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: OBSTETRIC AND PERINATAL OUTCOMES OF DIZYGOTIC TWIN PREGNANCIES RESULTING FROM IN VITRO FERTILIZATION VERSUS SPONTANEOUS CONCEPTION: A RETROSPECTIVE STUDY
Review round: 3
Reviewer: 2

Output:
Basic reporting: The English may benefit from some polishing but is understandable.
Literature references rather selected but adequate.
Article structure OK
As apparent from my detailed comments there are still unclear parts.
Experimental design: No comment, see omments to authors
Validity of the findings: Some questions about data interpretation remain.
Additional comments: The authors have made a number of changes in their manuscript. I still have some unanswered questions and comments.
Major comments
1.	Table 1 marks four significances: high maternal age and primiparity (which is a truism), mean gestational age, and “first cesarean section”. As I pointed out, the difference in mean gestational length is completely explained by the difference in gestational length determination for the two groups. It amounts to 0.34 weeks which means a little less than two and a half days. As now pointed out in the text (Line 205 but it should say 0.34 weeks, not days) this is reasonably an effect of the fact that culture time (2-5 days) was not included in the pregnancy length estimates for IVF twins. If this cannot be adjusted in the analysis, I suggest that a note is placed in Table 1 explaining the apparently significant difference.
2.	The second finding in Table 1 is an increased risk for First Cesarean Section. In my previous review I pointed out that this is a direct effect of the fact that there are more primiparous mothers in the IFV group than in the SCB group – if a woman has her first delivery and C-section, then it must be her first C-section! One therefore has to restrict the comparison to women with at least one previous delivery (and then this finding disappears).
3.	Table 4 in the previous manuscript has been removed as far as I can see but is still mentioned in line 174. According to this text and Table 3 there should be three malformed or stillborn infants in the SC group among them (according to Table 4 in the previous edition) one is a neonatal death. If one wants to add this into the analysis one should call it perinatal death and not stillbirth. In the IVF group there were four stillbirths and three malformed infants. The latter may (judge from the previous Table 4) be two heart defects and one jejunal atresia. But in Table 3, there are three and 12 cases listed and I think it should be three and seven, not twelve. Then – if we accept the debatable method to add the two very different outcomes perinatal deaths and malformations – we have 3/221 and 7/247 which (using exact Fisher analysis) gives a p value of 0.37 and an OR of 2.1 with a 95% CI of 0.5-12.7 thus far from statistically significant and with very low precision in the estimate. One gets roughly the same values if on uses the number of twins instead of number of twin births in the denominators. The mark of statistical significance in Table 3 thus seems incorrect. In Line 174 it is said that NICU admission is not statistically significantly increased – but in Table 3 it is marked as significant. Actually, as far as I can understand, this is the only convincing difference between the two groups. The odds ratio for NICU transfer of at least one of the twins is 2.2 (95% CI 1.3-3.7). Another matter is what can be the explanation to this.
4.	I cannot anywhere in the manuscript find any comment on the fact that there are ties between data for the two twins in any twin pair why statistical analyses get complicated, notably in relatively small materials. Let us take the example of stillbirths. There were four stillbirths in the IVF material – were they in four different pairs or were two of them in the same pair? Obviously the risk for one of the twins to be stillborn is increased if the other is stillborn.
5.	I also think that it would be useful if the authors point out that some of the findings of no difference between SC and IVF twins could be due to the limited material, notably in some of the outcomes listed in Table 2, e.g., placenta previa. The odds ratio for this is 2.2 (95% CI 0.7-8.0) – the study thus does not exclude an eight time increased risk! So be careful to say that your study shows that there is no increased risk, as stated in the Abstract. What you can say is that your study did not reveal an increased risk.
Minor comment
1.	Generally you have given authors names in Italian style in the manuscript but in the first paragraph of the Introduction it is not.
2.	Line 95: In the IVF-conceived---, line 108: in the first trimester, line 126 exposed part--, line 160 Remove And.


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: OBSTETRIC AND PERINATAL OUTCOMES OF DIZYGOTIC TWIN PREGNANCIES RESULTING FROM IN VITRO FERTILIZATION VERSUS SPONTANEOUS CONCEPTION: A RETROSPECTIVE STUDY
Review round: 4
Reviewer: 1

Output:
Basic reporting: Adequate. No more comments.
Experimental design: Adequate. No more comments.
Validity of the findings: Adequate. No more comments.
Additional comments: Authors did not correct the Abstract as suggested. It is still short and uninformative. In the previous revision I have written the whole abstract as it is supposed to be in the text. The only thing that authors had to do was to copy and paste it in their text. But, they did not do that. Authors need to correct the abstract as suggested.


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: OBSTETRIC AND PERINATAL OUTCOMES OF DIZYGOTIC TWIN PREGNANCIES RESULTING FROM IN VITRO FERTILIZATION VERSUS SPONTANEOUS CONCEPTION: A RETROSPECTIVE STUDY
Review round: 4
Reviewer: 2

Output:
Basic reporting: See previous reviews
Experimental design: See previous review
Validity of the findings: What is still missing is that the authors have not (as far as I can find) pointed out the relatively small size of the material which makes the "negative" outcome of the study less valuable. This will, however, be obvious for every reader of the paper.
Additional comments: The majority of my concerns have been answered and the text has been adequately modified.


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: RAPID SCREENING MUTATIONS OF FIRST-LINE-DRUG-RESISTANT GENES IN MYCOBACTERIUM TUBERCULOSIS STRAINS BY ALLELE-SPECIFIC REAL-TIME QUANTITATIVE PCR
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written. Some minors errors are present as follows:

1.	Line 18: “detection” instead of “detecting”
2.	Line 19: AS-PCR is defined on Line 22, yet mentioned here first. Maybe a good idea to define it here.
3.	Line 25: “Specificity” not “Specific”
4.	Line 27-29: The meaning of the sentence is not conveyed clearly. Maybe consider re-writing it.
5.	Line 41: “$” instead of “dollars”
6.	Line 70: use of upper case letters in the name of the hospital “Kunming Third People’s Hospital” and not “Kunming third People’s Hospital”
7.	Line 74: “a total” not “totally”
8.	Lines 74-76: These seem to be out of place here. Maybe they need to be moved into the end of the introduction or to the beginning of the results to validate why the authors chose to create primers for the specific alleles they studied.
9.	Line 85: “in” not “at”
10.	Line 86: In the description of the contents of 2×TSINGKE master mix, the amounts and the units are after the component “….(….DNA polymerase 1 U，MgCl2 1.5 mM….)…”. Conventionally, the amounts are written before the units, for example”….(…. 1 U DNA polymerase，1.5 mM MgCl2…..)…”
11.	Line 108: “…..optimizing the conditions of these primers….” should probably read “….optimizing the conditions of PCR with these primers….”
12.	Line 112: does “high quality” mean “high fidelity”? If not, the authors should define what it means.
13.	Also check spacing between words and brackets.

Use of references:

1.	Line 107: A reference seems to be missing here.

Comments about figures and tables:

1.	Figures 4 and S2: The numbers on the axes and the labels are too small.
2.	Figure 2: Why does each gel have so many samples? There is no explanation in the text or the figure legend.
3.	Figure S1: the lower bracket with the size of the product is confusing. Could the authors use arrows instead as they have done for the markers?
4.	Tables: check spacing between words
Experimental design: The experiments are well described. However, there are some aspects that could use more information. My comments are as follows for the Methods section of the manuscript:

1.	Lines 103-104: A sensitivity assay is mentioned, but there is no description of how the sensitivity was assessed. The text in the results suggests that the sensitivity was determined based on the appearance and intensity of the product on the gel. One sentence stating this would be helpful here.

