to change to “most” or give particular examples of “normal” fishes. “preciseness enough” advise to change to “correct”.
Line 56 “base” I advise to change to “based”. Line 62 also.
Line 63 “Here, the nuclear” I advise to change to “Here, the results of nuclear loci” because not all DNA microsatellites represent nuclear genes. “comparative analyzed” I advise to change to “compared and analyzed”.
Line 64 “insight” I advise to change to “insights”.

Materials & Methods
How exactly 180 swamp eels were collected? With net? Other method? Due to the fact that in Lines 73-74 is first mention of DT, FC, HN, WW, GC and WJ samples it is necessary to explain more about these samples. As I understood, for all mtDNA and DNA microsatellite markers were used same PCR conditions. Is that right? Please write how mtDNA samples were prepared for DNA sequencing, with what apparatus DNA sequencing and microsatellite allele length determination were carried out. Singletons were sequenced with both primers from the same pair or using one primer? It is necessary to clarify what primers were used for DNA sequencing of mtDNA genes. It is also important to write in Materials & Methods that sequences of mtDNA genes were combined to one sequence. Why six populations were combined to one group in Line 97? What is the reason for this calculation? In Line 98 it is not clear what is the meaning of genetic variation. Alleles, Ho, He? Actually, Ho and He should be mentioned in this section and not only in Results section. Due to the fact that I could not see many possible links between different haplotypes in MJ haplotype network I suspect that not only MJ option was used. If that is the case then in Lines 110-111 additional reference should be given. Finally, in this section should be presented information about deletions and insertions in mtDNA genes and clarified whether this data were used in Dnasp and Network calculations.

Minor changes:
Line 72 “eel” I advise to change to “swamp eel”.
Line 80 “designed primers”. Newly designed? By Your research group or other researchers? It is necessary to present primers DNA sequences or references.
Line 81 References should be written in chronological order.
Line 92 Provide reference or link.
Line 96 “perform” I advise to change to “performed” or “conducted”.
Line 99 “estimated to” I advise to change to “estimated in order to”.
Line 104 “reflected” I advise to change to “reflect”.
Line 105 “revealed” I advise to change to “show”.
Line 111-113 Structure program is created for DNA microsatellite data analysis. Therefore, it should be mentioned that in this case genetic structure was analyzed using only DNA microsatellite data.

Results
It is not correct to state that genetic diversity of M. albus is high based on finding that from 180 sequences 86 were distinct haplotypes. High compared to what? Due to the fact that this is Results section it is enough to write “relatively high genetic diversity” but in Discussion it is necessary to compare obtained result with the genetic diversity of other species. Similarly, statement about high genetic diversity using 10 DNA microsatellite loci should be corrected. In Line 123 it is written “MJ method”. However, this is not clear enough how Clades were determined from MJ haplotype network. Actually, it is obvious that this time main criteria for determination of Clades was mutation steps between haplotypes. This information probably should be written in Materials & Methods. It would be interesting to know whether same result is obtained using NJ method but I will not ask this additional analysis. Despite the fact that haplotype 5 (H-5) is most common in the current study that does not mean that this haplotype is the ancestral haplotype. This is only an assumption. It would be interesting to find out whether sequence of related species to M. albus would group with H-5 or other haplotypes in haplotype network. Once again I will not require this additional analysis during this revision. What I will require is the examples from literature of determined huge Fst values (~0.9431) in the context of intraspecific genetic diversity in Discussion. What alternatives to gene flow could be given in Lines 151-152?

Minor changes:
Lines 123-124 “contained five groups”. Please clarify. Five from six populations?
Line 128 In case this is about genetic differentiation, link to Fst results needed. If not then no changes required.
Line 130 “was range” I advise to change to “ranged”.
Line 131 “result” I advise to change to “results”.
Line 132 “population” I advise to change to “populations”.
Line 133 link to Fst and Nm results needed.
Line 134 “main stream” I advise to change to “mainstream”.
Lines 137-138 “significant negative” I advise to change to “significant (p<0.01) but negative”.
Line 139 “while” I advise to change to “because”.
Line 140 text should be corrected because one FC Tajima D value was significant.
Line 142 “estimated” I advise to change to “indicated”.
Line 149 “Structure results” link to results needed.
Line 150 “respected” I advise to change to “represented”, “indicated” or “shown”.
Line 156 “result” I advise to change to “obtained result”. “suggested” I advise to change to “suggested that”.

Discussion
It would be interesting to see possible explanations why Hd and Pi values of FC population are smallest. Similarly, explanations why Pi and Ho values of DT population are highest. In Lines 164-165 text should be modified because it is not correct to write it this way. Actually, during this study obtained Hd = 0.6620-0.9793 and Pi = 0.0017-0.0148 while Liang et al (2016) obtained Hd = 0.708 and Pi = 0.002. So, what explanation could be given for greater Pi values during this study compared to Liang et al (2016) research? It is necessary to clarify and expand text in Lines 171-172, 177-178, 197-200, 204, 215. Indeed, explain these terms: “sex-biased dispersal”, “genetic admixture”, “incomplete inheritance of ancestral polymorphisms”, “selective pressure regulated”. Also improve English in Lines 204, 215-216, 216-218. Finally, compare your results with results of additional genetic investigations of M. albus.

Minor changes:
Lines 166-167 “significant” I advise to change to “crucial” or “noticeable”.
Line 169 Need links to results.
Line 173 “expansion” Where?
Lines 175-176 “common fish” Give particular examples. In what environment movement is weak? In water or on ground?
Lines 176-177 “other species” Clarify from what taxa? Same order or higher taxa?
Lines 181-182 “population” I advise to change to “populations”. Species name should be indicated. Alternative: “tributary population structure” I advise to change to “genetic structure of swamp eel”.
Lines 190-191 Need to write obtained Fst value.
Lines 191-194 Need to write obtained Fst values.
Line 195 “studies”. In case it is only the current research, I advise to change to “study”. In case it is also other studies, provide references.
Line 200 “This study” I advise to change to “During this study”.
Line 201 “mainstream populations” I advise to change to “mainstream population”.
Line 205 “suggested” I advise to change to “suggest that”.
Lines 212-214 “was suffered” I advise to change to “could experience”. It is not clear how many eggs produce swamp eels and common fishes.

Conclusions
As mentioned earlier, it is not correct to just write “high genetic diversity”.

Minor changes:
Lines 220-221 “demographic genetic variation” Explain. Maybe it is reasonable to write “demographic and genetic variations”?
Line 223 “Each tributary population” Only investigated during this work or all?
Lines 224-226 Text after “population genetic structure” should be removed or appear in new sentence.
Lines 226-228 “insight” I advise to change to “insights”. “population genetic research” I advise to change to “population genetic structure and research”.

References
All references in text could be found in reference list.

Figures and Tables
Figure 2 This figure should be improved by adding information about smallest (n=1) and largest (n=?) circles.“methods” I advise to change to “method”. “number of species” I advise to change to “number of sequences”. Black dots represent mv?
Figure 3 I advise to not write COI and Cyt b because sequences of these genes were combined. It is not indicated what species history.
Table 1 could be combined with Figure 1 but I will not require that. In case it is not enough support information that these six samples truly are six populations, “populations” I advise to change to “samples”.
Table 2 Add total values. It would be better to write “mitochondrial data” or “mitochondrial sequence” and not “mitochondrial genes” because there were no calculations of genetic diversity quantitative parameters of separate mtDNA genes.

Minor changes:
Figure 4 “gentic” I advise to change to “genetic”.
Table 4 “diaonal” I advise to change to “diagonal”. It is necessary to write what populations.
Table 5 “test” I advise to change to “tests”.

Raw Data
I advise to add information which individual COI and Cyt b sequences represent which haplotypes.


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: POPULATION GENETICS OF SWAMP EEL IN THE YANGTZE RIVER: COMPARATIVE ANALYSES BETWEEN MITOCHONDRIAL AND MICROSATELLITE DATA PROVIDE NOVEL INSIGHTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is generally well-written and presents interesting and relevant data.
The introduction could, in my view, be very much improved. The authors cite the relevant literature (in what concerns previous phylogenetic studies) but fail to provide important information on the biology of the species and its habitat. In my opinion, life-history (e.g. dispersal of larvae and adults) and environmental parameters (e.g. dynamics of the Yangtze River) are of crucial importance to understand the results. Some details are introduced in the discussion but in my view that should be done in a straightforward line of reasoning since the introduction.
Methods are adequate and results are sound but in my view the discussion also needs improvement mainly in the interpretation of the results and relevant literature.
Experimental design: My main concern relates with the use of the population concept. In this study populations are equivalent to sampling locations and not related with the phylogeographic consistency. Perhaps that should be revaluated throughout the manuscript, including the statement in lines 168-171.
Additionally, also in my opinion the authors should, if possible and for comparative purposes, retrieve the sequences already made available in Genbank for this sampling locations. That would also improve the data set and provide a wider comparative approach at least for mitochondrial markers. Considering mitochondrial markers with concatenate them since the beginning? We cannot therefore assess the diversity for each marker…

Concerning the conclusions they should be toned down because the results, in my view, do not support them entirely.
Validity of the findings: no comment
Additional comments: The manuscript “Genetic research of six swamp eel (Monopterus albus) populations in the Yangtze River based on mitochondrial and microsatellite markers”, written by Zhou and colleagues, focuses on the phylogeographic structure of this highly commercially species on the referred river.

