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: DIFFERENT DISTANT METASTASIS PATTERNS BASED ON TUMOR SIZE COULD BE FOUND IN EXTENSIVE-STAGE SMALL CELL LUNG CANCER PATIENTS: A LARGE, POPULATION-BASED SEER STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper was well writtern. They found that the difference could be found between tumor size and distant metastasis pattern in ESSCLC.
Experimental design: 1. Please added the survival analysis in the study.
2. Added the flow chart of data selection.
Validity of the findings: All the results support the conclusion.
Additional comments: 1. Make all the limition of SEER database in the disscusion.
2. Clinical tumor size of small cell lung cancer was sometimes difficult to calculate, please add it to the limitation.


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: DIFFERENT DISTANT METASTASIS PATTERNS BASED ON TUMOR SIZE COULD BE FOUND IN EXTENSIVE-STAGE SMALL CELL LUNG CANCER PATIENTS: A LARGE, POPULATION-BASED SEER STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper has been improved substantially.
Experimental design: The paper has been improved substantially.
Validity of the findings: The paper has been improved substantially.
Additional comments: The authors addressed most comments in a satisfactory way and the paper has been 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: DIFFERENT DISTANT METASTASIS PATTERNS BASED ON TUMOR SIZE COULD BE FOUND IN EXTENSIVE-STAGE SMALL CELL LUNG CANCER PATIENTS: A LARGE, POPULATION-BASED SEER STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This article was well orgnaized and well writtern.


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: SWITCHING BETWEEN STANDARD CORAL REEF BENTHIC MONITORING PROTOCOLS IS COMPLICATED: PROOF OF CONCEPT
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper provides a clear and detailed comparison of two established methods for assessing coral reefs. It provides an excellent example of the pit-falls associated with changing a method halfway through a monitoring programme and the work required to fully understand the magnitude of these changes.

The readability of the abstract could be improved, for example the fourth sentence of the abstract is very long. Otherwise, the writing style is very good.
Experimental design: The paper presents the information in a clear format and the statistical analyses appear appropriate for the objectives of the study. The discussion adequately explains why discrepancies have appeared between the two coral methods.

How did the authors select the number of random points to use in CPCe? A rationale for the use of 25 points would be helpful. This is touched upon in the discussion from line 361 onwards. I think a clearer statement in the methods explaining the aligned of points between methods would be useful at that point. Although 25 points have been selected here, will the monitoring also use 25 points (which doesn’t seem many to me)? If not, what are additional implications of increasing this number on detection etc?

All aspects of the abundance (via cover) has been covered in great detail by following the Altman & Bland / Bland & Altman comparison method. However, I’m left wondering about the detection of species and the reporting of site richness. It would be interesting to see a table summarising the richness at each reef type according to both methods. I suspect richness is not a monitoring requirement (as you have only used broad Reef Check groupings in the paper) but is of general interest (especially as you say most analysis is done to species level).

An estimate on the additional processing time required for PQ would also be interesting when compared with the chain method.
Validity of the findings: The specific change in methods is perhaps only an issue in one country (and consequently one specific set of reef morphologies / species assemblages) which limits its interest elsewhere. It might help if the authors look for other countries also having to make similar changes. However, I like the analysis and how it has been related to specific environmental and biological aspects.

I would have liked a little more discussion on whether this move to PQ fundamentally supports the objectives of the monitoring (i.e. was the change sufficiently informative to jeopardise a time-series…which I assume it was). Also, the relative pros and cons of both methods would be interesting, i.e. replication vs processing time, detection of rare events (e.g. disease and human litter), transparency etc. I appreciate that much of this is covered in other publications but a brief summary relating to local considerations and objectives would be interesting.
Additional comments: This paper provides a clear and detailed comparison of two established methods for assessing coral reefs. It provides an excellent example of the pit-falls associated with changing a method halfway through a monitoring programme and the work required to fully understand the magnitude of these changes. The specific change in methods is perhaps only an issue in one country (and consequently one specific set of reef morphologies / species assemblages) which limits its interest elsewhere. However, I like the analysis and how it has been related to specific environmental and biological aspects.
The paper presents the information in a clear format and the statistical analyses appear appropriate for the objectives of the study. The discussion adequately explains why discrepancies have appeared between the two coral methods.
The readability of the abstract could be improved, for example the fourth sentence of the abstract is very long. Otherwise, the writing style is very good.
How did the authors select the number of random points to use in CPCe? A rationale for the use of 25 points would be helpful. This is touched upon in the discussion from line 361 onwards. I think a clearer statement in the methods explaining the aligned of points between methods would be useful at that point. Although 25 points have been selected here, will the monitoring also use 25 points (which doesn’t seem many to me)? If not, what are additional implications of increasing this number on detection etc?
All aspects of the abundance (via cover) has been covered in great detail by following the Altman & Bland / Bland & Altman comparison method. However, I’m left wondering about the detection of species and the reporting of site richness. It would be interesting to see a table summarising the richness at each reef type according to both methods. I suspect richness is not a monitoring requirement (as you have only used broad Reef Check groupings in the paper) but is of general interest (especially as you say most analysis is done to species level).
An estimate on the additional processing time required for PQ would also be interesting when compared with the chain method.
I would have liked a little more discussion on whether this move to PQ fundamentally supports the objectives of the monitoring (i.e. was the change sufficiently informative to jeopardise a time-series…which I assume it was). Also, the relative pros and cons of both methods would be interesting, i.e. replication vs processing time, detection of rare events (e.g. disease and human litter), transparency etc. I appreciate that much of this is covered in other publications but a brief summary relating to local considerations and objectives would be interesting.


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: SWITCHING BETWEEN STANDARD CORAL REEF BENTHIC MONITORING PROTOCOLS IS COMPLICATED: PROOF OF CONCEPT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is interesting and well written and the subject and research question clearly defined.

The paper as submitted meets the required standard for publication as outlined by PeerJ.

I have made some minor suggestions and noted a few minor typos or errors.

The results section would benefit from a simple summary table of the key findings for the reader, that included the main information. It would be useful to see at a glance which method underestimated or overestimated abundance for each component, within each type of habitat and the conversion required from CPI to PQ.
Line 17: minor typo; should be ‘There are a number ‘
Line 45- not sure being on the ‘frontline’ of the climate change crisis is appropriate wording, you mean that they are highly sensitive?
Line 153: typo should be approach not approached
Line 432- taut not taught, also noted in earlier section but I haven’t recorded line number.
Figure 1- clear and useful
Figure 2 – if retained can be small scale it doesn’t add much of value.
other tables, figures are fine as is the supplementary material.

Appropriate raw data has been made available.
Experimental design: The introduction would have benefited from a sentence or two on the driver behind switching to photoquadrat methods, I assume they are preferred because they also create a long-term photographic method and can be reanalysed? May be more cost-effective, particularly if automated image recognition can be applied in the future?

Other than this caveat, the research question was well defined and set in context. Approaches to integrate time-series data are necessary and assessments of their validity important, so that this study is relevant and meaningful.

The experimental approach provided a rigorous comparison between methods to what appears to be a high technical standard. The analysis and discussion of results were clear and thorough.

The methods were described with sufficient detail to allow replication.
Validity of the findings: The paper provides an assessment of two different methods of monitoring coral reef components. The question is relevant and driven by management requirements. The investigation demonstrated clearly the limitations in comparing methods and the adjustments required to integrate the findings from the different methods. As such it is a clear and useful experiment, the paper fully demonstrates the need for the research, fully articulates the question, addresses this and clearly states the conclusions.

 A summary table in the results section would support the reader in rapidly evaluating the results.

The conclusion section was well-written and fully evaluated the findings and discussed the potential underlying drivers of the observed variation between methods. The implications were discussed and the technical detail was presented clearly and without jargon. This research would allow an investigator or organisation to select a method, understand its limitations and support decision making around switching methods. This work was clearly necessary to support on-going monitoring and will have application elsewhere.

All underlying data have been provided.
Additional comments: I found this paper easy to read, interesting and enjoyed reviewing, thank-you.