2.	A real-time PCR was performed. However, there is no description of the method. A method description mentioning the templates, the primers, their respective concentrations and the reagents used would be helpful in reproducing the results.
Validity of the findings: The results are presented well and the conclusions are well described. However, in Line 27, the authors claim "....sensitivity of third-quarters primers was 500 copy numbers....", whereas in Line 124 and 125 it is said "....half of these primers could amplify an observable..." . Could the authors clarify which one is correct?
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: RAPID SCREENING MUTATIONS OF FIRST-LINE-DRUG-RESISTANT GENES IN MYCOBACTERIUM TUBERCULOSIS STRAINS BY ALLELE-SPECIFIC REAL-TIME QUANTITATIVE PCR
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present work "Rapidly screening mutations of first-line-drug-resistant genes in Mycobacterium tuberculosis strains by allele-specific real-time quantitative PCR" is a methodologically well conducted study addressing an important topic on the field of tuberculosis research as the design of faster and accurate methods for drug resistance profiling of strains is. The authors described a new protocol based or single nucleotide polimorphysm detection by real time PCR from M. tuberculosis isolates.
However, I have several major concerns about how this work is presented on the current version:
1. The information provided as background on the introduction is really poor. On lines 55-60 the authors simple describe the genes responsible for drug resistance but do not detailed the main mutations described worldwide that explain drug resistance on TB isolates. For instance, KatG 315 mutation is responsible for approximately 60% of isoniazide resistance and mutations 531, 516 and 526 at hotspot on rpoB are responsible for 90% or more of rifampicine resistance. This information is crucial to de included for all he 4 genes on this study.
2. It is only clear to me which mutations and why were included on this study after reading a reference (Li et al, 2017) detailed only on methods. Results section (lines 107 and 108) starts with authors saying that 16 mutations included on this work were described on a previous study, that must me cited again. On that paper, a quite unique mutation frequency distribution is described by the same authors on this paper for the Yunnan region of China. I am assuming that based on that findings those mutations where the one selected to develop this new protocol. However,  this point is mandatory to be address on this manuscript introduction and discussion sections, otherwise the reader will not understand why these mutations were selected when they are not the most common one worldwide. So far, a table with the codon location for all the mutations included should be somewhere on the manuscript (I am assuming the mutations codon are the one on the name of the primers on supplementary figure 1). Additionally, the authors need to explain what proportion of each kind of drug resistance is caused by the mutations included on the study. Most common mutations like the detailed on paragraph above were not selected so the future impact of this new protocol would be limited to the Yunnan region and that should be said. Some kind of comparison and contextualization with Gene-Xpert is missing too, at least regarding mutations on rpoB, and how improve "local" protocols based on particularly epidemiological scenarios like on Yunnan are neccessary.
3. The articule structure needs to be deeply changed. While on figure 1 all the genes studied are included it looks like the authors only show one of the mutation on rpsL as "an example" or something like that, while the rest of the mutations on rpsL and even the rest of the genes are included as supplementary material. This makes the article really difficult to read. Additionally, fundamental data simply cannot be presented as supplementary material. Instead of showing 14 samples run for rspL mutation, authors can show in figures on the paper 1 or 2 samples for each mutation of each gene and then a supplementary figure for the 14 samples for each mutation and each gene.
4. No clear explanation is made of why only 13 of the 16 mutations are tested on the real time PCR protocols. On line 24 of the abstract, authors says that "intutuively" they selected 13 mutations. What do the mean by "intutitevely'?

Additionally, other minor comments:
1. I will recommend to review grammar and vocabulary on the whole text by a native speaker. I am not a native english speaker but I believe there are corrections that needs to me made. For example, I believe the title is wrongly written, and instead of "rapidly screening" it should be "rapid screening". On line 48, "persons" is incorrect, it should be people.
2. In the abstract, line 27, instead of saying "third quarters" and "the others", please simple say the number of mutations with the different copy number level of mutations. Otherwise sounds not accurate.
3. On the introduction, line 41. It is not possible that 8 million dollars is the worldwide expense on TB. 8 billion perhaps? Please correct.
4. Please, update epidemiology TB data used on introduction using World TB report from 2018 or at least 2017. But authors are citing 2015 report.
5. Figure design might be improved. Use of red markers is unnecesary. Instead of 1 and 2 directly use wt (wild type) and mut (mutants). No need of M for DNA ladders and no need to include DL2000 ladder on the legend.
6. Figure 1. NC is not stated on the figure legend as Negative control.
7. Supplementary material. Figure and table legends are poorly detailed.
Experimental design: The experimental design is methodologically well address despite the lack of conceptualization described on the "Basic reporting" section. But several minor comments deserve to be done:
1. Better description of samples used on the study. Pulmonary and extrapulmonary? Age range? Also I believed that the 14 samples used must be the ones described and not the 57 isolates used for 16 mutations identification on the previous paper (Li et al, 2017).
2. Line 82. When describing primer designing, at 3', "non complementary nucleotide" is the correct term better than "random".
3. On figure 2, why 14 samples are included on panels A and B but only 7 in panels C and D?
4. On supplementary figure, for KatG mutations not always the copy number tested were the same for the different mutations. Is there any reason for that? For the other genes, all the mutations are tested with same copy numbers alternatives.
Validity of the findings: Regarding the validity of the findings, although data is robust and conclusion well stated, like in the introduction, results are not properly discussed and contextualized. All the comments made on point 1 and 2 at the "Basic reporting" section should be taking in consideration for rewriting the discussion.
Additional comments: No additional comments


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

Input:
Title: RAPID SCREENING MUTATIONS OF FIRST-LINE-DRUG-RESISTANT GENES IN MYCOBACTERIUM TUBERCULOSIS STRAINS BY ALLELE-SPECIFIC REAL-TIME QUANTITATIVE PCR
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all the concerns raised previously. Please check all spellings.
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: RAPID SCREENING MUTATIONS OF FIRST-LINE-DRUG-RESISTANT GENES IN MYCOBACTERIUM TUBERCULOSIS STRAINS BY ALLELE-SPECIFIC REAL-TIME QUANTITATIVE PCR
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have properly address on the response letter and on the manuscript all the major and minor concerns detailed on the first review. Introduction and discussion have been extraordinarly improved.
Experimental design: The authors have properly address on the response letter and on the manuscript all the major and minor concerns detailed on the first review regarding the experimental design.
Validity of the findings: Discussion has being greatly improved following reviewers suggestions allowing readers a better conceptualization of the importance of this research findings.
Additional comments: No additional comments


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

Input:
Title: UNDERSTANDING THE EXPERIENCE OF PSYCHOPATHOLOGY AFTER INTIMATE PARTNER VIOLENCE: THE ROLE OF PERSONALITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some sentences need to be written more clearly, for example, In line 133-136, page 9.
Experimental design: 1. In line 190, page 10. The sample size is 50 for IPV group and 50 for control. Do you have a sample size calculation? Or you should emphasize it on the limitation.

2. In line 192, page 10. Women in the IPV group were recruited from projects whose main goal was to support victims of intimate violence. I am concerning the representative of the sample, how about these women were recruited? One of the most possible reason, maybe these women have psychopathological symptoms and join the projects looking for help. How about other women who were victims without looking for help?
Validity of the findings: In line 242 of page 12, more detail of the demographic variable should be given, you’d better list in a table.
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: UNDERSTANDING THE EXPERIENCE OF PSYCHOPATHOLOGY AFTER INTIMATE PARTNER VIOLENCE: THE ROLE OF PERSONALITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting study focusing on the association between personality and psychopathological symptoms in women victims of intimate partner violence.
a.	The writing needs further revision for English language. For example, on Line 183, there are two aims of this study, and it should be “the aims of the present article were…”.
b.	In the introduction on Line 59 and 66, there should be citations for the prevalence of IPV in Western Europe and the Cloninger’s model.
c.	Because the participants were recruited in Portugal, what is the prevalence of IPV against women there, including physical, psychological and sexual violence?
d.	The structure conforms to PeerJ standards.
e.	The authors mentioned there are figures in the current manuscript, but there were not figures.
f.	All the titles for tables should be more concise. In tables, “NS”,”HA”,”PS” and so on should use their full spells, or list the full spell under the table. Meanwhile, the authors did not explain what NS1, HA1, PS1 and etc. stood for. If these meant different levels of each dimensions, the explanation should be showed in method section.
g.	The raw data is supplied.
Experimental design: a.	The study fits the Scope of the journal.
b.	The research question is defined.
However, I am concerned that will the victimization have direct or indirect impact on personality and interfere with the association between personality and psychopathological symptoms?
The research questions identified knowledge gap.
c.	The survey was conducted following an ethical standard.
d.	Methods described are sufficient. But here are my comments on the methods as following:
	There are no clear inclusion and exclusion criteria for both IPV victims and the comparison group.
	How did the authors determine the sample size for victims and non-victims?
	If victims and non-victims were paired to have the same sample size, what was the pairing process?
	The authors stated in the manuscript that they did not randomly recruit participants for control group. This could interfere the validity of results.
	The alpha values of TCI-R and BSI in this study should be reported.
	On line 227 to 229, the authors stated that they randomly selected half of participants to complete TCI-R first. Please explain the reason.
Validity of the findings: a.	Line 243 to 249, the authors described the sociodemographic information for both groups. However, it was only stated as “of similar age, occupational and educational status” without providing any statistical values to support the conclusion.
The distribution of marriage status, education, occupation should be compared between two groups.