The swamp eel is a very commercial species which has already been phylogenetically evaluated with various markers. The present study provides a comparative approach between different markers using 180 individuals from six eel populations in the Yangtze River, namely two mitochondrial genes and ten microsatellite loci.

Main results reveal:

- high diversity for all populations;
- significant differentiation for three tributary populations (probably due to seasonal cut-off;
- strong gene flow among the mainstream populations (no isolation by distance);
 - discrepancy between mitochondrial and microsatellite results.

The manuscript is generally well-written and presents interesting and relevant data.
The introduction could, in my view, be very much improved. The authors cite the relevant literature (in what concerns previous phylogenetic studies) but fail to provide important information on the biology of the species and its habitat. In my opinion, life-history (e.g. dispersal of larvae and adults) and environmental parameters (e.g. dynamics of the Yangtze River) are of crucial importance to understand the results. Some details are introduced in the discussion but in my view that should be done in a straightforward line of reasoning since the introduction.
Methods are adequate and results are sound but in my view the discussion also needs improvement mainly in the interpretation of the results and relevant literature.

My main concern relates with the use of the population concept. In this study populations are equivalent to sampling locations and not related with the phylogeographic consistency. Perhaps that should be revaluated throughout the manuscript, including the statement in lines 168-171.
Additionally, also in my opinion the authors should, if possible and for comparative purposes, retrieve the sequences already made available in Genbank for this sampling locations. That would also improve the data set and provide a wider comparative approach at least for mitochondrial markers. Considering mitochondrial markers with concatenate them since the beginning? We cannot therefore assess the diversity for each marker…

Concerning the conclusions they should be toned down because the results, in my view, do not support them entirely.

Minor comments

Line 52-53: “However, as the typical sex reversal fish, Monopterus albus has its unique life history (Liu, 1944; Qu, 2018).” Perhaps it should be “However, as a typical sex reversal fish, Monopterus albus has a unique life history (Liu, 1944; Qu, 2018).
Line 72: replace 180 by One hundred and eighty at the beginning of the sentence.

I sincerely hope that my suggestions can help the authors to improve the paper.

Joana Robalo
(jrobalo@ispa.pt)


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: POPULATION GENETICS OF SWAMP EEL IN THE YANGTZE RIVER: COMPARATIVE ANALYSES BETWEEN MITOCHONDRIAL AND MICROSATELLITE DATA PROVIDE NOVEL INSIGHTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Last time I wrote many comments and suggestions for authors. Consequently, this time I will try to be more concise. I really like that it was helpful and how authors solved revision problems. This is was good learning experience for authors and me. The overall quality of the manuscript increased significantly.

Basic Reporting
In general, I had no problems with English of the manuscript but English language is not my native language. Therefore, I advise Editor to evaluate whether current English of the manuscript is sufficient for journal requirements. I checked presented MJ haplotype network files and found that in general there is no problem with this data. However, I noticed one problem regarding interpretation of this data: haplotype network configuration doesn't support idea that haplotype H-47 belong to Clade C. Actually, H-47 is only two mutations away from H-30 that belong to different Clade but 24 mutational steps away from H-42. This problem should be fixed before acceptance.
Experimental design: Authors improved manuscript quality and now I think that there are no problems concerning research question, identification of the knowledge gap and statements how research fills this gap. All methods and procedures are described with sufficient information with the exception of information about calculations regarding deletions and insertions. However, I want to ask why authors decided to reduce number of DNA microsatellite loci from 10 to 8.
Validity of the findings: In general, after revision conclusions part looks better.
Additional comments: Congratulations. Great work. You really improved your manuscript quality. I really like how you solved revision problems. Some questions remained unanswered but I have no problem with that because it is not important from the perspective of the current manuscript. I consider that your work should be published in this journal after minor but essential changes.

Main Issue
Haplotype network configuration doesn't support idea that haplotype H-47 belong to Clade C. Actually, H-47 is only 2 mutations away from H-30 that belong to Clade D but 24 mutational steps away from H-42 (Clade C). This problem should be fixed before acceptance.

Abstract, Introduction and M&M
I wrote that it was not necessary to make big changes in the Abstract text but I have to admit that after revision it looks better. I also see significant good improvement in the Introduction. It is good that now in the Introduction is explained that two genes sequences were combined to one but I think that the same thing should be done in M&M where is first mention about these genes. You can argue that it is explained later in M&M where mentioned DNASTAR but in order to avoid confusion for the readers I advise to make these changes. You convinced me that you used only MJ option. However, this leads to my suggestion to not write "parsimony network" because MP option was not used. I still have to ask to present information in the manuscript regarding deletions and insertions during different calculations. What is the reason behind reduction of available data from 10 to 8 DNA microsatellites?

Results, Discussion, Conclusions
As I understood the non-significant values of SSD of main stem and DT indicate that sudden expansion could not be rejected. Therefore, significant values of SSD of FC and HN indicate that sudden expansion could be rejected. Is that right? If yes then what explanation you can give it for FC and HN SSD results? This 0.46 MYA was calculated by different way than data presented in Figure 3? Thank you for answers regarding huge Fst values but I still have to ask the examples from literature (not necessarily of Monopterus in investigated basin but animal species in general, preferably fish species) of determined huge Fst values (~0.9431) in the context of intraspecific genetic diversity in Discussion. It is true that mtDNA is maternally inherited but there are rare cases where animals mtDNA could be inherited paternally. I advise you to try to investigate this in your future Monopterus studies.

Figures and Tables
As I understood in Figure 2 lines without numbers between different haplotypes mean that it is only one mutational step between them. Is that correct? That should be explained to reader. In Figure 3 it is not explained what HPD (readers should understand every picture and table without munuscript text). Explain to readers what SSD mean in Table 4. Also change "mainstream" to "main stem".

Minor changes:
Line 33 "cause" I advise to change to "caused".
Line 50 "over exploitation" I advise to change to "overexploitation".
Line 98 "m mol/LM" it seems that it should be: "m mol/L".
Line 102 "Genotype" I advise to change to "Genotypes".
Line 248 "factors" I advise to change to "factor".


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: POPULATION GENETICS OF SWAMP EEL IN THE YANGTZE RIVER: COMPARATIVE ANALYSES BETWEEN MITOCHONDRIAL AND MICROSATELLITE DATA PROVIDE NOVEL INSIGHTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript "Population genetics of swamp eel in the Yangtze River: comparative analyses between mitochondrial and microsatellite data provide novel insights" is now in its second review. In my opinion the manuscript is very much improved. Nevertheless, it still presents some english imprecisions (e.g check the conclusions).
It should have a final revision by a native speacker.
Concerning my previous reviews, all points were rebutted accordingly.
Therefore my opinion is that, after english corrections, the manuscript may be published in its present form.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript "Population genetics of swamp eel in the Yangtze River: comparative analyses between mitochondrial and microsatellite data provide novel insights" is now in its second review. In my opinion the manuscript is very much improved. Nevertheless, it still presents some english imprecisions (e.g check the conclusions).
It should have a final revision by a native speacker.
Concerning my previous reviews, all points were rebutted accordingly.
Therefore my opinion is that, after english corrections, the manuscript may be published in its present form.


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: GLOBAL TRANSCRIPTOME ANALYSIS OF ALFALFA REVEALS SIX KEY BIOLOGICAL PROCESSES OF SENESCENT LEAVES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper aims to provide new information on the genetic basis of senescence development for future applications in plant breeding.The authors provide annotation method with transcriptomics tools in order to observe and offer a novel interpretation of the molecular mechanisms of leaf senescence in alfalfa. The bioinformatics pipeline involves several standard transcriptomics tools such as bowtie2, primer primer 5, and DESeq2. The provided annotations deserve attentions in this respect. However, there are several questions that should be answered in order to improve this manuscript, and the authors should revise it with incorporating the answer of these questions:

Extensive editing of English language and style are required.  English used in this manuscript was not clear and ambiguous. As for example: Line: 15,16, 28, 71, 80 (maize should not be italic) 81, 100, 101 and many other lines. The author should use professional English Language.