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: SWITCHING BETWEEN STANDARD CORAL REEF BENTHIC MONITORING PROTOCOLS IS COMPLICATED: PROOF OF CONCEPT
Review round: 1
Reviewer: 3

Output:
Basic reporting: I found the manuscript clear and unambiguous in describing the potential issues of switching between coral benthic monitoring methodologies without undertaking the necessary comparative study. The intro and background were well thought through and the references cited comprehensive. The layout of the methods, results, and discussion was well done with minimal redundant or over reporting of irrelevant results. Figures were excellent and high quality. Figure 3, in particular, was very well thought through and is an efficient and effective manner of summarizing the results and not typical for previous coral benthic method comparison studies. Table 1 and supplemental tables were also well done.
However, I found table 2 somewhat difficult to understand due in part to the poor description in the results/discussion/caption- why were the N values so different for gorgonians and lower for macroalgae on patch reefs?. Overall, I did not think table 2 added a lot of information to the findings and felt that the discussion of the relative tradeoffs between changing methods and detecting significant change from a managers perspective could be strengthened (please see comments on discussion under Validity of Findings). For table 2, the inclusion of comparative median change values for the 2012-2017 using the 2017 CPI data would provide additional context to the results as presented. I would suggest either removing table 2 (but leaving a the description in the discussion (lines 312-327) of how the PQ method (without necessary conversions) would skew the observed 2012-2017 trends. If the authors wish the table to remain, I would suggest they include a similar column of data for the 2012-2017 CPI changes (median values-1st and 3rd quartiles) for the different reef types/benthic categories.
Experimental design: The study is original primary research relevant to coral reef managers and scientists and within the scope of the Journal. The research questions are well defined and it is articulated how this research fills a current knowledge gap. The study design and methods are well thought out and replicable based on the descriptions and raw data provided. Particularly novel (at least in the coral reef scientific realm) is the robust statistical analysis of differences between the two monitoring methods which is a significant improvement over previous coral monitoring methodological studies.
Validity of the findings: The findings show significant scaling and reef type differences between the two different methods of measuring benthic cover (PQ vs CPI). Previous studies have used simple correlation analysis of results obtained from different methods and the r2 values (often high when spanning a large % cover range) to suggest high compatibility or minimal differences between methods. By utilizing the Bland & Altman (1986) statistical approach, this study provides a much more detailed evaluation of true methodological differences and causes including sample size, scaling, and reef type classification issues. The conclusions area well stated and underscore the potential issues with switching between methodologies without developing necessary scaling and reef type relationships for each benthic class.
The discussion includes a lot of detail about the scaling relationships and potential causes of the differences between the two methods. The significantly higher estimates for gorgonians using the PQ method reinforces previous studies but for many of the other differences (e.g., macroalgae/turf algae) the causes area less clear and some of the relationships appear to contrast with previous studies (e.g. Rogers and Miller, 2001 found relative macroalgae was significantly higher using PQ compared to chain intercept). Observer variance is also minimized in this study as an important factor. However, depending on the procedures used, it may be more important at influencing measurement bias if only one person is doing the lab PQ point counting (as opposed to many observers underwater doing the CPI).
In general, there could be more detail on the practical recommendations of applying corrective measures to historic monitoring datasets to allow for newer PQ methods. In the discussion, lines 319-320 highlight that % cover change is significant from a management perspective when it is 25% or greater and that several of the benthic components exceeded this threshold from methodological bias alone.  The implication is that data comparisons between different methods should not be undertaken without the necessary comparative relationships in indicators properly quantified. However, it may not realistic to do comparative studies like this for every long-term monitoring area/reef type/site. So some guidance on applying corrective transformations measured on a subset of sites or from one region to another particularly to coral cover would be a welcomed addition. For example going into some detail on how historic CPI coral cover would be “adjusted using the relationships found in this study (constants, linear equations, etc..) to bring methodologically disparate datasets together based on the 21 sites examined in this study would be quite instructive.  More details on the tradeoffs (loss of power to detect change) at the site, reef type, or whole island scale after “adjusting” the historic 1982-2012 CPI datasets to being “PQ comparable” would also be welcomed.
Additional comments: Overall, I found the manuscript to be a solid contribution that exposes the inherent problems of switching between methods and highly recommend its publishing. The two main issues that could strengthen the manuscript are (1) Table 2- suggest ether removing this table or if it stays please add comparative CPI median metrics to allow for a clearer interpretation of the significance of substituting PQ data; (2) expanding the discussion to include some guidance on applying transformations to CPI or PQ datasets in Barbados to minimize the methodological bias quantified in this study.

Additional comments below are some additional suggestions and should not be seen as necessary to address in order to be published.
Why not include correlation plots between the two methods with r2 values (essentially cross plots of supplemental table PQ vs CPI raw data)? This would show a fairly high correlation (r2> 0.8) for most benthic classes across the range of sites but (as this paper goes on to uncover) is somewhat misleading in that these correlations are directly related to the range of % cover values and over look scaling and other issues. Including this would underscore that what on the surface appears to be “highly comparable” methods, is in fact full of significant differences that must be quantified and corrected for to minimize the methodological bias.

What about coral species specific comparison between the two methods? Were the number of coral species or relative proportion significantly different measured by the two methods significantly different?

Cost/benefit comparison between the two methods? Specifically, it would be helpful to know what you found for the relative tradeoffs between the two methods- time underwater vs time in the laboratory/entering data to obtain the results. Even a qualitative statement would be useful additional information for managers in deciding weather or not to switch from PCI to PQ.


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: IMPACT OF HABITAT LOSS ON THE DIVERSITY AND STRUCTURE OF ECOLOGICAL NETWORKS BETWEEN OXYURID NEMATODES AND SPUR-THIGHED TORTOISES (TESTUDO GRAECA L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	The language is primarily clear and unambiguous, and professional English is used throughout. There are some parts where further clarification is needed, and a few typos remain.  Minor English language finetuning is needed e.g. in lines 121-122; 208-209; 262. Throughout, the potentially inappropriate use of causal language should be amended where there is no clear causal relationship. E.g. L291 “consequences for”; L301 “significantly affected” refer to the association between nematode presence and growth rate, where the direction of the causality is likely, if anything, the opposite to that inferred in text.
•	The introduction is reasonably successful in demonstrating how the work fits into the broader field of knowledge, but the consideration of prior literature could be more balanced.
•	The structure of the paper is clear and easy to follow. The figures and tables are legible and appropriate, but lack some axis titles and legend detail.
•	Partial raw data has been shared, but a large proportion of essential information is missing.
•	The submission appears to be self-contained, but there is some mismatch between the hypotheses and results. Some relevant methodological details and results are apparently missing or unclear.

Detailed comments
The paper deals with species interactions and the way in which habitat degradation or loss influences ecological networks centered around a specific species of interest, a severely threatened tortoise. Considering ecological networks, with a focus on the effects of the environment on species communities and interactions, have been largely overlooked in conservation studies to date. The study is, therefore, well-motivated and uses a potentially interesting network approach to the topic. It is also generally fluently written and well-structured. However, I believe there are several quite problematic points in the methods and the interpretation of the results that render the study outcomes unreliable. These points must be justified and clarified for the paper to meet publication standard.

Introduction
The introduction reads well, makes interesting points, and describes the aims of the present study. The growth rate component could be better linked to prior literature and the study hypotheses, however. For example, it would be useful to add something about what factors are known to influence (tortoise) growth rate and through what mechanisms habitat loss and nematode infections should influence growth (see e.g. references below). Additionally, any information on the presumed mechanism mediating nematode abundance and richness effects on host health might be useful for making predictions about their associations.

I would also appreciate a little more information on the proposition in L 71-73:
“for herbivorous reptiles, hindgut nematodes might be parasitic but might also play a positive role in the digestion and assimilation of plant matter”
I believe this is quite an unusual suggestion for many researchers who generally perceive gut nematodes as parasitic throughout, and seen as this is an important part of the hypotheses tested, I would appreciate some additional support for the idea. E.g. it seems to me likely that beneficial effects of gut nematodes might operate (at least in part) via nematode-microbiota interactions in the gut. I missed some background on the hypothesized association between host growth and parasite infections, which is integral to the study.
Experimental design: •	The research question is provided and is meaningful, but could be clarified further. The study identifies a knowledge gap, but it is not entirely clear that the study is appropriately designed to fill that gap. A fundamental issue relevant to the collection of relevant data (the parasite life cycle) appears to have been overlooked, as the analyses are based on the prevalence of adult nematodes in fecal samples, instead of the more common (though also flawed) method of examining nematode eggs, or molecular identification methods. While some useful information could, nevertheless, be gained from the study on the prevalence of certain parasite species in different habitats, the measure may come with a high measurement error and risk of non-detection, rendering the results potentially quite unreliable. The potential consequences of this choice of method on the outcomes have not been, but should be discussed.
•	It is somewhat unclear whether the investigation was conducted rigorously and to a high technical standard, because the data collection methods are insufficiently described. There are some points of potential concern in the data analyses (see below).
•	Ethical standards in the field appear to have been followed appropriately.
•	The methods were not described with sufficient information to be reproducible by another investigator. For example, the measurement of individual growth rate (the primary outcome variable) has not been described. Furthermore, no information is provided about the treatment and analysis of the parasite samples (basis for the primary predictor variable). See details below.

Detailed comments
Information on the habitat, study species, and aspects of the parasites are appropriately described and relevant, but there are a few important details that should be clarified.

L184-188: given the importance of the growth metric for the outcomes of the study, I would like for more details here rather than referring to the Rodríguez-Caro et al. (2013) paper for all details. If I had to guess what this means, I would assume that growth measured using recapture data from change in size over these 3 months, but surely this would not be sufficient time to record growth in a meaningful way? Rodríguez-Caro et al. appear to have used repeated measures of the same individuals over a 4-yr period to derive the growth curves. Is the population used for this study long-term monitored? Please provide details on the measurements and growth rate calculation used in the present study.
If the growth estimates are derived from longitudinal data, how relevant is this metric for the point sampling of the nematode community, and would the causality relationship change? E.g. statement in L 300-301 “both the number of species and nematode abundance significantly affected growth rates”.