I suggest the authors make a table for the sociodemographic information.
b.	Line 251 to line 259:
	There is no Figure 1 in the manuscript.
	In Table 1, the author did not explain the meanings of each subgroup under NS, HA, RD, PS, SD, CO and ST, nor discuss the differences between victims and non-victims, most of which showed significant differences with p value under 0.05.
c.	Line 261 to 266:
	There is no Figure 2.
	In Table 2, if the authors decided to show t value, please keep all values in two decimals.
d.	Line 275 to Line 284:
The authors applied dependent regressions to examine the association between personality and psychopathological symptoms. However, the analysis missed several important factors:
	First of all, it should be more clearer according to Line 238 to 240 whether the regression only included victims.
	In the regression analysis, the authors did not consider the impact of sociodemographic factors including age, education, occupation, marriage, etc.
	The analysis did not consider the interactions between different personalities on symptoms.
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: UNDERSTANDING THE EXPERIENCE OF PSYCHOPATHOLOGY AFTER INTIMATE PARTNER VIOLENCE: THE ROLE OF PERSONALITY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Moreira et al. conducted a cross-sectional study to evaluate the differences in psychopathology symptoms between women victims and non-victims of IPV, and explore the associations between personality and psychopathological symptoms in women victims of IPV. Generally speaking, the manuscript is clearly presented and adds some new knowledge to existing literature, in my view.
Experimental design: Cross-sectional design.
Validity of the findings: The authors should report the power of the main analyses to support their conclusion.
Additional comments: 1. Were the women victims of IPV consecutively recruited from the projects?
2. The power of main analysis should be given.
3. You have performed many t-tests and correction analyses. I am wondering under what circumstances it is more appropriate to use a smaller alpha value instead of the standard 0.05.


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: UNDERSTANDING THE EXPERIENCE OF PSYCHOPATHOLOGY AFTER INTIMATE PARTNER VIOLENCE: THE ROLE OF PERSONALITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clear
Experimental design: Meet the standards
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: UNDERSTANDING THE EXPERIENCE OF PSYCHOPATHOLOGY AFTER INTIMATE PARTNER VIOLENCE: THE ROLE OF PERSONALITY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1. The power analysis is still unclear. You should provide the parameters which were used in the power analysis. Then, the reviewers can repeat your analysis.
2. In Tables 2 to 3, it seems that TCI and BSI scores between IPV and on-IPV are compared by independent-samples t-test but the study design is 1:1 matched case-control according to the descriptions on the methodology of this study. In this case, paried-samples t test should be used.


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: IOCBIO SPARKS DETECTION AND ANALYSIS SOFTWARE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present a new open-source software toolbox for detecting and analyzing calcium release events (called sparks) in confocal images of cardiomyocytes. Calcium sparks provide insights into the calcium handling within the cell which in turn informs the excitation-contraction dynamics in the cardiac cell. The software is claimed to improve that process because it is specialized to detect calcium sparks only.

The main finding is the detection rates of the proposed algorithm (Figure 5) and the comparison to that of the competing SparkMaster software (Figure 8). I found both of those figures rather uncoventional and hence somewhat difficult to interpret. I felt that data might be better shown as conventional Receiver-Operating-Characteristic (ROC) curves.

Otherwise, the paper seems to be technically sound although I felt that it would benefit from some reorganising to bring the GUI (Figure 6) and the detection rates (Figures 5 & 8) forward. Doing so would give the reader an early insight into main findings before getting into the details of the methods.
Experimental design: The principal outcome is software so experimental design is not really an issue.  As mentioned above, I suspect that ROC curves might be a better way to present the detection-rate data.

The experimental preparation of the mice cardiomyocytes is outside of my expertise, so I make no comment on those.
Validity of the findings: No comment
Additional comments: Minor points

line 50: I suggest making your GUI screenshot (Fig 6) the first Figure in your paper. You could describe it here at line 50. That would give the reader an immediate feel for your software before getting into the details of the algorithms.

line 52: Better wording might be: "Lastly, the output is saved to an SQL database for inter-operability with third-party applications."

Figure 3: I think you can omit Figure 3. The reader is unlikely to care about the details of the database schema.

line 100: "used animals" -> "animals used" or "animal data"

line 193: grammar: "statistical measures are reported as"

Figure 4: This is a nice Figure.

line 202-203: I did not understand this sentence. "As a result, the signal-to-noise ratio (SNR) of the background (here, mean value divided by standard deviation is used as SNR) is defined by its mean value."

Figure 5B: The caption is confusing. Can you please clarify. I suspect that a Receiver-operating-characteristic (ROC) curve might be more appropriate.

line 224: Suggest rewording to " The time series data for the entire recording is shown in the central of the GUI. It can display large datasets. For example, our studies routinely used 512 x 60000 samples."

Discussion: I suggest rewriting to recapitulate what you FOUND rather than what you DID. For example (lines 348-350) We analyzed the sparks detection algorithm and found that sparks were detected reliably under xxxx conditions. In comparison with SparkMaster, both algorithms detected the same true positive but our software detected far less false positives (perhaps even quantify this with some numbers).


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: IOCBIO SPARKS DETECTION AND ANALYSIS SOFTWARE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Laasmaa et al. are proposing an interesting new software for analyzing calcium sparks which fulfill a lack of open source automated software specifically designed for Ca2+ sparks detection/analysis. This new software has many improvements in comparison with the most used Sparkmaster plugin, and it is more versatile than the software actually available. I found particularly interesting, inter alia, that the user can examine each spark individually and decide to include new ones of to exclude/eliminate not desired false sparks for example. It also allows data storage/handling, and a friendlier GUI than other spark detection software available.
While the proposed software by the authors emerges as an interesting alternative to the actual widely used software, I have the following comments on the manuscript.

Minor comments:
1. Line 7 of first paragraph of introduction: Ca2+ sparks are defined as the fluorescent event detected by confocal microscopy, and not the SR Ca2+ release itself. So, when defining Ca2+ sparks in line 30, use “Ca2+ sparks” after "change in fluorescence of a Ca2+ sensitive dye”.
2. Delete double “the” in Line 339
3. Delete double “the” in Lines 394-395
Experimental design: When opening a file, the software estimates the background image F0 by excluding autodetected sparks. But if the x,t image contains not only sparks but one or more calcium waves, the F0 estimation will generate an error in Fo estimation. Although something related with this is described under Software limitations (paced cells), please include instructions for the user, or discuss the situation in which in the same image coexist Ca2+ sparks and Ca2+ waves, or even with an isolated spontaneous calcium transient.
Validity of the findings: When comparing Iocbio to Sparkmaster, the authors only compare detected number of Ca2+ sparks, Ca2+ spark amplitude and false positive rate. It would be useful to compare the Ca2+ spark morphology results (e.g. FDHM, FWHM, and so on) obtained with the author´s software in the presence/absence of Isoprenaline, with the same images analyzed with Sparkmaster. This will help to validate the novel software.
Additional comments: N/A


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

Input:
Title: COMPARATIVE ANALYSIS AND CHARACTERIZATION OF THE GUT MICROBIOTA OF FOUR FARMED SNAKES FROM SOUTHERN CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This English used in this manuscript is generally ok although I would encourage the authors to use active rather than passive voice.

The Introduction is not adequate and in several instances inappropriate references are used. I have indicated areas in need of improvement and suggestions for doing so directly in comments on the pdf.
Experimental design: The methods are insufficient to replicate the study. Specific details are included in the comments on the pdf but in brief:

Sampling design is not thoroughly explained.
Abundance data should be rarefied or justification provided why is was not.
Validity of the findings: I cannot adequately assess the validity of findings without more thorough explanation of methodology, but I have attempted to address specific concerns I have with comments directly on the pdf.

In general the statistical analyses performed should be adequately rigorous to address the authors' aims.
Additional comments: This is an extremely interesting paper and the authors should be commended for their effort. However I find some fatal flaws in the description of methodology, insufficient or inaccurate background information and overstated conclusions that prevent me from recommending the paper in its current form. However, with major revisions I hope to see this study resubmitted if the flaws in methodology can be addressed.


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

Input:
Title: COMPARATIVE ANALYSIS AND CHARACTERIZATION OF THE GUT MICROBIOTA OF FOUR FARMED SNAKES FROM SOUTHERN CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The major objective of this study was to assess whether differences in gut microbiota composition of 4 farmed snake species correlated with the host species.

Despite the poor representation of reptiles, particularly snakes in the microbiota literature, this study is purely descriptive and shows a very superficial analysis of the microbiota composition, which fails to meet the high technical standards.

The raw data are not available.
Experimental design: Major flaw of this study are the lack of a clear hypothesis and the methods.
Methods are poorly described and show several inconsistencies, raising serious doubts about the reliability of the data and robustness of the analyses.

Below are some major concerns.

The Authors indicated that fecal “samples from the same species were pooled in the same centrifuge tube”, while the results of gut microbiota are shown for individual specimens.

Also, the Authors indicated the use of a paired-end approach for amplicon sequencing (line 95), but the results indicate an “average length of 252 bp” (line 141). Please clarify.

The analysis of the raw data is minimally described. How were the “paired-end  reads”  merged? How were they filtered? Were singletons removed?  How were the sequences aligned for tree construction? All these important steps need to be indicated. The full pipeline used should be explained.

The confidence threshold of 0.60 for taxonomic assignment of OTUs is extremely low. The minimum acceptable threshold is usually 0.80 (line 104).  Also, the RDP set 14 database is outdated.

