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: HABITAT ASSOCIATION IN THE CRITICALLY ENDANGERED MANGSHAN PIT VIPER (PROTOBOTHROPS MANGSHANENSIS), A SPECIES ENDEMIC TO CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article could use some proof-reading and copy-editing to improve the sentence structure and word choice. I have included some suggestions in the attached PDF.
Experimental design: The methods seem appropriate, but I feel some elaboration is needed. In general, more information on the transects is needed, clarity on sample sizes, and more detail about the statistical tests. This is detailed in my attached PDF.
Validity of the findings: The main facet I think needs to be included is a discussion of detection probability. As these snakes were encountered opportunistically along transects, do the results represent an overall view of habitat selection, or rather is the selection biased towards snakes that are basking and thus easily (relatively) observed? Also, how does the time of day impact these observations? On hot days, are snakes more or less likely to be out basking, etc.

In my own research (in prep), I found that snakes located via radio-telemetry were more likely to be found in areas of dense vegetation relative to snakes found during timed meanders, and that many snakes were completely concealed and thus would not have been found in transect surveys.

I think the results of this paper are still valid and important, but they need to be framed in the context of imperfect detectability.
Additional comments: This is a great paper that documents important natural history and habitat selection data that will be important for conservation. Please see my attached changes for suggestions to improve the manuscript.


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

Input:
Title: HABITAT ASSOCIATION IN THE CRITICALLY ENDANGERED MANGSHAN PIT VIPER (PROTOBOTHROPS MANGSHANENSIS), A SPECIES ENDEMIC TO CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Introduction – The introduction is generally well-organized and flows well. The authors start broadly and then focus down to the species of concern and objectives of the study, and reference appropriate studies throughout. There are several aspects, however, that could be improved.

1. I recommend that the grammar and style of this section be carefully edited, as there are many instances where the terminology used within a sentence is not quite stylistically cohesive. The pieces are mostly there, but the connections and writing style could be improved. For example, lines 40-41 I would change to: Effective conservation and management of species depends on an understanding of habitat use and selection, particularly if these aspects change seasonally. This is especially the case for species susceptible to habitat loss or fragmentation. Asking a colleague familiar with the topics explored in the background to reshape sentences would serve to greatly strengthen this section, in my opinion.

2. The first paragraph could benefit from a direct connection to why an understanding of habitat use is critical to species conservation and management. This really sets the stage for the whole introduction and importance of the study. The pieces are there, but the direct connections are not, so a reader unfamiliar with these fields may be left guessing at the significance. For example, such data could assist in the establishment of appropriately sized or placed reserves. Or…if the resources are limited and patchily distributed across the landscape, the identification and protection of movement corridors would be critical to population persistence. Something like this to really emphasize the connection for the reader. I say this, because all species require multiple resources for their survival, but the real issue relates to actually attaining those resources in a landscape shaped by humans.

3. The authors do a good job establishing the need for this particular study in terms of how it adds to our current understanding of Mangshan Pitviper ecology (lines 63-67). This paragraph could be further strengthened by mentioning current threats to population persistence (does habitat fragmentation or alteration affect movement or survival? What role does illegal collection play? - mentioned later in the discussion). Include anything relevant to the motivation to collect the habitat data that you did in this study. Specifically, any mention to the interest in testing for seasonal effects is currently missing in this section. More context needs to be provided in this regard.

Detailed comments:
•	Line 38, habitat or habitat types
•	Line 46, change poisonous to venomous
•	Line 51, instead of retreating, use the term refugia
•	Line 58, I think that mentioning this species is limited to just a single mountain range would be useful for the reader (change to: limited to just 10,500 ha on a single mountain range).
•	Table 1 caption states twelve ecological variables, but there are currently only 11. There could be up to 14 in this case because I suggest including the categorical variables, but some may be removed if correlated. Just be consistent with text and table captions.
•       Figure 1 title – change control to random plots.
•	Figure 1 – it is difficult to distinguish between the locations at the current resolution – I count around 40 observations but know there are double that number. Adjusting the different shapes could help, or perhaps zoom in a bit more? The colors could also be difficult to distinguish between for some readers.
Experimental design: Objectives - The objectives need to be clarified and fine-tuned based on the methodology used in the study. Based on my understanding, I think that there are just two objectives: 1) identify the habitat features associated with P. mangshanensis occurrence across the study area 2) determine if the association with these features varies across season. The approach does not allow for the investigation of the actual mechanism underlying these associations. The final statement in the last paragraph is extremely broad and should be revised to include testable predictions related to these objectives.

*Note that in my rephrasing of the objectives I use the term association and occurrence, not habitat selection. We cannot simply assume that where we find an animal (occurrence) is equivalent to selection or preference, unless we have detailed behavioral data to back the observations up. I think that given the nature of the data here, stick with occurrence and association.

Methods – My major concerns with this section of the manuscript include 1) aspects of data collection and lack of sufficient detail, which caused me to pose a number of questions while reading and 2) approaches used to analyze the data. A great strength - the use of active voice for the majority of this section – there are only a few places that are passive. I recommend active voice for clarity, and the authors did a great job here. I have organized my comments and questions according to the different subsections below.

Study Area: Please include the names of the dominant tree and shrub species here.

Survey Methods: Provide a citation for the type of transect surveys conducted. Provide detailed methodology for repeatability of the transects – e.g. length, number of surveyors, initial placement, were they time-constrained, etc. Out of curiosity, were any of the individuals observed over the 4-year period recaptures, or were the 83 locations the only observations? If you had more observations, how did you determine which locations to use? I am really impressed with the sample size for such a rare snake!

My greatest confusion and issue relate to lines 92 – 94. Were the 83 locations gathered throughout 2012-2016, but the habitat data measured later in 2015 and 2016? I checked the raw data, but there are no dates for the individual snakes. If this is the case, I assume that you revisited the locations during the time of year when the individual was initially encountered, but several years later? As currently written, it seems there are different plots picked for each season and then surveys conducted within the plots themselves during those times. These details need to be laid out more clearly. After rereading several times, I think that the snake observations predate collection of the habitat data. If this is the case, there are several potential issues. While some habitat variables remain consistent over time (e.g. elevation, slope location), others likely vary (e.g. aspects associated with vegetation including canopy cover and height). If the study area is such that changes would be negligible, provide support for this. Otherwise, you need to acknowledge that such variables have likely changed since the individuals were detected.

Pick one term, either random or available for the “paired” plots and use throughout the rest of the manuscript. I think that random may be more appropriate based on the difficulty in truly describing what is available to individuals.

Habitat Variables: I am curious about your previous research – I think that it would be useful to summarize related projects for the reader. For example, which specific aspects of previous research influenced you to include certain habitat measures in this particular study? If exploratory investigations revealed tendencies for this species to occur within certain habitat types, for example, this is also useful to point out in the second to last paragraph of the introduction. The reader would then realize that while associations between the species and habitat have been observed, the details had not been rigorously investigated prior to this manuscript. Remove the numbers that precede each of the habitat variables and write out each as their own descriptive sentence. I suggest this simply because there are many of them.

Please provide clarification with regards to the following variables: gravel (how did you determine the cutoffs for the three categories? This detail could be added to table S1 or the text), slope aspect (where exactly did you walk the slope? At the precise location of the snake or across the entire plot?), slope location (what were the specific elevational cutoffs for the three categories?) distance to water (was the distance calculated from the center of the plot, and does water refer to any type, or a specific category?), fallen log density (did the logs also fall within a specific length?), canopy cover (how did you estimate vertical projection?), and deciduous cover (how did you specifically measure this? via a visual estimate?).

I don’t think that table S1 is necessary if you keep the details in the text. Either move all details to the table or add missing details to what is already in the text. If you keep the table, I think that it would be interesting to include the proportions of used and available observations that fell within each category.

Specific comments:
•	Line 106 – Include the word categorical before habitat variables.
•	Line 106 – I suggest just using aspect for this categorical variable as slope and aspect are two different features of the landscape. Or, you could write out aspect of the slope.

Statistical Analyses: I have several concerns with the approaches used to analyze the data, and some of the tests are redundant and can be removed. My main issue is that I don’t think that the current methodology accurately compares habitat use across seasons. Please note that I am not a statistician, and there are likely other ways to analyze the data, but I suggest an approach that I am familiar with and have used for similar data. Perhaps another reviewer or the associate editor recommend a different strategy. In addition, for the logistic regression model, the assumptions of independence are violated because the used and available plots are paired, so plot pair would need to be incorporated as a random effect.

1) To truly evaluate if habitat use varies across seasons, you need to test the effect of season in a single model. Currently, you are comparing used versus available for each season separately. Therefore, you cannot compare the relative support of the models to one another because the models are comprised of different subsets of data. In addition, when running the models separately, you reduce your power due to lower sample sizes, increasing the likelihood that effects are due to chance. The general rule of thumb is to have at least 10 samples for each variable that you include in a model. When running the models separately, there are 14 total variables, but only up to 60 observations. If you include all of the observations in a single regression analysis, then you have a sufficient sample size for the number of variables you wish to test.