L201: ditto, at least a brief description of the parasite sample processing and data collection is needed here.

L205-207: Please specify which of these species belong to Oxyurida and Ascaridida, or other orders. This is relevant for the rest of the paragraph, where you focus on these two orders.

L207-209: I find the decision to exclude eggs a potentially very problematic issue, given the life cycles of these nematodes. At least in mammals, intact adult oxyurids should only be found in feces under very heavy infestation or possibly during severe gastrointestinal distress (e.g. prolonged diarrhea), whereas the eggs are actively deposited in the perineum. As adult oxyurids females migrate to the perineum, dead adult worms might occasionally be discovered in feces, but this is generally a very unreliable way of diagnosing an ongoing infection. Oxyurid infections are, therefore, quite difficult to assess reliably without the “perianal sticky-tape test” for eggs. Adult ascarid worms should be found in feces even less often because they live in the small intestine and dead worms should generally be broken down in digestion, whereas eggs are actively deposited to be dispersed in feces.
While I understand that the eggs cannot be visually identified to species level and therefore cannot be used to assess species abundance/diversity, the choice to focus on adults may severely bias the results. The potential consequences of this decision for the results and conclusions should, therefore, be clearly outlined and justified, and the findings discussed strictly in the light of these restrictions.
For example, how does the choice of method relate to the relative apparent abundance/diversity of oxyurids relative to other nematodes? How would this be reflected in the rank-abundance plots? And what would it do to the nestedness/ community analyses if a large number of actually prevalent species are not observed?
There may be no reason to assume that the adult worm shedding should vary according to habitat disturbance, but if only a small proportion of nematode species are found in feces in the adult stage, it may be practically impossible to say anything about the actual nematode communities or differences in those communities among sites.
Additionally, statements such as L 317-318 “dominant nematodes infecting tortoises throughout the landscapes were T. dentata and T. longicollis” should be amended throughout to acknowledge this constraint (i.e., here, the adult nematodes most commonly found in the feces were of the species…).

L 226-229: “Body weight and carapace length are strongly correlated to tortoise´s age (Rodríguez-Caro et al. 2013), whereas age classes were not homogeneously distributed across landscapes; therefore, weight and carapace length were not used further in the analyses related to the impact of nematode infestation on body traits across habitat types.”
The rationale of this decision is not obvious to me – why not include these traits as covariates in the analysis instead? Also, please remind the reader which body traits are considered if not the weight and length; I am a little confused at this point as the body measures, as far as I have understood, were L 183-185: “age estimated from the carapace growth ring (Rodríguez-Caro et al., 2015), body weight (g) and carapace length”. This seems to leave nothing to associate the nematode infections with. Please clarify this.

Please clarify whether tortoises were sampled multiple times each, and if so, how you treated the repeated measures in each of the analyses. Did you select one sampling event to assess parasite load or pool several samples per individual?

Given the relatively long sampling window, it would be useful to test and mention whether the nematode community changed over the season, and whether this might influence your network analyses.

L236-237: Please clarify why these were log-transformed. Note also that both measures may be considered as counts, which should ideally not be log-transformed. See e.g. O’hara, Robert B., and D. Johan Kotze. "Do not log‐transform count data." Methods in Ecology and Evolution 1.2 (2010): 118-122.

L245-246: I would suggest reconsidering the wording here to make this easier to grasp, and use careful and consistent wording throughout this sentence (and later in the paper where talking about this concept). Please clarify what this refers to - the relative abundance of each nematode species (where - per individual, site, or entire sample?) against the ranked abundance of those same species (where?)? This sounds initially as if the relative abundance was plotted against itself, and the absence of axis titles in the figure doesn’t help comprehension. I continue to have trouble with this sentence, though going over the paragraph multiple times (I think) I managed figure out what this means.

L277-285: Please indicate why you calculated each of these metrics (especially what additional information was to be gained from each of them)

L279: Please clarify what exactly “number of cells in the matrix” and “interaction diversity” refer to in this context

L285-287: Please remove repetition here
Validity of the findings: •	Based on the omissions in the method description, I am not fully convinced that the data are robust, statistically sound, and controlled. Only partial data used in the analyses have been made available.
•	The conclusion are linked to original research question, but in my opinion they occasionally venture quite far beyond the supporting results, e.g. L382-384. Speculation has not always been identified as such. See details below.

Detailed comments

Results
It would also be useful to have some more information on the tortoises that presented with no adult nematodes, relative to those that had them. Were they more commonly found in one or the other habitat type, did their growth rates differ?

Please tone down language implying causal relationship between growth rates and nematode load. Instead of “consequences”, effects”, I suggest using “association” or “relationship”. Appropriate reporting is already seen in e.g. L311-313.

L294: presumably this should be between 0 and 10 species (rather than 1 - 10)

L308-309: These statistics (richness, abundance) do not seem to be the correct ones for this sentence, please check.

L346: What does this antagonistic interaction refer to? Antagonism between the different nematode species in the community (as opposed to antagonism between host and nematodes)? It would be interesting to explain this further in the discussion.

Figure 2: Indicate whether this is excluding those tortoises that had zero nematodes. I believe it would be useful to include those individuals here for completeness.
Please clarify what the numbers in the figure panels after the r-coefficient refer to, i.e. what does this sequence mean: y= … - …(x)

Figure 3: bolding is not clear from figure - consider adding a color code. The axes are not titled, please amend.

Table 1 legend: “oh” -> “of”.
Does “density of nematodes” mean abundance? Please use uniform terminology in text and tables.

Discussion
L349-355: In light of the constraints mentioned above, I would be willing to cautiously trust the finding of similar nematode communities (of those species that can be observed as adults in feces) among habitat types. For the rest of the paragraph (tortoise growth – habitat – nematode community associations), it seems to me unlikely that this is the most parsimonious explanation for the observed associations. It seems to me substantially more probable, that the habitat disturbance alters the food availability, stress, movement ranges or other factors that influence both the growth of the animals, and consequently their exposure/susceptibility to nematode infections. Independently of growth, nematode abundance and diversity in populations may also be influenced by features of the environment, including population density and frequency of interactions e.g. through patchy resource distribution. These alternative explanations cannot be distinguished based on the presented data. I suggest refraining from drawing strong causal conclusions, such as using the word “impact” when describing the observed associations.

I agree that it is an interesting possibility that certain nematodes could promote the subsistence of their host species by facilitating food digestibility. However, it may be premature to draw such a conclusion from the present study, and therefore, I suggest toning down the discussion around the “symbiotic” relationship of nematodes with tortoises, which is carried through much of the discussion.
E.g. L 382-384: This seems to me to be a long leap of faith given the available data, while there are other, perhaps better supported potential explanations.

First, an apparent positive effects of nematode richness on growth could also arise through the influence of a shared third variable, such as a more varied diet. Second, there is a well-known association between host size and parasite load, which might explain at least part of your findings. Third, you may want to consider (and should at least mention) the more classical explanation: that the intestinal communities of organisms are determined via the patterns of acquisition from the environment, and the capacity of the host to get rid of unwanted organisms. For example, using the statements from the abstract:
1)	“At low habitat loss, nematode infestation was positively associated with growth rates (suggesting a mutualistic nematode-tortoise relationship)”, could alternatively be explained through higher resource seeking activity such as broader habitat use by those individuals that facilitated a faster growth but simultaneously exposed the individual to more nematode eggs, regardless of their parasitic/commensal/symbiotic nature.
2)	“The association became negative at high habitat loss (suggesting a parasitic relationship)” could also be explained with a lowered capacity of individuals to cope with a parasite infection in more challenging conditions, or those individuals with low growth rates may have poorer acquired immunity to parasites etc.
3)	“No relationship was observed when habitat loss was intermediate (suggesting a commensal relationship)” could also indicate that the hosts were able to sufficiently allocate resources to growth and parasite resistance that parasite acquisition and resistance balanced out.

This is only to point out, that there are alternative, previously supported hypotheses that could explain the observed patterns, even if all nematodes were parasitic. I suggest including these potential alternative explanations in your discussion and evaluating the support for each alternative with your results and the literature, perhaps suggesting further studies that could be used to distinguish between the potential explanations.

Data
Please also provide the tortoise body measures, growth rate, etc. that were used in the analyses in the data file.
Additional comments: Potentially useful references
Adamson, Martin. "Evolutionary patterns in life histories of Oxyurida." International Journal for Parasitology 24.8 (1994): 1167-1177.

Aho, John M. "Helminth communities of amphibians and reptiles: comparative approaches to understanding patterns and processes." Parasite communities: patterns and processes. Springer, Dordrecht, 1990. 157-195.

Arneberg, Per. "Host population density and body mass as determinants of species richness in parasite communities: comparative analyses of directly transmitted nematodes of mammals." Ecography 25.1 (2002): 88-94.