The background was sufficient. But the references in many places were old. Authors should use more related new references.
Experimental design: Besides this fundamental criticism, in the Material and Method section, the authors need to specify in more detail the plant growth conditions and the age of the samples leaves.

In line 159 :Did you use default parameters for Primer Primer 5? Please expose all the parameters that you used!
Validity of the findings: Figures were relevant and well described to the content of this article. But the resolution should be better. The title of Y-axis of Fig 2A should be in right direction.
Additional comments: 1. Is there any biological explanation why those KEGGS have the most significant enrichment?
2. What is the version of your reference genome? For example, human genome has version number as well (eg.hg19).
3. Line 169-171 and Line 175-177 should be moved to materials and method part
4. Please change the title at line: 187
5. Why did you take 12 genes not more?
6. The discussion is weak. The conclusions are vague. Line 377-379: How the novel ribosomal protein genes expressed at high levels in mature leaves and are expressed at lower levels in young and senescent leaves are related to leaf senescence.
7. Extensive editing of English language and style are required for all part of this manuscript with new reference.


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: GLOBAL TRANSCRIPTOME ANALYSIS OF ALFALFA REVEALS SIX KEY BIOLOGICAL PROCESSES OF SENESCENT LEAVES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general, the manuscript meets most of the acceptability criteria. It is only suggested to revise the writing of English by a specialized service to improve understanding in some passages, especially in the Discussion section.

It is suggested to omit from the Introduction section the paragraph that explains the methodology and the preliminary results (lines 84 to 95). That information can be considered in the Summary.

It is also suggested to enrich the background with a description of the transcriptomic analyzes in alfalfa related publications, the status of the reference genome and its complexity, among other aspects that account for the state of the RNASeq technique in this plant model.

It is generally recommended to leave the conclusions of each analysis presented in the Results section as part of the discussion.

In the DEG analysis (Fig 2) it is necessary to declare which is the reference condition in each comparison.

It is recommended to incorporate a table that can summarize the abundant functional information coming from the classification of significant GO terms in all the comparisons as part of the main tables of the manuscript. The supplementary figure in tree format by category CC, MF and BP could remain but the size of the letter needs to be increased.
Finally, as a general recommendation, it is known that numbers greater than 10 can be expressed in digits (example: line 424)
Experimental design: The experimental design of the study is appropriate. I only have one comment about the plant material. Only the quantification of chlorophyll is mentioned as an indicator for the stage of development of the three study conditions of alfalfa. Fig. 1 shows the same values of chlorophyll a and b between the Young and Senescent stages. It is suggested to improve the description of the phenological characterization of the three conditions studied, in order to demonstrate a clear physiological and metabolic differentiation between the three selected stages of development. In any case, the low amount of DEG shared among the three conditions (Fig 2B, 60 genes) suggests that the three transcriptomes are sufficiently different.
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: TOWARD A CLINICAL REAL TIME TISSUE ABLATION TECHNOLOGY: COMBINING ELECTROPORATION AND ELECTROLYSIS (E2)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written, refers to relevant literature.
Several figures are used, however a sumamry table reporting different experimental conditions would help the reader.
Experimental design: Experimental conditions are properly described.
Validity of the findings: The methodology is properly decribed and the results interesting.
Beside figures, the authors should make an effort to summarize experimental conditions and results in a table. It might help to evidence the validity of the new technology to quantify the results (i.e. by mesurement of the ablated area) in the different experimental conditions.
The authors might consider to evidence cell induced apoptosis by specific stainign of the samples.
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: TOWARD A CLINICAL REAL TIME TISSUE ABLATION TECHNOLOGY: COMBINING ELECTROPORATION AND ELECTROLYSIS (E2)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is very well written and the length is adequate. It includes a sufficient background and appropriate literature references.
The structure of the article is conform to the format of standard sections. Figures are relevant but they should be graphically improved (see Comments for the authors). The article is self-contained with relevant results to hypotheses.
Experimental design: The content of the study is original and well designed and should make a nice addition to the literature. Methods are described in detail.
Validity of the findings: Impact and novelty are achieved and the conclusions are well stated, linked to original reserach and translational research questions, however the data should be supported by statistical analysis.
Additional comments: Gunther and colleagues presented an interesting work describing a successful and clinically relevant real time tissue ablation technology based on the combination of electroporation and electrolysis. In general, this work can represent an advance in the knowledge about the treatment of solid malignancies based on the use of irreversible electroporation (IRE) and reversible electroporation with chemotoxic drugs, called electrochemotherapy (ECT).

The manuscript is very well written and theorically well designed, however I have the following major concerns regarding this work.

Point 1.
Qualitative data are not associated to any statistical test so the results are not statistically sound and controlled. Please, try to add a statistical analysis of the data emerging from the histological study.

Point 2.
Lines 142-143: The authors cite 19 lesions but it is not provided any description of them. Please, give a short definition of the type of lesions identified in the three pig livers.

Point 3.
Figure 1 and Figure 2 should be inverted in the text following the numerical order.

Point 4.
Overall, the figure need to be graphically implemented. Figure 1, Figure 2C, Figure 3 (third column): please, increase the size of text.

Point 5.
In the legend of Figure 2B a brief description of what the ultrasound images show should be added.

Point 6.
At line 241 the authors tell about “condensation of hepatocyte cytoplasm, condensed nuclei…” but these features are not visible in the histological sections included in the figures at 20x magnification. 40x magnification images can be suggested.

Point 7.
Supplementary files are not specified and visible.

Point 8.
Lines 365-366: the description of Figure 8 does not correspond to the real content of the figure.

In addition, few orthographic errors should be addressed in order to make this work ready for publication. Moreover the English language should be improved to ensure that an international audience can clearly understand your text. Some examples where the language could be improved include:
 such as the following ones:
- lines 53-54, 239-240, 243-244, please revise the verb form
- lines 78-79, 350-351, please revise the English form
- lines 85, please consider to use the form “Marshall et al”
- lines 109-110, please revise the English form
- please, use the extended form of the acronyms only the first time they appear in the text, e.g. ECT, Masson’s trhicrome staining (Mas), KCI, etc.


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

Input:
Title: TOWARD A CLINICAL REAL TIME TISSUE ABLATION TECHNOLOGY: COMBINING ELECTROPORATION AND ELECTROLYSIS (E2)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed all my concerns.


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: EXPERIMENTAL EVALUATION OF GENOMIC DNA DEGRADATION RATES FOR THE PATHOGEN PSEUDOGYMNOASCUS DESTRUCTANS (PD) IN BAT GUANO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled "Experimental evaluation of genomic DNA degradation rates for the pathogen Pseudogymnoascus destructs (Pd) in bat guano" describes an experiment aimed at measuring Pd in bat guano over time.

The main laboratory procedure in this manuscript, qPCR assay for Pd, is not properly described and/or cited. There are several published qPCR assays for Pd in the literature, it seems that the authors likely used the method described in Muller et al. (2013) -- however, it is not cited. Additionally the probe used for qPCR is not described either.

If the widely used Muller et al (2013) assay was used herein, then there are some problems with limit of detection described in this paper, as the lowest limit of detection in the Muller et al (2013) qPCR assay was reported to be 3.3 fg. Here the authors claim they could measure 0.195 fg, which is outside the detection limits of the assay.  Moreover, the authors state that a standard curve was run for each qPCR experiment using purified gDNA from Pd in 1:10 serial dilutions from 1000 fg/ul to 1 fg/ul -- and that 5 ul was used for each qPCR assay --> so that means the limit of detection in this assay cannot be below 5 fg, the lowest concentration in the standard curve.

Lines 137-180:  What is a positive result? what is a negative result?
Experimental design: The research question addressed here was to measure the genomic DNA degradation rate in bat guano over time.  The authors spiked Pd DNA into autoclaved bat guano and then incubated these samples in ambient temperature at multiple roosting sites. DNA was measure every 14 days from guano samples in triplicate.  While the main research question is to answer DNA stability/detection in guano samples, there are no laboratory controls to give the results context, ie is DNA stability in bat guano better or worse than leaving a sample on a bench top for 70 days? different temperatures, etc.