2) The approach that I suggest: Run a multinomial logistic regression model with a random effect to explore if habitat associations of individuals if any and which variables are associated with the used versus the available locations (why I refer to this as objective 1 in my comments above – are the snakes selecting for certain habitat features compared to what is available to them in general), and if these associations varied across the seasons (objective 2 above). Include all the data, with 4 response variable categories, the available or random locations and then snake observations grouped by season (0 = all available or random points, 1 = spring locations, 2 = summer locations, and 3 = fall locations). Use the same approach for model evaluation as you describe to identify the best-fit models but run an ANOVA on the best-fit models to view P-values and to evaluate the significance of each explanatory variable and associated coefficients to show the direction of the relationship (Menard 2002). This will produce a table similar to table 1, but with all available data combined in one column, and will include the associated coefficients. Significant variables in the available column reveal differences between the used vs. available locations in general, and significant variables within each month group show if any seasonal differences exist. You will need to recalculate the means for the used plots.

I have never used discrimination analysis, so I cannot comment on the appropriateness of its use here, but I think that using the approach I have described above and looking at which variables are significant for the random points should get at this.

Running all of the paired tests results in multiple comparisons, and as such, some differences may be due to chance alone. This is why I suggest running the regression model instead.

3) Before deciding which variables to include in the regression model, run the Pearson correlation tests to identify and remove correlated variables. You explain doing this, just move this information to the first paragraph. You also want to include the categorical habitat variables, which the regression analysis can accommodate. Just be sure to check if any are correlated first (use Chi-square test of independence) and be aware that you need to calculate dummy variables due to more than just two categories.

Specific comments:
•       How did you build your regression models? For example, did you use a forward-selection backward-elimination approach?
•       Were any of the variables not normal, and if so, did you transform the data, and how?
•       % correct classified included in table 2 is not mentioned in the methodology section.
Validity of the findings: Results – I think that the reason some variables detected as significant during paired comparisons are not included in the best-fit models comes down to the problem of multiple comparisons and sample size. The first time I read through the manuscript, I was confused about the differences in table 1 and table 2 in terms of variables significantly associated with snake presence.

Specific comments:
•	Lines 165-169 – Change to: analysis revealed a positive association between fallen log density and snake presence and a negative association between…
•	I suggest listing the variables in order of their significance OR alphabetically and then be consistent across all sections using this same approach.
•	State the direction or relationship of the variables when comparing random locations with occupied ones.
•	Line 193 – change proving to suggests. I agree that it is likely that the snakes are actively selecting the logs, but these data show associations. If radio-telemetry or mark-recapture data revealed snakes repeatedly on logs, then that would reveal actual selection or preference.
•	Line 201 – According to the current results in table 1, shrub height is also significant in all three seasons.
•	Lines 204-211 – If you decide to keep this analysis, could you provide more clarity on what these results mean? As I have previously mentioned, I am unfamiliar with the approach, and this is confusing to me.
•	Figure 1 title – change control to random plots.
•	Figure 1 – it is difficult to distinguish between the locations at the current resolution – I count around 40 observations but know there are double that number. Adjusting the different shapes could help, or perhaps zoom in a bit more? The colors could also be difficult to distinguish between for some readers.
•	Figure 2 – Wonderful addition to include these images! What a stunning species!

Discussion – Be clear in the discussion when referring to other studies versus data reported here within all paragraphs.

1) The first paragraph needs some improvement. Again, these data do not show preference, but rather association. There seem to be two separate arguments within this paragraph that just need to be clarified. The snakes are ambush feeders – they lie in wait until prey appear – but then, they may use caudal luring to attract the prey at that point. Their whole body doesn’t resemble a food item of their prey (line 240), unless the prey they consume also consume lichens, rather their tail does. That is the body region that resembles a grub. If they use a strategy similar to Copperheads (a species that I have worked with), their camouflage allows them to blend into the habitats traversed by their prey during the preys’ foraging movements, thereby potentially increasing the likelihood of an encounter. Maybe I am being nit-picky here but am trying to provide suggestions on how to help clarify the points you are making.

Specific comments:
Line 122 – change shelter to refugia.
Lines 228-229 – you can’t state the snakes were giving logs priority unless you have detailed telemetry data or mark-recapture observations, in my opinion. You CAN say that field observations confirmed strong associations of individuals with the logs.
Line 241 – Based on other literature that I have read about this species, scientists state lichen not moss.
Line 252 – So you do have telemetry data? Very cool! Share this fact with the reader in the methods when you explain why you selected the variables that you did. This likely explains why you state “snakes preferentially crawled near logs” on line 230, so included based on unpublished telemetry data there as well (if it is unpublished, otherwise provide a reference to that study).
Lines 248-255 – Very interesting that you did not observe the snakes drinking water, but visually noticed the increased presence of small mammals!

Conclusions – 2 out of the 3 recommendations are great! I don’t clearly see the connection between the first recommendation and the results reported here. Is the increased likelihood of human/snake contact due to the fact that the snakes are using more “open” areas in the spring?
Additional comments: Figure 2 – Wonderful addition to include these images! What a stunning species!


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

Input:
Title: HABITAT ASSOCIATION IN THE CRITICALLY ENDANGERED MANGSHAN PIT VIPER (PROTOBOTHROPS MANGSHANENSIS), A SPECIES ENDEMIC TO CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Some details could be refined to more clearly communicate meaning.
Experimental design: I am not qualified to evaluate the details of the anlaysis.
Validity of the findings: Ten meters is a substantial distance and includes a lot of hiding spots. A snake under dense cover is going to be more detectable than a snake in the open next to or on top of a log. Given the focus of the manuscript on identifying important microhabitat features, it is important to consider how this variation in detection probability influenced observations and why I brought it up in my initial review. I’m glad to see there is now some general information in the discussion about how detectability may have affected observations, but it is worth considering including some specifics based on the results. For example, is a snake by a log more detectable? How would that have affected data. The authors now state that a number of measures were incorporated to address detection issues (for example, standardizing start times and observation method) but I’m not sure how they are relevant. Ideally detection probability is incorporated into analysis, but if this is not possible, then it needs to be discussed to a greater extent.

Further justification is needed as to why it is acceptable to quantify microhabitat at a site up to three years after a snake was spotted there. Stating that it reveals their preferences only to “a certain extent” is not informative.

There is still no information about how/why it is appropriate to quantify habitat selection at only one spatial scale given most snakes likely select habitat at multiple spatial scales. Is it possible that something important or relevant to management is overlooked, why or why not?

Specifically how many individual snakes were observed?

Promoting the value of tourism has little to do with the study so I’m not sure why it is a part of a conclusion.
Additional comments: The manuscript is much improved.


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

Input:
Title: HABITAT ASSOCIATION IN THE CRITICALLY ENDANGERED MANGSHAN PIT VIPER (PROTOBOTHROPS MANGSHANENSIS), A SPECIES ENDEMIC TO CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: I find no problems with the basic reporting. The article is well written in excellent English. I have provided some additional suggestions on word choice below. The literature cited and background/context is sufficiently developed in the Introduction. The tables, figures, and raw data are appropriate. The results and hypotheses are connected and relevant.
Experimental design: The methods are improved from the previous submission and adequately address the concerns and suggestions of reviewers. I have not used random forest methods, so I don’t feel qualified to comment on the specifics of this technique. The supplemental tables allow for replication in the field if needed. Please see attached document for specific comments.
Validity of the findings: The conclusions are well stated, are linked to the original research question, and are limited to supporting results. I found no indication of social spiders in the raw data. I feel the authors should include additional discussion on lag time (in years) between early snake detections and habitat surveys, as well as a more detailed discussion of the potential impact of imperfect detection on their surveys. Overall, I suggest changing the use of "occurrences" to "detections" throughout. Please see attached document for specific comments.
Additional comments: I enjoyed reading the revised manuscript.


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

Input:
Title: ANALYSIS OF DEUBIQUITINASE OTUD5 AS A BIOMARKER AND THERAPEUTIC TARGET FOR CERVICAL CANCER BY BIOINFORMATIC ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article needs linguistic improvement
Experimental design: No comment. Seems scientifically sound
Validity of the findings: I would like to suggest authors to prove the bioinformatic data with experimental works, like protein level expression of OTUD5.
Additional comments: No additional comments


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

Input:
Title: ANALYSIS OF DEUBIQUITINASE OTUD5 AS A BIOMARKER AND THERAPEUTIC TARGET FOR CERVICAL CANCER BY BIOINFORMATIC ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language should be improved to ensure that an international audience can clearly understand the text. It gets extremely difficult in the 'Discussion' section to understand what authors are trying to say. The language of all the sections needs to be improved.

Lot of acronyms have been used throughout the manuscript, even in the abstract (e.g. OTU, EMT, WDR-45 etc.) , without their full form. Please provide full form of all the acronyms used.

Literature references should be provided in the introduction and discussion section (e.g. Line 50, 302 etc.)
Experimental design: In the present study, using bioinformatic analysis, the authors have shown that lower expression of OTUD5 is associated with poor prognosis of Cervical Cancer and it's expression is associated with tumor stage, metastatic nodes and tumor subtypes.

In the results section 3.1, data analysis from TCGA database shows that there are missense mutations and copy number variations and deletions in OTUD5 gene in Cervical Cancer patients. Authors need to comment on the the upstream and downstream regulatory pathways which might be responsible for their observation.