The Authors indicate that a principal component analysis (PCA) was used, while they clearly performed a principal coordinate analysis (PcoA).

Dominant species were defined as those with relative abundance greater than 10% (line 114). This seems a personal choice and should be justified.

Line 147: “pooled sample”? Unclear, since individual gut microbiota composition is shown.
Validity of the findings: The main hypothesis, that is that host species is a major determinant in the structuring the gut microbiota of these four snakes species was not statistically tested. Results indicated “the PCA showed that the gut microbiotas from the same group were more similar to each other than to the gut microbiotas from different groups” (line 179-180). This is just a visual observation, and as shown in Figure S6 (which should go into the main manuscript) Ec and Da are indistinguishable. Also, giving that the host species sampled came from two distinct farms, factors associated to the distinct environments (despite a similar diet and habitat condition) can be important variables. This should be considered and discussed.

Among the several indexes used to estimate alpha diversity, which should be limited to no more than two to avoid redundancy, simpson’s diversity shows an opposite pattern with respect to all others. This is clearly an error in plotting, but surprisingly the Authors report these results without commenting.

Figure S1 shows a rank curve for evenness that is obscure. How can you assess evenness from this figure, if not sequencing depth is reported?

The Discussion is limited to a comparison of taxonomic composition of these four snakes species to published data from other vertebrates.  Lizards, which are the reptiles with the most extensive microbial dataset currently available are not properly integrated into the discussion.
Additional comments: Overall, I suggest the Authors to perform a more thoroughly analysis of the data, displaying the details of the pipeline and testing with statistical analyses their major objective.


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

Input:
Title: COMPARATIVE ANALYSIS AND CHARACTERIZATION OF THE GUT MICROBIOTA OF FOUR FARMED SNAKES FROM SOUTHERN CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: In line 68, replace “southern China and teste that host species...” with “southern China and to evaluate whether host species...”

In the Introduction section, lines 49 - 58 the authors refer to gut Microbiome studies on birds and mammals. The inclusion of studies on reptiles (other than snakes), by virtue of their phylogenetic relationship, would potentially add greater value to the paper.
Experimental design: There is a need to perhaps include a statement relating to the feeding of the snakes (line 76) particularly in the context that: (i) one of the species was collected from a different site and (ii) that the regimen or proportions of feeding included multiple food sources (chicks and mice). The authors should indicate whether the feeding was entirely random in terms of these alternate sources and whether there is any indication of whether the same feeding patterns were applied at both sites of collection. This is with respect to the fact that the gut Microbiome composition in other spp. have been shown to be affected by different levels of protein and fat content, which would be the case for chicks and mice.

Line 85. The authors indicate that the individual samples were collected and then spooled according to species, followed by downstream freezing prior to DNA extraction. They should indicate that the individual samples were also treated in the same manner as the pooled samples, if indeed this was the case.

Line 99. Authors to indicate how the sequences were filtered and the low quality reads removed.

Lines 114 and 115. What was the basis for selecting the respective cutoff values. If taken  from known studies, provide references.

Line 116. Is there an available references in support of the methodology employed.

In the section: “Comparisons of gut microbiotas among groups” starting at line 158, there is mention of analyses conducted in Qiime and Mothur. Do the authors have specific motivation for implementing different analyses using different packages.
Validity of the findings: Line 177. The observation of the lower diversity of gut microbiota in sample  Ec could would be explored in the context that it is from a different site as the other three. The potential impact should be stated, in order to indicate that the authors are aware of it.

An evaluation of the number of reads generated for each of the individual samples indicate an average of 33059 reads (Table S1). What is the implication of this relatively low read number on the overall findings that were reported. How does this number compare to other studies of a similar nature. How does this number correlate with the overall diversity that was sampled versus the total available biodiversity (eg. Include a representation via a rarefaction curve).
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: COMPARATIVE ANALYSIS AND CHARACTERIZATION OF THE GUT MICROBIOTA OF FOUR FARMED SNAKES FROM SOUTHERN CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: The authors in their response to the initial review (point number 8, from reviewer 3) indicate that the use of different software for analyses does not affect results of the analyses. They however fail to indicate the basis for this statement. Could they provide other supporting material (references) supporting this.
Validity of the findings: The authors in their response to the initial review (point number 10, from reviewer 3) indicate that the number of reads evaluated in this study is similar to a number of other studies, which they mention. They, however, do not provide the references to these studies. Could they perhaps include this as part of their discussion, with the necessary references to support their work.
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: TRANSCRIPTIONAL FACTOR REGULATION NETWORK AND COMPETITIVE ENDOGENOUS RNA (CERNA) NETWORK DETERMINING RESPONSE OF ESOPHAGEAL SQUAMOUS CELL CARCINOMAS TO NEOADJUVANT CHEMORADIOTHERAPY
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The English should be improved, for example, accurately use the abbreviations.
2. More details of the status of nCRT in ESCC should be introduced.
3. The figure legend should be detailed. Presently it is too simple to describe the figures. Figure 1 is confusing, which samples are responder samples. The number of samples in figure1A is not consistent with the manuscript. No figure 6 is shown.
Experimental design: 1. More information of the two microarray data should be described.
Validity of the findings: no comment
Additional comments: This article shows the TF regulation network and ceRNA network determining response of ESCC to nCRT. nCRT followed by surgery benefits survival for patients with ESCC, but the clinical outcomes of nCRT are heterogeneous. The theme is interesting and this analytical work can increase understanding of molecular mechanisms determining nCRT response of ESCC.


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: TRANSCRIPTIONAL FACTOR REGULATION NETWORK AND COMPETITIVE ENDOGENOUS RNA (CERNA) NETWORK DETERMINING RESPONSE OF ESOPHAGEAL SQUAMOUS CELL CARCINOMAS TO NEOADJUVANT CHEMORADIOTHERAPY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well structured and the written english is clear. The lack of molecular biomarkers of neoadjuvant chemoradiotherapy response in esophageal cancer is a relevant theme in the oncology field and the authors provide a good background to explain the need of more knowledge in this area. The number of literature references also seem suitable for this type of research article.
Experimental design: The research question is well defined, and fits the aims and scope of the journal.
In terms of study design and methodology, the study was well panned and the methods were described with sufficient detail.
The authors should clarify the "Materials and Methods" section in the Abstract since they say that "RNA sequencing data of GSE59974 and GSE45670 were analyzed..." and then in the "Material and Methods" section of the manuscript they talk about "Microarray data". Since sequencing data is different from microarray data the authors should correct this.
Validity of the findings: The results were very interesting and they should be validated in esophageal cancer patients in the future.
Additional comments: The manuscript was very good and covered an emergent theme in the oncology field, I recommend its 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: TRANSCRIPTIONAL FACTOR REGULATION NETWORK AND COMPETITIVE ENDOGENOUS RNA (CERNA) NETWORK DETERMINING RESPONSE OF ESOPHAGEAL SQUAMOUS CELL CARCINOMAS TO NEOADJUVANT CHEMORADIOTHERAPY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, the manuscript is a well designed observational study but needs some work in the following areas
1) There are several times when the authors use the terms genes and mRNA/lincRNA or miRNA interchangeably.  Please use mRNA or miRNA or lincRNA consistently
2)  Several sentences are incomplete fragments that can limit reader understanding. Please revise (e.g line 210-212 "And growth factor....")
3) The figure legends are unsatisfactory. E.g. Figure 1 just says heatmap of two arrays without a color key, or sample description. Additionally, the reader needs to go back to materials and methods to figure out the array numbers (Viz. GSE 45670 and GSE 59974) . Please include descriptions that are more comprehensive
4) Given that neoadjuvant chemoradiotherapy(nCRT) is the subject of the review, very little background or details have been provided on this topic.
5) Tables captions need work. E.g. Table 1 uses acronyms that are not defined in legend (BP,CC MF)
Experimental design: This manuscript is within the scope of the journal and the methods are described with clarity
While the research question is defined, the authors have not contextualized their findings in the results or discussion. They need to work on conveying the significance of the results( see more in note to authors)
Validity of the findings: The findings while valid provide no clear biomarkers to separate responders from non-responders to nsCRT nor do the authors provide any further analyses to support their applicability. Some tools to enhance the value of the findings include
1) calculating the predictive power of the suggested biomarkers in separating responders from non-responders,
2) Making a heatmap of mRNA, miRNA and lincRNA identified in your network to compare responders and non-responders
3) Discuss the results from theses analyses and any additional data that might supplement this work
This will help answer their research question of using RNA transcripts to determine response to ESCC more definitively.
Additional comments: I believe that the authors have made a strong attempt to understand the interactions between mRNA-miRNA and lnRNA expression in Esophageal squamous cell carcnomas(ESCC).
Overall, I think that the readability and applicability of the paper will be greatly improved by focusing
1) The questions that the authors are trying to address: are you identifying prognostic markers or performing a RNA network analysis?
2) The results section needs to be explained with more clarity. There are several instances where the authors say 'x' genes were identified without any explanation as to how that number was reached. Furthermore, the authors need to explain how they are selecting genes from one analyses such as DEG and using them for the next step like network building. One suggestion would be to create a flow chart to visualize the analyses and define/explain the input and output of each tool with it.
3) The authors dont explain the significance of any of their findings. e.g What does it mean to have 'x' genes be significant by this tool? Why is this particular analyses necessary.  The Gene ontology analyses are just stated without any explanation of the data
Some additional points include
-  Please provide a citation for STRING and the significance cut off for interactions in the materials and methods (lines 121-123).
- What was the input for STRING that resulted in the identification of 49 interacting proteins (Line 168)
- Could the authors use GSE numbers in conjunction with type of array so the reader can follow the results and discussion without needing to figure out the array type being referred to?
-  Both the supplementary figures lack a legend, figure titles and a corresponding explanation in the text.
- In figure 1; the heatmap needs a color key, and a horizontal bar over it showing which samples are responders and which are non-responders. Is the clustering supervised?
-Please provide the differential genes identified in your analyses as supplemental information
-  The authors use the terms "highly expressed" and "low expressed" several times (e.g line 171) do you mean up and down regulated?  If not define these terms
- The figures say figure 5 twice, replace second instance as fig 6