There are several occasions where the authors seem to use quantity (fg) interchangeably with concentration (fg/ul) -- these are important details and do not mean the same thing.  For example, the methods say that 5 concentrations of DNA (fg/ul) were inoculated into guano? What was the volume? How did you inoculate? What was total fg of DNA per mg of guano?  How does the DNA extraction yield impact the results? These details are necessary in order to quantify how much DNA can be extracted from bat guano in the first place, e.g. taking into account yield loss during DNA extraction might influence how you interpret the results.
Validity of the findings: Underlying qPCR data not provided. At a minimum, the standard curve used for interpretation of the qPCR results should be provided.

Figures 1 and 2 don't really add anything to manuscript.
Additional comments: No additional comments


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

Input:
Title: EXPERIMENTAL EVALUATION OF GENOMIC DNA DEGRADATION RATES FOR THE PATHOGEN PSEUDOGYMNOASCUS DESTRUCTANS (PD) IN BAT GUANO
Review round: 1
Reviewer: 2

Output:
Basic reporting: L227. Citation needed
Experimental design: L101-109. It is odd that actual environmental conditions were not measured at the five sampling locations, particularly temperature and relative humidity. It is also unclear how often guano samples from typical sampling sites are exposed to direct solar radiation or rain. Many roost sites (and consequently the guano) are relatively protected from each potential source of DNA degradation.
Validity of the findings: To my knowledge, it is unclear from the current literature whether Pd is viable from guano. If it is indeed viable, then one would posit that the DNA would persist longer.
Additional comments: This manuscript addresses an important question of the ability to detect Pd over time from environmental samples such as guano.

Minor Comments/Edits
L30. unclear who is doing the positing. Perhaps change to "it has been posited"?
L42. Change smallest to lowest
L53. Change cryophilic to psychrophilic
L66. Change bat to bats
L75. Citations confusing. As written it appears that these citations refer to Pd studies when in fact they are from other pathogens. Perhaps put these citations earlier in sentence (e.g. after the word guano) or rewrite for clarity.
L76. Muller et al. 2013 is the best citation for the qPCR. Should also be cited in the methods section. Muller, L. K., J. M. Lorch, D. L. Lindner, M. O’Connor, A. Gargas, and D. S. Blehert. 2013. Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans. Mycologia 105:253-259.

L83. Unclear where to put this but sampling from guano under real conditions may also detect Pd from the environment as it is shed off of bats.

An alternative to sampling guano could be to sample cave/hibernacula/bat house substrates, with Langwig et al. 2015 demonstrating the ability to detect Pd from cave walls and Dobony & Johnson 2018 from the surfaces of bat boxes.

Langwig, K. E., J. R. Hoyt, K. L. Parise, J. Kath, D. Kirk, W. F. Frick, J. T. Foster, and A. M. Kilpatrick. 2015. Invasion dynamics of white-nose syndrome fungus, Midwestern United States, 2012-2014. Emerging Infectious Diseases 21:1023-1026.

Dobony, C. A., and J. B. Johnson. 2018. Observed resiliency of little brown myotis to long-term white-nose syndrome exposure. Journal of Fish and Wildlife Management 9:168-179.

L113. More details are needed about how the inoculation was actually done. With a pipette? The DNA was dripped onto the pellet? Spread evenly? What was the process?

L116. "on a the"?
L118. Change ending to ended
L123. Change inside to in
L131, 132. Change Taqman to TaqMan
L133. Are these the conditions of Muller et al. 2013? If so, cite here.
L137. What was your cycle threshold value? any amplification before Ct of 40?
L177. Not sure what you mean by "weight", guano sample weight? Also, I believe it should be referred to as mass?
L190. This is misleading as stated. Collecting wing tissue is only needed for confirmation of WNS, not detection of Pd. Noninvasive sampling such as guano testing cannot confirm WNS. Thus, a wing punch is not typically used for Pd detection.
L204. Again, I'm confused about what weight means here
L205. The point of this sentence is unclear. And isn't intracellular DNA only repaired in living cells?
L207. Delete were
L207. Change during to from
L213. A more complete description of the sites would be helpful. It was surprising to see that some of these sites were in the sun.
L220. Change is to was
L233-234. This is only true if the WNS community can agree on testing standards for Pd assays, Ct values, etc. Currently some states are trying to refute Pd detections for a variety of reasons. For example, if guano sampling typically only detects low levels of Pd then some states may not consider these to be true detections.

Figure 2. Was this modeled data? If so, you should state that.
Table 1. Unclear what the values for each site are referring to? The legend needs more details.


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

Input:
Title: EXPERIMENTAL EVALUATION OF GENOMIC DNA DEGRADATION RATES FOR THE PATHOGEN PSEUDOGYMNOASCUS DESTRUCTANS (PD) IN BAT GUANO
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed my major concerns with this revision.


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: EXPERIMENTAL EVALUATION OF GENOMIC DNA DEGRADATION RATES FOR THE PATHOGEN PSEUDOGYMNOASCUS DESTRUCTANS (PD) IN BAT GUANO
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed all of my concerns.


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

Input:
Title: A COMPREHENSIVE ANALYSIS FOR ASSOCIATIONS BETWEEN MULTIPLE MICRORNAS AND PROGNOSIS OF OSTEOSARCOMA PATIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: The authors should list the primer sequences of the included miRNAs and U6.
The authors should explain why they studied the included miRNAs and why they did not study other miRNAs. I think it is not possible to study miRNAs randomly and there are thousands of miRNAs in human. It is not enough that the authors just depended on the reports of previously published articles.
There are two types of mature miR-21 and please make it clear that miR-21-3p or miR-21-5p the authors used. Similarly, the authors should make it clear which type they studied about miR-214, miR-433, miR-30c, miR-34a, miR-101, miR-133a, miR-539, miR-218.
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: A COMPREHENSIVE ANALYSIS FOR ASSOCIATIONS BETWEEN MULTIPLE MICRORNAS AND PROGNOSIS OF OSTEOSARCOMA PATIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Figures
- I recommend combining figures 3 through 8 into one figure.
- Please make table including the results of cox regression.

Literature references, sufficient filed background/context
- The authors cited Cheng D et al (MicroRNAs with prognostic significance in osteosarcoma~) and they focused on each microRNAs. Please cite Kim YH et al., 2017 and discuss the risk of pooled microRNAs in osteosarcoma patients. In addition, why don't you combine microRNAs to make stronger prognostic predictions or diagnostic markers?

- In the discussion, please discuss the results (microRNAs expression and prognostic significance) in detail. Now the authors focused on previous molecular studies.

Raw data shared
- If the authors share the qRT-PCR and paired clinical data, it will help to many researchers.
Experimental design: Methods
- Please describe in detail.
- In survival curve, they might use the qRT-PCR data from tumour tissue. How about to use differences or fold change between tumour and paired normal tissues.
- In cox regression, please describe how they use expression data as categorical or continuous value.
Validity of the findings: - There is one cohort containing microRNAs and clincial data (GSE30934). By using the cohort, please identify the association between chemotherapy response and microRNA expression.

- If possible, the authors check the microRNAs function through microRNA transfection.
Additional comments: Thank you for giving a chance for reviewing the manuscript. The manuscript is very interesting, however, there are some scientific and technical issues to address.


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

Input:
Title: A COMPREHENSIVE ANALYSIS FOR ASSOCIATIONS BETWEEN MULTIPLE MICRORNAS AND PROGNOSIS OF OSTEOSARCOMA PATIENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Osteosarcoma (OS) mainly arises from the metaphysis of the long bones and it is the most common primary bone malignancy in adolescents and young adults. Despite advances in disease management and treatment, the long-term prognosis of patients with OS is still poor.
However, the authors should clearly explain
1.	What is the gap in the existing literature and why do they need to use miRNA as the prognostic markers?
2.	What is the reason(s) for the authors only to look at the few miRNAs mentioned in this study? It is ideal and unbiased to have done transcriptome profiling of miRNA array for the patient samples they posses from which they could have bioinformatically narrowed-down to pin point which amongst the miRNAs are relevant to prognosis. If they have the tumor and control samples it would be a better study to do the RNA seq for miRNAs.