In the results section 3.2, authors need to comment on why there is no difference in OTUD5 expression in HPV infected patients.

In the results section 3.3, authors have studied genes, which are correlated with OTUD5 expression in Cervical Cancer. Four highly positively correlated genes: USP11, RBM10, WDR45 and GRIPAP1 were chosen for further analysis.To clarify, their relationship with OTUD5, authors have knocked down OTUD5 in HeLa cells and showed that they are positively correlated. However, they need to comment on the fact that how this correlation affects poor prognosis of CESC? What happens to OTUD5 expression if USP11, RBM10, WDR45, GRPAP1 is knocked down? From this data, can we draw any conclusion on how these genes are related to each other and lead to poor prognosis?

In Results section 3.4 and 3.5, authors have described the miRNAs and transcription factors that regulate mRNA expression of OTUD5 and proteins that interact with OTUD5. They describe their observation but need to discuss how these regulation of OTUD5 expression and protein-protein interaction might cause poor prognosis of CESC.
Validity of the findings: I thank authors for providing all the underlying data and statistical analysis. However, they need to comment on their observation. Only stating the data is not sufficient.
Additional comments: No additional comments


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

Input:
Title: ANALYSIS OF DEUBIQUITINASE OTUD5 AS A BIOMARKER AND THERAPEUTIC TARGET FOR CERVICAL CANCER BY BIOINFORMATIC ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Significant efforts are needed to improve the grammar in the manuscript as well as the general use of English (some sentences do not make sense - perhaps an error in translation?).  Please also consider one of the software available to check and correct grammar. Additionally, it is highly recommended that authors find a colleague or a collaborator with good writing skills in English and have the manuscript reviewed. There are several instances where sentences that do not make any sense logically or scientifically.
	- The first word in the abstract, abbreviation OTU needs to be defined.
	- First sentence in abstract:
	"OTU deubiquitinase 5 (OTUD5), as a member of the OTU family, was previously reported play important roles in DNA repair and immunity." 'to' is missing before 'play'.
	Define abbreviations: PI3K-AKT, EMT
	Add some information and/or descriptive terms about the genes being discussed: WDR40, USP11, GRIPAP1, and RBM1 to improve scientific relevance.
	Avoid repetition of words such as 'Furthermore' in consecutive sentences
	The title of the manuscript needs to be changed too, in its current format it is not scientifically accurate. Consider something along the lines of
	"Bioinformatics analysis reveals deubiquitinase OTUD5... "
	Introductions are to be used as an opportunity to clearly set background for the research done or the results being presented. In several instances author go into expressing opinions or use sentences that read more like a discussion than an introduction. Also mostly these are unnecessary interpretations/expression of opinions. Just state facts and cite relevant literature.
	Example:
	Line 40-42:
	Although it has entered the era of targeted therapy and immunotherapy, traditional surgery, radiotherapy, and chemotherapy are still the most common treatments for cervical cancer.

	This sentence is unnecessary in the introduction. Neither the treatment options or the trends in the field are a focus of this manuscript.

	Check grammar:
	43-44

	Authors need to rewrite the introduction. Very little to no information is provided about the cellular role of otulins and ubiquitination.
	Include in the introduction:
	Otulins
	Ubiquitination machinery
	he roles associated with ubiquitination status of the cellular proteins
	Background information on OTU5
	known interactions of otulins, particularly OTU5

	Reread the manuscript and update citations. Appropriate citations are missing in several instances.
Experimental design: There are serious flaws in the experimental design:
	- No efforts have been done, or authors have not mentioned, whether the databases they are using have the curated information that would be appropriate to make scientific conclusions.
	- The datasets the authors have used as control is significantly smaller than the CESC samples, invalidating several of the results based on these datasets.
	Figure 1B: Not a meaningful comparison if taken into account the number of samples in control vs CESC.
	Also due to such a high variability in transcript values in CESC, it cannot be conclusively interpreted that the expression of OTUD5 in cervical cancer tissues was lower than that in normal tissue. A subset of samples in CESC has the exact identical distribution to that of normal samples.

	- Figure 1A:
	It is unclear what data the authors are plotting and interpreting in Figure 1A. 'Survival of OTUD5' is not a scientifically sound observation or a measurement. Explain the rationale, observation, and the plot further.
	Figure 2A:
	Again, high variability in the number of samples in each dataset.
	In Stage 1 and Stage 2 samples, n = 161 and 69, respectively, the transcript level of OTUD5 range from 20 to 80, this is contracted against the 'normal', n=3. This is not a valid comparison (1) due to the high variability of transcript levels and (2) high variability of the number of samples in the datasets being compared. This weakens, if not invalidates, the interpretation of data.
	Authors need to include some control that is known to maintain stable expression and this can be used for meaningful comparison.

	Figure 3A:
	Levels of other proteins are compared against that of OTUD5 to identify the genes whose expression is positively or negatively correlated with that of OTUD5. But, Figure 1 shows that OTUD5 expression is highly variable in CESC samples. In such a case, comparing the gene expression profile for other genes to that of OTUD5 would not yield any meaningful results. All dependent results discussed in figure 3 are shaky.

	Possible solution - include a housekeeping gene or a gene known to have a stable expression as a control.
Validity of the findings: Due to the flaws identified in the 'experimental design' section above, several results and conclusions are invalid without the proper controls.
	Also due to the high variability of sample sizes, the comparisons are meaningless as identified above.
Additional comments: No additional comments


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

Input:
Title: ANALYSIS OF DEUBIQUITINASE OTUD5 AS A BIOMARKER AND THERAPEUTIC TARGET FOR CERVICAL CANCER BY BIOINFORMATIC ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English language of the manuscript has been improved from the previous version. However, it still need more work.

Please provide literature reference at line 301 in the discussion section.
Experimental design: All the comments on the previous version is addressed in the current version of the manuscript.
Validity of the findings: No comments
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: MICROBIAL COMMUNITY ANALYSIS IN THE GILLS OF ABALONES SUGGESTED POSSIBLE DOMINANCE OF EPSILONPROTEOBACTERIUM IN HALIOTIS GIGANTEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Many landmark studies on microbial community on and/or in the gill of invertebrate have been reported, so novel association is expected in marine invertebrates. Authors found unique microbial community dominated by Epsilonproteobacteria on the gill of Haliotis gigantea, and visualized those cells by whole cell in situ hybridization technique. It is very interesting study, but is needed for the publication.
Experimental design: Experiments reported in this manuscript were based on standard methodology.
However, authors only used abalone individuals form market. This might have mislead of natural microbial community of abalone.
Validity of the findings: FISH images were not sufficient to provide robust findings of presence of epsilonproteobacterial cells.
Additional comments: Many landmark studies on microbial community on and/or in the gill of invertebrate has been reported, so novel association is expected in marine invertebrates. Authors found unique microbial community dominated by Epsilonproteobacteria on the gill of Haliotis gigantea, and visualized those cells by whole cell in situ hybridization technique. It is very interesting study, but is needed for the publication.

Majors
1. Title is too long, should be simple and concise.
2. Abstract should be shortened.
3. It was wondering epsilonbacterial cells were active or not, how much sequences were matched obtained from meta16S analysis and actual single cell on the gill.
4. It was also wondering the reason why only some Haliotis gigantea individuals harbored unique Epsilonproteobacteria. Authors should confirm the presence of Epsilonproteobacteria in wild animals.
5. Authors should add anatomical description of circulation system of abalone. Authors also describe species specific features of these system, in particular in Haliotis gigantea.
6. Authors also should confirm no microbes in hemolymph of animals used in this study.
7. In fig.4, cell localization was difficult to be confirmed. Eps333 and Eub338 merged picture was necessary.
8. English must be improved.

Minors
1. L152: the subtitle should be shortened.
2. L204: a period after 80 should be changed to comma.


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: MICROBIAL COMMUNITY ANALYSIS IN THE GILLS OF ABALONES SUGGESTED POSSIBLE DOMINANCE OF EPSILONPROTEOBACTERIUM IN HALIOTIS GIGANTEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Have you checked the authors data deposition statement? Yes, we can access to the data
Can you access the deposited data? Yes
Has the data been deposited correctly? Yes BUT in NCBI, the accession number corresponding to the stone sample, for the organism description it is written sediment metagenome, why? If it’s stones’ sample.
"DRX188994 Sample: Stones SAMD00192396 • DRS114354 • Organism: sediment metagenome”
Is the deposition information noted in the manuscript? Yes
Experimental design: Most of the manuscript is clear and well written in professional, unambiguous language.
The structure of the manuscript is well conforms to PeerJ standards, discipline norm, except the materials and methods section which needs to be improved in term of clarity (see comments below).

The main aim and rationale of the study need to be clearer explain as well as the focus on the specific Epsilonproteobacteria taxa.

