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: SYSTEMATIC REVIEW AND META-ANALYSIS OF 50 YEARS OF CORAL DISEASE RESEARCH VISUALIZED THROUGH THE SCOPE OF NETWORK THEORY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written but the discussion needs to be more fully developed.
Experimental design: This appears to be a standard approach.  My only concern is that they define "white syndrome" as tissue discoloration and loss.   White syndrome is a tissue loss disease regardless of whether the lesion has discolorations or not.  Not sure if that would effect the categorization of their clusters or not.
Validity of the findings: Findings are interesting and valid.  However, they need to be presented in a much clearer manner.
Additional comments: This is an interesting approach with the intent to determine where research efforts have been in coral disease research identifying potential gaps.  The concept is interesting and I think there is valuable information presented.  Table 1 is clear and informative although I would request a full Table with all of the information even as a supplemental file.  From the full Table one could better evaluation which corals, regions, etc are completely missing.  The authors, however, need a better visual representation of their findings (Fig. 5-7) or perhaps better, more detailed information in the Figure legends.  Perhaps something along the lines of Fig. 2?   In order for this paper to be useful there needs to be a better explanation of what these “communities” are and what they tell us in terms of research gaps.  Discussion needs to be developed more fully explaining the major new understanding of coral disease research efforts.  For example, in the discussion it is stated that “For example, Black Band Disease occupies a preponderant place in coral disease research. “ but they don’t explain how the Figures support this.  Is it because BBD is linked across 5 clusters (c2, c4, c5, c12, c14)?  If so, then they need to spell that out so readers can better understand these results.


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

Input:
Title: SYSTEMATIC REVIEW AND META-ANALYSIS OF 50 YEARS OF CORAL DISEASE RESEARCH VISUALIZED THROUGH THE SCOPE OF NETWORK THEORY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The Revised article is acceptable for publication
Experimental design: Appropriate Experimental Design
Validity of the findings: Appropriate
Additional comments: The revised manuscript is acceptable for publication. No further issues are 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: SYSTEMATIC REVIEW AND META-ANALYSIS OF 50 YEARS OF CORAL DISEASE RESEARCH VISUALIZED THROUGH THE SCOPE OF NETWORK THEORY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Addition of the supplementary materials was really helpful
Experimental design: no comment
Validity of the findings: The authors have done a good job clarifying and expanding upon the relevance of their findings to the field of coral disease research.
Additional comments: This revised manuscript is much more easily understood and will be a nice addition to our knowledge of the state of coral disease research.


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: PTMPHINDER: AN R PACKAGE FOR PTM SITE LOCALIZATION AND MOTIF EXTRACTION FROM PROTEOMIC DATASETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No addition comments.
Experimental design: Methods are now described, although this does reveal how trivial the software is.

It's good to see that support for other PTMs (beyond phosphorylation) has now been added.

A PSI standard (mzTab) is now mentioned as future work, though what role it would play is not specified.

The authors' argument that "we would like to finalize the publication of our tool prior to uploading it to BioConductor to ensure that it is thoroughly tested and optimized" is concerning as it implies the software may not be fit for purpose at the present time.
Validity of the findings: Basic validation has now been added.
Additional comments: I do not expect this to be a high impact paper. The methods are straightforward, the software is simple (169 lines of R code) and without incorporation into a package such as BioConductor it will not be widely used.

Having said all that, none of these issues appear to preclude this revised version of the paper from being accepted for publication in PeerJ.


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: FUTURE CO2-INDUCED SEAWATER ACIDIFICATION MEDIATES THE PHYSIOLOGICAL PERFORMANCE OF A GREEN ALGA ULVA LINZA IN DIFFERENT PHOTOPERIODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Needs clarification at many points.
Experimental design: No Comments
Validity of the findings: No comment
Additional comments: This manuscript reports innovative and significant results, but needs significant improvement, as indicated below.

Line 28. Is the 28% 48% of the total anthopogenic CO2 that has been produced, or 48% of what is not in the atmosphere?

Line 33. ‘ocean acidification’, measured as the decrease in surface ocean pH since the pre-industrial Holocene, is not the cause of the perturbations of the inorganic carbon system, but is one of the outcomes of introducing more carbon dioxide, along with an increase in bicarbonate and decrease in dissolved carbonate.

Line 39. Reference needed for the macroalgal contribution to marine primary productivity.

Line 51. Ulva australis is not ‘giant’, nor is it a kelp: a kelp is a member of the Laminariales, or, more broadly, including some members of the Desmarestiales and Fucales. ‘giant kelp’ should be attached to Macrocystis pyrifera.

Lines 58-59. Clarify the positive and negative effects.

Line 62 .’longer’ rather than ‘increased’.

Line 65. ‘increase’ rather than ‘upregulate’; this latter term is best reserved for the changes in mRNA and protein in cells.

Line 71. There is an implication here that green tides were first observed in the Yellow Sea in 2007, rather than earlier elsewhere in the world ocean. Clarify. See, for example, Taylor 1999 Botanical Journal of Scotland 51: 193-203.

Line 74. Ulva (including Enteromorpha) is not the only genus of green tide alga. See Tsutsui et al. 2015 Int Aquat Res 7: 47-62.

Lines 77-78. This single examples contrasts with the effects (increased, no change, decreased) on growth of Ulva species mentioned on lines 43-52.

Line 89. By what criteria (morphological? molecular?) was the alga identified?

Line 91.’Healthy’ by what criteria.

Line 97. ‘adults’ by what criteria?

Line 108. ‘were not obtained’:  does this mean there was no variation in fresh weight, or that fresh weights we not determined?

Lines 153-154. Why could not absolute ETR values be determined? It is not difficult to measure the absorptance of Ulva (Longstaff et al. 2002 Photosynthesis Research  74: 281-293).

Line 160. Why were photosynthesis measurements made, as well are those of respiration?

Line 189. What is meant by ‘strong’ in this context?

Lines 199-200. Mention the species of Ulva that were used.

Line 202. ‘algal physiology’ includes a very range of activities. Be more specific.

Lines 203-215. This argument requires that the decreased external pH increases the influx rate of H+ and/or increases the energy cost H+ efflux in  maintaining the pH of intracellular compartments.


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: FUTURE CO2-INDUCED SEAWATER ACIDIFICATION MEDIATES THE PHYSIOLOGICAL PERFORMANCE OF A GREEN ALGA ULVA LINZA IN DIFFERENT PHOTOPERIODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well written, clear, and concise. However, it is too concise in some parts of the paper and I think there is potential to add more to the discussion section to thoroughly cover all aspects of the results of this experiment. While the grammar is okay overall, I found many small errors. I pointed out some in the notes below, but there are more, and this paper should be edited before resubmission.
Experimental design: The research question of this paper is relevant to understanding how green tide will be affected by future ocean acidification. Many environmental factors can alter the effect of OA on Ulva spp, the primary bloom forming species in the Yellow Sea, including light. Understanding the physiology by doing lab experiments can help us understand what could happen in the natural environment and can contribute to our understanding of algal physiology. This paper collected algae from a natural site and subjected it to three photoperiods and two CO2 levels to understand the effects on growth, chlorophyll fluorescence, respiration, and chlorophyll. The experimental design is appropriate, although some of the methods for the alteration of CO2 could be described in greater detail, particularly the carbonate chemistry.
Validity of the findings: The finding of an interaction between CO2 and photoperiod on the growth of Ulva is valid, but the discussion lacks a comprehensive interpretation of the results. The authors did attempt to connect various physiological responses to the conditions of the experiment and compare it to the results of similar research, but it was superficial and did not explore the details that would help readers understand the meaning of the results in the context of the great body of knowledge.
Additional comments: Abstract

The abstract does a good job at explaining the main findings from the experiment and their significance.

Lines 15-16: Describe how the growth and Yield responses are similar. In the graphs, the patterns are similar in the 8:16 and 16:8 photoperiods, but not the 12:12 photoperiod and the statistical significance of the results differ, so the statement is misleading without clarification.
Line 17: change “Non-fluorescent chemical” to “Non-photochemical”

Introduction

Line 31: change “04” to “0.4”
Lines 33-35: Munday et al. 2011 citation is not in literature cited.  The effects of OA on calcareous plankton and especially fishes are not relevant to this study and should be taken out anyways. Check that all cited literature is in the literature cited.
Lines 35-36: change “no-calcareous” to “non-calcareous” and add a citation to this sentence and state why they may benefit.
Line 39: cite the original paper for “macroalgae accounts for 10%...”
Line 41: cite or combine with next sentence
Line 43: Change “concentration” to “concentrating”
Line 43: Explain why the influence of OA on macroalgae is species-specific based on the previous sentences or other references. Before you state that most macroalgae use CCMs, so that does not naturally lead to the conclusion that “Therefore, the influence of OA…is species-specific”. Explain why in greater detail.
Line 43: change “positively” to “positive”
Line 44: change “influences” to “influences on the growth”
Line 51: Remove part about the giant kelp Ulva australis and add before Macrocystis
Line 52: change “reduces” to “reduced”
Lines 55-57: after temperature and nutrients you could add “light” since that is what is paper focuses on. Make sure to include an appropriate citation.
Lines 58-61: It isn’t immediately clear what you mean by positive effects in the daytime and negative impacts during the night. You could expand on this a little by talking about diurnal fluctuations of pH due to photosynthesis and respiration.
Line 63: Elaborate on what you mean by strengthening the control on the CCM
Line 64: You need do work on developing a convincing argument for why the light dark cycle is important in OA. You touched on a few reasons but lack the detail for why it might have an effect. If you expand more on a few points from this paragraph such as CCMs and diurnal fluctuations in pH it would be more convincing.
Lines 64 -66: The sentence about pH upregulation in kelp forests seems random and not within the scope of this paper.
Line 67: By “opportunities” do you mean “growth”?
Lines 68: Which species were tested by Nielsen 1997 and Rost et al. 2003?
Lines 80-83: In your hypothesis you mention the down-regulation of CCMs but did not include any background information or previous studies that show this occurs. You could add that to the previous paragraph when I mentioned you could add more detail.