Few sentences were not clear:
1. In the abstract and result please use the full form for any abbreviation.
2. The meaning of the sentences in line 110 is not clear. Please elaborate.
Experimental design: 1. It is unclear from this manuscript the gap in the existing literature and why do they need to use miRNA as the prognostic markers.
2. Please read Zhang et al 2015, Clinica Chimica ACTA where a list of miRNAs with their expression and relation OS are presented in table1. The premise of selectively choosing few miRNA in this study remains unclear.
3. The authors have performed qRT-PCR for OS and control samples to analyze the expression levels of the miRNAs. Could you please plot graphical plots to show the result along with the table you provided? Please plot ΔΔCt with standard deviation or any meaningful way.
4.	If you have the details of these OS tumors as in table 1, could you also specify what stages of the OS tumors were used to perform qRT-PCR (was done by pooling all the stages/grades of OS together or there were separate samples per stage/grade of OS)? It is important to know the expression pattern over the progression of the disease. Provide elaborate explanation.
5.	 Also, could you provide evidence of the expression of miRNAs in the metastatic and recurrent tumor samples.
6.	As a proof of principle, please use TCGA datasets OS patient samples to analyze and find whether the specific miRNAs are differentially regulated between OS and control samples in an independent dataset.
7.	Method section is very poorly written. As I can understand that this is a bioinformatics-based manuscript, please provide detail description of all the computational methods used.
8.	What is the purpose of selecting p-value <0.1 for multivariate analysis?
9.	Each of the K-M survival plots should be indicative of no. of patients in each cohort and median time of survival. Very important please provide data as Tables for all the figures and keep the color code same all throughout (eg. Hig expression as green and low expression as blue maintain same color for all graphs).
10.	What is the cut-off used to segregate low vs high miRNA expression and why?
Validity of the findings: Data analysis shows miRNA 21 and 214 (high expression) while 34a, 133a, 539 (low expression) are poor prognostic markers for OS.
Please refer to questions in the above panel.
Additional comments: The study is a short bioinformatics one. It could be potentially better with answers to the above mentioned questions. An independant study using the TCGA OS dataset will provide not only proof of principle to your study but also an in-depth overview of miRNAs in OS patient survival and prognosis.


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

Input:
Title: A COMPREHENSIVE ANALYSIS FOR ASSOCIATIONS BETWEEN MULTIPLE MICRORNAS AND PROGNOSIS OF OSTEOSARCOMA PATIENTS
Review round: 2
Reviewer: 1

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


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

Input:
Title: A COMPREHENSIVE ANALYSIS FOR ASSOCIATIONS BETWEEN MULTIPLE MICRORNAS AND PROGNOSIS OF OSTEOSARCOMA PATIENTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors revised the manuscript very well.
Experimental design: The authors revised the manuscript very well in Experimental design.
Validity of the findings: The authors revised the manuscript very well.
Additional comments: The authors revised the manuscript very well.


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

Input:
Title: A COMPREHENSIVE ANALYSIS FOR ASSOCIATIONS BETWEEN MULTIPLE MICRORNAS AND PROGNOSIS OF OSTEOSARCOMA PATIENTS
Review round: 2
Reviewer: 3

Output:
Basic reporting: Overall, the revised manuscript has certainly improved.
Authors successfully answered most of the questions, however they were unable to address few questions because of insufficient funds as the first author mentioned. Minor changes like spellings the authors can take care at the time of proof-read.
I do think the study is suitable for PeerJ journal submission.
Experimental design: Overall, the revised manuscript has certainly improved.
Authors successfully answered most of the questions, however they were unable to address few questions because of insufficient funds as the first author mentioned. Minor changes like spellings the authors can take care at the time of proof-read.
I do think the study is suitable for PeerJ journal submission.
Validity of the findings: Overall, the revised manuscript has certainly improved.
Authors successfully answered most of the questions, however they were unable to address few questions because of insufficient funds as the first author mentioned. Minor changes like spellings the authors can take care at the time of proof-read.
I do think the study is suitable for PeerJ journal submission.
Additional comments: Overall, the revised manuscript has certainly improved.
Authors successfully answered most of the questions, however they were unable to address few questions because of insufficient funds as the first author mentioned. Minor changes like spellings the authors can take care at the time of proof-read.
I do think the study is suitable for PeerJ journal submission.


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: ORGANIC ACIDS AND 2,4-DI-TERT-BUTYLPHENOL: MAJOR COMPOUNDS OF WEISSELLA CONFUSA WM36 CELL-FREE SUPERNATANT AGAINST GROWTH, SURVIVAL AND VIRULENCE OF SALMONELLA TYPHI
Review round: 1
Reviewer: 1

Output:
Basic reporting: Pelyuntha et al, evaluated antimicrobial proprieties of cell-free culture supernatant from Weissella confuse a lactic bacteria in where other groups have been found the same proprieties, however, authors shown that this strain is able to produce high levels of antimicrobial molecules during its growth, which may be relevant in the development of probiotics. Nevertheless, there are some data that should be reviewed:
Experimental design: No comment
Validity of the findings: 1.-As conclusion of the of metabolites in CFCS by GC-MS analysis, the authors argue that the main component is “2,4 DTBP, with the highest % peak area of 59.24”, however these results do not shown peaks from lactic acid and acetic acid, that should be in a high levels. What causes that these substances cannot be detected by GC-MS? Both organic acids were not reported in the table 3.
2.-The authors propose that organic acids are the main responsible for antimicrobial activity. Because addition of alkali can increase pH and neutralize the acids, however when pH is modified, also many chemical groups from other molecules (not only acids) could lose its positive charges or even modify its structure. If one of these molecules were the main antibacterial component, it could lose its antibacterial activity? What additional evidence could you provide in order to demonstrate that organic acids are in fact the responsible for this activity?
3.-I recommend to review results about HPLC and quantification of acetic and lactic acids, because 261mM and 266 mM from theses acids, would represent approximately 1.6% and 2.4% (w/v) respectively. That is a minimum of 4% of these acids in the supernatant. It is possible that Weissella confuse can produce those levels of organic acid in medium MRS under the experimental incubation conditions performed in this work? Could you explain and show some evidences (may be articles agree with your data) that support your results.
Additional comments: No additional comments


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

Input:
Title: ORGANIC ACIDS AND 2,4-DI-TERT-BUTYLPHENOL: MAJOR COMPOUNDS OF WEISSELLA CONFUSA WM36 CELL-FREE SUPERNATANT AGAINST GROWTH, SURVIVAL AND VIRULENCE OF SALMONELLA TYPHI
Review round: 1
Reviewer: 2

Output:
Basic reporting: In Pelyuntha et al, the authors examine the effect of acid organics and 2,4,Di-tert-butylphenol of Weissella confusa WM36 under general pathogenesis of Salmonella Typhi. They found that adding the cell-free culture supernatant of W. confusa leads to decreased growth, biofilm formation, motility, and expression of virulence genes (mainly those that code for the type 3 secretion system effector proteins and quorum sensing. Besides, they found the main antibacterial compounds present in the supernatant of W. confusa, which they suggest could be good candidates molecules against typhoid fever.
Experimental design: The experiments are rigorous and the data presented clearly show an effect of the cell-free culture supernatant of W. confusa on growth, biofilm formation, motility and gene expression of some virulence factors of Salmonella Typhi.
Validity of the findings: No comment
Additional comments: The study is straight-forward and written in a clear, logical manner, and the data appear to be rigorous. My major concern is that the data cannot be extrapolated to infection, as adding the cell-free culture supernatant of Weissella confusa to broth cultures is very different than the bacteria interacting with organic acids and 2,4 DTBP. However, this paper could be promising for further studies, which analyze more the action of W. confusa metabolites against typhoid fever. Overall, authors used appropriate methods and report results in accessible form. Some of their interpretations could be modified/expanded.


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: ORGANIC ACIDS AND 2,4-DI-TERT-BUTYLPHENOL: MAJOR COMPOUNDS OF WEISSELLA CONFUSA WM36 CELL-FREE SUPERNATANT AGAINST GROWTH, SURVIVAL AND VIRULENCE OF SALMONELLA TYPHI
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The new manuscript fulfill all recommendations and suggestions of both reviewers, so I consider that now this paper is suitable 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: ORGANIC ACIDS AND 2,4-DI-TERT-BUTYLPHENOL: MAJOR COMPOUNDS OF WEISSELLA CONFUSA WM36 CELL-FREE SUPERNATANT AGAINST GROWTH, SURVIVAL AND VIRULENCE OF SALMONELLA TYPHI
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The results describe how the hypothesis raised during the project was fulfilled. It is a very complete and interesting 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: INTEGRATED BIOINFORMATICS ANALYSIS OF POTENTIAL PATHWAY BIOMARKERS USING ABNORMAL PROTEINS IN CLUBFOOT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The expression of the article is basically clear, with some points needing polishing, such as line 144.The overall structure of the article is clear, many tables and figures are good for easily understanding.
Experimental design: 1.The research idea of this article is simple and clear.However, the methods used in the 8 articles are different, and the location of the materials is also different. How much does this influence the experimental results?It needs to be explained in detail.
2.The principle and value of each step of the experimental method should be explained in more detail.
Validity of the findings: In this paper, only a few methods are used to obtain the possible important proteins. It would be more convincing if additional experiments were conducted to verify the results or the data from the database were still used for secondary verification.
Additional comments: The overall design of the article is novel and creative, clear results, sufficient analysis and discussion, detailed results are very valuable.However, the methods used in the 8 articles are different and the location of the materials is different, which should be explained.Secondly, it is better to increase the workload for the second verification of the results, so that the results are more convincing.