The Figures are relevant, with high quality, well labelled & described.
Raw data supplied (see PeerJ policy) are available

Intro & background to show context. Literature well referenced & relevant.
The introduction described well the state of the art of the gills’ symbiont topic with relevant and right amount of references but in my opinion, focuses too much on deep sea hydrothermal or cold seep systems, even if the comparison between shallow and deep sea on lines 82-86 is well appropriated.
From line 67 to 71, on the Solemyidae description, it could be good to add few references about the symbiotic sulfur-oxidizing symbionts and to link that with the Thyasiridae description:
Dmytrenko, Oleg, et al. "The genome of the intracellular bacterium of the coastal bivalve, Solemya velum: a blueprint for thriving in and out of symbiosis." BMC genomics 15.1 (2014): 924 ; see also other papers
Also, the authors described the Solemyidae and the Thyasiridae and their symbionts’ roles but then, somehow it felt like the description of the species used in this study is lacking. Indeed, several information about their life’s styles, previous evidences of symbiont in the gill of specimens of the same family etc… will be beneficial for the manuscript.
Validity of the findings: The findings are well explained. Statistical analysis are done when it's possible. The conclusions are coherent with the results shown in the manuscript however some words about H. discus, H. diversicolor, T. cornutus could be a good improvement of the conclusion.

There are some discrepancy between the text and the phylogenetic tree: with the tree lines: 276-279 “BLAST analysis of the uncultured epsilonproteobacterial sequence revealed that the sequence could be assigned to various described Epsilonproteobacteria species, such as Arcobacter canalis strain F138-33 (87.43% identity), Arcobacter marinus strain CL-S1 (87.19% identity), Sulfurovum lithotrophicum strain 42BKT (87.01% identity) and Helicobacter pullorum strain ATCC51801 (86.78% identity), but these identity scores were low” BUT in the phylogenetic tree (Figure 2), clone Hgig1 is closest to Sulfurimonas autotrophica and Sulfuricurvum kujiense and bit further from Arcobacter nitrofigilis. That differs from the manuscript. Also, why not using Arcobacter canalis, Arcobacter marinus, Sulfurovum lithotrophicum, Helicobacter pullorum to do the phylogenetic tree??
Additional comments: The manuscript “Microbial community analysis in the gills of coastal shellfish and molecular identification of the potentially dominant epsilonproteobacterium on the gills of Haliotis gigantea” presents a relevant and interesting study, describing the gill bacterial composition of 4 species of gastropds (Haliotis gigantea, H. discus, H. diversicolor and Turbo cornutus) and 2 species of molluscs species (Meretrix lusoria and Cyclina sinensis) using high throughput sequencing, cloning-sequencing and microscopic methods. Besides, the authors revealed the evidence of a specific taxon of uncultivated Epsilonproteobacteria closely affiliated to Helicobacteraceae family, using specific primers designed for this purpose, in the gill of the abalone Haliotis gigantea
The manuscript flow well and it well written and the topic is really interesting.
However, there is several main points, that should be added or clarified before the paper is recommended for publication

Major comments:
The manuscript will really gain in clarity if the main aims of this study were clearly described in the introduction: why studying these specific species, why comparing molluscs and gastropods Which scientific gaps this study ambitions to explore. That will really help the readers to understand the study and its rationale.
Also, the authors should highlight why they did a specific focus on the uncultured Epsilonproteobacteria detection in different tissues of Haliotis gigantea. Is this specific taxon also present in the others abalone gills (Haliotis discus and H. diversicolor)? Did the authors checked that using the specific primers that they designed for the uncultivated Epsilonproteobacteria detected in Haliotis gigantea?

The manuscript: discussion and conclusion mostly, will gain in relevance if the authors dig deeper in the analysis of the gill microbiome, meaning not only the classes but maybe until the family or genus level when possible. For instance, the histogram shows only the classes with a relative abundance >10% and in the text, only few genera are highlighted as Vibrio, Arcobacter. Then, for example, I wonder what are their relative abundance within the Gammaproteobacteria and Epsilonproteobacteria respectively.

The manuscript will improve a lot if the authors can highlight both the core microbiome and variable microbiome, up to the genus level, in the gills of Haliotis gigantea itself and in H. diversicolor and compare and point out some similarity or dissimilarity among the core microbiote of these 2 species belonging to the same genus.
See for example: Neu, Alexander T., Eric E. Allen, and Kaustuv Roy. "Diversity and composition of intertidal gastropod microbiomes across a major marine biogeographic boundary." Environmental microbiology reports 11.3 (2019): 434-447.
Reich, Inga, et al. "16S rRNA sequencing reveals likely beneficial core microbes within faecal samples of the EU protected slug Geomalacus maculosus." Scientific reports 8.1 (2018): 1-9.
Hernandez-Agreda, Alejandra, Ruth D. Gates, and Tracy D. Ainsworth. "Defining the core microbiome in corals’ microbial soup." Trends in Microbiology 25.2 (2017): 125-140.

The material and method need some clarifications:
Does sample Hgig1 corresponds to the pool of 5 specimen collected at the farm? Not clear
“Other abalone specimens (H. gigantea, H. discus and H. diversicolor) and Turbo cornutus were obtained from a fish-market in Mie, Japan, from April 2017 to May 2019”: Any idea of where they come from? Where had they been collected?
“Gill tissues from H. discus (n=3), H. diversicolor (n=5), and T. cornutus (n=3) specimens were pooled into three tubes, one for each species (sample code: Hdis1, Hdiv1, and Tcor1)”: Can you explain why you have decided to pool the gill of each specie together?
H. discus specimens corresponding to samples Hdis2-4: I Can’t reckon where come from the sample 1 for H Discus? Line 100 H. discus n=3; line 103 sample name Hdis2-4 so no Hdis1 in the manuscript BUT in graph yes there is a sample Hdis 1 and in NCBI as well. Here some explanations are missing.
“Seawater and stone samples were collected from Minami-ise” Some indications are missing as the sampling point coordinates, when have they been collected, Which year? How have they have been collected Are these samples been collected close to the Farming Fishery Center, from the Farming Fishery Center?
Also, line 369: “sediments smelt …”why did you investigated the microbial diversity in the stones and not in the sediment and to look for potential symbionts inside?
Obtaining the 16S rRNA gene sequences of the uncultured epsilonproteobacterium by cloning:
“The phylogenetic tree of representative members of Epsilonproteobacteria inferred from 16S rRNA gene sequences was estimated by the Maximum Likelihood method using MEGA 7.0 (Kumar et al. 2016).” That’s not clear, I get that the authors wanted to obtain the almost full length of 16S rRNA gene in order to get a fair idea of the affiliation and taxonomy of this uncultured epsilonproteobacterium taxon but here I missed a link because the authors used universal primers that target all the Bacteria and not only the epsilonproteobacteria. Also, why only the epsilonproteobacteria are in the phylogenetic tree? How did the authors checked that send only Epsilonproteobactria clones to be sequenced?
Nested PCR analysis and sequencing of the uncultured epsilonproteobacterium:
“The nested PCR assay targeted 356 bp of the uncultured epsilonproteobacterial 16S rRNA gene using bacterial universal primers 27F and 1492R, as well as the uncultured epsilonproteobacterium-specific primers, Eps222F (5'-CGCTAAGAGATTGGACTATAT-3') and Eps578R (5'-GACTTAATAGGACACCTACATACC-3') (designed in this study).” Here the sentence is not clear, does that mean that the authors have used the same PCR products as for the cloning-sequencing? About the Eps222F/ Eps578R primers, more precisions are needed: are these primers been design for the NGS data, from the almost full length 16S rRNA gene? Specificity of the primers?
“Sequencing and phylogenetic analysis were performed as previously described in the subsection “Obtaining the 16S rRNA gene sequences of the uncultured epsilonproteobacterium using cloning method” above” then, why only one sequence of 1436 pb has been deposited in GenBank (LC511979.1). There is no accession number for the nested PCR data.
“Four out of sixteen cloned sequences were closely related to the sequence of the uncultured epsilonproteobacterium obtained from the 16S rRNA gene amplicon sequencing undertaken in this study. The longest sequence length was 1436 bp (LC511979).” How many clones have been sequenced? How did the authors checked they send only epsilonproteobacteria clones to be sequenced? Why only 16 sequences: it’s very few especially if the nested PCR have been done from the DNA from the gills of the 5 organisms? If the sequences were closely related, what is the percentage of similarity? Why only 1 accession number? Why only 1 sequence in the phylogenetic tree? How many clones did the authors managed to obtain per Hgig sample?

Minor comments:
Abstract:
-“Recently, it has been reported that chemosynthetic bacteria in the gills of some shallow-water bivalves have the ability to fix nitrogen carbon, and synthesize amino acids for their hosts.” This sentence is not useful as the study is not dealing with the metabolic activities of the symbionts and neither on which compounds the symbionts can supply to their hosts.
- “Microbiome analysis suggested that the gills of H. gigantea, M. lusoria and C. sinensis each have unique bacterial community structures that differ from those in the surrounding environment”; add detected between differ and those in the surrounding… In addition, some results about the microbial communities inhabiting the gills of Haliotis discus, H. diversicolor and Turbo cornutus would be appreciated.
-Few words about the core and variable microbiome within Haliotis gigantea and H. diversicolor will improve a lot the abstract.