Materials and Methods

1.	What kind of post-hoc analysis was done?
2.	Were carbonate chemistry parameters measured and how was pH monitored? What was the pH of the different treatments
3.	What kind of curve did you use to fit your RLC? In Fig. 3, it almost looks like there could be photoinhibition occurring at PAR 1000. See Ralph and Gademann 2005 for advice on curve fitting using RLC.

Line 97: change “approximate” to “approximately”
Line 103: How was pH measured to make sure the variation was maintained to between ± 0.05?
Line 105: Its not clear if the experiment lasted 6 days and the measurements were taken on the last 3 days or if the experiment was 9 days and the measurements were taken on the last 3 days.
Lines 107-111:  Did you average the RGR across all the 2-day time periods throughout the experiment or only during the last three days as stated previously?
Line 120: change “Non-fluorescent chemical” to “Non-photochemical”
Line 127: How long was the respiration rate measured for?
Line 135: There is what appears to be a spot for a reference that needs to be filled in after SPSS

Results

Line 143: How can elevated CO2 and the light dark cycle both have the greatest effects? This is not clear.
Line 146: be consistent with using L:D or light/dark cycle
Line 152: It doesn’t really look like NPQ increase from 12:12 to 16:8. So be more specific about the results in the results section and describe what happen during each photoperiod.
Line 159: Same comment as above. You need to describe all your results here, not just what generally happened, because the exceptions may be important.

Discussion

1.	The discussion seems a little sparse as it stands now. You didn’t mention the results from the Rapid light curve in your discussion.
2.	I think it would be important to discuss the findings of Li et al. 2018. Their study was similar to yours but did not find an interacting effect using the species Ulva prolifera. What do you think these differences in results were caused by?
3.	I think more thought needs to be put into the discussion by comparing your results to other similar studies and by connecting various physiological mechanisms. This has been done well already, but I think there is room to expand.

Line 182-189: There is a lot of variation in how photoperiod affects different species which is described here. Is there life history features of the different types of algae that cause it to have different optimal growths at different photoperiods? Like are some fast-growing or slow growing or what kind of habitat are they typically found in? Season might play an important role here to. If the algae are collected from a natural environment and then used in a lab experiment, they may still be acclimated to the natural photoperiod. All these aspects could potentially be covered in the discussion, because the discussion at present is very lacking in detail and is only two paragraphs long.
Line 210-212: Its not clear how saved energy from down regulated CCMs could damage the algal photosystem. You should elaborate.

Figures

1.	Describe what the lines and letters over the bars represent.
2.	Is Fig. 3 necessary? It’s difficult to differentiate between the different treatments, so either make it clear (maybe by separating HC and LC treatments?) or remove it since the information from the graph is summarized in table 3.


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

Input:
Title: EFFECTS OF PROLONGED CONTINUOUS COMPUTER GAMING ON PHYSICAL AND OCULAR SYMPTOMS AND BINOCULAR VISION FUNCTIONS IN YOUNG HEALTHY INDIVIDUALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The quality of technical writing of the manuscript is excellent, and the prose is easy to read.  Relevant previous literature is cited.  There are some important omissions, noted below, which appear to have tackled a very similar question.

Minor issues:

line 61. 24 % *of* children

line 185.  Given that body posture can be frequently changed during game play, it is unclear why higher scores should be obtained for physical symptoms than ocular symptoms (apart from tired eyes).
Experimental design: The research question, and experimental methods employed to investigate it, are both well defined.  However, as discussed in further detail below, it has not been convincingly demonstrated that this research fills an identified knowledge gap.  The authors should clarify how their study provides a greater understanding of the effects of short- or prolonged computer gameplay on visual function, as differentiated from that of VDT exposure that does not involve computer gaming.
Validity of the findings: The results reported appear to be reasonable, and the comparisons to similar results from related studies were helpful to put the findings in context of previous literature.  There are several issues, however, that should be addressed to make clear the contribution of the findings:

1. Surprisingly, the authors have not addressed what would seem to be the most obvious question, regarding whether there is a difference in the effects of prolonged VDT exposure from non-gaming use, e.g., of programmers.  Are the observed effects in fact due to gameplay activity or do they manifest equally in any interactive (or perhaps even passive) screen-viewing activity?

It is well established that VDT use affects visual function.  As one example, cited by the authors, (Qu XM, Chu RY, Wang L, Yao PJ, Liu JR. 2005. Effects of short-term VDT usage on visual functions. Zhonghua Yan Ke Za Zhi 41(11):986–989 DOI: 10.3760/j:issn:0412-4081.2005.11.007) conclude that ``Short-term VDT work does have a significantly greater temporarily [sic] effect on visual function, tear film quality and visual quality.''  However, as described by other literature, similar effects are observed in other (non-VDT) near field work.  Studies by Yeow and Taylor report that ``there are no significant differences in type, number and frequency of the work-related symptoms between VDT users and non-VDT users'' (P.T. Yeow, S.P. Taylor, The effects of long-term VDT usage on the nature and incidence of asthenopic symptoms, Applied Ergonomics, Volume 21, Issue 4, 1990, https://doi.org/10.1016/0003-6870(90)90199-8) and ``Results show that VDT work does not have a significantly greater effect on visual function than non-VDT work.'' (Yeow and Taylor, Effects of short-term VDT usage on visual functions, Optom Vis Sci. 1989 Jul;66(7):459-66).

2. Beyond the direct reporting of the experimental results, a meaningful discussion of the implications of the data would have strengthened the manuscript.  Instead, the Conclusions state that the ``visual fatigue caused by long-term VDT use may tend to accumulate over time.''  The duration of the experiment was, of course, limited to a single 4-hour game session, and thus, this hypothesis is offered without substantiation or supporting citations.

3. Apart from blink rate, for which data were reported only before and during gameplay, all of the experimental measures returned to baseline values by the following morning.  The authors should therefore elaborate on the contention that their study provides ``the first direct evidence of changes in physical and ocular health and visual functions after prolonged continuous computer gaming''.  One might argue, to the contrary, that these functions manifested changes only during and immediately following gameplay, when such physiological effects are entirely anticipated, just as one's heart rate and respiration increase during periods of physical activity.  The more important question, I would argue, is recovery time: do these effects persist beyond a typical non-VDT-centric-activity ocular recovery time following cessation of the activity?  If not, this warrants comment.
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF PROLONGED CONTINUOUS COMPUTER GAMING ON PHYSICAL AND OCULAR SYMPTOMS AND BINOCULAR VISION FUNCTIONS IN YOUNG HEALTHY INDIVIDUALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. On table 1, authors should identify between each group the differences were found statistically significant.
2. The meaning of the negative and positive  values for phoria should be in the text.
Experimental design: 1. The major drawback of the study is the lack of a control group.
1.Why VA was measured with the phoropter? This is not a normal vieweing condition.
2.The PPA was measured monocularly or binocularly
3. Why all the accommodation parameters were measures binocularly? Any change in binocular vision will have impact in the binocular accommodation parameters even if the accommodation will not suffer of any change.
4. In the methods it is not explained that measurements were repeated in the next morning.
Validity of the findings: The authors state that " Although these changes are usually temporary, visual fatigue caused by long-term VDT use may tend to accumulate over time". However, this can not be concluded by this study.
They also say that "ergonomically designed work spaces are desirable to minimize physical discomfort after long VDT tasks" but no study was done to evaluate it.
Authors should rewrite their conclusions.
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: EFFECTS OF PROLONGED CONTINUOUS COMPUTER GAMING ON PHYSICAL AND OCULAR SYMPTOMS AND BINOCULAR VISION FUNCTIONS IN YOUNG HEALTHY INDIVIDUALS
Review round: 1
Reviewer: 3

Output:
Basic reporting: See comments
Experimental design: See comments
Validity of the findings: See comments
Additional comments: Review of the manuscript: “Effects of prolonged continuous computer gaming on physical and ocular health and visual functions in young healthy individuals”

The manuscript reports results of a study investigating effects of 4 hours of continuous computer gaming (“the task”) in certain aspects of the visual system. The study shows that near phoria changed after the task and that was also accompanied by an increased score in serf-reported visual discomfort and changes in blinking rate. The results of the study are interesting and likely to attract the attention of the scientific and clinical community. The presentation of the findings can be improved to help readers that may be unfamiliar with some of the parameters measured. It is unclear from the text which was/were the hypothesis or hypotheses. During the discussion and conclusions, there is a tendency to go beyond what the results show or can prove. I have a few suggestions that will significantly improve the manuscript.