Benavides JA, Huchard E, Pettorelli N, King AJ, Brown ME, Archer CE, et al. From parasite encounter to infection: multiple-scale drivers of parasite richness in a wild social primate population. Am J Phys Anthropol. 2012;147:52–63.

Bordes F, Morand S, Kelt DA, Van Vuren DH. Home range and parasite diversity in mammals. Am Nat. 2009;173:467–74.

Coop, R. L., and I. Kyriazakis. "Nutrition–parasite interaction." Veterinary parasitology 84.3-4 (1999): 187-204.

Morand, Serge, and Robert Poulin. "Density, body mass and parasite species richness of terrestrial mammals." Evolutionary Ecology 12.6 (1998): 717-727.

Poulin, Robert, and Mario George-Nascimento. "The scaling of total parasite biomass with host body mass." International journal for parasitology 37.3-4 (2007): 359-364.

Stephenson, Lani S., Michael C. Latham, and E. A. Ottesen. "Malnutrition and parasitic helminth infections." Parasitology 121.S1 (2000): S23-S38.

Wilson K, Bjørnstad O, Dobson A, Merler S, Poglayen G, Randolph S, et al. Heterogeneities in macroparasite infections: patterns and processes. In: Hudson PJ, Rizzoli A, Grenfell BT, Heesterbeek H, Dobson AP, editors. The ecology of wildlife diseases. Oxford: Oxford University Press; 2002. p. 6–44.


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: IMPACT OF HABITAT LOSS ON THE DIVERSITY AND STRUCTURE OF ECOLOGICAL NETWORKS BETWEEN OXYURID NEMATODES AND SPUR-THIGHED TORTOISES (TESTUDO GRAECA L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well-written and interesting study on the structure of nematode-tortoise networks and the structural changes within these networks with habitat loss. I particularly liked the point that the authors relate the degree of nematode infestation to tortoises’ growth rates, showing that the relationship changes from positive to neutral to negative with increasing habitat loss. This interesting finding indicates that changes in the interactions between nematodes and tortoise individuals with increasing habitat loss affect tortoise’ fitness.

This being said, I think the manuscript would be even stronger if the way network structure affects the fitness of tortoises was even more directly tested. At present, the authors analyze the interactions only at network level (e.g., with measures of nestedness or connectance). Reading the manuscript, I wondered if instead it would be helpful to analyze also species-level attributes of network structure. For example, they could test whether species degree (the number of nematodes affecting each tortoise), species strength or measures of species specialization (e.g., Blüthgen’s d’; a measure of how specialized a species or individual interacts within the community) can be directly related to the growth rates of the studied tortoise individuals. In my view, this would make an even stronger point that network structure directly relates to the fitness of interacting individuals, something which is indeed very rarely studied.

I provide more detailed and minor comments below, and hope they are helpful in further strengthening this interesting manuscript.

Abstract:
L41: Do you mean that rarity thus equals specialization? But this is not specialization measured at the species level but with network-level indices?
L43: In how far is connectance related to network size (e.g., the number of interacting species)? This two are usually strongly negatively correlated. Did you consider correcting for this, for instance using null model analysis? (see e.g., Dalsgaard et al. 2017, Ecography).

Introduction:
L57: This should be “Fahrig”.
L57: Note that Lenore Fahrig herself appears pretty critical about the very general statement that habitat fragmentation has primarily negative effects on biodiversity (see her recent interesting comment in Global Ecology and Biogeography 2019; 28:33-41).
L79: What kind of animal species?
L80-83: Also here, it would be interesting to know which species you are referring to.
L84ff: This paragraph comes rather abruptly. When reading the manuscript, I got the feeling it would be easier to understand your design if the region were first descript (this is the part from L111ff). You may consider moving this section before the part that starts in line 84. You could then finish the Introduction with the paragraph on network ecology (L95-110), before listing you hypothesis.
L122-123: It is unclear what you mean by “diverge with the level of habitat loss”, try to be more specific.

Methods:
L147-150: What is the scale at which you defined scrubland cover? For example, was there a predefined radius surrounding your study populations at which you conducted the landscape composition analysis?
L150-151: Where habitat loss, fragmentation and intensification related to each other, and could this possibly confound your results on the effects of habitat loss?
L154-160: I was very surprised to read that 76% landscape cover with suitable habitat is referred to as “high habitat loss” – to my naïve understanding, this would still look like a landscape with very high amounts of natural habitat remaining. I presume that tortoises may see this differently though, but please justify why a loss of 24% suitable habitat is already considered as the “high loss” category, and how this refers to tortoise biology.
L187: If space allows, shortly describe the Bertalanffy model for non-experts.
L208: Word missing between “because” and “the lack of”.
L209-219: These information would be better placed in the Introduction, to motivate the hypothesis of the study and explain why contrasting effects of nematode infection on growth rates can be expected.

Statistics:
In general I found the statistical analysis sound. However, I wondered if you could have used GLMs instead of log(x+1)-transforming the data (L236-237) (e.g., using a Poisson or negative-binomial distribution).

L256-258: How large was the proportion of individuals considered in the interaction network analysis?
L260-265: As far as I understand, these are then weighted interaction networks (not binary, which is great), perhaps include this point.
L268: If the networks are weighted, why not use the a weighted measure of nestedness such as wNODF?
L274: Please describe how ‘Null Model II’ works, for example, how is the community matrix randomized (e.g., with fixed connectance and/or fixed marginal sums)? This can be very important to the result (Dormann et al. 2017, Annual Review of Ecol. Evol. Syst.)
L274ff: Referring to the null model, this could also be used to standardize the network parameters, which may overcome artifacts by differences in network size across localities with different amount of habitat loss. (using a standardization such as SES = (obs_nestedness – mean(random_nestedness)) / sd(random_nestedness).
L277-280: As commented before, I would expect connectance and interaction diversity to be strongly correlated to network size.

Results:
L290: “some aspects” is vague, please be more specific.
L294: Provide means and a measure of spread (e.g. standard errors) throughout.
Figure 2: The symbols are pretty small and hard to discern (particularly when overlapping). If possible, increase their size or use colors to indicate the habitat types. Additionally, including confidence intervals of the regression fits would be helpful.
Figure 4: Please include a legend that displays how the number of interactions corresponds to the width of the boxes.

Discussion:
L370ff: But your landscape do not appear to be heavily modified in the sense that high amounts of natural/suitable habitat have been lost, when compared to most other studies on habitat loss.
L379-387: Here, more discussion and analysis how this contrasting infestation effects result would be great – possibly network analysis at the species-level could help here.
L400-404: These studies appear to contradict each other – how can any fragment regardless of its size support viable populations, and fragmentation still negatively affect them?
L405ff: Does this also mean that critical thresholds of habitat loss on tortoise survival remain unclear? Are there landscapes with higher amount of habitat loss than considered in your study in which large tortoise populations remain?
L430-435: See comment above why species-level indices may be even more informative.
Experimental design: see basic report above.
Validity of the findings: see basic report above.
Additional comments: see basic report above.


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: IMPACT OF HABITAT LOSS ON THE DIVERSITY AND STRUCTURE OF ECOLOGICAL NETWORKS BETWEEN OXYURID NEMATODES AND SPUR-THIGHED TORTOISES (TESTUDO GRAECA L.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Ok, see below
Experimental design: Ok, see below
Validity of the findings: I thank the authors for their efforts to clarify points that were unclear in the previous manuscript draft. They have successfully responded to most of my smaller comments and the added appendix and details on the measurement of growth rates significantly clarified the methods. Unfortunately, there has been little change to the two major points that I brought up in my previous review. Rather, the issues are more obvious to me now that the unclear points have been clarified.

FIRST, I'm sorry but I simply cannot buy into the suggested "effect" or “influence” of nematode loads on growth rates. Word choice aside, the assumption of an effect of current nematode loads on past tortoise growth is difficult to justify. The authors have not succeeded in convincing me that this is not a problem (although I appreciated the additions to L465-480, which helped balance the discussion!)
I ask the authors to please reconsider what is being implied by suggesting that certain nematode communities influence(d) the tortoises’ growth etc.. Unless I have completely misunderstood the way the study was conducted, this is not a trivial detail but has significant implications for the ecological relevance of the study. I’ll try to make my thinking clearer.
You are measuring adult Oxyurid nematodes in (adult) tortoises. You are associating this nematode load with growth rate over the past n years of the tortoise’s life. Over this time, the tortoise may or many not have been carrying certain species of nematodes. Nematode loads increase with age, as you point out, but there is also turnover in both the environment and within the host, so at times the nematode load may be higher, others lower, and the tortoises have likely gone through such phases in their lifetime. We cannot know the numbers or prevalences of those nematodes when the growth occurred, because a single sample was taken at the very end of the growth progression and nematode accumulation over the animal's lifespan to date.