Introduction:
The introduction described well the state of the art of the gills’ symbiont topic with relevant and right amount of references but in my opinion, focuses too much on deep sea hydrothermal or cold seep systems, even if the comparison between shallow and deep sea on lines 82-86 is well appropriated.
From line 67 to 71, on the Solemyidae description, it could be good to add few references about the symbiotic sulfur-oxidizing symbionts and to link that with the Thyasiridae description:
Dmytrenko, Oleg, et al. "The genome of the intracellular bacterium of the coastal bivalve, Solemya velum: a blueprint for thriving in and out of symbiosis." BMC genomics 15.1 (2014): 924 ; see also other papers
Also, the authors described the Solemyidae and the Thyasiridae and their symbionts’ roles but then, somehow it felt like the description of the species used in this study is lacking. Indeed, several information about their life’s styles, previous evidences of symbiont in the gill of specimens of the same family etc… will

Results:
“Greengenes database”: Which version

“These sequences were observed in all abalone specimens, but were not dominant, except in Hgig5 (68.4%).”, any idea why Hgig 5 is so different from the others

“PCoA”: Utilization of which software, R? Need to be described in the Materials & Methods

Discrepancy between the text and the phylogenetic tree: with the tree lines: 276-279 “BLAST analysis of the uncultured epsilonproteobacterial sequence revealed that the sequence could be assigned to various described Epsilonproteobacteria species, such as Arcobacter canalis strain F138-33 (87.43% identity), Arcobacter marinus strain CL-S1 (87.19% identity), Sulfurovum lithotrophicum strain 42BKT (87.01% identity) and Helicobacter pullorum strain ATCC51801 (86.78% identity), but these identity scores were low” BUT in the phylogenetic tree (Figure 2), clone Hgig1 is closest to Sulfurimonas autotrophica and Sulfuricurvum kujiense and bit further from Arcobacter nitrofigilis. That differs from the manuscript. Also, why not using Arcobacter canalis, Arcobacter marinus, Sulfurovum lithotrophicum, Helicobacter pullorum to do the phylogenetic tree??

Discussion:
“This assumption was also supported by the finding that the gills of Hgig6 gave a clearer band than the gills of Hgig4 in the PCR assay using the same concentration of DNA template.” qPCR is needed to infer the number of representatives of this specific taxon in each gill’ abalone.

References:
Newton ILG, et al., is followed by Katoh K et all and Kuwahara H, et al is followed by On S et al., : Check this order.

Wickham, 2016 in the manuscript while it’s 2009 in the ref Wickham H. 2009. ggplot2: Elegant Graphics for Data Analysis. New York: Springer. DOI: 10.1007/978-0-387-98141-3 in the reference list

Figure 2: species name in italic


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: MICROBIAL COMMUNITY ANALYSIS IN THE GILLS OF ABALONES SUGGESTED POSSIBLE DOMINANCE OF EPSILONPROTEOBACTERIUM IN HALIOTIS GIGANTEA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript informs bacterial community compositions in bivalve and gastropods, particularly epsilonproteobacterium in Haliotis gigantea. Several investigation approaches were performed to elucidate the bacterial communities including 16S rRNA amplicon sequencing, PCR and fluorescence In-situ hybridization (FISH). The manuscript is written in good English language even though some modification is needed. It also conforms to PeerJ standard including accession of DNA sequence and data. However, introduction and background of the study need to be reformulated clearly. The authors mention epsilonproteobacterium in the title; but the importance of this bacterial group is missing in the introduction section. The authors also mentioned bacterial community in stone but they did not discuss why and how the information from that sample influence/support the objective. Furthermore, figure quality and tables need to be improved.
Experimental design: Despite of varied methods and data, I observed an essential issue on sampling design. It is still not clear whether H. gigantea specimens were the same origin/nature. It is mentioned that the H. gigantea specimens were collected from farming fisheries center and fish markets. However, it is important to clarify if they are wild specimens or domesticated origin. In addition, it is also important to mention how long the specimens stayed in the fish markets which may affect bacterial community compositions in H. gigantea. Furthermore, there is an unequal samples if Hgig1 (cultured giant abalone, N=5) is a pool samples, while other Hgig samples (Hgig2-6) were prepared individually. In my opinion, this will affect 16S rRNA amplicon sequencing analyses. Please also see detail information which I provide separately (together with annotated PDF file in last page) .
Validity of the findings: The results have been described in a descriptive way. The authors conclude the study based on the result that that they have and it is in accordance to their objective.
Additional comments: The study deliver a promising result about bacterial community compositions in shallow water bivalves and gastropods. Please find my detail comment in the last page of PDF file!


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: MICROBIAL COMMUNITY ANALYSIS IN THE GILLS OF ABALONES SUGGESTED POSSIBLE DOMINANCE OF EPSILONPROTEOBACTERIUM IN HALIOTIS GIGANTEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: this is second review, all points raised in the first review were fixed.
Experimental design: this is second review, all points raised in the first review were fixed.
Validity of the findings: this is second review, all points raised in the first review were fixed.
Additional comments: this is second review, all points raised in the first review were fixed.


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: MICROBIAL COMMUNITY ANALYSIS IN THE GILLS OF ABALONES SUGGESTED POSSIBLE DOMINANCE OF EPSILONPROTEOBACTERIUM IN HALIOTIS GIGANTEA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Microbial community analysis in the gills of abalones 1 suggested possible dominance of epsilonproteobacterium in Haliotis gigantea” presents an interesting study based on various approaches: NGS, cloning-sequencing, FISH microscopy in the aim to investigate the microbial diversity inhabiting invertebrates’ gills. The authors have highlighted that the microbial diversity inhabiting the gills of Haliotis gigantean is dominated by an unknow OUT affiliated to the Epsilonproteobacteria (Helicobacteracea) while this OUT in not or very little detected in the other abalone species studied. The FISH analysis done with specific primers proved that the uncultivated Epsilonproteobacteria cells are located on the gills. The authors have also exhibited that core microbiome co-own between the 3 abalone species is relatively restraint with only 2 commons OTUs, 1 affiliated to the Spirochaetaceae family and 1 to Mycoplasma genera.
This study is relevant for the both the environmental and aquaculture field. Overall it is a very interesting study, well written which can be published.
Experimental design: Wild and cultured abalones were used for this study and molecular approach was used to describe the microbial community in the gills, foot and gut. The specimen replicates varied between 3 and 5 allowing statistical analysis. Classical methods were used: NGS, cloning-sequencing and FISH microscopy.
Validity of the findings: The results have been well described and the findings have been well explained and discussed when possible. Statistical analyses were done when it was possible. The conclusion is coherent with the results shown in the manuscript and fit the objectives of this study.
Additional comments: This version of the manuscript is clearer especially the materials and methods. Also, the objective of the study is now well described. By removing the data about the 2 molluscs species Meretrix lusoria and Cyclina sinensis, the authors allowed the manuscript to be sharper and to bring out the main conclusion about the Espilonproteobacteria dominance in the gill of H. gigantea.
In fine, this study brought new highlights into the microbial symbiosis in the gills of the coastal abalone.

There are few minor comments to fix:
Line 80 add “role” between important and for
Line 108-109 : should be “Solemya velum, affiliated to the Solemyidae family”
Line 152 : should be Fifty milliliters of seawater were filtered instead of was filtered
Line 411-412 : “The genus Vibrio (Gammaproteobacteria) was observed in all samples” instead of were
Line 452 : “The unclassified Spirochaetaceae was abundant in” instead of abundance
Line 553: “Four out of sixteen cloned sequences were completely matched” remove were
Line 670-671 : should be “where they are often associated with invertebrate hosts”
Line 698 : “gigantea in in direct” remove one in


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: MICROENVIRONMENT-RELATED GENE TNFSF13B PREDICTS POOR PROGNOSIS IN KIDNEY RENAL CLEAR CELL CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The written English is readable and satisfactory. There are some typos in the manuscript (line 22, 113 etc.).
Literature references are sufficient in terms of background coverage and biological validation. However, the citation style in the text is confusing. For example, in line 53, there are four references cited by only using first letter of authors, this mistake is repeated in whole manuscript.
Resolution of some figures is poor, e.g., Figure 3 (all fonts in small captions are  unreadable), Figure 7B.
Experimental design: The manuscript did not develop a novel algorithm for RNA-sequencing analysis or for biological network analysis, so it might not be considered as a novel research paper.
This study could be interpreted as a case-study. The found biomarker might initiate new in-vivo / vitro studies for KIRC treatment. However, this fact does not change being a case-study type of manuscript.

The method section needs improvements in terms of following questions and suggestions:
1. How were down-regulated genes selected by using only LogFC > 1 cut-off?
2. In line 100-101, "the number of connections of gene node" should be expressed as "degree". This definition should be reflected to the rest of the manuscript.
3. In line 102, identification of a hub node in a PPI network is bounded by nodes with a higher than 5 connections (degree). How was this cutoff "5" chosen?
4. The details of ESTIMATE algorithm can be explained in the beginning of the method section. Although it is used as a tool, it could be helpful to give more technical details to replicate this study.
Validity of the findings: There are some questions about explanations of results in this study:
1. In line 143, authors wrote that "the KIRC samples were divided into high score group and low score group". What are the numerical ranges of these high and low scores? How did they set these score values?
2. In line 172, the construct PPI sub-network covers 53 nodes and 110 edges. why did it not cover 95 of DEGs identified in the previous step?
Additional comments: The study performs a network-based analysis of KIRC patient RNA-sequencing samples to identify some early stage diagnostic markers by considering the tumor microenvironment. Verification of identified markers on the independent datasets is remarkable analysis of the study.