# Title
The title is misleading and should be changed. The study is not investigating any health or disease issues but instead is investigating changes in self-reported discomfort – which does not correspond necessarily to any unhealthy state. Visual function is also misleading because, for example, visual acuity is not part of the main results. I suggest replacing it by “binocular vision function”. A good example of the phrasing is what is written in line 82: …” … computer gaming on physical and ocular symptoms and binocular vision in young …” which is what was actually done in the study.

# Introduction
The text is full of vague statements that should be removed or replaced by concrete contents, some examples:
line 43 - “In today’s generation”, what is today’s generation?

Line 52-54 – it seem like authors are trying to suggest that people should stop using computers, maybe this sentences should be re-written;

line 62 – “Computer gaming addiction can cause health and mental issues”, what is the relationship of this sentence with poor visual ergonomics? Ergonomics seem to be the topic of the paragraph (not mental health);

line 77 – “Computer overuse has been deteriorating the visual environment year after year”, please specify the meaning of “deteriorating the visual environment”, “Actually, most individuals addicted to computer games”, What defines an addicted person?

Line 82 – “Moreover, most studies related to computer use and vision have individually evaluated physical and ocular symptoms and binocular vision.”, if this is the main justification for this study, this evidence should be given in the introduction. As it is this statement comes from nowhere;

# Methods
Line 95 – “Sample size was estimated based on the difference of computer-using time between a 3.6 ± 96 2.2 hours per day and a 2.4 ± 1.6 hours per day for internet-addicted and non-addicted Korea adolescents in the previous study.” Is not clear why and how does the sample size comparing addicted and non-addicted user has been used if there is only one group in the study.

Line 109 – an English version of the questionnaire should be included in the extra material and a clear reference should be given;

Line 116 – 128 – here there are different tests for different “visual functions”. I recommend that authors define what each function mean before explaining the measurement. For example, accommodative and vergence facility are two variables only defined in discussion. Also, a brief explanation why these measurements are important should be included, here or in introduction. In general, the introduction is very vague about which vision problems can arise because of gaming;

Line 128 – the words “finally, the” should be removed from the text and the method of counting explained. I assume that the camera does not count blinks (complete / incomplete blinks should be defined);

# Results
The statistical tests used seem adequate. Although, given the amount of results presented, normative values for each test presented in the same table as the experimental data (Table 2) would improve the readability of the results/manuscript;

Is not clear what do the plus or minus sign means throughout all the results -- that should be described;

Line 163-174, I assume all results comparing “before-after-day after” mentioned here are included in Figure 1, therefore this figure should be mentioned before reporting the findings;

Also, lines 163-166 are about anova results and, I presume, lines 171-174 still anova findings. Describe the anova model, e.g. which factors were used. The correlation results in between anaova outcomes should be removed and all anova findings reported together. The text saying “ no change” should be reviewed. Authors should be aware that there were changes, but they weren’t statistically significant – please read more about this here: https://www.ncbi.nlm.nih.gov/pubmed/7647644. In addition, for example in line 164 “whereas horizontal phoria at near changed from −3.73 ± 3.93”, which test has been used? p-value? The same for other significant results;

What is the different between these results? And what is the difference between “shift” and “change”: Lines 173-174: “Distance phoria showed no shift during the game (repeated measures ANOVA; F = 0.61, p = 0.610)”. and Line163: “Horizontal phoria at distance showed no change after the gaming session (F = 1.67, p = 0.193)”;

# Discussion
Line 178: Can authors please explain in the manuscript the meaning of “ impaired visual functions”? If they don’t have a simple explanation, maybe should consider re-writing.

Lines 200-202: Is unclear for me why authors bring NPA in line 199 and then conclude that their findings are related with intra and extraocular muscles fatigue, a better connection between the findings and the causes should be given;

Paragraph line 203 - 233, discusses poor illumination but authors do not explain how that applies to their study. Authors should relate the discussion with illumination in the room where the experiment took part and/or the luminance of the screen. This paragraph needs further clarification and connection between previous findings and the findings of the present study;

Lines 229-233, ideas are very foggy here and the final recommendation is unsupported by the findings. This part of the discussion needs further work and authors should be cautious about the recommendation they do based on their results;

Line 238-243: authors need to explain why they consider that the sample was small when they use more subjects that the sample size that they computed and described in methods. Also, I cannot understand which “physical and ocular health” aspects changed during the study. Why do further studies need to include people with binocular disorders is also unclear to me. It seem that this single paragraph puts together the limitations and the main findings of the study, which is somewhat contradicting.

Lines 252-257, It is unclear for me as reviewer what support does this study provides to the set of recommendations given. Unless authors can clarify what they mean what why these lines should be part of the manuscript, I recommend them to be removed.


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

Input:
Title: EFFECTS OF PROLONGED CONTINUOUS COMPUTER GAMING ON PHYSICAL AND OCULAR SYMPTOMS AND BINOCULAR VISION FUNCTIONS IN YOUNG HEALTHY INDIVIDUALS
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: I remain uncertain as what contribution the present work offers to the literature. The table (R.1) provided in rebuttal provides a succinct comparison of the author's approach and findings with that of prior literature cited. However, neither this table, nor the following text that elaborates on this body of work, establishes an unexpected difference in ocular discomfort ("temporary effect on visual function") from the "accelerated" (4 hours without break) exposure to game-based VDT activity vs. that of Qu et al.'s short-term (1 hour) VDT exposure.  The possible exception is that of physical (non-ocular) discomfort, which was, I would guess, not investigated by Qu et al.  This problem is exacerbated by the lack of a comparison group, e.g., non-gaming VDT users, exposed to a display for similar periods of time, that would otherwise plausibly offer an opportunity to explore such a comparison.  Since the present manuscript does not focus predominantly on physical effects, the question remains: "what does a reader learn about the impact of VDT exposure (whether for one hour or four hours; whether for gaming or non-gaming activities) that is not already known from the literature?"

With regard to the point I raised in my initial review, the claim that the study "provide[s] the first direct evidence of changes in physical and ocular health and visual functions after prolonged continuous computer gaming" seems possibly hyperbolic, given the past evidence for, at the very least, such ocular effects from extended VDT exposure.  Moreover, these results would seem, intuitively, consistent with expectations for any activity demanding long-term visual attention in a seated position.  However, I appreciate the addition of the references to visual function recovery time in other studies, as well as the addition of Murata et al. (1996) as a citation to accumulation of visual fatigue over time.


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

Input:
Title: EFFECTS OF PROLONGED CONTINUOUS COMPUTER GAMING ON PHYSICAL AND OCULAR SYMPTOMS AND BINOCULAR VISION FUNCTIONS IN YOUNG HEALTHY INDIVIDUALS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This reviwed version is a much better version of the work.
Experimental design: Although authors have answer to all my concerns, I still have some issues regarding this part of the work

1. On table 2, authors should identify between each group the differences were found statistically significant.
Validity of the findings: N.A.
Additional comments: This reviwed version is a much better version of the work.


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

Input:
Title: EFFECTS OF PROLONGED CONTINUOUS COMPUTER GAMING ON PHYSICAL AND OCULAR SYMPTOMS AND BINOCULAR VISION FUNCTIONS IN YOUNG HEALTHY INDIVIDUALS
Review round: 2
Reviewer: 3

Output:
Basic reporting: (1.1) In my previous review I asked the authors to add the expected values for variables in Table 2, and authors answered "normative values for each test could not be determined". This is a misinterpretation of my request, expected values for binocular and accommodative functions exist and this reference https://www.ncbi.nlm.nih.gov/pubmed/28095087 is an example. I still think expected values for binocular and accommodative functions should be in Table 2 to certify that participants in the current study showed baseline values within the expected range.

(1.2) line 98 in all the manuscript - what is the meaning of "± 2.2" is 2.2 the standard error?, standard deviation of the mean? the inter-quartile range? please specify before using.

(1.3) This statement would fit better in discussion: "Our results are not limited to gaming activities, but applicable under the environment of overuse of personal computers for the purpose of computer game after the college students’ work day."

(1.4) Blinking rates should be presented with a central tendency measure (e.g. mean) and a measure dispersion (e.g. standard deviation);

(1.5) Figure 1, what do the symbols represent? mean values?

(1.6) Lines 163-184, it would be easier to understand the results if  mean differences for statistically significant changes are reported;
Experimental design: (2.1) Sample size calculation is unclear, authors use hours-of-computer-use (HCP) obtained from addicted and non-addicted internet users from a previous study to determine the sample size needed to test their hypothesis. In the current study HCP are fixed (not an outcome mesure); therefore, I cannot understand why authors use such a variable (HCP) to determine the sample size. Also, the hypothesis of the study should be more clear if authors want readers to understand their sample size calculations.