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: TRANSCRIPTIONAL FACTOR REGULATION NETWORK AND COMPETITIVE ENDOGENOUS RNA (CERNA) NETWORK DETERMINING RESPONSE OF ESOPHAGEAL SQUAMOUS CELL CARCINOMAS TO NEOADJUVANT CHEMORADIOTHERAPY
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comments
Validity of the findings: No comments
Additional comments: I believe that the authors have sufficiently addressed a majority of my concerns. I request the authors to clarify the following in the manuscript
1)	time points at which samples were analyzed. Are your microarray samples from patients at baseline( i.e prior to receiving any nCRT?). If yes, then you are studying predictive networks while if they are post treatment, you are really looking at mechanisms for non-response. Please clarify that in your paper, particularly in the discussion section
2)	Figure 5B in particular is hard to read. Could you please upload a higher resolution version of it?
3)	In STRING, you have identified a number of “hub” genes including BMP4( discussed), ALB and EPHB2. However, you focus on only BMP2. Is it because it is a transcription factor? If not, could you clarify if there is anything known about the other hubs in ESCC?


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: UP-REGULATION OF GRB2-ASSOCIATED BINDER 1 PROMOTES HEPATOCYTE GROWTH FACTOR-INDUCED ENDOTHELIAL PROGENITOR CELL PROLIFERATION AND MIGRATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The text is clear throughout without need for correction.

For the most part, there is sufficient background. However, on lines 93-95, you mention the fluorescent staining assessment of EPCs. You end with "...as described previously". Please supply a reference for this statement.
Experimental design: In the methods section (lines 153-161), the authors describe both immunoprecipitation and western blotting, yet it is unclear where the IP was performed and to what purpose. This needs to be clarified as it appears to me from the figures presented that only western blot was performed.

On what basis were SHP and ERK1/2 chosen as key downstream indicators of Gab1 signalling? Was PI3K/Akt Which residues of the target signalling proteins were the phospho-specific antibodies directed towards?

Why was 20ng/ml HGF chosen? The amount of ligand has a significant effect on the cellular readout. Has this concentration been optimised previously (if so, can this be referenced)?

EPC migration was performed in EGM-2 without FBS. It is unclear whether earlier experiments assessing proliferation were also performed without FBS. This should be clarified. If there is a difference between the two methods, this should be explained.

Did the authors assess the level of the MET receptor on the EPCs? As this is the major receptor through which HGF signals, it would be useful to ensure that this is consistent between the groups.

EGM-2 medium contains a number of different growth factors pertinent to endothelial cell growth (e.g. VEGF, EGF, FGF etc.). Many of the receptor tyrosine kinases through which these growth factors signal also utilise Gab1 (as mentioned by the authors). Overexpression of Gab1 in the absence of HGF increases proliferation to a similar extent to control + HGF. Can the authors provide an explanation as to why HGF has a much larger effect than all other Gab1-related growth factors combined?

At 96 and 120 hours, the OD values have dropped back significantly so that the AD-Gab1+HGF is no longer different to the other conditions. If the OD represents a surrogate marker of the increase in the number of cells over time, then this suggests a significant loss of cells between 72 and 96 hours. Can the authors provide some explanation as to what has happened? Is this due to overproliferation and therefore lack of nutritional support in the remaining media? Promotion of proliferation is a major outcome measure in the study but it must be sustainable and the new cells must be healthy if it is to have any benefit for TEHV.
Validity of the findings: I have no problems with the validity of the findings, although further clarity is needed in areas mentioned above.

More information is required about the samples to determine the robustness of the method. How many cords were utilised in the study? In the statistical analysis section it is stated that "All these tests were performed on at least three replicates." - were these technical replicates or independent biological replicates from n=3 different cords? This is important information that needs to be included.

Conclusions support the results provided.
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: UP-REGULATION OF GRB2-ASSOCIATED BINDER 1 PROMOTES HEPATOCYTE GROWTH FACTOR-INDUCED ENDOTHELIAL PROGENITOR CELL PROLIFERATION AND MIGRATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: 1.	Isolation of EPCs.  EPCs are known to be a heterogeneous population of cells. The authors use the uptake of acLDL and binding of UEA-1 as markers of EPCs. This should be further verified  by expression of CD31 and VEGFR-2 (KDR).

2.	Adenoviral mediated overexpression of Gab1. It is not clear why the authors need to overexpress Gab1 in the EPCs.  Endogenous expression of Gab1 in EPCs is shown in Fig.1I and Fig.4A.  Indeed, Fig.4A appears to show hardly any increase in Gab1 in the Ad-Gab1 cells compared with the untransfected cells. Fig.1G is confusing as it appears that a MOI of 20 is chosen based on cell proliferation rather than expression of Gab1. It seems strange that this effect on proliferation is not dose-dependent as a MOI of 24 appears to show no increase in proliferation?

The reliance on overexpression of Gab1 to generate results is a major flaw in the study.  It would be better to study the effect of manipulating endogenous Gab1 in the EPCs, using siRNA mediated gene silencing, as an initial strategy before progressing to over expression of Gab1.  Furthermore, overexpression of Gab1 appears to stimulate only a very small increase in HGF-stimulated proliferation (Fig.2A) and HGF-stimulated migration (Fig.3A) compared with HGF stimulation of control transfected cells? What about using more complex angiogenesis assays such as a tubular morphogenesis assay on a fibroblast monolayer (Richards M et al 2016 Methods Mol Biol 1430:159-166). This would allow the analysis of vascular structures with the EPCs.
 It is hard to see what the clinical significance of this data is.  Are the authors advocating overexpression of Gab1 in EPCs before infusion back into patients? What would be the advantage of this procedure considering the very small effects of Gab1 overexpression?
Validity of the findings: Overall, I think that the strategy employed to analyse Gab1 effects is flawed and not relevant to normal physiology. The authors should utilise a system where endogenous levels of the Gab1 protein can be utilised rather than an artificial system based on overexpression.
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: UP-REGULATION OF GRB2-ASSOCIATED BINDER 1 PROMOTES HEPATOCYTE GROWTH FACTOR-INDUCED ENDOTHELIAL PROGENITOR CELL PROLIFERATION AND MIGRATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The required changes have been made and are acceptable to improve the clarity of the paper.
Experimental design: Inappropriate methodological statements have been removed as requested and the descriptions are now clear enough to allow replication.

However, it is unclear how much effect Gab1 overexpression has on other signalling pathways. This should be investigated further to determine the specificity of the proposed method. Whilst the results obtained by the authors shows promise for increasing proliferation, the wider context must be considered.
Validity of the findings: The findings provide some additional, albeit incremental, advance in our current understanding and conclusions are reasonable.
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: UP-REGULATION OF GRB2-ASSOCIATED BINDER 1 PROMOTES HEPATOCYTE GROWTH FACTOR-INDUCED ENDOTHELIAL PROGENITOR CELL PROLIFERATION AND MIGRATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Manuscript has been improved
Additional comments: I believe that the authors have made a number of revisions to the manuscript to improve the data presented and that this manuscript is now 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: EVALUATION OF A TOXOID FUSION PROTEIN VACCINE PRODUCED IN PLANTS TO PROTECT POULTRY AGAINST NECROTIC ENTERITIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Evaluation of a toxoid fusion protein vaccine produced in plants to protect poultry against necrotic enteritis.  The authors developed a fusion protein combining a non-toxic carboxyl-terminal domain of atoxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE, using a DNA sequence that was codon-optimized for Nicotiana in broilers.  Immunized birds produced a strong serum IgY response against both the plant produced PlcCNetB protein and against bacterially produced His-PlcC and His-NetB and were significantly protected against a subsequent in-feed challenge with virulent C. perfringenswhen treated with the suggesing that a plant-produced PlcC-NetB toxoid is a promising vaccine.  The article is well written and clear and has sufficient field background/context.  It has professional structure, figs, and tables.
Experimental design: The research queestion is well defined and the experimental design well established.  Authors perfomred a rigorous research and provide in their material and methods section, sufficient detail so other researchers could replicate their work.
Validity of the findings: The study is novel and the reuslt resented are robust, statistically sound, and controlled.  Their conclusion are well stated, linked to original research question  limited to supporting results.
Additional comments: Study well conducted and written.