Authors did not develop a novel analysis algorithm for RNA-sequencing or biological network analysis, they applied well-known algorithms and online data sources. So, this study could be interpreted as a case-study. The identified biomarker, TNFSF13B, was analyzed in other cancers, but not mainly kidney one. This finding could have contribution to initiate other experimental studies for KIRC treatment.

This manuscript can be evaluated as new research paper, when it performs analysis for another cancer type by applying the same computational pipeline and considering effects of the tumor microenvironment.


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: MICROENVIRONMENT-RELATED GENE TNFSF13B PREDICTS POOR PROGNOSIS IN KIDNEY RENAL CLEAR CELL CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. No need to mention the abbreviations of terms that are mentioned only once in the abstract.
2. Most references were cited using first name (or an abbreviation), year for example line 40, 46 and 53. Please revise. References to data or tools should be cited on the first mention for example TCGA (line 48).
3. TCGA is currently hosted by GDC. A direct link the current host would be the most helpful.
4. A brief explanation of what the ESTIMATE algorithm does might be needed. Especially what each of the scores means exactly in the context of the study.
5. The description of the differential expression analysis (line 86) is not sufficient. The section should mention input data, preprocessing, quality controls and the comparisons that were attempted.
6. References to some of the tools (R packages) were not included in the text. For example, heatmap, beeswarm, ggplot2 and R itself. If no papers/books are recommended by the creators to cite, please cite them as software.
7. On some occasions, the authors didn’t mention the specific tools that were used to conduct certain steps of the analysis. For example, calculating the node degrees (line 101) and the survival curves (line 104).
8. The heatmaps (Figure 3A&B) are not very informative. Different scaling, color schemes or clustering might be helpful in showing the trends in the expression profiles. The labels (rows and columns) are very small. Alternative graphs such as volcano plots might be considered to show the difference in expression between low and high scores.
9. The functional enrichment analysis reported in Figure 3E&F was only briefly mentioned in the text (line 69). The findings of this analysis should be described in the results or removed if not essential to the study. In addition, the x-axis labels of those two figures are missing.
10. Figures 5A-F (right) were referred to in the text as scatter maps (line 183). Those look more like a box plot with point or jitter overlayed on top. Also, the unified scale of the y-axis in those plots (0-80) makes it difficult to see the spread of the points. Rescaling or different y ranges might be useful.
11. The authors state that the cutt-off of the statistical significance in expression between low and high scores were based on log FC and (p)-value (line 161-162). Adjusting for multiple testing using something like FDR should be considered or stated if already used. Moreover, it seems like the intersection between immune and stromal score-based differentially expressed genes rather than the “interaction” (line 163) was used to select the genes of interest.
Experimental design: 1. The section describing “selection of hub genes with oncomine database” is confusing. First, the specific aim of that step of the analysis is not clear. How can hub genes be selected based on a meta-analysis? Second, the datasets on which the meta-analysis was based were neither cited nor described properly. Finally, the verification eluded to (line 121) was based on the same datasets.
2. Figure 5 shows the prognostic values of six hub genes. These predictors were not shown to be independent. In addition, the relation of these factors to other clinical characteristics of the samples/patients can be shown to support or explain their values.
3. Although 95 differentially expressed genes were suggested to be of interest, only 53 are found in the protein-protein interaction network. There seems to be an unacknowledged loss of information in this step.
4. The selection of TNFSF13B as a “hub gene” from previously declared six “hub genes” is unjustified or at best unexplained.
5. Figure 6B-E show an upward trend of the centered rations of the gene of interest with the tumor stage/grade but no correlation stats were reported as claimed in the text (line 192).
Validity of the findings: This study stresses the importance of the tumor microenvironment in renal carcinoma. However, due to the lack of sufficient details of the methodology (above, basic reporting) and some arbitrary analysis decisions (above, experimental design), the presented findings doesn’t seem to justify the claims of the study. The above mentioned issues can be summarized in two points. First, some of the analysis were taken as support for the findings of the preceding steps. For example, it is not clear why a meta-analysis of gene expression in some datasets would support the importance of highly connected genes in a network that was derived from differentially expressed genes between samples with different immune or stromal scores. Second, some of the claims were not rigorously investigated. For example, the prognostic values of the six “hub genes” were not tested in a multivariate analysis and were not shown to be independent predictors. Another example was to claim the a gene of interest is correlated with tumor grade or stage without showing a correlation statistics.
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: MICROENVIRONMENT-RELATED GENE TNFSF13B PREDICTS POOR PROGNOSIS IN KIDNEY RENAL CLEAR CELL CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: Overall the research question is meaningful and the methods used to address it are sound.
However the methods section should be further clarified:
1.	How are hub genes found? It seems PPI and survival analysis is used as 1 method and Oncomine database is used as another. However, it’s not clear how the information from the 2 methods is integrated. It would be helpful if details are provided about number of genes found by all methods, overlap between them and significance of overlap.
2.	Version of all software should be mentioned along with the libraries/parameters used. E.g.: R version?, GSEA version?, limma version? Etc.
3.	For GSEA analysis give more details. What gene sets were included/excluded. E.g. greater than 15 or less than?
4.	Line 87 mentions LogFC. What is the base here?
5.	Give details about ESTIMATE algorithm. How are the 3 scores calculated: immune, stromal, ESTIMATE.
Validity of the findings: Overall the authors have mined the TCGA database and gotten some interesting results for KIRC. However the manuscript in its current form is difficult to follow and many of the analysis seem arbitrary. It would greatly help if the authors could provide a rationale for some of their choices. E.g.: explain why hub genes should be identified through their method, difference between the 2 methods and the reason for taking the intersection.
Some other analysis/text that are unclear:
1.	The authors wanted to determine the role of immune and stromal cells to the tumor microenvironment in KIRC. They found survival to be anti-correlated to immune score however not stromal scores. It’s important to explain over here the differences between the different scores.
2.	It’s also not clear why the authors took an intersection of the differential genes found by immune & stromal scores when stromal scores show no differences between stages. These 95 genes should be provided in a table.
3.	Its not clear why the authors only select TNFS13B as a hub gene in the end when earlier they had found 23 hub genes. What happened to the rest?
4.	Also its confusing how hub genes seem to have multiple names in the manuscript: e.g: central node genes, hub nodes etc.
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: MICROENVIRONMENT-RELATED GENE TNFSF13B PREDICTS POOR PROGNOSIS IN KIDNEY RENAL CLEAR CELL CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English proof-reading elevated the language of manuscript. The correction of basic typos helps to increase quality.

Improving the resolution of figures also helps to interpret the major results of the study.
Experimental design: The suggestions made for explanation of the ESTIMATE algorithm was taken seriously and added an extra paragraph.

The MCODE algorithm was not mentioned at all in the original submission. Due to some questions about network clustering etc. authors also added the usage purpose of this algorithm into manuscript, this extension also found to be helpful, since it puts together the whole picture in analysis stage.
Validity of the findings: The statistical questions of reviewers about the interpretation of the results were generally replied and introduced necessary data and sections into the manuscript.
Additional comments: I thank to authors; they took comments seriously and updated the original manuscript with the light of several suggestions. The better explanation of applied analysis definitely improved the quality of paper, the representation of results makes an easier understanding of the study.


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

Input:
Title: MICROENVIRONMENT-RELATED GENE TNFSF13B PREDICTS POOR PROGNOSIS IN KIDNEY RENAL CLEAR CELL CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Citation for some of the tools are still missing. For example, the “survival” and “survminer” R packages. Moreover, in addition to mentioning the analysis tools, a brief description of the goal, input and output of the analysis is always useful.
The reported three analyses in place of Oncomine were not fully described in the methods section. More specifically, which groups were compared in the differential expression and which genes were included in the survival and cox regression? Finally, no rationale was given as to why the intersection of seemingly very different analysis is of particular importance.
In the univariate and the multivariate analyses, TNFSF13B had a very small effect size or non-significant one. Despite being shown to predict survival in KM analysis, the cox analysis does not support this finding.
The authors claim that TNFSF13B is a potential biomarker and that it is related to the tumor microenvironment. The first point is not strongly supported by the revised analysis. The second point is only touched on briefly. The findings that many genes are differentially expressed between groups of different stromal/immune scores is not surprising. TNFSF13B is not shown to be representative or key to these differences.


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: MICROENVIRONMENT-RELATED GENE TNFSF13B PREDICTS POOR PROGNOSIS IN KIDNEY RENAL CLEAR CELL CARCINOMA
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: The points raised by me have been addressed sufficiently.
Validity of the findings: The authors have significantly improved the manuscript and the conclusions are well supported.
Additional comments: There is a recent study published on TNFSF13B and KIRC that on a brief glance seems complementary and very close to your work. (TNFSF13B correlated with poor prognosis and immune infiltrates in KIRC: a study based on TCGA data..by Song Li, Junqing Hou, Zhen Wang, Junkai Chang, Weibo Xu). You should add that reference and discuss any agreement/disagreement in results.