(2.2) How can participants can be randomly enrolled when there is only one group? please clarify

(2.3) Given (2.2) I am unable to understand the limitations described in lines 265-268
Validity of the findings: 'no comment'
Additional comments: GC1: authors should verify if "divergent shift" is the correct term to express the observed changes in phorias;

GC2: The last sentence of the abstract starting "and highlight the need for ..." is not supported by the current study and author should delete it;

GC3: Reference Ahn appears with years 2017 in the text and 2007 in the reference list, please correct;

GC4: unless authors can defend the use of the expression "deterioration of the visual environment" this expression should be replaced by a more objective one;


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

Input:
Title: EFFECTS OF PROLONGED CONTINUOUS COMPUTER GAMING ON PHYSICAL AND OCULAR SYMPTOMS AND BINOCULAR VISION FUNCTIONS IN YOUNG HEALTHY INDIVIDUALS
Review round: 3
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


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: DIETARY SUPPLEMENTATION WITH PROBIOTICS REGULATES GUT MICROBIOTA STRUCTURE AND FUNCTION IN NILE TILAPIA EXPOSED TO ALUMINUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language is not at a professional level and the text is written in a way not easy to follow. I suggest seeking professional help in order to prepare the manuscript in an acceptable form.
The use of the literature is not sufficient, as much irrelevant information is included.
The figures are acceptable as well as the table, but more information is required in order to be more comprehensive.
Experimental design: The research falls into the journal aims and scope. However, the research question is not very well defined, and the background given is not quite sufficient. The methods described need to be presented in more details.
Validity of the findings: The statistical analysis is not performed properly. More analysis is required in order to be able to reach to the right conclusions. There is a lot of speculation in the discussion, which can be decreased in order to discuss the relevant data.
Additional comments: The authors present a work related to the effects of probiotic dietary supplementation with L.plantarum on the gut microbiota and aluminium toxicity alleviation in the Nile tilapia. The aim of the study is interesting overall, but there are several major concerns that have to be addressed properly in order to reach the right conclusions.
1. The language is not at a professional level and the text is written in a way not easy to follow. I suggest seeking professional help in order to prepare the manuscript in an acceptable form.

2. The abstract should be re-written. The authors should include a brief intro, then state their scientific question and then describe the experimental setup, results and conclusions. It is not easy to follow the way is currently written. Moreover, the authors should not use abbreviations at this stage.

3. The introduction is not informative enough, while it includes irrelevant information. The authors should organise their introduction in a more comprehensive manner in order to:
a. Show why the used the aluminium and how is it relevant to study for fish? Where does Al pollution come from? The reference to Mexico is not relevant to the main question the authors ask.
b. Were there studies before showing that it has an impact on the gut microbiota?
c. Why did they choose to use probiotics and why the specific strain?
d. Why did they choose tilapia as an experimental animal?
e. The rationale of the study should be better explained, and the question better defined.
Moreover, the literature used is not always relevant.

4. In the materials and methods, the description of the experimental setup is insufficient. There are several issues concerning the statistical support in the study and the indices measured in order to reach the conclusions. The widely accepted indices to evaluate changes in the diversity and richness (i.e. Shannon H index and observed OTUs) are not used, and the authors reach to conclusions just by speculating from a descriptive analysis. Therefore, I recommend the authors to use widely accepted measures to evaluate the impact on the microbial diversity and richness. Moreover, when comparing the abundances of specific taxa, parametric methods are not accepted since the normality of the data distribution is not assumed. The authors should use non-parametric tests to evaluate the impact of the different treatments on the abundance of specific taxa, but also for the beta-diversity (principal coordinate analysis, PCoA), Permanova test should be applied. The authors should also state which metric they used for the PCoA.
Several others points that also need to be revised:
Line 88. How did the authors select this concentration and how did they check it in the feeds.
Lines 100-104. How many tanks did they authors use per group? The way is written is quite confusing.  How did the authors succeed the Al concentration in the water and what are overall the acceptable limits? Was is in excess in the water?
Lines 105-106. The sentence is not clear. How did the authors collect the faeces in order to make sure they were not affected by the Al concentration in the water?
Line 108. Which part of the intestine was selected? It has been recently shown that different compositions of microbiota can exist between different parts of the Nile tilapia gut and these can be affected by diet or other components, thus it is important to understand that there were no shifts in the microbiome. Where there any intestinal contents after 24h? Did the authors select only the gut digesta or also they sampled the tissue as well?

Lines 115-118. Please refer to the exact method used and add if there is any reference. Moreover, be precise on the exact quantities and methods used.

Line 120. What exact quantity of faeces did the authors use for the DNA extraction?

Line 125. The expression of the copy number per g of faeces can only be used if the authors know the exact amount of faeces used for the DNA extraction.

Table 2. Where did these primers come from? Did the authors design them or originated from another study? Please give more details.

Line 132. Which MiSeq sequencing platform was used?

Line 138 – 141. The authors should perform extra data analysis since the use of ANOVA is not accepted for the microbiome data, non-parametric tests should be applied.

5. The results section needs also support with statistics. I would recommend for Tables 3 and 4, the authors to perform a time-point comparison in order to compare the changes across time. Use different indications for these comparisons within the table (i.e. capital letters instead of small ones).
Section 3.3 needs to be re-written after performing the non-parametric analysis for both the PCoA analysis and for each specific taxa. Moreover, the authors should describe their outcome in the OTU (species)-level for a better resolution. Microbial diversity indices should be also used (i.e. Shannon H index or dominance or richness). Furthermore, there is a major confusion between phyla and genera in the text in lines 187 to 196, this section should be also re-written and better describe the results.

6. The discussion section is too extensive, without proper support based on the data presented. It should be re-written to discuss only the relevant results.
Line 234. How can the authors be sure that the excretion of Al is because of the L.plantarum? Maybe the change in the microbial diversity may have caused this effect.
Lines 235. The authors need a reference to support the study on the mice.
Lines 237-240. This sentence is not clear.
Lines 241-245. The authors have no proper statistics to discuss these results.
Line 247-253. This part is very speculative.
Line 253. Pseudomonas is the genera level. How do the authors know that this is relevant to the probiotic in the previous study? The most probable explanation for the increase in the Flavobacterium seems to be the good Al tolerance. The authors cannot support the other hypothesis with the Pseudomonas.
Lines 256-267. This paragraph is not relevant to the results of the study. Please remove.
Lines 277-278. The authors did not test for diversity analysis. Please first do so.
Lines 278-281. Too speculative.
Lines 281-286. This does not seem to be relevant information with the study.
Lines 300-303. In their previous study, the authors tested the Al-induced tissue damages. Why didn’t the authors check that as well in the present study? It would give a more relevant information on the actual performance of the fish as well as if the changes in the microbial diversity would be indeed beneficial.

7. The figure legends should be more informative. Especially the one presenting the principal coordinate analysis. Which metric did the authors use for this type of analysis?