How is it possible to infer a causal effect of this current nematode load on the growth of the animal at any time in the past? Simple logical reasoning of cause and effect suggests that a cause comes before effect, thus the causal relationship here could only be assumed to be that fast-growing animals accumulated and retained more nematodes over time (or that fast-growing animals more easily shed Oxyurids into feces when stressed by handling). The ecological implications including those for the host-nematode relationships are very different in these scenarios, and I think it is misleading to suggest any effect of nematodes on the host (especially the host's past life) based on the present study.

Given this problem, an attempt to infer the type of relationship between host and nematode community is not valid because of the impossibility to infer a causal effect of current nematode load on past growth and current size. Furthermore, the richness and total abundance of shedded adult Oxyurids seems to me a poor indicator of the host-nematode interaction type because it does not take into consideration the potentially differing roles of the different Oxyurid species.

Given these uncertainties, I dare to propose that the paper would actually be stronger if everything related to aim 2, L149- (and interpretation in L459-480, determining whether the nematode community in any one of the environment types was mutualistic, commensal or parasitic) is dropped from the paper. I agree that it is a very interesting possibility that there are different kinds of interactions at play and this warrants further study but unfortunately this retrospective speculation cannot really accomplish that goal in my opinion. Perhaps the paper could simply report the network analysis as that part seems to be sound and would likely make for a sufficiently strong stand-alone paper. I'm sorry I can't be more positive about this.

SECOND, I had also suggested including information on (and implications of) the ecology of nematodes beyond their potential roles as interaction partners with the host. The authors have kindly added a statement to L540- about the limitation of excluding oxyurid eggs, but the more general implications of using adult nematode shedding has not been recognized. At the very least, you should point out to less informed readers that the measure of adult nematodes in feces may or may not be reflective of the actual nematode communities inhabiting the host gut. This really is important for your network analysis. Shedding of adult nematodes is not very common and may signal digestive problems (perhaps including stress defecation due to handling). Normally, adult nematodes may not be detected in feces even if there are many of them in the digestive tract. It is a simple thing to acknowledge this limitation in your study e.g. in the last paragraph of the discussion (the study is still a valid first pass at the problem!) and e.g. propose molecular studies identifying all nematodes present in the feces based on DNA barcoding.

However, as you now clarified in the methods (L247) that all the nematodes you detected were Oxyurids, the situation is a little clearer. Oxyurid adults are probably easier to detect because they are located lower in the digestive tract. Therefore, you may have good representation of Oxyurids (possible other taxa may be underrepresented). However, as you refer to “nematodes” only throughout the paper, this fact may be lost on many readers. I suggest you clarify and reiterate in the results that only Oxyurid adults were found and point out that e.g. the network analyses all refer to Oxyurid nematodes only. ALso, calling them "endosymbionts" in the title is not founded, given that there are these multiple possible ways of interacting, better just refer to "Oxyurid nematodes" for clarity.
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: IMPACT OF HABITAT LOSS ON THE DIVERSITY AND STRUCTURE OF ECOLOGICAL NETWORKS BETWEEN OXYURID NEMATODES AND SPUR-THIGHED TORTOISES (TESTUDO GRAECA L.)
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I thank the authors for the additional edits and patient clarifications. I hope that these have also made the paper accessible to a broader audience. I have not further concerns about the paper and I believe that in its current state it will make a valuable contribution to the field.

I have a few very minor final suggestions:

A minor English language issue: The wording changes to “association” have led to several instances where the prepositions are a bit strange. Instead of “association on” this should usually be “association with”.

L121-138: Consider restructuring this paragraph. E.g. I suggest moving the sentence in L128-130 (introducing the nematode taxa) to the beginning of the paragraph (currently first mention of ascarids seems abrupt) and rewording something in the final 2 sentences; the statements do not seem entirely logical to me.

L375: Instead of frequency, should this be number/count/diversity/richness?

L440-443: I suggest reconsidering this is long and complex sentence, perhaps split in two.

L502 -> Another/additional (possibly more parsimonious?) explanation might be stronger selective disappearance in degraded habitats, i.e. animals that were not able to grow fast enough had lower survival, and perhaps never made it to adulthood.


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: CO-EXPRESSION ANALYSIS AND CERNA NETWORK REVEAL EIGHT NOVEL POTENTIAL LNCRNA BIOMARKERS IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript consists of lots of grammatical errors which hinders the comprehension. The authors should address this.

The authors have provided a comprehensive background of the subject with sufficient literature references.

The authors need to improve the presentation structure of figures and tables. In Figure1 the authors should mentioned what each color in the volcano plot represent.
Experimental design: The authors should mention the rationale for selecting only the microRNAs with first 10 and last 10 FC values for subsequent analysis.
Validity of the findings: The authors should discuss about the impact and novelty of their study, including the limitations of the study.
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: CO-EXPRESSION ANALYSIS AND CERNA NETWORK REVEAL EIGHT NOVEL POTENTIAL LNCRNA BIOMARKERS IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is easy to follow, but the background of HCC and WGCNA is no sufficient.
The article structure, figures and table layout is good.
Experimental design: The experimental design lack the validation experiment.
Validity of the findings: No comment.
Additional comments: This article aim to reveal potential novel lncRNA biomarkers in HCC. The whole layout of manuscript is reasonable. But the manuscript lack the verification of key targets.


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: CO-EXPRESSION ANALYSIS AND CERNA NETWORK REVEAL EIGHT NOVEL POTENTIAL LNCRNA BIOMARKERS IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study is clear, and can do some good impact to the current knowledge.
But we need to consider adding and correcting few things to make it ready for publication in case you consider it and agree with my simple opinion.

-Language:The language is acceptable and well written. But, would recommend few more revisions and correct few minor things.

-The Title:(Co-expression analysis and ceRNA network reveals potential eight novel lncRNA biomarkers in hepatocellular carcinoma) Please remove the (S) from the verb(reveals)to make it Grammatically correct.

-introduction: Hepatocellular cancer [HCC] is the most common primary liver cancer in the world, with high degree of malignancy? they need to define the (degree of malignancy?) and embed the answer in your introduction text.

-In the introduction/ Lines: 2-3: (is the second highest leading cause of death after cardiovascular disease) please correct this part using the following link CDC reports:
https://www.cdc.gov/nchs/data/nvsr/nvsr67/nvsr67_06.pdf

-In the introduction fifth and sixth lines/L5-6:
[primary hepatocellular carcinoma (PLC) is considered to be one of the most common malignant tumors, and about 90% of these tumors are hepatocellular carcinoma (HCC)]!
Please consider making some correction to the expression way of the whole sentence and the (abbreviation PLC) and you might consider using the term (primary hepatic cancer) or (primary liver cancer). Because the HCC is the subdivision of that.

-In the introduction: Lines 22 and 23:
 (Additionally, functional enrichment analysis shows that these eight novel lncRNA play
23 an important role in the development and progression of liver cancer.)
Please refer to the parts of your study supporting this fact so that would be stronger introduction.

-Materials and Methods: Please provide more details about using WGCNA and hypergeometric test analysis?

-Data preprocessing and differential gene selection:
Lines(26-27) (The clinical data, RNAseq data and microRNA data of LIHC were downloaded from the TCGA database): Please consider Spelling out acronyms the first time you use them, even if they are commonly used ones, for example: The Cancer Genome Atlas(TCGA).

-The (Volcano plots) of differentially expressed RNAs already used which is a very nice work, great add to the manuscript.

-You have to add more details to the (Heatmap plots) of differentially expressed mRNAs.
 / More details about the axis representing the samples?

-Add more details explaining and simplifying your tables and figures in the captions please. Especially Figure 1. (Different expression of miRNA and lncRNA).

-They should consider adding small paragraph summarizing the conclusion, although it’s already included in different parts of the results, but it would be a great add to the manuscript.
Experimental design: -Materials and Methods: details about how they used(WGCNA and hypergeometric) test analysis?

-We need more details to the (Heatmap plots) of differentially expressed mRNAs.
 / More details about the axis representing the samples.

-More details explaining and simplifying your tables and figures in the captions please. Especially Figure 1. (Different expression of miRNA and lncRNA).

-Summarizing the conclusion by the end would be a good add.
Validity of the findings: Please consider doing some statistical review by a specialist and reviewing the validity of the graphs.
Additional comments: Dear authors,

I reviewed your work (Co-expression analysis and ceRNA network reveals potential
eight novel lncRNA biomarkers in hepatocellular carcinoma) It was interesting, but there were some problems to be solved. I want to review the revised paper again.

Minor points. We need requiring further details and corrections:

-The Title:(Co-expression analysis and ceRNA network reveals potential eight novel lncRNA biomarkers in hepatocellular carcinoma) Please remove the ( S ) from the verb(reveals)to make it Grammatically correct.

-introduction:
Hepatocellular cancer [HCC] is the most common primary liver cancer in the world, with high degree of malignancy?  Please define the (degree of malignancy?) and embed the answer in your introduction text.