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: INTEGRATED BIOINFORMATICS ANALYSIS OF POTENTIAL PATHWAY BIOMARKERS USING ABNORMAL PROTEINS IN CLUBFOOT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article written by the authors is very clear, unambiguous and technically, but there need more carefully in paper. It’s better to replace “lead to” with “including” or “such as” at lines 51, because there is no causality. “Ponseti” should be lowercase at lines 58 and 60.
Experimental design: 1.PubMed and Science Direct described in the paper are commonly used by the bioinformatics community, but But why you don't try other databases? Such as TCGA and EBI. Please explain it.
2.It's better to supply a figure of PPI.
Validity of the findings: The problem addressed by the authors is important. Their key findings are beneficial to the medicine community to some extent.
Additional comments: No commens.


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: INTEGRATED BIOINFORMATICS ANALYSIS OF POTENTIAL PATHWAY BIOMARKERS USING ABNORMAL PROTEINS IN CLUBFOOT
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this paper, the authors provided a bioinformatic analysis for identifying potential biomarkers for clubfoot. The paper need be revised before publication, especially in language. The expresssion could be improved. I suggest invite a native speaker to check the main text for avoiding grammer mistakes.
Experimental design: The experimental design of this paper is okay. While it is can be improved. For instance, the identified biomarkers are based on a single dataset. I suggest use another similar datasets to check the consistency in the experiments.
Validity of the findings: The findings of biomarker need be validated. For instance, the identified biomakers for classifying the samples. The application of biomarkers in this paper is for diagnosis. The reader might have the wonder of the classification performance of these identified biomarkers. I suggest add a section about the justification of these identified biomarkers.
Additional comments: The authors provided many GO enrichment fucntional analyses, such as the figures. However, it is still not clear the dysfunctions of the deformity. Moreover, the newly findings in genetics about clubfoot need be summarized in the descriptions about the search in PubMed. The figures need be presented in higher resolution. Many figures can not be recognized. Please also refer to my former comments for revising the paper.


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

Input:
Title: INTEGRATED BIOINFORMATICS ANALYSIS OF POTENTIAL PATHWAY BIOMARKERS USING ABNORMAL PROTEINS IN CLUBFOOT
Review round: 1
Reviewer: 4

Output:
Basic reporting: Cai et al. present a bioinformatics analysis of genes implicated in developmental malformation (clubfoot). The authors performed a literature search, identified genes reported to be associated with the condition, and applied a range of bioinformatics approaches on those genes, including enrichment analysis, protein-protein interaction analysis, and others.

Shedding light on the pathogenesis of clubfoot is a worthwhile endeavor, given the high incidence of the condition. However, I have several major reservations on the manuscript:

- Goal of the project: It is not clear to me what the goal of the analysis is, and what actionable insights the large number of pathways that are predicted to be associated with the condition can provide. There is no overarching hypothesis that ties together the plethora of tools and approaches that were utilized.

- Approach: The authors performed a literature search to extract genes associated with the condition. However, the criteria that were used to retain or discard a PubMed or Science Direct entry are not specified. For example, on what grounds did the authors rejected 1015 articles? Study bias and selection bias are factors that need to be carefully considered here, especially when performing enrichment analysis.
It is also not clear what kind of associations were found between the genes they curated and the disease. Is it differential expression, mutation, or other?
Further, only a cursory description of the parameters used by each tool were given, and no rationale was provided for choosing a particular tool over another.

- Style: the manuscript has several typos throughout and many non-idiomatic expressions.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: INTEGRATED BIOINFORMATICS ANALYSIS OF POTENTIAL PATHWAY BIOMARKERS USING ABNORMAL PROTEINS IN CLUBFOOT
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this version, the authors have addressed most of my former comments. For all the comments received, the authors have revised the manuscript accordingly. The identified pathway biomarkers are with the potential in development of clinical applications.
Experimental design: The authors searched in literature to collect the genes related to clubfoot. Then the paper implements enriched analysis to identify pathways. Then the biomarkers have been identified by classification.
Validity of the findings: The authors need strengthen the justification of these novel biomarkers. This is very important in biomarker discovery. The authors need play attention to this point.
Additional comments: In this version, the authors have addressed most of my former comments. I appreciate the responses. I think this version is much better than the former one. I support its publication. The authors should review the pathway biomarkers because this is an important concept. Moreover, the validation the identified biomarkers is very important in clinics.


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: INTEGRATED BIOINFORMATICS ANALYSIS OF POTENTIAL PATHWAY BIOMARKERS USING ABNORMAL PROTEINS IN CLUBFOOT
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: No additional comments


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

Input:
Title: GINI COEFFICIENTS FOR MEASURING THE DISTRIBUTION OF SEXUALLY TRANSMITTED INFECTIONS AMONG INDIVIDUALS WITH DIFFERENT LEVELS OF SEXUAL ACTIVITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is clearly written, and well referenced. The paper is also well-structured, and assumptions and results are well described and presented.
Experimental design: The research question and objectives are well posed and clearly addressed. The analyses seemed to have been performed rigorously, and the right data was used.
Validity of the findings: Although the general idea (using Gini coefficient to make inference of biological parameters that determine STI transmission and to assess the impact of control measures) is interesting and potentially useful, the data used is still limited and therefore the uncertainties too large to make any strong conclusions, which has been stated as a limitation by the authors. Perhaps the authors should suggest/describe what data would be needed to be able to make stronger predictions/conclusions.
Additional comments: I think this is an interesting paper that makes a clear contribution to the field, thus I support its publication. However, I left a few comments throughout the paper for the authors to consider.


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: GINI COEFFICIENTS FOR MEASURING THE DISTRIBUTION OF SEXUALLY TRANSMITTED INFECTIONS AMONG INDIVIDUALS WITH DIFFERENT LEVELS OF SEXUAL ACTIVITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The document is consistent with the basic reporting guidelines, with the exceptions listed below.
The language of 80:83 could be improved for clarity and substance. Specifically (in order of importance):
1. Misc: It is not generally clear when the comparison of indices is in time, or when it is being compared to the ODE data.
2. 80:81 it would be helpful to clarify the comparison over time.
3. 82:83 is too long and the intent is unclear.
4. References in 163:167 (discussed later as well)
Experimental design: 1. Raw data showing the simulated Gini index and the true Gini index have not been provided.
2. It is not clear how and where the Gini index is used to correlate data from the ODE.
3. The Gini index as an indicator of imbalance in the data set has not been addressed clearly. That a higher Gini index shows imbalance should clearly be explained.
Validity of the findings: 1. 165:166 The change in the model parameter, and subsequent change in output has been linked to education and condom usage in an unsubstantiated manner. It is imperative to discuss the parameters of a campaign which would cause such a change in the model parameter. (Lee WC. [4]) describes a clearer example of simulating a parameter change.
2. 163:165 is also unsubstantiated.
3. The interpretation and understanding which is to be drawn from 209:210 (point estimates) is not clearly explained.
4. No data has been provided to describe how changes in the ODE model are able to provide insights to the Gini coefficient
5. 211:215 as noted previously, the changes need to be explained better. How is the transmission probability factored into the Gini coefficient?
Additional comments: 1. The analysis might be improved by incorporating the interpretation of the Gini index given by Rogerson, P.A. Lett Spat Resour Sci (2013) 6: 109. (https://doi.org/10.1007/s12076-013-0091-x).
2. Natsal-2 is not mentioned in the acknowledgements, though Natsal-3 has been mentioned.
3. It would be nice, but not necessary to have a plot showing the Lorentz curves

I am convinced that the authors have acted in good faith and commend their well commented R scripts on Github. I am certain that after the revisions requested have been addressed satisfactorily, the paper should 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: GINI COEFFICIENTS FOR MEASURING THE DISTRIBUTION OF SEXUALLY TRANSMITTED INFECTIONS AMONG INDIVIDUALS WITH DIFFERENT LEVELS OF SEXUAL ACTIVITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK
Additional comments: None


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

Input:
Title: GINI COEFFICIENTS FOR MEASURING THE DISTRIBUTION OF SEXUALLY TRANSMITTED INFECTIONS AMONG INDIVIDUALS WITH DIFFERENT LEVELS OF SEXUAL ACTIVITY
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comments.
Experimental design: No Comments.
Validity of the findings: No comments.
Additional comments: The authors have expanded the manuscript in response to the previous reviews in a commendable manner. The work presented, on the introduction and usage of the Gini coefficient for disease transmission is now clear. The authors have clarified the aims and the study is reproducible (with well documented code), and is a worthy contribution to the existing literature. The simulated usage of the Gini coefficient as a predictive tool has also been described well.