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: DIETARY SUPPLEMENTATION WITH PROBIOTICS REGULATES GUT MICROBIOTA STRUCTURE AND FUNCTION IN NILE TILAPIA EXPOSED TO ALUMINUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1)	More references and background info are needed in the intro. More information on how aluminium exposed fish affects aquaculture and/or fisheries production and human consumption.
2)	I am confused about some word choices in the text. I suggest the authors use the full name for “aluminium” instead of “Al” as Al can be confused with the acronym AI (artificial intelligence). Also, the strain “CCFM639” of the probiotic should only be mentioned once in the abstract and methods since the authors should say “probiotic” instead, in my opinion. Genus names should be in italics font (e.g. Flavobacterium).
3)	Several spelling mistakes:
a.	Introduction: lite, healthy, metabolism, salmon salar (italics, also include common name: Atlantic salmon), decreased, andmetabolism, microbiota.Therefore,
4)	Reference missing from lines 52-54, 66-69 (especially rainbow trout), 71-73.
5)	Please define “safe strains” (line 61).
Experimental design: 1)	In general, more information is needed for future studies to replicate this work. The fish trial and microbiota analysis is sufficient, but the authors need to do more statistical analyses to back up their conclusions. The authors looked at the top 15 bacteria, but did not perform stats on the alpha or beta diversities that are commonly performed for microbiota data.
2)	Original primary research is within the Aims and Scope of the journal. Research question is well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.More info needed on lines 84 (MRS broth company), 86 (percent saline), 87 (basal diet composition and origin), 89 (how did you determine 10^8 CFU/g?), 95 (± SE or SD, please indicate), 96 (fed by hand or feeder?), 101 (freshwater?), 102 (how was the concentration of Al measured? Where was the Al from? How often was this checked?), 108 (how much faeces? Distal or proximal intestine? Squeezed or scrapped? Collected in sterile tubes?), 115 (company of the microwave? what percentage?), 116 (How long is a moment?), 118 (company of the AAS? Reference to the method?), 121 (kit company?), 122 (cDNA kit and qPCR machine and company?), 128 (kit company?), 131 (reference of primers), 132 (company and location of sequencer?), 135 (OUT to OTU), 140 (software company?).
3)	Remove “Healthy” (line 92) unless you have scored information of blood and immune parameters that you can include in the manuscript.
4)	What is the toxic level of Al for tilapia (lines 102-104)? Is 2.73 mg/L below toxic levels and is >20 mg/L toxic? Please make clear.
5)	Remove line 104 as this has been stated already.
6)	Table 2 is not necessary and the primers can be stated in the text.
7)	Please explain what “improved” means (lines 134 and 135).
8)	What database and technique did you use to align the sequences? Please explain. Did you remove OTUs that were mistakenly sequenced and related to chloroplasts and mitochondria (not bacteria)? This is a disadvantage of using short pair-end reads. Please see methods section of Huyben et al. (2018) Applied Microbiol doi:10.1111/jam.13738 and Gajardo et al (2016) Scientific Reports DOI: 10.1038/srep30893.
9)	Why were no multivariate statistics performed to look at the alpha and beta diversities (e.g. Shannon diversity and PERMANOVA)? You need to do this to provide evidence that the probiotic resulted in changes to the Al exposed fish. Also, was the data normally distributed? Were the data log transformed to be normalized? Please include this or a normality test, such as Shapiro-Wilk and/or Levene tests. P and F values should be reported as well, or at least in the supplemental table.
10)	Table 3 is more of a methodology and should be included in the methods section instead of results.
11)	Why is the AL+CCFM639 treatment have 8-10mg/L more Al than the Al only treatment? Could this not be equalised? Please explain.
12)	Wrong figure cited (line 237).
13)	Reference needed (line 247)
Validity of the findings: 1)	In general, the conclusions are not clear or well supported and imply that a positive effect and mechanism of the probiotic when this may not exist. Data are not robust as there is a small sample size for microbiota analyses (n=3) and limited amount of stats (no multivariate statistics, only an ANOVA). Most of the statements about changes in bacteria abundance are speculatitive and mostly refer to mammalian models and not fish, thus they authors need to be more careful.
2)	The statement that the two groups are significantly different is not supported with statistics. PERMANOVA or MANOVA should be performed to give evidence instead of subjective description of a PCo plot (lines 166-171). What similarity index was used to make the PCo plot? Why are the treatment abbreviations different in the plot?
3)	How was diversity reduced (line 176-178)? How did you measure this and did you use stats?
4)	How many sequences and OTUs per sample did you find and what was the variation (SD)? How many sequences were lost to unknown, eukaryotes, chloroplasts, etc? Were there any unclassified bacteria as it seems strange that all bacteria found were in the reference database? Please explain.
5)	Did you include a negative control of water or buffer in order to determine if the samples had contamination? For example, Duganella was found in high amounts (Fig 3) and has been listed as a common contaminant found in negative controls as according to Salter et al (2014) BMC Biology 2014, 12:87. This possibility should be at least stated in the Discussion.
6)	Please add the individual p-values for each OTU (line 189-196).
7)	“Components” should be replaced with “OTUs” in Fig 3. Why only 17 OTUs? Is this OTUs with >1% abundance? Please state.
8)	Units missing from Fig 4 and same comment as Fig 3.
9)	Reword “intimate” (line 213).
10)	More references needed (line 225), such as Ran et al. (2015) PLoS ONE 10(12): e0145448. Also, Giatsis et al (2015) Scientific Reports 5:18206.
11)	References needed (lines 235, 248, 257, 269, 275).
12)	Define “good Al tolerance” (line 255).
13)	Define “host” (line 257).
14)	The authors use the term “slightly increased the diversity and stability of the tilapia’s gut microbiota” (line 277-278). There is no statistical evidence to support this and stability of microbiota was not investigated as the 16S gut bacteria were only evaluated once at the end of the experiment and not over time. The authors should remove this statement from the discussion and conclusion unless they perform stats that show this. In my opinion the plots and abundance figures seem to show an effect of Al exposure, but no effect of the probiotic. There is natural variation of gut bacteria and a sample size of 3 at one time point is very small, thus a higher sample size or more time points are recommended.
15)	Define the “mechanism”. Is it that the probiotic binds Al and then the fish deficate it? Please clarify.
16)	The authors say that Al increased negative bacteria and decreased beneficial bacteria (line 292). The authors talk about some pathogenic bacteria, but more discussion of beneficial bacteria is needed. More information about common bacteria found in tilapia is also needed in the discussion.
17)	The conclusion talks about the probiotic “alleviating adverse effects” (line 296) but this was not evaluated in this study (eg growth, behaviour, cortisol). The phenomena of the probiotic binding Al and increasing more in the faeces than in the fish is not explained, but it is one of the main conclusions. Figure 5 is only referenced in the conclusion with no clear explanation. If this is what the authors are concluding, they need to analyse Al in the carcass, organs, intestinal lining and/or bones of the fish to see if the Al is depositing more in other tissues to conclude this possible mechanism of reducing Al exposure.
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: DIETARY SUPPLEMENTATION WITH PROBIOTICS REGULATES GUT MICROBIOTA STRUCTURE AND FUNCTION IN NILE TILAPIA EXPOSED TO ALUMINUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revisions improved the quality of the manuscript
Experimental design: I have several comments concerning the statistical analysis applied addressed below.
Validity of the findings: The authors performed all necessary analysis as requested in order to validate their results.
Additional comments: The authors revisions really improved the manuscript.
However, I do still have several comments concerning the presentation of the results.
Introduction:
Line 61: I would write this as : ‘research has been shown that concentration of aluminium ranging from 0.1 to 0.2 mg/L  can be harmful to fish’
Line 64-66: ‘decrease the number of Atlantic salmon’ do the authors mean cause mortalities? Please rephrase this sentence
Line 68-76: The authors are switching the topic to microorganisms – I suggest this part goes in another paragraph
Line 88-94: Move this part concerning the tilapia at the end of the introduction – directly after line 104
So according to the above points the intro should be as following, following line 68:
“Microorganisms produce a variety of metabolites that can have remarkable effects … the composition of intestinal microbiota (Wu et al. 2017; Zhai et al. 2017).
Lactic acid bacteria probiotics, which are generally derived from humans or food products…. control infection of tilapia by Edwardsiella tarda and Streptococcus agalactiae (Pirarat et al. 2006; Pirarat et al. 2015).
Our previous study showed that supplementation with probiotic L. plantarum CCFM639…… is an aluminum toxicity alleviation mechanism exerted by probiotics in tilapia.
Tilapia is one of the most important aquatic species in aquaculture worldwide ……excellent stress sensitivity (Zheng et al. 2016).”

Methods: In the 2.6 Data analysis section, what type of t-test was it applied to compare between treatments?

Results: Section 3.3 Intestinal microbial diversity and composition
please replace ‘In the alpha diversity analyses, the microbiomes of tilapia in 639 only and Al + 639 groups were richer than those in the control group (Figure 1).’ with ‘Concerning alpha diversity microbiome analysis, Shannon and Chao1 indices were found higher in the 639 only and the Al+639 treatment groups compared to the control’.
Please replace: ‘As shown in Figure 2, principal coordinate 1 (PC1) and PC2 were 19.88% and 65.75%, respectively. After treatment with aluminum, the results for the Al-only group and Al + 639 group were mainly concentrated in the upper right quadrant and differed significantly from those of the control group (lower right quadrant).’ With ‘As shown in Figure 2, Principal Coordinate Analysis based on Unweighted unifrac distance indicated a significant clustering between the Al treated versus non-treated groups.’
‘Moreover, the results for the 639 only group were concentrated mainly in the lower left quadrant and also differed significantly from those of the control group.’ Was the separation of 639 with control significant? Did the authors perform a post-hoc Permanova test?
‘The results of principal coordinate analysis show that aluminum treatment and the addition of CCFM639 had the greatest effect on the fecal microbiota composition in tilapia.’ In figure 2 I can see a main separation due to the Al exposure. The authors provide a P-value that shows overall a significant clustering but it doesn’t tell what happens between the treatments. The authors show verify this.


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

Input:
Title: DIETARY SUPPLEMENTATION WITH PROBIOTICS REGULATES GUT MICROBIOTA STRUCTURE AND FUNCTION IN NILE TILAPIA EXPOSED TO ALUMINUM
Review round: 2
Reviewer: 2

Output:
Basic reporting: All criticisms have been acknowledged and revised.
Experimental design: All criticisms have been acknowledged and revised.
Validity of the findings: All criticisms have been acknowledged and revised.
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: DIETARY SUPPLEMENTATION WITH PROBIOTICS REGULATES GUT MICROBIOTA STRUCTURE AND FUNCTION IN NILE TILAPIA EXPOSED TO ALUMINUM
Review round: 3
Reviewer: 1

Output:
Basic reporting: The revisions improved the manuscript, but there is still room for the language improvement. However, I leave this to the editor to decide.
Experimental design: Sufficient
Validity of the findings: Sufficient
Additional comments: The revisions improve the manuscript.
I have minor suggestions:
- Move Line 98-100 'The aim of the study was to explore whether the regulation of gut microbiota is an aluminum toxicity alleviation mechanism exerted by probiotics in tilapia.' as the last sentence of the introduction.
- Line 192: The authors used Tukey t-test. Did they test for normality of their data? A parametric posthoc test needs to fullfil spefic terms. The authors need to verify if this is the case otherwise they need to apply non-parametric analysis.


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

Input:
Title: COMPARISON OF THERAPEUTIC EFFECTS OF DIFFERENT MESENCHYMAL STEM CELLS ON RHEUMATOID ARTHRITIS IN MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well organized with clear scientific writing.
Experimental design: The research question and methods are well defined with hypothesis driven.
Validity of the findings: No comment
Additional comments: In this manuscript, the authors tried to compare different origins of MSCs in their immunomodulation ability, and used RA as an animal model to evaluate the treatment outcomes. Overall, the topic is important in the field, and the results are consistent through whole manuscript. I only have some suggestions in Discussion section.