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

Input:
Title: EVALUATION OF A TOXOID FUSION PROTEIN VACCINE PRODUCED IN PLANTS TO PROTECT POULTRY AGAINST NECROTIC ENTERITIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: the authors have designed a fusion gene expressed in plant leaves for the production of netB an alpha toxoid, which was shown to be immunogenic and partially protective when injected into broilers. Literature references in the introduction should be used with care. Refering to the mouse model of gas gangrene is of no use in the context of necrotic enteritis in broilers.Not all references have shown documented protection against necrotic enteritis lesions, and if they did, protection was always partial.
Parts of the materials and methods are mentioned in the results section.
Line 352: Fig. 5 should read Fig. 6.
the authors mention high yield as an advantage of their expression system, however, the yield is not reported in the results section.
Line 388: NG mentioned for the first time. What is this?
Line 430: Please read paper 29 carefully: effectiveness is questionable.
Relevance of results: the progress compared to what is reported in literature with both antigens separately in other expression systems is limited, whit actually no difference in protection.
Experimental design: Research question is not clearly defined. This is the application of different currently available tools to improve immunogenicity of vaccine antigens against necrotic enteritis in broilers.
The methods are described with sufficient detail.
Validity of the findings: Data and statistics are sufficiently sound.
Novelty lies only in the application of the plant expression system to C. perfringens antigens.
conclusions are well stated.
Additional comments: No additional comments


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

Input:
Title: EVALUATION OF A TOXOID FUSION PROTEIN VACCINE PRODUCED IN PLANTS TO PROTECT POULTRY AGAINST NECROTIC ENTERITIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Clear and unambiguous, professional English.  Well written and well designed experiments.
Experimental design: Well designed experiments well within the scope and aims of the journal.  The only issue I found was that the number of birds/treatment was not include din the text of the M&M.  Otherwise, the experimental detail was more than sufficient.
Validity of the findings: All findings were documented and repeated. The results were solid and believable.
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: EVALUATION OF A TOXOID FUSION PROTEIN VACCINE PRODUCED IN PLANTS TO PROTECT POULTRY AGAINST NECROTIC ENTERITIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: the authors responded to the comments correctly.
I have no further comments.
Experimental design: O.K.
Validity of the findings: O.K.
Additional comments: no further comments


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

Input:
Title: COMPARISON OF THE FECAL MICROBIOTA OF TWO FREE-RANGING CHINESE SUBSPECIES OF THE LEOPARD (PANTHERA PARDUS) USING HIGH-THROUGHPUT SEQUENCING
Review round: 1
Reviewer: 1

Output:
Basic reporting: General recommendations:
♣	Please revise all in-text reference formatting and adapt to meet the author’s instructions provided by the journal.
E.g. L46: (Nicholson, Holmes et al. 2012) is the wrong formatting. Should be (Nicholson et al., 2012)
♣	Please revise the full reference list formatting and adapt to meet the author’s instructions provided by the journal.
E.g.  JB, C., V. P, H. H, W. T, H. BM, A.-G. GA, T. DA, L. HT, T. BV and M. VV (2016). "Captivity humanizes the primate microbiome." Proceedings of the National Academy of Sciences of the United States of America 113 (37): 10376. > Surnames should not be abbreviated.
E.g.  Katherine R Amato, Carl J Yeoman, Angela Kent, Nicoletta Righini, Franck Carbonero, Alejandro Estrada, H Rex, Gaskins, Rebecca M Stumpf, Suleyman Yildirim, Manolito Torralba, Marcus Gillis, Brenda A Wilson, Karen E Nelson, Bryan A White and S. R. Leigh (2013). "Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes." Isme Journal 7 (7): 1344-1353. > Abbreviate author names, use correct Journal abbreviations, …
L623: remove abstract from reference!
TIP: Use a decent referencing program with a plugin for your writing program (e.g. Mendeley, EndNote, Reference Manager)
♣	Please consider an alternative title that more precisely describes the study. “Analysis of gut microbiota for” is too general nowadays. Try to refer to the type of analysis (diversity analyses) and two different subspecies of leopards. The latter is important given the emphasis throughout the manuscript on similarities and differences between both subspecies.
E.g. Fecal microbial diversity composition in free-ranging North Chinese and Amur leopards as revealed by Ion Torrent 16S rRNA gene sequencing
E.g. Phylogenetic diversity analysis of fecal microbial communities in free-ranging North Chinese and Amur leopards as determined by ...
♣	Capitalize ‘north Chinese leopard’ and change throughout manuscript to North Chinese leopard
♣	Introduction: Clarify more precisely the current findings on the relationship between conservation (and associated important factors of it) and gut microbial composition.
♣	Figures: increase quality of all figures and rewrite figure legends more detailed
♣	Figure 3b: there is a marked difference in relative abundance at Phylum level resulting from unweighted or weighted Unifrac distances (e.g. Proteobacteria). Please elaborate on that in your results or reconsider the use of either one or the other, depending upon your research objective.

Detailed manuscript review:
INTRODUCTION
♣	L45: Change ‘The characterization of gut bacterial community’ into ‘Characterization of gut bacterial communities’
♣	L47: remove following part ‘the gut bacterial community structures of a species can vary substantially when the environment changes from wild to captivity (ref)’ and refer to this aspect at the end of this alinea. Thus, first state some general well-known aspects of host-microbiota associations (e.g. role in diet, host health, and physiology, act as a barrier against pathogens, …) and then refer to association microbiota with wild/captive settings.
♣	L47: Question: Please elaborate on findings from reference provided in L47 (JB et al., 2016). Given you work on samples from free-ranging animals and state throughout the manuscript that the information gathered could be useful for conservation strategies, it is necessary to adapt your introduction more towards this aspect. Moreover, this then leads into alinea two starting at L56.
♣	L56: ‘The gut microbiota analysis’ … Remove ‘the’ because there is no such thing as “The gut microbiota analysis” and change into more specified wording e.g. gut microbial diversity analyses or deciphering gut microbial composition and function, …
♣	L59-L61: unclear how exactly these aspects (host habitat, diet and gut microbiota) are related. Write less generally the findings from Amato et al., 2015
♣	L61-L63: Likewise, state the findings from Gomez et al., 2015 less general! Replace “Increased anthropogenic pressure could also be distinguished via the composition of the gut bacterial community” > how was the relationship between the increase of anthropogenic pressure and gut bacterial community composition (did this increase, change, decrease, …?)
♣	L63: Besides. And disease-associated bacteria. Replace by ‘Additionally, it has been shown that the detection of pathogenic bacteria …’.
♣	L65-66: Reformulate by emphasizing that knowledge on gut microbial composition can contribute to conservation strategies by its impact on different factors important for conservation (habit fragmentation, pathogen load, …)
♣	L69: Remove in the past century
♣	L75-L76: rephrase to ‘Once patrolling from Northeast China to southernmost portions of the Russian Far East and the Korean peninsula, …’
♣	L79: rephrase to ‘The North Chinese leopard originally distributed North and Central China. However, an accurate distribution area and population size still remain unclear …’
♣	L83-86: rephrase to clarify following sentence ‘included the North Chinese leopard in Amur leopard on account of the obscure biogeographical barrier’. Why are they subspecies in the leopard clade?
♣	L89: Question: Why are leopards elusive in their feeding behavior? Is ‘elusive’ the correct term to use in this context?
♣	L89: rephrase and correct the following sentence ‘it is acknowledged that the gut microbiota relates to adaptive evolution to a high purine and fat diet of carnivores’. Please clarify and specify what aspect of the gut microbiota: composition, richness, functional pathways, … has been shown to adapt following an evolution to a high purine and fat diet? Consider including additional references, this has been shown by different authors.
♣	L95: change ‘gut bacterial communities’ to ‘fecal bacterial communities’
♣	L96: change ‘hyper-variable’ to ‘hypervariable’
♣	L97-L98: remove this part of the sentence ‘and discussed the implications … these endangered species.’ This has not been discussed in-depth in the discussion section. Suggestion to state that this study aims to provide the first benchmark of gut microbial diversity in leopards that potentially can contribute to further conservation research.