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: EVOLUTION OF SEXUAL SIZE DIMORPHISM IN THE WING MUSCULATURE OF DROSOPHILA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well written manuscript that is easily read and understood and conforms to PeerJ standards. The aims and conclusions of the study are clearly stated and well referenced. The figures are excellent quality and readily interpreted with fully explanatory legends. The raw numerical data has been supplied but not any of the original morphology studies. As such the numerical data do corresponds with the data used in the manuscript but the lack of the original morphological data is a bit of an issue.
Experimental design: The manuscript presents an interesting and original piece of primary research that fits comfortably with the scope of the journal. Whilst the work is primarily descriptive it addresses a fascinating and important area of evolutionary biology. The core approach is to use comparative and quantitative morphology to shed light on a subject that will be of interest not only to a specialist audience but also a more general scientific and even lay audience. The quality of the work is exceptional, it is a simple concept executed well and rigorously analysed. The methodologies are adequately described with sufficient detail. I am not qualified to comment on the phylogenetic methodologies used but I am fully familiar with morphometric studies. If I were to have a criticism is that the paper does not show any images of the muscles studied and it isn’t possible to assess what the muscles look like nor see how they differ in size and shape.  I am sure that the shape of the muscles may also provide interesting insights into the evolution of song. The paper could be improved by the inclusion of a figure showing how the muscles differ in proportion and shape even in there is nothing interesting in their shapes.
Validity of the findings: The lack of morphology aside the results are still very interesting and provide insight into a much-studied aspect of Drosophila biology. it is without doubt an interesting and important contribution that will stimulate further study.

I have no issues with grammar or the written English and have no changes to recommend.

In summary the work is simple and elegant in concept, excellent in execution and presentation. I would recommend acceptance with a suggestion of the additional anatomical figure.
Additional comments: I would encourage the authors to consider showing some of the morphological data, even if the analysis is qualitative.


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: EVOLUTION OF SEXUAL SIZE DIMORPHISM IN THE WING MUSCULATURE OF DROSOPHILA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Well written article
Experimental design: Well written.
Validity of the findings: Strong results, but  discussions speculative in parts.
Additional comments: In this manuscript, Tracy et al provide fascinating comparative insights into the structures of 4 steering muscles (hg1-4 muscles) which are associated with the actuation of the posterior notal wing process in Drosophila species. Previous work by Shirangi et al discovered the role of one of these muscles (hg1) in generating the ‘sine’ song during courtship in the case of D. melanogaster. This muscle was found to be sexually dimorphic, as it is much larger in males than in females.

In this manuscript, the authors extend this study by conducting a thorough exploration of size variation in hg1-4 muscles across 19 Drosophilid species. Their study finds a great degree of species-specific variation and size dimorphism in these muscles. Of these, hg1 muscles are most size dimorphic in the Oriental lineage (which diversified most recently). In comparison, the hg2 muscles are usually monomorphic, but in a few cases show size dimorphism and female enlargement. Similarly, hg3 muscles also show size dimorphism in some clades, but are usually monomorphic. In contrast to these, the hg4 muscle is entirely monomorphic in all species.

The authors discuss what these findings might mean in terms of their role in courtship song diversification and speciation. It must be said that the discussion is very speculative and often veers into the ‘why’ territory, but I did not feel that any of it was unreasonable. Indeed, it is rare to see studies that throw light on evolution of behavioural traits (in this case, courtship song) using carefully conducted morphological studies. Because the authors had already developed insights about hg1’s role in courtship song, their study and inferences are convincing, the methods are sound, and the paper is very well-written. As such I have no major comments or criticisms of the paper, and recommend it for publication.

I did note a few minor points that occurred to me while reading the manuscript:

1.	Line 256-260: “It is also possible that …. may have evolved to prevent dimorphism in a monomorphic behaviour”. This entire paragraph is much too speculative and should be omitted in my opinion. It is true that steering muscles also play a role in flight, and hence any ‘song-specific’ changes in their structure must potentially trade-off with flight performance. The only problem is: we don’t fully understand their role in flight. A recent paper by Lindsay et al (which is cited in this paper) demonstrated the functional organization of these steering muscles into two specific functional groups - the larger phasically activated muscles that actuate the wing movements required during sudden turns, and the smaller tonically-activated muscles whose role is subtler but continuous. Lindsay et al find that hg1 and hg3 are phasic muscles, whereas this paper shows significant size dimorphism in these muscles, which may in principle influence flight. The zeroth-order experiment would be to knock out these muscles individually or alter their relative sizes, and test the effect on flight performance which I hope will be conducted in due course. Also, the authors often write sentences of the form “X evolved for Y purpose”. Such sentence construction is problematic and should be avoided, as it suggests directionality and purpose to evolution.

2.	The entire discussion on song production emphasizes the role of muscles, but entirely ignores exoskeletal structures and their biomechanics which also must co-evolve with these muscles. Actuation of these inter-connected skeletal structures dictates how wings move in response to muscle activity. These have not been dealt with in the paper, but a sentence or two to convey their importance may be worth including.

3.	Line 254: “raise a hypothesis”?  Sounds awkward.

4.	Fig 2: I felt that choosing a Cohen's d value of 1.5 as the cut-off between dimorphic and monomorphic was somewhat arbitrary, although I do not entirely disagree with that choice in cases for which there was a clear gap in size variation. However, in the case of hg1, there is no clear gap and hence the difference between mono- and dimorphic seems trickier to nail. Can the authors please comment on this?


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: AUTOPHAGY-RELATED GENES PROGNOSIS SIGNATURE AS POTENTIAL PREDICTIVE MARKERS FOR IMMUNOTHERAPY IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, the authors demonstrated that many Autophagy-related genes are differentially expressed in hepatocellular carcinoma (HCC). The authors further showed the potential involvement of  PI3K-AKT-mTOR signaling pathway candidates in HCC and concluded that the prognostic effects of ATGs could be useful.
Experimental design: All experiments were designed clearly.
Validity of the findings: The results of the bioinformatic studies were well stated. However, most of the findings were either well-expected or well known in the field. May be, supplementation of these findings with in vitro/in vivo studies could be interesting and useful for the manuscript.
Additional comments: This manuscript looked at the potential use of ATGs as a marker for Hepatocellular carcinoma. The role of autophagy in HCC is already reported and well documented. Previous studies clearly established the inhibitory effect of ATGs on PI3K-AKT-mTOR signaling, which in turn may promote tumor progression. In this study, analyzing RNA-seq transcriptome profiles of HCC patients unsurprisingly enriched multiple differentially expressed genes in the ATG family. This enrichment is well expected from the already published results and unfortunately lacks novelty. However, the differential investigation of biomarkers using immune cell infiltration is relatively new and required to be investigated more, especially in today's world. My suggestion to the authors is to either concentrate on the novel part of the manuscript and try to build a story on it or to follow up on the claims within vitro/in vivo results.


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

Input:
Title: AUTOPHAGY-RELATED GENES PROGNOSIS SIGNATURE AS POTENTIAL PREDICTIVE MARKERS FOR IMMUNOTHERAPY IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Mao et al. characterized differentially expression of multiple autophagy related genes in tumor and normal tissue, and proposed the expression can be used as prognostic marker for certain liver cancer. The authors further showed that these upregulation of ATG is related to immune cell infiltration, which provide a mechanism basis for cancer immunotherapy strategies. In principle, this is an interesting study.