1. As the authors mentioned that macrophage plays a major role in RA, the authors should discuss some potential mechanisms, in which how systemic MSC therapy inhibits macrophage activity?

2. In the first paragraph of Discussion, the authors claimed that systemic MSC infusion may be able to migrate into the damaged tissue. However, several recent publications have shown that paracrine effects, such as cell-cell interaction and cellular component transfer, likely be the major reasons in systemic MSC therapy. Please discuss.

3. The reason of using SHED in this study is not convincing. Craniofacial region-derived MSCs have unique advantages, such as easily accessible, outstanding proliferation ability, and so on. Please revise in the Discussion.


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

Input:
Title: COMPARISON OF THERAPEUTIC EFFECTS OF DIFFERENT MESENCHYMAL STEM CELLS ON RHEUMATOID ARTHRITIS IN MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Article text is clear and easily understandable throughout.  There are some minor errors as below
Abstract:  Line 31: disease; Line 36: in mice; Line 38 and 47: MSC-treated
Introduction:
Line 55: Using “kind of chronic“ is unclear. Please address
Line 58:.. “articular or bone margin..
Line 65: “immune dysfunction”
Line 75: space after RA
Line 84: MSC
Line 88: MSC populations above…
Methods
Line 129: Use “1x106”
Results
Line 192 &199: MSC
Line 193: …the most obvious…
Discussion
Line 226: MSC
Line 238 and 240:  …considered to have….reported to have…

Minor Issues:
Literature references, sufficient field background/context provided.
Introduction is written well and referenced appropriately for the most part.  However, some studies that indicate that there are scenarios where the use of MSCs is not always positive in the context of RA, particularly when the cells are applied at a point where inflammation is high.  Please provide references here.
Line 128: The authors should justify the use of the 6 surface markers use to characterise MSC populations (Line 128) and the selection made.
Line 142: Provide a reference for the scoring system used here rather than end of paragraph.
Line 166-167: “current standard being used” is not sufficient to describe a scoring system and is this scheme used by a lot of publications?  Cite if so.

Professional article structure, figures, tables. Raw data shared.

Submission format is acceptable. Figures are relevant and images are of an acceptable quality and legible for the most part. The use of “remarkably” in some figure legends is inappropriate and should be changed. Raw data is shared as supplemental.
Please use more appropriate language – for example in figure 1 there are small although significant reductions in inflammatory cytokine levels and should be described as such with references to the statistical analysis performed.  Please also indicate the number of mice used (n=?) in all figures where relevant.

Minor Comments
Figure 2: Indicate what collagen type used; Use “MSC treatment …to different extents”.  Please indicate the number of replicates used where relevant in all figures as well
Figure 3:  Please label the graphs representing histological scores as B and D and address the legend appropriately.
Rephrase “remarkable”.
 “… cytokine expression in hind limb ankle joints.”
“… UC treatment..”
Y-axis legends are not easily readable and font should be increased.
 Figure 4 and 5:
“MSC treatment…… . UC treatment….. BMSC treatment”
(D).  “SHED had….”.
Re-phrase “remarkable”.
Experimental design: The research described has some novelty with respect to its comparative nature but is iterative.
The research question is defined and has some relevance to the field in the context of the cell comparison performed. This particular comparison has not been performed previously to my knowledge.
Ethical approvals for pre-clinical experiments was sought with a reference to same in Methods.  Overall, methods used were well described.
Validity of the findings: Submission format is acceptable. Figures are relevant and images are of an acceptable quality and legible for the most part. The use of “remarkably” in some figure legends is inappropriate and should be changed. Raw data is shared as supplemental.
Please use more appropriate language – for example in figure 1 there are small although significant reductions in inflammatory cytokine levels and should be described as such with references to the statistical analysis performed.  Please also indicate the number of mice used (n=6) in all figures where relevant.

Minor Comments
Figure 2: Indicate what collagen type used; Use “MSC treatment …to different extents”.  Please indicate the number of replicates used where relevant in all figures as well
Figure 3:  Please label the graphs representing histological scores as B and D and address the legend appropriately.
Rephrase “remarkable”.
 “… cytokine expression in hind limb ankle joints.”
“… UC treatment..”
Y-axis legends are not easily readable and font should be increased.
 Figure 4 and 5:
“MSC treatment…… . UC treatment….. BMSC treatment”
(D).  “SHED had….”.
Re-phrase “remarkable”.
Additional comments: Overall, the results found were valid with statistical significance and some novelty exists. The number of animals (n=6) did seem low for the model but significant data was generated.
Data is robust and statistics are handled well.

Conclusions are well stated, linked to original research question & limited to supporting results.  The use of remarkable should be deleted here as well though (line 260) – a clear over statement of data.  A statistical reference should be sufficient throughout.


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

Input:
Title: COMPARISON OF THERAPEUTIC EFFECTS OF DIFFERENT MESENCHYMAL STEM CELLS ON RHEUMATOID ARTHRITIS IN MICE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors are presenting a work that aims at clarifying that several sources of MSC provide different outcomes regarding RA treatment. This subject is clinically relevant and with great impact in helping the decision on the MSCs selection for future clinical use. However, the approach adopted was not innovative, nor presents new methodologies, or possible mechanism that could increase the knowledge in the field.
Nevertheless, given the relevance of the work some crucial improvements are suggested.

Overall, the English language should be improved to ensure that an international audience can clearly understand your text. For example, the authors should avoid terms such as “is a kind” (line 56).
The structure of the article is under an acceptable format of ‘standard sections’ and the figures are relevant to the content of the article. Nevertheless, the resolution of the graph Fig 2B needs to be improved in order to distinguish the labeling and the figure legends could be improved.
Experimental design: The original research is within the aims and scope of the journal; and the research question is relevant. However, it has been briefly support by the current knowledge and the some technical issues have been identified, as follows:

INTRODUCTION

1. The introduction needs more detail as well. I suggest that you improve the description at lines 78-85 to provide more justification for using MSC as a therapeutic strategy for RA. Specifically, you should detail the mechanisms by which MSC can indeed be a better alternative regarding the current treatment options. Moreover, the clinical aspects of RA should also be detailed and clearly articulated with the potential effect of the MSCs.

2. SHED cells are derived from adult tissues therefore it is not clear what the authors meant by “SHED are obtained from relatively primitive tissues” and “with a greater potential for differentiation” (lines 94-95). Please comment.

MATERIALS and METHODS

3. The materials and methods section should be greatly improved and detailed. Specifically:

4- The ethical issues regarding the MSCs collection are missing.

5- The isolation and characterization of the MSCs must be more detailed. The methodologies adopted have not been justified nor supported with literature.

6- The establishment of experimental CIA model has only been briefly described in the manuscript (lines 136-137). This information should be included in the materials and methods section in detail.

7- The authors should justify the MSCs administration conditions. Specifically, the authors should justify the quantity of cells that were administered, the timing (why day 30?), and the number of times the cells were administered (only once?).

8-	The timing and collection of mice serum for measuring the levels of TNF-α and IL-1β are missing. The same applies for the joint samples, that are not mentioned in the M&M section and are described only for the first time in the results section (line 197).

RESULTS SECTION

The first subsection from the results (“Flow cytometry evaluation of surface markers of MSCs”) is very limited. The authors should perform a more in deep analyses to confirm that the isolated cells are indeed MSCs. Namely, that the cells fulfill the ISCT criteria, ie, show three lineage differentiation capacity and are positive for MSC specific markers. Otherwise, with the current available data it is not clear that the isolated cells are MSCs. Importantly, the purity of the isolated populations is also not shown.

The subsections “MSCs treatment reduced disease severity score in RA”, “MSCs treatment reduced inflammatory responses in CIA”, “MSCs treatment prevented tissue damage in CIA”, should be merged and the results articulated.

DISCUSSION SECTION

Some sentences are not supported by literature data, nor from results obtained by the authors (e.g.  The sentence “UCs have the strongest immunosuppressive ability, possibly because they are more primitive cells compared to BMSCs and SHED.” (Lines 245-246)).
Validity of the findings: 9. Overall, the methodology adopted, and results obtained are briefly addressed or missing, which limits the interpretation of the data. For example, the purity of the cell population obtained after the isolation process was not evaluated. This would be important for relating the effects observed with the administered cells. Moreover, the MSCs were administered i.v. therefore the authors should monitor the cells within the organism (to decipher the mechanism of action of the cells it is important to follow cells survival during the experiment time, if the cells migrate to the lesions site, or if cells are kept in other tissues (e.g. the lung)).
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: COMPARISON OF THERAPEUTIC EFFECTS OF DIFFERENT MESENCHYMAL STEM CELLS ON RHEUMATOID ARTHRITIS IN MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall, the authors have addressed all the reviewr's request and concerns. There has been a good effort ot improve the manuscript that has now a clear and appropriate scientific language. The introduction section, M&M, results and discussion have also been greatly improved and detailed. Therefore, I believe the manuscript is acceptable for publication.
Experimental design: Please see the comment above.
Validity of the findings: Please see the comment above.
Additional comments: Please see the comment 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: A SIMPLE MODEL OF A GROWING TUMOUR
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is clearly written and presented, with appropriate referencing.
Experimental design: In general the tumour model seems clear. However, I didn't follow the description of the PK models. Eg I'm not entirely clear what the authors mean by 'simple decay (K-PD), a step model, or a one-compartment model'. Some elaboration of the models proposed, the mathematical description and the extraction of the parameters from xenograft experiments for the evaluation would be helpful.
Validity of the findings: The investigation of the combinations and dose ordering effects is an interesting aspect of the manuscript.
Additional comments: Although I think the work is good quality, I'm not clear what it adds scientifically. The model looks like a hybrid Simeoni/cell cycle model with perhaps some simplifications. Both of these are already described in the literature and hence this paper is not a major step forward in tumour modelling, in my opinion. I'm not sure about the value of linking the paper to a simulator is, unless the model can be accessed and edited? Eg if the authors could publish the code in an open source software, then I think that this may be of utility and interest.