-In the introduction/ Lines: 2-3: (is the second highest leading cause of death after cardiovascular disease) please correct this part using the following link CDC reports:
https://www.cdc.gov/nchs/data/nvsr/nvsr67/nvsr67_06.pdf

-In the introduction fifth and sixth lines/L5-6:
[primary hepatocellular carcinoma (PLC) is considered to be one of the most common malignant tumors, and about 90% of these tumors are hepatocellular carcinoma (HCC)]!
Please consider making some correction to the expression way of the whole sentence and the (abbreviation PLC) and you might consider using the term (primary hepatic cancer) or (primary liver cancer).

-In the introduction: Lines 22 and 23:
 (Additionally, functional enrichment analysis shows that these eight novel lncRNA play
23 an important role in the development and progression of liver cancer.)
Please refer to the parts of your study supporting this fact so that would be stronger introduction.

-Materials and Methods: Please provide more details about using WGCNA and hypergeometric test analysis?

-Data preprocessing and differential gene selection:
Lines(26-27) (The clinical data, RNAseq data and microRNA data of LIHC were downloaded from the TCGA database): Please consider Spelling out acronyms the first time you use them, even if they are commonly used ones, for example: The Cancer Genome Atlas(TCGA).

-The (Volcano plots) of differentially expressed RNAs already used which is a very nice work in your paper.

-You have to add more details to the (Heatmap plots) of differentially expressed mRNAs.
 / More details about the axis representing the samples?

-Add more details explaining and simplifying your tables and figures in the captions please. Especially Figure 1. (Different expression of miRNA and lncRNA).

-Results: Are well stated.

-Please consider adding small paragraph summarizing the conclusion, although it’s already included in different parts of the results, but it would be a great add to your 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: CO-EXPRESSION ANALYSIS AND CERNA NETWORK REVEAL EIGHT NOVEL POTENTIAL LNCRNA BIOMARKERS IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 4

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this paper, Zou et al. performed integrated analysis of ceRNAs in Hepatocelluar carcinoma (HCC), and identified eight lncRNAs which were involved in important pathways. This is interesting work for research of HCC. However, some issues must be addressed as follows:
1. At the Abstract part, authors should revised to provide more description about Research methods, major Results, Significance, and Conclusion, not Introduction. The statements in the abstract that introduce HCC, ceRNA and lncRNA can be concatenated into one or two sentences.
2. The next sentence in the line 8 of Introduction should not be a transitional relationship with the previous sentence. “However” is inappropriate.
3. The sentence in lines 29 and 30 in 2.1 is somewhat confusing. How to get CeRNA? Method for cutoff 0.01?
4. The sentence beginning with “Specific” in line 39 of 2.2 needs to enumerate all the detailed formula descriptions used in the study, rather than special cases. What is co-expression effect?
In this 2.2, authors utilized RNA22 method to performed miRNA-target identification. Why not use miRcode、TargetScan or miRanda methods. Please explain it.
5. In 2.3, authors should describe the mechanism of action of WGCNA more clearly.
6. 2.5 The first two sentences are repeated and can be appropriately modified.
7. The meaning of “eigengenes” (Line 71) in 3.2? Figure 2 Since the meaning of the red box is explained, the meaning of the blue box should also be explained. And why the module analysis is performed in the Results? The conclusion of this part?
The relation between lncRNA modules and TNM stage must be explained clearly, and many sentences in this section belong to Methods part, not Results part.
8. Construction of a ceRNA network of lncRNA part belong to Methods Part, not Results part. The authors must revise this part and explain the biological meanings of the ceRNA network.
9. what is the relation and difference between networks from Figure 3 and Figure 4. And how to get eight lncRNA-cored networks in Figure4.
10. The description of lncRNA in the legend in Figure 4 does not match the figure (the triangle is in the figure, the arrow shape in the figure), and the description of lncRNA and mRNA should be the same shape or color.
11. Should the C diagram in Figure 5 be presented separately as Figure 6.


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

Input:
Title: CO-EXPRESSION ANALYSIS AND CERNA NETWORK REVEAL EIGHT NOVEL POTENTIAL LNCRNA BIOMARKERS IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: This paper is clear and unambiguous, professional English used throughout.
Experimental design: The paper had a  unequivocal aim and scope.
Validity of the findings: This study makes sense in the field of liver cancer research.
Additional comments: This study is very meaningful in the field of liver cancer research, it is recommended to accept.


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: CO-EXPRESSION ANALYSIS AND CERNA NETWORK REVEAL EIGHT NOVEL POTENTIAL LNCRNA BIOMARKERS IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: More biological meanings of these eight lncRNAs were missing.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this revision, the authors addressed some of my comments, however, most issues were not addressed or revised. So major revision need to be performed again before publication.

Q1. For the Abstract issue, the authors have re-written this part, including the background, methods, results and conclusions. I think the background part is too long than others.

Q3. For DE genes, the cutoff was defined as FDR<0.01 and |logFC|>1. However, for DE miRNAs, the totally different cutoff was defined. Why? Explain it, or discuss it.

Q4. What is the definition “co-expression effect”? The word “effect” is confusing, whether other articles used this word and provided the reference. More importantly, other miRNA-target databases must be discussed in your manuscript.

Q7. The section 3.2 was totally not revised. This part must be revised to provide more description about the Figure 2 or conclusions, and more References must be added to explain these results.

Q8. For one biological network, the basic attributes, such as degree and betweeness, must be displayed and analyzed. But this analysis was missing.

New comments. The description or explanations for the eight lncRNAs should added, and more references must be added to explain the relationship between these lncRNAs and hepatocellular carcinoma. Whether some lncRNAs have be identified as tumor bio-marker? And which lncRNA is novel bio-marker for tumor or hepatocellular carcinoma? The authors must display it.


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: THE IMPACT OF STORAGE CONDITIONS ON HUMAN STOOL 16S RRNA MICROBIOME COMPOSITION AND DIVERSITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Well written and good literature review.
Experimental design: Good technical approach in an important topical area of research
Validity of the findings: The sample size for the study is too small to be meaningful
Additional comments: The authors set to identify the effect of storage conditions on the microbiome of three children from Uganda. The authors used five different collection methods, including raw, ethanol, RNAlater and OMNIgene.GUT. The authors then analysed the 16s rRNA poriles using the V1-V2 regions. There were significant differences were observed in the composition and diversity between preservation methods. However, they could still observe clustering that was driven my child rather than sample preparation method. The authors are particularly interested in how temperature and storage could affect the microbiota and how this relates to samples from field studies, where there are more likely to be fluctuations in the storage collection and conditions which may alter the microbiota. The authors address important questions about the variation in samples depending on collection and storage before processing. The data they present shows the importance of standardizing collection within a study.

Major comments:
The n=3 for most of the samples (raw, ethanol, RNAlater) and n=2 for the OMNIgene.GUT is very low. How robust is this?
Line 231~: “In total 87 stool samples were collected for analysis” >How are these 87 comprised? Are these different times of the 3 samples, in the different storage methods? Please insert a table of these samples.
Figure 1: All children have a distinct microbiome by PCA (Line 236), however on figure 1 you also see a clustering of the RNA later samples. (Fig 1b). Please discuss the clustering – there is similarity between child B and A for ethanol and raw too. (this is partly mentioned in lines 265-267)
Figure 3: the mixture of genera, phylum, family and order is not clear. This needs to be separated out by taxonomic levels
The authors should carry out ANOSIM analysis to determine whether the differences in the population are due to the child or the method.
Lines 257-285: Please quantify this conclusion that inter-storage preservation methods were relatively stable over time. A population analysis over time would answer this.
Lines 277-279: this is most likely not an increase in relative abundance of these genera (Sediminibactreium and Rummeliibacillus), but due to a drop in other taxa which then unmasks this difference. What is the alpha and beta diversity at these time points?
Minor points:
Methods
Line 185 :  “and then reads less 186 than 250 bp in length were discarded” What was the length of the V1-V2 PCR? The forward and reverse reads should cover the 453bp fragment?

Line 201-203: “Higher taxonomic groups were 201 excluded where it was assumed that a lower taxonomic group was accountable for the observed 202 change. These situations were where a higher taxonomic group had a less significant or equal 203 change in relative abundance compared to a lower taxonomic group classified to the higher 204 taxonomy by LEfSe analysis.”
This needs further clarification and reasoning.
Line 222: “Principal Coordinates Analysis (PCoA) plots” or principle component analysis?
Lines 311-315: the OMNIgene was only performed on two samples to this is too few to analyse


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: THE IMPACT OF STORAGE CONDITIONS ON HUMAN STOOL 16S RRNA MICROBIOME COMPOSITION AND DIVERSITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written.
Experimental design: Generally speaking, the methods are well described and experiments are well conducted. One limitation is the small sample size. (Only three individuals)
Validity of the findings: I may have missed the Accession numbers, but it does not appear that the data sets from this work have been uploaded to a public database (i.e., 16s sequences FASTQ data).
Additional comments: Generally speaking, the analyses the authors did are pretty straightforward .A moderate degree of revision is needed.  Please consider the following comments intended to improve the manuscript.