1, Fig.1A, In x-axis, what are the color codes cyan and red stand for? Is data regarding gene expression of normal tissue also included?
2, Fig. 1B, the GO and KEGG data is not showing in a  standard fashion. Please modify it to show a bar plot with p-value.
3, Page 12, lane 124, the figure legend should not be inserted into the main text.
Experimental design: 4, The work is largely based on the bioinformatic analysis. To ensure the reproducibility, the authors should include the original code of all the bioinformatic analysis in the supplemental data.
Validity of the findings: 5, Page 15, lane 165. The authors wrote that “the above results fully demonstrate that the prognosis risk signature is the driving factor for the malignant progression of the tumor”, which is overstatement of the data . Actually, all analysis showing here can only provide a correlation between tumor malignancy and  these markers.
6, Page 15, lane 173-174. I disagree the title used here “Autophagy-related genes promote HCC progression by inhibiting PI3K-AKT-mTOR signaling. As discussed above, the analysis itself doesn’t support this claim.  Please modify the title as well as the conclusion in lane 192-193, “we further confirmed that autophagy promotes the malignant process of HCC by inhibiting PI3K-AKT-mTOR signaling.”
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: AUTOPHAGY-RELATED GENES PROGNOSIS SIGNATURE AS POTENTIAL PREDICTIVE MARKERS FOR IMMUNOTHERAPY IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors clearly reported the study.
Experimental design: The experiment is well designed.
Validity of the findings: All the original codes is now provided.
Additional comments: The authors have well addressed my concerns.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF NADPH OXIDASE GENES IN RESPONSE TO ABA AND ABIOTIC STRESSES, AND IN FIBRE FORMATION IN GOSSYPIUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The text is well write, however need to check some minor changes, particularly in the correct way to write the scientific names.
The references used are adequate.
The results are relevant in understanding the role that Rbohs play during the development of fibers in Gossypium.
Line 168> change “everage” by “average”
Line 174: change “V” by “VI”, in the phylogenetic tree the subfamily VI contained 6 member, while subfamily V contains 18 members.
Line 241: write the scientific names in cursive.
Line 251: change “Cacao” by “cacao”; “Communis” by “communis”; “Trichocarpa” by “trichocarpa”
Line 252: change “Distachyon” by “distachyon”, “Sativa” by “sativa”
Line 255: change “Distachyon” by “distachyon”; “Sativa” by “sativa”
In the Figure 5 change “R. Communis” by “R. communis”
Line 277: use another synonymous of “rose” maybe “increased”
Line 357: change “Cacao” by “cacao”; “Communis” by “communis”; “Trichocarpa” by “trichocarpa”; “Distachyon” by “distachyon”; “Sativa” by “sativa”
Experimental design: The methods are well described.
Validity of the findings: The data shows are clear,

The conclusions are well documented according with the results, however additional information about the effects of abiotic and ABA treatments could help to understand the role of Rbohs on growth and development of fiber in Gossypium.
Additional comments: The present study by Zhang and collaborators “Genome-wide identification and expression analysis of NADPH oxidase genes in response to ABA and abiotic stresses, and in fibre formation in Gossypium”, characterizes NADPH oxidases (Rbohs) in different Gossypium species (G. arboretum, G. raimondii, G. hirsutum and G. barbadense). The results indicated a preferential expression in the floral tissue, which the author suggests a possible role of Rbohs in the fibers formation and abiotic responses.
The document is well write, the authors need to correct the writing of scientific names, the author don`t shows whether the abiotic stress and ABA treatment have an effect on development of fiber in Gossypium only the levels of expression of Rbohs that could help to understand the role of Rbohs.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF NADPH OXIDASE GENES IN RESPONSE TO ABA AND ABIOTIC STRESSES, AND IN FIBRE FORMATION IN GOSSYPIUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this work Gaofeng Zhang et. al. analyze Rboh gene family in four species from Gossypium genus, additionally they evaluate this gene family gene expression in G. hirsutum. The aim and scope of the article is very interesting and relevant. The manuscript is clear and unambiguous, and well writing.

The authors provide enough references and field background; however, the authors could take into account the next references:

Li, P. T., Chen, T. T., Lu, Q. W., Ge, Q., Gong, W. K., Liu, A. Y., ... & Li, S. Q. (2019). Transcriptomic and biochemical analysis of upland cotton (Gossypium hirsutum) and a chromosome segment substitution line from G. hirsutum× G. barbadense in response to Verticillium dahliae infection. BMC plant biology, 19(1), 19.

Wang, F., Chen, Z. H., Liu, X., Shabala, L., Yu, M., Zhou, M., ... & Shabala, S. (2019). The loss of RBOHD function modulates root adaptive responses to combined hypoxia and salinity stress in Arabidopsis. Environmental and experimental botany, 158, 125-135.

Zhang, F., Zhu, G., Du, L., Shang, X., Cheng, C., Yang, B., ... & Guo, W. (2016). Genetic regulation of salt stress tolerance revealed by RNA-Seq in cotton diploid wild species, Gossypium davidsonii. Scientific reports, 6, 20582.

The structure of the article has a standard format. The figures are relevant and help to understand the paper. However, the author should be considerate represent figures 6-10 as tables, for a better results interpretation. The paper presents enough ‘self-contained’ results to represent an appropriate ‘unit of publication’.
Experimental design: Bioinformatics and experimental findings are original and within Aims and Scope of the journal. The identified knowledge gap (knowledge about genomic information and genetic evolution of Rbohs gene families in Gossypium genus) is well filled, following high technical standard. However, the phylogenetic reconstruction should be better described.
Validity of the findings: The majority of the results and findings are well supported. However, there are some issues that must be clarified.

Gene identification was made using similarity, nevertheless in the multiple species phylogeny (Figure 5), this classification did not totally match, for example, F4 genes (Family VII in Figure 1) clusters with AtRbohH (and AtRbohJ), so this suggest that this group might be RbohH or RbohJ-like proteins. F cluster (family II) groups whit AtRbohF and AtRbohI, so this is not clear if family II might be RbohF or RbohF proteins. A table with similarity values from Blast search (with three best hits) must be added as supplemental material to clarify last two points.

Family number legends from Figure 1 must be show in Figure 5.
OTUS legends in Figure 5 must be the same that legends used in Figure 1.
Bootstrap values (>70) in Figures 1 and 5 must be showed.

It is widely recommended to perform a statistics analysis of expression profiles/patterns experiments to determine significant differences between treatments.
Additional comments: No additional comments


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF NADPH OXIDASE GENES IN RESPONSE TO ABA AND ABIOTIC STRESSES, AND IN FIBRE FORMATION IN GOSSYPIUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF NADPH OXIDASE GENES IN RESPONSE TO ABA AND ABIOTIC STRESSES, AND IN FIBRE FORMATION IN GOSSYPIUM
Review round: 2
Reviewer: 2

Output:
Basic reporting: In this work Gaofeng Zhang et. al. analyzed Rboh gene family in four species from Gossypium genus, additionally they evaluate this gene family gene expression in G. hirsutum. The aim and scope of the article is very interesting and relevant. The manuscript is clear and unambiguous, and well writing.

The authors provide enough references and field background and include those ones that were recommended.

The structure of this document version has a standard format. The figures are relevant and help to understand the paper and added information that was required as supplementary materials.

The paper presents enough ‘self-contained’ results to represent an appropriate ‘unit of publication’.
Experimental design: Bioinformatics and experimental findings are original and within Aims and Scope of the journal. The identified knowledge gap (knowledge about genomic information and genetic evolution of Rbohs gene families in Gossypium genus) is well filled, following high technical standard.

In this version, the authors describe the methodology in a good manner as it was required.

It is recommended to perform an anova statistical analysis.
Validity of the findings: The majority of the results and findings are well supported. The authors made the corrections that were required. However, the new information has allowed us to observe some issues that must be addressed before the document is accepted.

1.	Phylogenetic analysis (Figure 5) and Blast results (Table S7, lines 43-45-Cotton_A_13344, and lines 129-132, Gorai.008G199100) showed that group VII (Gossypium Rbohs J) formed a new paralogous group since AtRbohH and AtRbohJ clustered together with Gossypium Rbohs group VI (Gossypium Rbohs H); group VI and VII are well supported by a high bootstrap values, suggesting that are reliable groups. Furthermore, group VII proteins domains analysis (Figure 1C) sustain this idea. In this case, the sequence alignment length of the Rbohs with AtRbohJ and the Maximum-likelihood values (that must be showed) do not validate the result presented since this values can be explained due to evolutive distance of all AtRboh proteins, therefore, tree topology must be taken in count and Gossypium Rbohs J must be renamed (as author decided) as a new kind of Rbohs proteins.

2.	In the same way tree topology showed that Gossypium Rbohs C (group V) is also a new paralogous group and must be renamed as a new kind of Rbohs proteins.

3.	It is widely recommended performing an ANOVA statistical analysis of expression profiles/patterns experiments to determine significant differences between treatments since with Student’s t test it is not possible to analyze between different tissue and time.

Minor issue:
•	In lines 341-342 is mentioned that GaRbohJ, GrRbohF-1, GrRbohF-3 and GrRbohD-1 had no orthologs in G. barbadense, however list of proteins appear GbRbohJD, GbRbohF-1A, GbRbohF-3A and GbRbohD-1A, this proteins and tree topology shows that are orthologs.
Additional comments: No additional comments