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

Input:
Title: A SIMPLE MODEL OF A GROWING TUMOUR
Review round: 1
Reviewer: 2

Output:
Basic reporting: Orell and Mistry ms describes a mathematical model of a growing tumour to simulate and predict the effect of treatment on xenograft studies and potentially translate them to the the clinic. Their model uses parameters that can be observed or reasonably well approximated which is a strong argument.

The English is in general good, although in several instances it could do with more formality and/or clarity. (e.g. line 171 “we recap the argument here”, please review)

The introduction and background are weak. There is no real background given in the introduction to understand what other models have been developed in the past, how useful these have been, and what are in the authors opinions the pros and cons of each method. There is some background given in the methods but it’s really little and not placed appropriately.

The literature cited is relevant, but maybe again on the light side.

The figures could be improved (thicker lines, larger labels). Also, it ismy opinion that the ms requires more figures  (see general comments below).

The data that has been used comes from published articles. It has not been provided here as .xls or other format.
Experimental design: The primary research question appears to be the possibility of predicting combination xenograft experiments based on control and monotherapy data, using knowledge of cell-cycle dependent drug effects.

It is not clear though how the research fills an identified knowledge gap due to the almost absent literature review and discussions.

The investigation was performed to a good technical standard but it will benefit from more elaboration at all levels.

Methods are described appropriately, although several details are missing (see general comments below).
Validity of the findings: The findings appear valid to this reviewer.
Additional comments: Regarding agent-based modelling approaches, the authors should note that these cannot be easily parametrized using available data.

Line 81. The authors refer to 2 other ODEs approach but highlight that “such models may not accurately capture the time-dependent effect of certain drugs”. It’s not clear why this is stated, as the referenced papers are ODEs system where the cell cycle is modelled, and drug targets are also cell cycle dependent.

Line 87. Similar comments. It is stated “The CellCycler model addresses this problem by dividing the cell cycle up into a number of separate compartments of equal length”. I believe this is what have also been done in at least one of the referenced papers.

I assume “figure below” (l.110) is figure 1, in which the volume in compartment 1 is shown following a perturbation to that compartment alone after one cell doubling period of 24 hours. It is not clear to me what type of perturbation has been introduced. It is said “the magnitude is scaled to the number of compartments, so the total size of the perturbation is the same in terms of total volume”. Please be more mathematically explicit.

What do the authors mean by “blurring effect”? Why is this “equivalent to saying that the cell population has a variable doubling time”? It is difficult to comment on what follow without these details.

Regarding drug effect, “repairable” and “apoptotic” states are introduced. A diagram of the proposed model would help. Have the author analysed the effect of modelling these as parallel (r-Main-a), rather than concatenated (Main-r-a)? Why this choice?

Please review or elaborate a bit more, as it results to me that if V=4 pi r2 d, then dr/dt=1/(2r) not 1/(4 pi r2).

Please remind that the resulting growth equation is only valid once the tumor has grown enough and a necrotic core has developed. What would be appropriate R0 size for that to hold? Please elaborate on how a scientist would assess that for his/her xenograft model.

Similarly, please explain/discuss how the user would chose typical fractions for G1, S and M phases.

Important information (e.g. the schematic of the compartment PK models) are given in the online documentation. These should be incorporated in the paper supplementary material.

When referring to Dose/Volume (l.211), please precise this is the drug volume of distribution, to avoid confusion with tumur outer layers volume and/or cell populations volumes.

Please note that the drug concentration in the tumour is in many cases different from plasma pk – the PK models. The simple decay or 1-compartment PK models implemented here are not appropriate in these cases. It would be good to consider adding at least a 2 (and maybe even 3) compartment models.

In practice, how would a user decide on apoptosis, damage and repair rates? I would imagine that caspases, gamaH2AX and repairs biomarkers should be used. However, in practice, the information available is only based on proliferation assays which only display the aggregate effects of these rates. Please comment and elaborate.

How are the “schedule” and “week repeated” entries in the shiny app defined?

l.219 “The results can be compared with experimental results by using the “Read data from file” option.” I could not find this option/button in the app??

l.220 A sample file, consisting of data from an adenoid cystic carcinoma study, is currently loaded by default for demonstration purposes.” As mentioned, could not find it. Please also provide some background information about the data.

l.222 “If the user selects “show linear fit” then the plot will include a linear interpolation, with estimates for initial volume and growing layer thickness given in a table.” What type of data is being referred to? Tumor growth data? What is being estimated exactly, the initial tumor volume? How is the growing layer thickness defined then? Please give details of interpolations and fitting procedures.

l. 255 “The overall simulation time was three weeks, which is the typical length of a xenograft
experiment.” 3 weeks is a good average duration but not typical. There are many instances of longer (months) and even shorter experiments. Maybe change to “often used” or else instead.

The repair rate is fixed for both drugs – I appreciate the choice in the absence of information at this level, however, is there really evidence that repair rates in different phases are the same?

Case study 1: I assume the apoptosis rate for the EGFRi is set to 0.0 (not present in table 1)?

Plots of the results with all the details about the simulation parameters should be included to enable users to understand and replicate them. At the moment table 1 does not contain all the entries requires in the app and table 2 gives some summary results but there are no plots of the simulations.

The case is not discussed enough. There are only 2 sentences starting on l.257 and more mechanistic insights, together with plots of population dynamics, should be given to understand the different regimens results.

Case study 2:

Again, shall the reader assume that the apoptosis rate for the CDK4/6i is set to 0.0 (not present in table 3)?

Figure 2 shows fitting of the tumour radius to the data but there are no details of what has been parametrized and how the “calibration” has been performed.

I don’t think that anything is offered in the app for fitting/calibration purposes. Is the expectation that the user would have performed his own model fittings with alternative software prior to simulating the model with CellCycler?

The authors note (l.280) that “It is noticeable in Figure 2 that the CellCycler is underestimating the diameter curves after the treatment has stopped, post day 21. However, we are using the mean value at each experimental time-point and so this must be taken into account when judging the model fits.” Why using mean data would imply difficulties in capturing post-treatment kinetics. Also, standard deviations from the experimental data should be added in Figure 2.

Figure 3 also needs experimental standard deviations.

Case study 3
Most of previous comments apply here too.

Overall, it would have been better to have examples where combinations predictions work and examples where they also don’t work. Mechanistic insights would benefit the manuscript.

Appendix

l.363-365 The phrasing is a bit strange. The authors should simply start from VT=VT0*exp(k1-k2)t being solution to the ode, hence the relationship with the doubling time.


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

Input:
Title: A SIMPLE MODEL OF A GROWING TUMOUR
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors comment in the introduction that 'keeping the model simple makes the results easier to interpret'. I think this is not necessarily true (eg where the simple model cannot simulate a novel observed dataset and/or give a clear link that gives mechanistic insight) and they should consider acknowledging the weaknesses of overly simple models. They note themselves that '. There are no shortcuts, which is why the CellCycler uses a large number of equations', and it looks to me like the GitHub code is some 700 or so lines of code.The authors note themselves that simple models do not give sufficient insight, elaboration is needed to describe the data they present and in all likelihood further mechanistic detail will be required in the future. So, perhaps the title ought to be 'A more mechanistic model of tumour growth'?  Nevertheless, I agree that this tool could be helpful in the education of non-specialists and perhaps preclinical experimental design. Minor suggestions; I think that the K-PD might mislead some of the potential user group. iv bolus would be a more common description, in my opinion. 2 compartment PK is probably the most commonly observed PK profile, so perhaps adding this model would be helpful? Also, the app isn't clear about units; most drug discovery scientists use CL rather that rate constants and so this might cause some confusion. It is not clear to me whether the PK relates to free or total drug; if the latter then free fraction needs to be accounted for. Overall, I think this is good work and it is very positive that the authors intend to publish the code.