A more nuanced conclusion seems desirable.  A lot of literature already compared 16S fecal sample collection methods/storage conditions.    What is the major difference between this study and previous 16S methodology comparison studies? What’s the unique/new finding of this study?
Also, the authors could further discuss the meaning of their findings. Why the tested methods gave different results? What is the mechanism?

Other specific comments:

-	The QIIME-OTU picking method (with greengenes 13.8 database) which the authors current using is acceptable, but a little bit old, especially for this kind of methodology paper. Since the QIIME2 platform is public available for a long time (a couple of years), it is recommended to use the better algorithm/pipeline,  like deblur- sOTU method.

-	According to supplement FigS1,  the  observed OTU curves did not reach  plateau even at 10000 sequences per sample. So it would be good to explain the rarefaction level which used for the downstream analyses , at least in the Method section.

-	Most of the recent 16S  microbiome studies are targeting on V4 region , or V3-V4 regions.   Please explain why your targeting on V1-V2 regions in the discussion section.

-	For the alpha- diversity analysis,  other indices (eg. PD-whole tree) are needed  to better describe more aspects/features of the data.  Never the less,  these alpha- diversity analyses should be performed at multiple level (eg. genus level, and  otu level).

-	Fig.1. Title. For beta-diversity weighted/ unweighted UniFrac distance analyses,  did the authors use Principal Coordinates Analysis (PcoA) rather than Principal component analysis (PCA)?  Please clarify.  Here PcoA is recommended.

-	Fig.1.  For 3D figures,  it could be misleading if these figures  are not displayed in exact same angle and direction.  It is strongly recommended to display Fig1A and Fig1B in exact same manner.     Further more, additional analyses like PERMANOVA should be used to provide more evidence.

-	Fig. S2. It is strongly recommended to display FigS2A and FigS2B in exact same manner.  And add the PERMANOVA R square value to give more information.

-	Fig.2B.  Should add the figure legend to show the name of  these genera, at least the top 10 genera.   Put these information in supplemental is not good enough.

-	Fig.3.  It is not informative to put all different taxa level together and order them by LDA score value.    It would be much better to separate this figure by taxa level .   An additional phylogenetic tree with highlighted significant taxa would be even better.

-	Line23.    Typo. “However…”
-	Line 115.  Please double check.  “300g each”???  The reader will be shocked.
-	Line 233, “In total 87 stool samples……. there was an average of 233 67,575 (range 19,083 – 466,807) reads per sample (n = 85)”. It looks like two samples were excluded.   If so , what's the criteria? Please describe.
-	Line306, “Compared to the null species richness model” ,about species richness model, please show more details of the modeling results in Table S6.


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: THE IMPACT OF STORAGE CONDITIONS ON HUMAN STOOL 16S RRNA MICROBIOME COMPOSITION AND DIVERSITY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study "The impact of storage conditions on human stool 16S rRNA microbiome composition and diversity" is interesting and very important addition to understand how the method selection affects the results. In general the language is clear, but polishing is needed in some parts. The structure of the article is fine, but some figures need efforts so they would not seem just like a default outputs of a program. Also, supplementary material are very raw!
Raw data not shared! Did not find any notes where is the sequencing data deposited.
Experimental design: Although the research question is well defined, the methods are not (see general comments). In general the bioinformatics and statistics part is poorly outlined, perhaps indicating the un-complete understanding of the performed analyses.
Validity of the findings: Based on poory outlined method section, I have no comments here.
Additional comments: L112 – it weird to say specimen for a stool. Perhaps simply “one stool sample”.
L 115 – each sample (outer surface, central inner and mixed stool) was 300 g??? A child’s stool can’t be ca 900 grams.
L118 – RNAlater and EtOH samples also fozen? Why???
L123 – “an attempt was made to keep samples in cool …” – was this just an attempt or did it really happen? Not clear.
L126 – how long was the freeze-thaw cycle?
L138 – “weighing process” – please specify here how many grams were included to the DNA extraction?
L147 – odd primers. provide primer names! Did not find those primers in Alcon-Giner et al. 2017.
L169 – provide indices
L171 – 8 cycles seems a bit low. Why only 8 cycles?
L182 – fix to 2 x 300 bp
L183 – “up to 100,000 reads per sample” specify reagent kit instead.
L184 – no need to specify the Python download link
L184 – cutadapt uses Python3, not 2.7! And I guess you ran cutadapt through some Linux system, not through Python itself?
L185 – “reads trimmed to a minimum quality score of 20” - not saying that you should analyze the data again, but in my opinion this is really bad quality trimming habit. Just average and only q>20 means that probably your reads still contain very high proportion of errors
E.g. the average q score of (1,4,7,9,10,20,20,20,30,30,30,30,30,40,40,40,40,40,40,40,40,30,30,30,30,10,10,9,5,5,5,4,3,2,2,2) is 20.5! But obviously the read has lot of errors.
L187 – “PANDAseq in Python version 2.7” – it’s very misleading if you say that pandaseq works like this on python!
L190 – “Operational Taxonomic Units were assigned with 97% clustering” – which algorithm? do not see that in CodeS1 file.
L195 – how was the UniFrac distance calculated?
L204 – “more significant p value” -> higher p value
L218 – “Backward elimination was used for sequential removal of non-significant variables (p ≥ 0.05)” – this is not how the backward elimination works, by just p value. Was the AIC model selection used with the backward elimination strategy?

Methods: Why did authors choose 2-step PCR and in between also purifying the samples? Very laborious. Any specific reasons for that?
Any measures taken to account for unequal sequencing depth between samples? Any transformations for the raw read count prior making similarity matrix?

L238 – report results of the statistics here as well.
L242-244 – how can an ordination method say statistical significance?
L289 – if PCA does not show any “apparent” clustering, it does not mean immediately that there is no difference between ‘groups’! This has to be tested in order to say something about “significance”.
L304 – “(p = 2.871e-13)” – p < 0.001 is enough.

CodeS1 file:
poorly organized script file.
 “-q minimum quality (20 for pilot will increase to 25 for main study)” – what is a pilot, what is main? Carefully edit your files before submitting.
CodeS2: please edit for better vizualisation.

Supplementary tables should have legend inside the 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: THE IMPACT OF STORAGE CONDITIONS ON HUMAN STOOL 16S RRNA MICROBIOME COMPOSITION AND DIVERSITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: well written
Experimental design: Now fine
Validity of the findings: Important technical development that should have wide appeal to the microbiome community
Additional comments: The authors have addressed the issues raised


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

Input:
Title: THE IMPACT OF STORAGE CONDITIONS ON HUMAN STOOL 16S RRNA MICROBIOME COMPOSITION AND DIVERSITY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have read the revised manuscript, and the authors have considered the comment and suggestions that I raised. The revisions that they made to the manuscript are very effective in addressing the remaining concerns. Unless noted otherwise from editors or co-reviewer, the manuscript could be accepted for publication.


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

Input:
Title: PROPOFOL INHIBITS STROMATOXIN-1-SENSITIVE VOLTAGE-DEPENDENT K+ CHANNELS IN PANCREATIC Β-CELLS AND ENHANCES INSULIN SECRETION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper investigated the mechanism by which propofol induce insulin secretion.  Because propofol is used in perioperative patients and that glucose control is important in such patients, the data shown in this study is potentially interesting and beneficial.  However, the paper is missing several critical information.
Experimental design: The most critical point of this study is that (as authors also mentioned in discussion as limitation), authors only performed experiments in cell line.  This reviewer does not understand why authors used both MIN-6 and INS-1.  Authors should simply isolate islets from mice and perform batch incubation for insulin secretion and use primary beta cells for electrophysiological study.

Also, measurement of blood glucose in vivo under propofol treatment would be critical information that should be included in this paper.  If propofol affects short term insulin secretion, it should show immediate change in glucose level in mice.

Also it is unnecessary to perform insulin secretion experiment in cell line using sucrose and maltose.  Did authors wanted to compensate osmotic pressure of incubation solution? If so, it should be noted in the manuscript.

This reviewer do not understand why authors measured SUR2 for qRT-PCR assay  (which did authors measured SUR2A or 2B? or both???).  Authors should know that beta-cell KATP channel is composed of Kir6.2 and SUR1, not SUR2.

Because propofol induced insulin secretion in low glucose condition, the underlying mechanism for insulin secretion by propofol is unlikely to be only by the Kv channel current inhibition. In low glucose concentration (in hyperpolarized state), Kv channel is closed so propofol cannot enhance insulin secretion through inhibition of Kv channel.  Also, there is confusion in description about contribution of KATP channel in this paper.  In Result section (line 241), authors mentioned that "This evidence suggests that propofol affects ion channels such as ATP-sensitive potassium channels which are otherwise insensitive to glibenclamide and diazoxide  (???, very confusing description and hard to understand....)"  but in discussion section, authors mention"KATP channels are not involved in modulation of IS and GSIS by propofol (line 373)".  This is very confusing.  In order to clarify this, authors should simply measure the KATP channel current with and without propofol in both whole cell and perforated patch mode.
Validity of the findings: If authors can show more clearly about underlying mechanism for insulin secretion induced by propofol, this study is very interesting and clinically important.
Additional comments: The study itself is interesting and contain important information.  However, in the present form, this study can only say that propofol can affect Kv channel activity in cell line.  It is not clear whether this Kv channel modulation truly affects insulin secretion.  With present data there is still a possibility that propofol is affecting KATP channel or any other factor that may influence insulin secretion.