MATERIAL AND METHODS
♣	L101: Rephrase to ‘Opportunistic fecal sampling occurred in the period from December 2016 to March 2017 in the two distribution areas from the leopards.’
♣	L155: Change ‘could help preserve …’ to the more academic wording ‘contributed to the preservation of gut microbes in the fecal samples’.
♣	L155: What is considered a “fresh” fecal sample by the team? This is of utmost importance when analyzing afterward the fecal bacterial composition. (cfr. General recommendations)
♣	L116: I assume storage at -80℃ refers to laboratory storage. What were in-field storage and transport conditions? Were samples collected on ice, dry ice?
♣	L118: Total bacterial genomic DNA
♣	L118-L119: Please add methodologies for the check of DNA quantity and quality after DNA extraction procedures.
♣	L121: hypervariable
♣	L125: ‘and15μL ddH2O’ > insert a space
♣	L133: Question: What quality control procedures were applied?
♣	L134: change ‘Life Ion S5TMXL’ to correct brand name ‘Life Ion S5TM XL’
♣	L139-L140: Remove ‘Chimeric sequences can contribute to … excluded (ref)’. This should not be mentioned in the Material and Methods section. Just state immediately how chimeric sequences were checked for and eliminated.
♣	L142-L143: Formulate more appropriately. Chimeric sequences were detected by the UCHIME algorithm and the chimeras were removed using the ChimeraSlayer utility (references).
♣	L144: Rephrase to ‘For all samples, OTUs were generated from clean reads via Uparse v…’
♣	L149: This sentence is unclear and not necessary ‘all taxonomic information was obtained to form the composition of the gut bacterial community’. Later on in the manuscript, the authors state that many OTUs remained unclassified so not all taxonomic information was obtained. Please remove the sentence.
♣	L145-L150: Please rephrase more correctly this methodology. E.g. Using Mothur (Schloss et al., 2009), sequences were annotated against the SILVA SSUrRNA database (http://www.arb-silva.de) (reference) and aligned by MUSCLE (ref).
♣	L153: Rephrase to ‘calculated and analyzed in QIIME’
♣	L154: The Rarefaction curves and rank abundance curves were constructed in R (Version 2.15.3).
♣	L155 – L157: I do not agree with the statistical methods applied. Please reconsider these. One should start by identifying the nature of the distribution of values of the indices. Knowing the nature of the data in microbial ecology and the small sample size, it seems highly unlikely to me that these data are distributed normally and homogeneous. Moreover, why use then both parametric and non-parametric test?!
♣	L157: correct name of the test is Wilcoxon Test (wilcox.test is the command line in R)
♣	L158: Do not capitalize beta-diversity
♣	L158-L159: Please rephrase more correctly this methodology. E.g. Using QIIME pipeline (version), beta-diversity was assessed by calculation of Unifrac distances and subsequently visualized by principal component analysis (PCoA). Phylogenetic trees were also built using UPGMA (unweighted pair group method with arithmetic mean)
♣	L159: UPGMA = unweighted pair group method with arithmetic mean
♣	L159-L163: I would recommend to choose one visualization analyses and not include all three of them i.e. PCA, PCoA, and NMDS! Moreover, the results and discussion do not mention potential different outcomes of these methodologies.
♣	L163-L166: Question: Why do you choose these statistical methods? Why use parametric and non-parametric tests for paired group testing and then ANOSIM (parametric method) to test this same data.

RESULTS
♣	L167: Question: Can you explain an LDA score of 4?
♣	L174: were > are shown
♣	L174: Do not capitalize rarefaction curves
♣	L174: What do you mean by “the stable values”? To my knowledge, rarefaction curves do not need to reach a certain value but show a pattern of plateau formation
♣	L176: use an alternative verb for ‘comprehended’
♣	L176: Do not capitalize rank abundance curves
♣	L178-179: Please move this sentence to Materials and Methods
♣	L181: change ‘we identified’ to ‘X number of OTUs (%) could be taxonomically assigned to 28 phyla, 55 classes, … .’. I would like to see the percentage of OTUs that could be taxonomically assigned. Since most of the results are partitioned into Amur leopard vs North Chinese leopard, please report also the number of sequences recovered for each sample set separately. I would like to know if there was a difference between that because this will also impact your discussion on the percentage of taxonomic groups present.
♣	L185: were also contributed to > Other phyla included Proteobacteria (x%), …
♣	L186 and L188: please remove underscore and use proper bacterial nomenclature (please check also elsewhere in the manuscript)
♣	L196: Remove sentence (should be in Materials and Methods)
♣	L197-L198: Please mention these sample codes for both groups in Materials and Methods already.
♣	L200-201: Remove sentence ‘This analysis was generated using both weighted Unifrac distance and unweighted Unifrac distance respectively.’ This again refers to Material and Methods, avoid redundant information.
♣	L203-L205: Remove the first sentence since it encompasses an explanation about a boxplot, one should know what it represents. No need to cover that in the manuscript.
♣	L206-L213: Rephrase this whole section and report the results observed from the methodologies instead of rewriting the methodology. This is of utmost importance for a result section.
♣	L222: froorth??
♣	L224: Question: What does that mean ‘the stress value was 0.110’? Is that relevant to mention in the manuscript result section??
♣	L227-L228: Remove that first sentence, it is a methodology description. F.ex. Fron LEfSE it was shown that the relative abundance of Bacillaceae is remarkably higher …

DISCUSSION
♣	L23: for > in
♣	L233-L238: Remove these sentences. This is a repetition from the introduction and not directly relevant to the outcome of your study since you do not link any metadata except for host species to your data.
♣	L238-L240: Question: Why are these studies important for the conservation of endangered species?! What relationships have been found between factors of conservation and gut microbiota?! How can you apply knowledge on gut microbial composition to species conservation?
♣	L243-245: Question: Why is this analysis imperative for research in wild leopard conservation? (cfr previous comment, elaborate more specifically on the application of this knowledge to species conservation)
♣	L243-245: Question: Why would we consider these analyses necessary if obtaining samples is actually not easy and hardly feasible. With this statement ‘…tremendous hardship in obtaining non-invasive samples or intestinal part of endangered leopard has impeded the characterization of their gut microbiota’, you undermine your own study and this contradicts with the outcome of your rarefaction and rank-abundance curves.
♣	L245-L247: Question: Finding multiple unclassified genera, is this due to small sample size or methodology or reference database?! Knowing current databases already include sequences from many different animals (including different large carnivores), how many novel sequences (%) would you then expect to find in leopards?!
♣	L250: Please rephrase to ‘which is in accordance with the vertebrate gut microbial diversity described by many other studies’ and add reference Ley et al., 2008 (https://www.nature.com/articles/nrmicro1978)
♣	L253-L254: Please clarify: ‘no significant difference in phyla composition? Relative abundance?
♣	L261: insert space for ‘some studies reported that’
♣	L262: ’is associated’ instead of ‘was associated’
♣	L263: Please correct to ‘the tendency of an increase in Firmicutes and a decrease in Bacteroidetes…’
♣	L266: futher > further
♣	L265-L267: It is already a well-known given in the field that the proportion of Firmicutes/Bacteroidetes might reflect differences in dietary pattern. Please elaborate on that and be as specific as possible in your discussion. As it is stated now, it is too vague knowing the body of literature out there.
♣	L268-L273: Remove this section out of the discussion unless you actually discuss these findings in the light of other studies. As it is written know, it is a reporting of results, which should occur in the result section.
♣	L280: ‘..a relatively small proportion compared to this family in the gut microbiota of wild leopards’.
♣	L280: Clarify the phrase above, it is not clear as written now. Did you mean Lachnospiraceae constituted a relatively small proportion in your sample set compared to snow leopards and wolves instead of wild leopards?! Also include reference there.
♣	L280: Question: Any idea on why you observe a difference between leopards and snow leopards and wolves?
♣	L283: the gut microbiota was affected by > gut microbial composition is affected by
♣	L288: Question: Elaborate on this and add additional references that underpin the impact of protein content in the diet on Clostridium populations in carnivores [e.g. Becker et al., 2014 (in cheetahs), Schwab et al. 2011 (in grizzly bears), Zentek et al., 2003 (in dogs and cats)]
♣	L289-L292: Remove sentence ‘In addition, research on …’ as it does not fully fit into the discussion. Such parameters (e.g. sepsis, inflammation, antibiotics) have not been measured in your study.
♣	L297: Include more references on the finding of Clostridium perfringens in strict carnivores (non-domestic such as cats and dogs). Cfr previously mentioned references
♣	L294-L299: Suggestion to include some background findings on the presence of gastrointestinal diseases in wild or captive leopards.
♣	L302: include a space before your reference
♣	L317: Rephrase so it is more clear that the G+C content leads to a bias because of the implication of a high G+C content on methodologies
♣	L322-L323: Please remove sentence on storage conditions unless you put your results in light of those findings from Maukonen et al., 2012. Moreover, if you consider your storage conditions to be suboptimal for assessment for microbial diversity, why only discuss in the light of Bacteroidetes?
♣	L328-L331: Please be more specific on what nutrients and pathogens. All gut bacteria somehow contribute to the uptake of nutrients for the host and competition with pathogens (natural colonization barrier)
♣	L331-L332: Please discuss your findings in the light of the total observed microbial composition in relation to leopard health, not only Bacteroides
♣	L344-L345: Please report critically: how much change in bacterial composition do you expect to see that could be attributed to nutritional differences between both host species?! Maybe this difference cannot be observed because it does not distinguishable from normal variation in their gut microbial diversity or sample size is too small or …?
♣	L348: Knowing the differences in their diet, would it have been advisable to also analyze the feces for prey composition?!
♣	L353: Please explain “internal factors”
♣	L354: Please remove either free-ranging or wild, they mean the same.
♣	L360: cfr previous remark, if you question your sampling storage conditions, your whole study is questioned. Nowadays, good guidelines exist to storage fresh fecal samples for next-gen sequencing diversity studies. If proper storage is assured, it should be of a lesser impact then the multitude of other variables influencing microbial gut composition.
Experimental design: This is an original primary research within the Aims and Scope of the journal. Although the research question is well defined, the manuscript lacks in the discussion section a critical reflection of these results in the light of conservation of leopards. Please be more precise, too many statements are too general about gut microbial diversity.