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

Input:
Title: MICROENVIRONMENT-RELATED GENE TNFSF13B PREDICTS POOR PROGNOSIS IN KIDNEY RENAL CLEAR CELL CARCINOMA
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised manuscript addresses most of the issues raised in the review. Two issues remain however.
1. If a software tool doesn’t have a directly related paper by the creator, it can be cited as a software program. Use the `citation(“<package name>”)` in R to get the recommended citation for a given package. Alternatively, many of the R packages have books or web pages that can be used as citations.
2. No rationale was given as to why the intersection of seemingly different analysis is of particular importance.


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: MICROENVIRONMENT-RELATED GENE TNFSF13B PREDICTS POOR PROGNOSIS IN KIDNEY RENAL CLEAR CELL CARCINOMA
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I want to thank the authors for incorporating all reviewer comments. The article is substantially improved and gives some interesting insights about the role of TNFSF13B.


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: CRANIAL OSTEOLOGY OF THE EARLY CRETACEOUS TURTLE PLEUROSTERNON BULLOCKII (PARACRYPTODIRA: PLEUROSTERNIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript by Evers, Rollot and Joyce is an outstanding contribution to comparative anatomy of turtles. The authors thoroughly redescribe Pleurosternon bullockii based on a CT-scan of a single well-preserved and partially articulated specimen and compare their findings to a good sample of other paracryptodires and the condition of other turtles, such as helochelydrids and pleurodires. The manuscript is well-written in perfect English and the discussion is well-supported by the data. The introduction covers the relevant context and the figures are beautifully present, and adequately illustrated the discussed topics. I have only some minor comments and suggestions which I provide on the attached commented PDF file. One of these needs to be addressed because it might be an error in the 3D segmentation of the described specimen. As such, I fully support the publication of this manuscript after this single issue is addressed by the authors.
The authors are welcomed to know my identity.
Experimental design: The authors use 3D reconstructions and renderings based on a micro-CT scan of a single specimen to build their redescription. All CT scan parameters are presented and the methods are well outlined.
Validity of the findings: The manuscript contains several important and novel anatomical findings and reinterpretations that will surely serve as the basis for novel phylogenetic interpretations of paracryptodires and turtles in general. I particularly enjoyed reading the comparisons between the described specimens and pleurodires, and I believe they will be very important for discussions in the turtle community. As such, this contribution will be of interest for experts in turtle anatomy and evolution.
Additional comments: As explained above, I have only some minor comments that are all available on the PDF file. It is worth to highlight just three of the most important ones.
(1) Between lines 276 and 279 of the manuscript PDF where you discuss the position of the jugal in the ventrolateral margin of the skull. You state that it forms a step-like margin between the maxilla and jugal, which is ok for your specimen, but you cite Glyptops as also possessing this feature. Although I've never seen a Glyptops specimen in person, based on its original description publication I find its margin very similar to that of Dorsetochelys, and not step-like. I might have not understood what you meant by a "step-like margin". Please, double-check this feature.
(2) You alternately used "cheek" and "lower temporal" emargination along the text. Please, choose one and be consistent all along it.
(3) The right and left premaxilla reconstructions seem conflicting to me. The left premaxilla is much larger, including an ascending process that forms part of the lateral rim of the external nare, which is shown on the right side as completely formed by the maxilla. Also, the anterior extension of the maxilla on the right side reaches the smaller premaxilla, which is compatible with the most anterior piece of the left premaxilla. This raises me the question of whether the second piece of the left premaxilla isn't, in fact, a piece of the maxilla instead, in which a crack was confused with a sutural contact. In fact, in all of the other taxa in your comparison sample, e.g. Compsemys, Dorsetochelys, Glyptops, and Arundelemys, the premaxilla does not extend so far posteriorly, which agrees with my interpretation above and also to the size and position of the right premaxilla. It would be very important for you to double-check this, so as to avoid misinterpreting its anatomy.


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: CRANIAL OSTEOLOGY OF THE EARLY CRETACEOUS TURTLE PLEUROSTERNON BULLOCKII (PARACRYPTODIRA: PLEUROSTERNIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I enjoyed reading this paper for several reasons: it is well-written (the occasional typo should be easy to correct using ‘spell-check’), exhaustingly referenced, and beautifully illustrated. The paper presents a detailed morphological description and analysis of a fossil turtle skull (Pleurosternon bullockii) from the Early Cretaceous of Europe, based on CT scans. The skeletal structures and associated soft anatomy (cranial nerves and blood vessels, jaw adductor musculature) is competently described in detail and exhaustively compared with a diversity of potentially closely related taxa. I am no expert in fine-grained turtle interrelationships, so I cannot comment critically on the choice of taxa employed in these comparisons. But the pleurodiran features shared by Pleurosternon bullockii are certainly particularly intriguing.
I think the authors do a particularly good job at a detailed analysis of cranial morphology in a suite of taxa that are compared with Pleurosternon bullockii, carefully evaluating the potential to derive from those descriptions and comparisons phylogenetically informative characters, without rushing ahead to present another global analysis of turtle interrelationships. The paper is exemplary for how to lay a solid partial groundwork for future such analyses. It is refreshing to see the authors concentrate on morphological details, rather than on character coding. I recommend the paper to be accepted for publication once the occasional typos have been cleaned up.


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: CRANIAL OSTEOLOGY OF THE EARLY CRETACEOUS TURTLE PLEUROSTERNON BULLOCKII (PARACRYPTODIRA: PLEUROSTERNIDAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: It is a good and well written paper that will be useful for the community interested in turtle morphological evolution. I have a series of small remarks that are listed in "general comments".
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is an interesting study documenting the cranial anatomy of Pleurosternon bullocki.  The paper is good, and my remarks concerns minor things that may help authors to tune the paper.

1. The authors argues that it is important for phylogenetic reconstruction, but they did not re-evaluate phylogenetic reconstructions with the characters they found and the similarity for Pleurodira. This is not a problem, but I think that these arguments could be likely dropped from the abstract and introduction since indeed better documenting morphology always help to better document phylogenies (but not only, they can also help to better understand the evolution of morphological functions). We have here a good redescripition of the skull of this species and there is no need to present that need as a solution for resolving a systematic problem since the paper is not demonstrating that we know better understand the relationship of paracryptodira and within that group. I am, however, fine with the discussion about character importance.

2. you often do some general comparisons to cryptodira and pleurodira without giving example of taxa; it should be good from the beginning if your comparison extend to cryptodira sensu stricto, or to pan cryptodira (and the same for pleurodira). You also do some comparison with Mesochelydia, maybe you should refer to Joyce 2017, when you do it.

3. Can the authors appreciate the size of the foramen stapedio-temporale and compare it with trionychids and carettochelyids?

4. l.216: the "crownward stem-lineage" term looks akward -> could you rephrase it?

5. l. 258: orbital ??? you probably mean orbit. I am not sure that sulcus would be a good term to describe a space.

6. l.255: Thus, we use terms from -> I suggest "we extend", and you can drop "although we note that it is incipient with respect to the pleurodire morphology".

7. l.260. You say that there is another similarity with pleurodires, but a few lines after you write "also an eclectic group of cryptodires" -> this similarity is therefore more likely to be not only shared with pleurodires, can you list cryptodira you are refering to (or provides a set of examples)?

8. l. 338: do you suggest it is destroyed or that it never existed ? maybe you can send the reader to the vomer part here "(see vomer section)".

9. l. 480: Well, but does that means that it had the same function than the processus TROCHLEARIS pterygoidei? Not sure that there is any functional homology here. It seems no from what you write juste after.

10. you are using "parabasisphenoid" and usually refer to this complex for the contact and comparison, I suggest to use basisphenoid or parasphenoid whenever you can since these two bones can be distinguished in that specimen.

11. l. 736-738 and next: does that change anything to select one or the other name ? does that mean that when the internal carotid artery enter anteriorly, we should stop homologize the opening with the foramen posterius canalis carotici interni ? or does that means that you want to homologize foramen posterius canalis carotici cerebralis with the canalis carotici interni as a whole ???? I think that the fpccc should be indicated on fig 7C. I have also a problem with fig 7E: how can we see faccc in this view ? it should be concealed by the lateral flange of the rbs (cf fig 7D)? You do not discuss the position of faccc in the text, I think it should be done. why not finally calling fpccc -> foramen ventralis canalis carotici cerebralis; and faccc : foramen dorsalis canalis carotici cerebralis ? Does the CT reveal the trajectory of the canal issuing from what you call faccc ?

12. l. 850...etc: it would be good to have a table summarizing similarities and differences among groups of paracryptodira that would be a good complement to the text.

13. l. 909: contact between Pterygoids and basioccipital : it was noted already in Gaffney 1979 for Glyptops; and Gaffney also noted this contact in Dorsetochelys (fig 28). Subsequent authors probably forgot about it, but it was already at least supposed that this contact was present in several mesozoic groups (not only in Baenids).

This is a signed review:
Julien CLAUDE
Institut des Sciences de l'Evolution de Montpellier.