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: PROPOFOL INHIBITS STROMATOXIN-1-SENSITIVE VOLTAGE-DEPENDENT K+ CHANNELS IN PANCREATIC Β-CELLS AND ENHANCES INSULIN SECRETION
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript the Authors studied the effect of Propofol, an intravenous anesthetic drug, on insulin secretion and suggests that it may act modulating potassium channel activity. However, there are some aspects that need to be clarified and reviewed.
Minor point:

- In the title, the Authors mention MIN6 cells as the in vitro model of pancreatic beta cells under study. However, INS-1 cells were studied as well., A more generic title would be appropriate to introduce the reader to the manuscript
Experimental design: Many experimental aspects need to be improved. My comments are as follows:
-	In my opinion it is not necessary to use different doses of propofol in each experiment. After doing an initial dose response evaluation, the more effective dose should be used. In the specific case, I believe that 25 µM may be fine. It is also somehow concerning that the same doses give different results in different experiments (compare for example Fig. 1a; 2a; 2c)
-	Why did the Authors choose 100 µM propofol to study cellular energy metabolism (fig. 5) if they previously demonstrated a negative effect of this dose on insulin secretion? and why did the Authors use two other different doses of Propofol to evaluate oxygen metabolism (fig 5)? As I suggested before, one Propofol dose should be chosen and used for all the evaluations.
-	Fig. 6., 1h of Propofol treatment is not sufficient to evaluate gene expression variations. Please, consider a longer time to investigate the effect of Propofol on gene expression. The best choice would be a time course assay.
-	Fig 6., why have the authors performed western blot experiments only for Kv2.1 ? Protein expression levels of the other targets may be also worth investigating. However, a longer treatment time would be preferable to investigate changes in protein expression levels.
-	Lines 296, 297, 298,299, 300. “Propofol increase the frequency of action potential in 10mM glucose from 0.32 to 0.84 at 10 µM and to 1.08 at 25 µM. Similar results were obtained with glibenclamide (100 µM). Application of additional propofono (25 µM) increased the frequency of the action potential from 0.35 with glibenclamide to 0.59 (fig. 7)…”  It does not seem that adding propofol improves the increment obtained when glibenclamide was used alone. Please, clarify this aspect.
Validity of the findings: The results obtained are really interesting and could have important clinical implications, since propofol and other anesthetic drugs are widely and routinely used in clinical practice every day. Indeed, if these data are confirmed, physicians should consider the hypoglycemic risks to which patients would be exposed. In this regard, I suggest the Authors to emphasize the importance of this aspect in the text.
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: PROPOFOL INHIBITS STROMATOXIN-1-SENSITIVE VOLTAGE-DEPENDENT K+ CHANNELS IN PANCREATIC Β-CELLS AND ENHANCES INSULIN SECRETION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors investigated propofol effects on glucose-induced insulin secretion, and they try to dissect the mechanism of action of this anaesthetic drug. The claim that propofol specifically blocks specifically stromatoxin-1-sensitive voltage-dependent K+ channels, by blocking Kv2.1 currents in MIN6 cells, and that the inhibition increases insulin secretion in response to glucose. They also do some test on INS-1 cells.
Literature citing is limited. It would be desirable to review other authors too.
The English language is not always clear, should be reviewed.
Expression of results is not convincing if the authors do not make clear how were the percentages calculated. It would be more convincing if they give the raw data in the bars.
One of the main problems of this manuscript is that they take MIN-6 cells as normal beta cells, and this is not exact. One of the main differences is that cell lines divide, while healthy cells do not. This concept is taken very far because, in the discussion, they consider that the effects they observed could be significant for the anaesthetic procedures in humans! They did not even took a mouse to check the facts in the whole animal. The model should develop hypoglycemia.
Experimental design: The experimental design was easy because they use cell lines, but it would be better to check the results in the whole animal, and single real beta cells.

In the experiments in the whole cell mode of the patch clamp technique is essential to take in consideration that glucose cannot be metabolised, cells are dialysed. Glucose will not affect currents under these conditions.

MIN6 cells do not respond too much to glucose; they could be in different moments of the cell cycle. The effect of propofol is variable. The drug without hypnotic effect also increases IS and GSIS, we could assume a not specific observation.

I would not expect too many changes in GLUT2 or the other channels expressed in the membrane of the cells after one-hour exposition to propofol.
Validity of the findings: In the experiments with glibenclamide and propofol, you conclude that propofol affects channels that are insensitive to glibenclamide; while there could be other explanations, it could be affecting calcium currents or other IS mechanisms.

They must discuss the limitations of the study.
Additional comments: The results are too many and expressed as a percentage of something that is not clear, are difficult to follow.
When you comment on the results in the text it would be more useful to manage as the percentage increase or decrease, better than the numbers that are already in the graphics.
The discussion could be more profound.


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: PROPOFOL INHIBITS STROMATOXIN-1-SENSITIVE VOLTAGE-DEPENDENT K+ CHANNELS IN PANCREATIC Β-CELLS AND ENHANCES INSULIN SECRETION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have improved according to my suggestion.
Experimental design: Designs are accurate and I have no further comment.
Validity of the findings: Interesting enough to be published.
Additional comments: I have no further comments.


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

Input:
Title: PROPOFOL INHIBITS STROMATOXIN-1-SENSITIVE VOLTAGE-DEPENDENT K+ CHANNELS IN PANCREATIC Β-CELLS AND ENHANCES INSULIN SECRETION
Review round: 2
Reviewer: 2

Output:
Basic reporting: ...
Experimental design: ...
Validity of the findings: ...
Additional comments: The Authors have not responded satisfactorily to my previously comments. In particular, my observations are as follows:

1)	The Authors write that the choice of 100uM of propofol was made because this concentration had a negative effect on insulin secretion both under 2mM glucose and for GSIS. However, the aim of this work was to evaluate the impact of propofol on insulin secretion at low and high glucose levels. For this reason, it would be preferable to use 25uM propofol to evaluate its effects on cellular energy metabolism.
2)	In my opinion, a time course with a range between 1 hour and 4 hour it is no enough to assess effects on expression levels. Please, set a time course at least up to 12 hours for the gene expression study and at least up to 48 hours to study protein expression.
3)	In my previous comment I did not mention Kv2.2 channel subunit. In fig. 6., the Authors show the impact of propofol and glucose concentration on the expression of glucose transporter 2, ion channels and insulin. Western blot was performed only for Kv2.1 protein. Why only for Kv2.1? Protein expression levels of Slc2a2, Cacna1c, Kcnj11, Kcnb1, Abcc8 and Ins1 may be also worth investigating. However, as stated in point 2, a longer and appropriate treatment time (at least 48hours) would be preferable to investigate changes of protein levels.


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: PROPOFOL INHIBITS STROMATOXIN-1-SENSITIVE VOLTAGE-DEPENDENT K+ CHANNELS IN PANCREATIC Β-CELLS AND ENHANCES INSULIN SECRETION
Review round: 2
Reviewer: 3

Output:
Basic reporting: This is a second review. I only want to state that the title must state tumoral cell lines because most of the work was done on them, and they have many differences from mice cells.
Experimental design: No more comments
Validity of the findings: Please comment on all the issues of the first review.
Additional comments: The manuscript is better, but I still have some doubts.
I want to see a mean IV curve, with the number of cells studied. They are still MIN cells instead of normal mouse cells. I do not understand this procedure.
The effects on the basal insulin secretion are severe, and that could cause hypoglycemia. I suggested this possibility in my first review, but you did not pay attention to it. The discussion is still missing that they work mostly on tumoral cell lines; you must give the limits to the study.
There are still many orthographic mistakes, especially on the corrected part of the manuscript.


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

Input:
Title: PROPOFOL INHIBITS STROMATOXIN-1-SENSITIVE VOLTAGE-DEPENDENT K+ CHANNELS IN PANCREATIC Β-CELLS AND ENHANCES INSULIN SECRETION
Review round: 3
Reviewer: 1

Output:
Basic reporting: ...
Experimental design: ...
Validity of the findings: ...
Additional comments: New figure 6 is not clear. I think that there has been a misunderstanding. The Authors have changed the terms of comparison of the gene expression. Indeed, in the previous figure all data were compared to 2 mM glucose / 0 uM propofol. In the new version of the figure the Authors compare all points to 25 mM glucose / 0 uM propofol.  Furthermore, since the Authors want also to investigate the effect of glucose on the expression genes, I suggest them to prepare two different graphs: in one they can show the effect of glucose on gene expression and in the other one the combined effect of glucose and propofol until 48h. Please, in this second graphs leave also original data with 1 and 4 hours expression levels (figure 6 of the previous version). Judging from the histograms height, it seems that there is some change during the time course.