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

Input:
Title: A SIMPLE MODEL OF A GROWING TUMOUR
Review round: 2
Reviewer: 2

Output:
Basic reporting: No further comments
Experimental design: No further comments
Validity of the findings: No further comments
Additional comments: Previous comments should have been addressed more thoroughly. 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: CONFINDR: RAPID DETECTION OF INTRASPECIES AND CROSS-SPECIES CONTAMINATION IN BACTERIAL WHOLE-GENOME SEQUENCE DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Specific comments

1)	Figure 1 Legend.  Missing closing parenthesis on “(Supplementary Table S3”.
Experimental design: Specific comments

1)	Coverage – The coverage evaluated in table S1 ranged from approximately 30X to 150X. Coverage can frequently exceed 300X-500X for isolates sequenced on a HiSeq or high-priority isolates that need to be quickly run on a MiSeq with a small number of samples. It would be helpful to evaluate the effect of coverage on uncontaminated isolates to ensure that sequencing artifacts don’t appear as contamination.
2)	Line 103 – Parameter settings for ART are not provided in the paper or in the supplementary material.
3)	The authors used version 3 chemistry at 2x300 sequencing conditions to generate their real data and also their simulated data (see point 2 above, simulation conditions not provided). Many public health laboratories are using 2x150 or 2x250 chemistry. The authors should evaluate the performance of their software with shorter reads. Note – if the simulation conditions are provided then it should not be necessary to submit the synthetic data to NCBI.
Validity of the findings: Specific comments

1)	Table S4. It would be helpful to include BioSample accessions along with metrics such as number of reads and skesa output (coverage depth, number of contigs, assembly size) for the SRA samples.  Some of these appear to have very high coverage and it’s not clear how this may influence the ConFindr results.
2)	Lines 189-191.  There are 1500 SRA accessions in table S4 but line 189 states 500 isolates. Is this a typo or do the 1500 SRA accessions include replicates?
3)	Figure 1. Standard error bars are shown but the number of replicates for each bin is not reported.
Additional comments: The paper is clear, well organized, and the software addresses a pressing need for assessment of contamination in multiplexed short read sequencing runs in public health laboratories.  The source code appears to be well written and the software documentation is complete.  Lastly, the software installed easily using BioConda.

ConFindr is available under the MIT license but the rMLST data base required to run the software is under a much more restrictive license (https://pubmlst.org/rmlst/rMLST_licence.pdf). Many public health laboratories may not qualify for the pubmlst academic use license. I strongly encourage the authors to distribute the software with a stripped-down version of an rMLST database compatible with the MIT license containing public sequences for Salmonella, Listeria, STECs, and other priority pathogens.  The bulk of the contamination events identified in SRA in the supplementary material appear to be intraspecies so having a smaller database with fewer species wouldn’t seem to have too much of an impact.

Unfortunately I’m not able to test the current version of ConFindr, 0.5.1, as my pubMLST request for rMLST access is still pending. I have tested the 0.4.3 version of the software and I can confirm that it produced results consistent with other methods for evaluating contamination.


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: CONFINDR: RAPID DETECTION OF INTRASPECIES AND CROSS-SPECIES CONTAMINATION IN BACTERIAL WHOLE-GENOME SEQUENCE DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clearly and professionally written, with attention to detail in text, figures and table. A good background is provided with relevant references throughout.
Experimental design: The manuscript clearly states the impetus for the work and distinguishes it from existing tools. Rigorous experiments are performed to validate the work and methods are well described, with source code available and datasets submitted to public repositories. The one major sticking point in availability is that, for the software to work, it seems necessary to email a single person named Keith and await a response granting access to a database. If this tool is to be widely incorporated into pipelines, as suggested, this does not seem practical or sustainable. Perhaps a static version of the database could be provided for quick start-up, with the license-restricted update left as an option?
Validity of the findings: The findings are for the most part well-supported. The one area where I felt that the claims went slightly further than the experiments showed was the superior sensitivity versus existing tools, Kraken and CheckM. These tools were given rather arbitrary cutoffs for positive identification. While the 0.5% cutoff for Kraken seems a lot lower than the 5% ConFindr requires for each SNV, these are not necessarily comparable. Since Kraken is a genome-wide classifier, many reads will simply be uninformative for closely-related strains, but a few classified to strain level could still be meaningful for finding contamination. Though CheckM is an MLST based method like ConFindr, its contamination percentages may simply be skewed by assembly rather than missing the contamination completely. Rather than anecdotal experience for needing to investigate CheckM's results, I would like to see some kind of analysis of false positives to justify the cutoffs for both CheckM and Kraken. A complete ROC may not be necessary, but perhaps at least loosen the thresholds until these tools are as sensitive as ConFindr and see whether false positives do indeed occur.
Additional comments: The manuscript is thoughtful, well-motivated, and clear. Though the central claim of improved sensitivity could use bolstering as described above, the manuscript in general benefits from a wide variety of well-documented experiments, and the generation and inclusion of real instrument data is appreciated.


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: CONFINDR: RAPID DETECTION OF INTRASPECIES AND CROSS-SPECIES CONTAMINATION IN BACTERIAL WHOLE-GENOME SEQUENCE DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revisions address the concerns I noted in my first review.  Unfortunately the authors were not able to resolve the database license issue for this release of their tool. Hopefully future versions of ConFindr will include a static database that is unencumbered by the pubmlst rMLST license.


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: CONFINDR: RAPID DETECTION OF INTRASPECIES AND CROSS-SPECIES CONTAMINATION IN BACTERIAL WHOLE-GENOME SEQUENCE DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revisions are written to the same high quality as the original submission.
Experimental design: The authors address the database licensing issues with plans to update the distribution with priority-specific databases in the future.
Validity of the findings: The authors have performed and described additional experiments showing that false positives occur when attempting to make CheckM and Kraken more sensitive, and that ConFindr's sensitivity and specificity are indeed superior.
Additional comments: Concerns in the original manuscript have been thoughtfully and thoroughly addressed.


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

Input:
Title: INTEGRATING SPECIES AND INTERACTIONS INTO SIMILARITY METRICS: A GRAPH THEORY-BASED APPROACH TO UNDERSTANDING COMMUNITY SIMILARITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, the authors examine how the graph edit distance (GED)
could be used to evaluate similarity between ecological communities.
According to me, this is an interesting topic well described in the
introduction, the methods are readable and the findings well reported.
Moreover, the manuscript is well structured and conforms to PeerJ standards.
Figures are relevant and of good quality. I however I did not understand where
the second figure (the pdf file `nyl.pdf`) is supposed to be.

A website to retrieve raw data is mentioned and I was able to use them.
However, given what the authors explain lines 111-113, I think it would
better if the authors could provide the cleaned data set. Also, as the authors only
used one of the data set avaiable on the Interaction Web Database, I
recommend to add the following link in the manuscript:
https://www.nceas.ucsb.edu/interactionweb/html/thomps_towns.html

As a non-native English speakers, I do not feel comfortable enough to evaluate
the quality of the English language used. That said, I think it is overall
clear and unambiguous, I was however able to detect one grammatical error in the
acknowledgment section, line 274: "We thank to Interaction Web Database of".
Experimental design: ## What beta-diversity diversity

It is indeed relevant to quantify how the estimations of dissimilarity based
on GED differ from the one based on a common index of similarity (here the
Jaccard index). However I believe that the authors should also
compare their method to the one developed by @poisot_dissimilarity_2012.
The authors first mention this study lines 74-75:

> [...], but there is also evidence showing a complete
lack of correlation between composition similarity and interaction similarity
(Poisot et al., 2012).

First, I think that more is done in this study. T. Poisot and colleagues actually
propose a method to quantify the dissimilarity between networks that takes
into account the classical beta-diversity as a component of the changes.
Second, given that this method actually has a very similar goal to the one
the authors develop in this manuscript, to me, they must carry out a meaningful
comparison of the two.

## Costs exploration

One important question related to the used of GED is how to determine
what are relevant costs in the context of communities dissimilarity?
In this manuscript, the authors explore 9 scenarios. I think this is
not enough, especially given that AMI (Adjusted Mutual Information metric) is
fairly variable among scenarios as highlighted lines 180-181:

> "It should be noted that one of the models where low cost was
assigned to interactions (values close to 0), the AMI values were higher (scenario 9; see Fig. 1)."

I believe the authors should further explore how AMI varies with costs scenarios.
For instance, they could set 5 scenarios for the cost associated with
"Flipping an interaction" (e.g. 0, .25, .5. .75 and 1) and then for each of these
scenarios they could create colored matrices that would represent how AMI
values vary with a large number of combinations of the two other costs (e.g.
from 0 to 1 with an increment of 0.01).

## Details about the method

Even though the methods section is overall clear, I have two questions that require
clarifications in the manuscript:

- Why is GED consistently higher than .6?

- Lines 148-149, the authors wrote: "We used 1,000 iterations for the calculations."
To what calculations the authors are referring to here and why 1,000 iterations are required?
Validity of the findings: To me, the findings reported here are valid and I have nothing to report in this section.
I have however suggested major modifications in the methods section that will likely
lead to new findings to report.
Additional comments: - lines 29-31:

> "The similarity between ecological communities has been one of the central topics in
 ecology as community structure can impact the functioning, biodiversity, and management of
 ecosystems."

 I believe this sentence should be dropped as it does not add anything to the
 narrative of the paper.

- line 109: "[..] from ca. The size of these networks varies from ca. 49 to 113 [...]"
I don't understand why "ca." is being used here.

- line 155: "[...] variation in cost operations on AMI [...]"  what the acronym not l.163

- Errors pertaining to the formatting of references:

  - lines 170-171: "(Core, 2015)" (by the way the R version should be mentioned).

  - lines 70-71: "overlapping (i.e. node overlapping index; (Strona and Veech, 2015, Strona et al. 2018; Zhang et
    al., 2016)".

  - l.138: "(Dehmer, 2010; Ibragim format"