Following changes are suggested to improve the technical standard. Take special care to the rationale of performed statistic tests and visualization analyses.

♣	Materials and Methods: Provide more information on fecal sample collection > what is a fresh sample, how much time after defecation was it collected? E.g. How long was the sample outside in the environment? Meteorological conditions during sampling? Include as well how the samples were collected: what (sterile?!) recipients were used?
♣	Material and Methods: I do not agree with the statistical analyses using parametric as well as non-parametric tests. The nature of these data and the small sample size should point towards a non-homogenous and non-normal distribution of the data. Test with assumptions of homogeneity and normal distribution should therefore not be used.
♣	Material and Methods: Quantitative (unweighted Unifrac) and qualitative (weighted Unifrac) beta-diversity measures lead to different insights into factors that structure microbial community composition. Assess to what extent unweighted or weighted Unifrac should be preferred for this study.
♣	Results: Please improve quality of figures 3a and 3b.
♣	Results: Reporting of results should be restricted to results, not include again Material and Methods.
♣	Results L214-L226: cfr. Material and Methods. Reconsider the value of using three different visualization analyses.
Validity of the findings: ♣	Discussion: When reporting from other studies, please use correct verb tense. (see also detailed manuscript review)
♣	Discussion: When reporting results from microbial diversity studies, please specify whether these findings were also obtained by culture-independent high-throughput sequencing. Some of the differences observed between microbial diversity studies might be mainly attributed to different technologies used!
♣	Conclusion: L361-L370 Please rewrite conclusions to include actual well stated results of this study, linked to the original research question.

Although we encourage microbial diversity studies in endangered host species, the authors should put their findings more in relation to specific aspects of conservation. Too often it remains to speculative or vague without an actual foundation of why this information should be relevant to collect from endangered species.
Additional comments: Thank you for your submission.
Please go carefully over the manuscript review comments ranging from important general comments to detailed grammatical comments.

Focus on how to increase the validity of findings by choosing the appropriate statistical test and visualization techniques (instead of performing different incomparable tests alongside each other), by increasing quality of figures and writing concise but informative figure legends.

Report results strictly in result section, and rephrase wording in discussion section so that the results are actually discussed and not repeated in a 'reporting' format.

Although the text generally flows and is written in proper scientific English, please consider remaining grammatical errors. Please take care of referencing (and the reference list) in the correct format, following the author's guidelines of the journal.


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: COMPARISON OF THE FECAL MICROBIOTA OF TWO FREE-RANGING CHINESE SUBSPECIES OF THE LEOPARD (PANTHERA PARDUS) USING HIGH-THROUGHPUT SEQUENCING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript “Analysis of gut microbiota for free-ranging leopards (Panthera pardus) in China using high-throughput sequencing” by Han et al. describes a comparison of the gut microbiota of two subspecies of leopards using faecal samples as a surrogate. The gut microbiome of carnivores is still not well understood today and the current study adds to that limited knowledge. Although I consider the study to be mostly solid, I was struggling to understand some analyses performed. The manuscript would benefit greatly from improved clarity of the text and figure quality. I would like to see the work published after a major revision.
Introduction, 3 pages:
Most of the first two paragraphs of the introduction (lines 45 -- 66) deal with topics not relevant to the current study. I would like to see the introduction and discussion much more focussed: a bit of background on microbiome in wild carnivores, why the authors expect differences between the two populations, etc.

Methods, 3.5 pages:
Line 145: “The species sequences were annotated” the authors are probably referring to the representative sequences of the OTUs. Also, how were the representative sequences chosen? E.g. most abundant or longest sequence per cluster?
150: “The sequences were then aligned using MUSCLE software (Version 3.8.31)(Edgar 2004) to construct the phylogenetic relationship between different OTUs.” How was the phylogenetic tree inferred?

Res, 3.5 pages
Description of relative abundances of phyla, families and genera in the text is quite lengthy and I feel a table would be more appropriate. I think it is important the authors provide their annotated OTU table as well as summary tables on the different taxonomic ranks so that readers can investigate taxa of interest.
Figures: There are 7 figures in total although the PDF generated states 8. There is redundant information in the figures, for example Figure 2 and Figure 3b illustrate relative phylum abundance. Figure 2 and 3 could be combined without losing information. Figure 5 could go into supplementary and Figure 7 could be excluded completely.
I also feel that figure quality should be improved. Figures blur when zooming in. I cannot tell if this is an issue with the original figures or the PDF.

Figure 1:
A colour scheme that makes it easy to distinguish the two groups would be helpful. Also, the legend describes what rarefaction and rank abundance curves are and not what the figure shows. This is also true for some of the other figures.

Figure 2:
Legend says “bacterial species” but shown are phyla.

Figure 3:
The title is misleading/not informative: “The dendrogram (a) and UPMGA clustering trees (b).” Shown is a heatmap and a bar chart of relative phyla abundance with the dendrogram of the clustering as added information.
a)	I do not understand the colour coding of the relative abundances. According to the legend the values range from -4 to 4. This means the values were scaled. How?
b)	I do not understand why phylum relative abundance is different between weighted and unweighted Unifrac. Using different distances can affect the clustering but why the abundances? The legend says “The unweighted Unifrac distance and weighted Unifrac distance were used to calculate the overall percentages of relative abundance among all samples at phylum level.” Distances are calculated based on the relative abundances, not the other way round. The legends of the two subplots differ in size.

Figure 5:
As no structure is revealed by the analysis, I feel this figure could go into supplementary information.

Figure 6:
Please change the colour scheme to accommodate for red-green blind readers.

Figure 7:
I question the relevance of this figure. Only three taxa were different. Do we really need a bar chart and a cladogram to show this?

Discussion: 6.5 pages.
The discussion is extensive and, in my opinion, not always relevant to the results. Often bacterial taxa from the current study are mentioned along with other studies that also found these taxa. The studies cited are then compared much like in a literature review.
Also, some parts are better suited for the results section, e.g. lines 268—273 describe results.
I suggest the authors concentrate on the main taxa and where they have been previously been described in other carnivores and on why one might expect differences in the microbiomes of the two subspecies (like they did in lines 339 – 353).

Conclusions
The conclusions do not refer to the findings of the paper.
Experimental design: There is no original field study approval document provided, only a text describing why the authors do not provide the original, i.e. that it is in Chinese. The editor needs to decide if this is acceptable.

There are different numbers of individuals in the two groups (8 vs. 13) which could be problematic for the statistical analyses. However, as no big claims about significant differences are made I do not consider this an issue. The authors should, however, discuss this issue as a potential explanation for the lack of statistically significant differences.
Validity of the findings: The main findings are descriptions of the predominant taxa in the two subspecies of leopards and a lack of statistically significant differences between the two groups (with the exception of the three taxa identified in the LDA Effect Size analysis). I consider these findings valid based on the current experimental design.
Additional comments: •	The title does not indicate that two subspecies are being compared. Suggestion: “Comparison of the fecal microbiota of two free-ranging Chinese subspecies of the leopard (Panthera pardus) using high-throughput sequencing”.
•	I was not able to access the raw sequence data in SRA under SRP149194.
•	As many other papers in the field faecal samples are used to study the gut microbiome. Faecal samples can be used as a proxy but I think this should be mentioned in the text. For example the first sentence of the abstract reads: ”The analysis of gut microbiota provides a non-invasive approach to understand the complex interactions between host species and their intestinal bacterial community.” This is not correct. Obtaining gut samples is invasive, collecting faecal samples and using them as a proxy for the gut is non-invasive.
•	References are not formatted correctly. Please refer to author instructions for the correct format.
•	In-text citations of multiple references should be separated by semicolons instead of commas.