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: CRANIAL OSTEOLOGY OF THE EARLY CRETACEOUS TURTLE PLEUROSTERNON BULLOCKII (PARACRYPTODIRA: PLEUROSTERNIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors answered to all my comments satisfactorily and, specifically, they revised the segmentation of the maxilla/premaxilla bones. I am satisfied with the revised manuscript and, as such, I recommend it for publication as it is.
Experimental design: No comments.
Validity of the findings: No comments.
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: INVESTIGATION OF MICROBIAL COMMUNITY INTERACTIONS BETWEEN LAKE WASHINGTON METHANOTROPHS USING GENOME-SCALE METABOLIC MODELING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written, clear and unambiguous. The introduction covered all the content needed to understand the results with the literature well-referenced and current. All figures are in good quality and well described. My concerns and suggestions are described in Comments for the author.
Experimental design: This work is within the scope of the journal and shows a well-designed and rigorous investigation with coherent methods to achieve the objectives. Methods were described with sufficient detail and information to replicate.
Validity of the findings: The microbial community interactions of methanotrophs is of interest to the field, so this study is a welcome addition.
Additional comments: This manuscript by Islam el al describes an effort for developing highly curated genome-scale metabolic models of Washington lake isolating methanotrophs and investigating microbial community interactions between them. The objective was clear and the method applied such as GSM reconstructions, flux analysis, community model formation which revealed some interesting insights into the interactions of methanotrophs in this microbial community. Based on this property the manuscript is of interest to the C1 community. However, in order to be more suitable for publication, the authors are encouraged to address the following concerns:
Major comments:
-	The reviewer concerns about how the biomass equation and non-growth associated maintenance of these models were generated. Please include in the Materials and Methods section.
-	There were three modes for methane oxidation including redox arm, direct coupling and uphill electrons. Which mode was included in these models of Methylobacter and Methylobacter?
-	One significant question the reviewer has is that Methylobacter Tundripaludum 21/22 and Methylomonas sp. LW13 are members of the Gammaproteobacteria (type I methanotroph) so that they should use RuMP cycle as the main pathway for formaldehyde assimilation. Additionally, they only possess an incomplete serine cycle, lacking reactions converting acetyl coenzyme A to glyoxylate. Therefore, the serine cycle cannot serve as the major carbon assimilation pathway in type I methanotrophs. However, as described in the manuscript, the flux analysis showed that all of the consumed methane enters the serine cycle for biomass formation and no carbon goes through RuMP cycle (HPS and PHI fluxes were zero). Please explain about this.
Minor comments:
-	Line 131-132: There were several accurate genome-scale metabolic models that have been developed in both type I and type II methanotrophs which improved our knowledge on C1 metabolism. These relevant papers could have been cited in the Introduction section in order to give a better picture of the current state of the art in this field.
-	Line 248-249: Please add the references for the Methylococcus capsulatus and Methanomonas methanica models.
-	Line 402-404: The author described Methylomonas have branched and a complete oxidative TCA cycle. The reviewer recommends the author to describe TCA cycle in Methylobacter in order to compare the TCA cycle between these methanotrophs under different conditions.


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: INVESTIGATION OF MICROBIAL COMMUNITY INTERACTIONS BETWEEN LAKE WASHINGTON METHANOTROPHS USING GENOME-SCALE METABOLIC MODELING
Review round: 1
Reviewer: 2

Output:
Basic reporting: To my knowledge, this is the first study on simulation of a methanotrophic community comprising of two type I methanotrophs. This topic is of great importance since it is experimentally challenging to resolve flux distributions of individual microbes in a bacterial community. This paper has a nice introduction regarding both the methanotrophic communities and simulation of cocultures via genome-scale models. Overall, the manuscript is well-written, and all figures and tables are well described.
Experimental design: Overall, the method and materials part has presented detailed information of how the genome-scale models were built and how the simulation was achieved via multi-layer optimization. I have a few questions:
1.	What is the community level objectivity exactly applied for all the growth conditions? Can you clarify that in the method part? Are there multiple community level objectives possible, which might lead to different results?
2.	How about the maintenance energy? Can you add some comments on what value(s) you have used? Or did you just let the model find whatever gives the optimal solution? If this is the case, you might need to check if the value is within reported ranges of energy cost by cell maintenance.
3.	Methanotrophs are known to produce exopolymeric substances (EPS), especially at a high cell density. Have you considered the EPS formation in the model?
Validity of the findings: With regard the findings, I have some major concerns, because some of them do not make sense for type I methanotrophs:

1.	For aerobic methanotrophs, the theoretical O2:CH4 consumption ratio is between 1 and 1.5. The pMMO may oxidize NH4 as well, so this number can be higher than 1.5. However, based on Figure 1 and 7, this ratio is predicted to be higher than 3 in general. This does not make sense. Please see the following research articles which either measured or predicted the O2/CH4 consumption ratios (ranging from 1.0 to 1.6) agreeable with theoretical values.
a.	Leak, D. J., Dalton, H., 1986. Growth yields of methanotrophs. Appl. Microbiol. Biotechnol. 23, 470-476.
b.	Gilman A, et al. 2015. Bioreactor performance parameters for an industrially-promising methanotroph Methylomicrobium buryatense 5GB1. Microb Cell Fact 14:182.
c.	de la Torre A, et al. 2015. Genome-scale metabolic reconstructions and theoretical investigation of methane conversion in Methylomicrobium buryatense strain 5G(B1). Microb Cell Fact 14:188.
d.	Lieven C, et al. 2018. A genome-scale metabolic model for Methylococcus capsulatus (Bath) suggests reduced efficiency electron transfer to the particulate methane monooxygenase.
e.	Fu Y, et al. 2019. Core metabolism shifts during growth on methanol versus methane in the methanotroph Methylomicrobium buryatense 5GB1. mBio

2.	For the type I methanotrophs, the ribulose monophosphate (RuMP) cycle should be the major route for formaldehyde assimilation. However, based on this study, this cycle remains inactive under all growth conditions for both strains: fluxes of H6P synthase are all 0.0 in figure 3-6, and the serine cycle is predicted to be the major carbon assimilation pathway in many cases. I don’t think this is correct. It has been reported that serine cycle carries a minimal flux during growth on methane for Methylomicrobium buryatense 5GB1C (Fu, et al. 2017). Although it can be more active in a different growth condition or in a different methanotroph, the serine cycle should not be the major pathway responsible for C1 assimilation in the type I methanotrophs.

In addition, the in vivo reaction rate of formaldehyde + THF->methylene-THF is reported to be low (Hai He, et al. 2020; Peyraud R, et al. 2011; Gregory J. Crowther, et al. 2007). But in your flux maps, this reaction is too active. Please note that this is not an enzymatic reaction but a spontaneous reaction. And evidence has shown that formate, instead of formaldehyde, is assimilated into the serine cycle (See ref c-d), although classic articles described formaldehyde as the branch point.

a.	Fu Y, et al. 2017. The oxidative TCA cycle operates during methanotrophic growth of the type I methanotroph Methylomicrobium buryatense 5GB1. Metab Eng 42:43–51.
b.	Hai He, et al. 2020. In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate. Metabolites, 10, 65.
c.	Gregory J. Crowther, et al. 2007. Formate as the Main Branch Point for Methylotrophic Metabolism in Methylobacterium extorquens AM1. J. BACTERIOL.
d.	Peyraud R, et al. 2011. Genome-scale reconstruction and system level investigation of the metabolic network of Methylobacterium extorquens AM1. BMC Syst Biol 5:189

3.	I am not fully convinced by your prediction of the formaldehyde exchange between Methylobacter and Methylomonas. According to Ingeborg Bussmann, et al., “formaldehyde production (0.2–7 mM) by methanotrophs was reported only during growth at high methanol and oxygen concentrations”. However, you have reported production and exchange of formaldehyde under oxygen limitation (Figure 1B). Some experimental evidence has to be presented for further validation.
a.	Ingeborg Bussmann, Monali Rahalkar & Bernhard Schink. Cultivation of methanotrophic bacteria in opposing gradients of methane and oxygen FEMS Microbiol Ecol 56 (2006) 331–344

Therefore, you have to further curate your models till the simulation makes physiological sense. Except the above papers, following papers may be useful to your study.

•	Akberdin IR, et al. 2018. Methane utilization in Methylomicrobium alcaliphilum 20ZR: a systems approach. Sci Rep 8:2512.
•	Gilman A, Fu Y, Hendershott M, Chu F, Puri AW, Smith AL, Pesesky M, Lieberman R, Beck DA, Lidstrom ME. 2017. Oxygen-limited metabolism in the methanotroph Methylomicrobium buryatense 5GB1C. PeerJ 5:e3945.
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: INVESTIGATION OF MICROBIAL COMMUNITY INTERACTIONS BETWEEN LAKE WASHINGTON METHANOTROPHS USING GENOME-SCALE METABOLIC MODELING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is well written, clear and unambiguous. The introduction covered all the content needed to understand the results with the literature well-referenced and current. All figures are in good quality and well described.
Experimental design: This work is within the scope of the journal and shows a well-designed and rigorous investigation with coherent methods to achieve the objectives. Methods were described with sufficient detail and information to replicate.
Validity of the findings: The microbial community interactions of methanotrophs was appropriately analyzed using  genome-scale metabolic modeling. The microbial community interactions of methanotrophs is of interest to the field, so this study is a welcome addition.
Additional comments: The authors have adequately addressed all concerns raised by reviewer. Thus, this revised manuscript can be accepted as is.