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: IS THE FUTURE ALREADY HERE? THE IMPACT OF CLIMATE CHANGE ON THE DISTRIBUTION OF THE EASTERN CORAL SNAKE (MICRURUS FULVIUS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Thanks for implementing suggested revisions in the methods, the strategy followed to generate the model is much clearer now compared to the previous edition.
Validity of the findings: no comment
Additional comments: I thank the authors for the successful revision of the MS by following the suggested revisions by the reviewers, the article did improve a lot. The response curve word document in Appendix 3 is corrupt, please fix the document with the correct version.


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: IS THE FUTURE ALREADY HERE? THE IMPACT OF CLIMATE CHANGE ON THE DISTRIBUTION OF THE EASTERN CORAL SNAKE (MICRURUS FULVIUS)
Review round: 2
Reviewer: 3

Output:
Basic reporting: Overall this paper is a pleasure to read. The authors are correct that little distribution work, with modeled or not, has included snakes, particularly a secretive but recognizable one such as the Eastern Coral Snake, so this paper is a solid contribution in that regard. Their approach is sufficiently well constructed and thoughtful to be a model not only for other snake species but other under-studied vertebrates with sufficient field occurrence data. Specifically matching occurrence years with modeled climate years and examining distributions with respect to future climate modeling (including the bracketing of projections) will be important in our rapidly changing world.

The authors do not discuss much of the natural history of these secretive snakes, which I view as both a challenge for modeling and also makes them good candidates since models can extrapolate distributions from limited sources. The discussion of the paper would feel more “complete” if they discuss their modeling in light of the natural history of Micrurus fulvius and how that may impact SDMs and their interpretation.

Their most interesting conclusion is that they may have shown with their future climate models that the snakes are already responding to climate change. They could improve their case with some easy fixes to figures (see below). Impact on Batesian mimic species, both the kingsnake and scarlet snake, is a fascinating discussion topic, and I am glad they included this.

Generally, the authors use available occurrence and climate datasets and methods that this is reproducible especially given the submitted locality sites used in the study.  I note the site data is only sites; no specific specimen information like GUID is provided which would make it difficult to verify any voucher specimens, but the authors make no claims to have done so either.

The authors responded thoroughly and appropriately to previous reviewer comments to which I had access.
Experimental design: Three main goals are stated:
Line 19: "Specifically, we: (1) identified the current range and suitable environment of M. fulvius in the Southeastern United States, (2) investigated the potential impacts of climate change on the distribution of M. fulvius, and (3) evaluated the utility of future models in predicting recent (2001-2015) records. "

Methods
SDM methods are explained well and are reasonable and justified; however, it’s confusing to have the authors refer to the sites in the SDM section of methods before talking about the occurrence data. I would suggest swapping the order. In other words, describe the locality data from museums, etc first then the environmental data and spatial extent since it is in part based on those localities.

They chose to use Maxent using k-folding (10 reps) which is one way of mitigating inherent model bias. My biggest issue while reading this initially was why the four parameterized versions of the current climate? Most studies caution against using all available variables to avoid overfitting in SDMs- the authors point this out (it appears that way in their Fig 3A) so I was expecting more discourse on methodology in the results.  They do address these differences in Results. It would have been expected that they select the most robust model balancing model performance statistics and extent of suitable habitat for the rest of the analysis and discussion, which in my opinion is the Reduced model. They chose to bracket their modelling with the Full model and Reduced model for the future climate models, which is acceptable.

Environmental layers include WorldClim 1.4, Soils (FAO), justifying their choice of resolution at 2.5 arcmin based on the uncertainty estimates of the species occurrence.

Line 184: a little description of the SDMToolbox would be helpful for readers who may not know about this set of python scripts for ArcGIS 10 (especially given the level of detail they went into for other aspects of their Methods). Please add the proper reference (Brown et al 2017). They do supply a weblink.

Line 190-191: references for “Maxent’s minimum location requirement” As far as I know Maxent itself has no minimum threshold but there are many references to what are effective minimum sample sizes, including Graham et al 2006 and other references already used in the paper to highlight some of the discussion on this topic. Could this be reworded to be less misleading?

Line 236: nice summary of GCMs and RCPs. They choose a 4.5 RCP (low emissions scenario) — are they underestimating climate impact? They bracket a high and low future projections by choosing MIROC-ESM and GISS-E2-R but still may be underestimations.

They tested model performance with AUC and TSS (Cohen’s Kappa) as well as used a Wilcoxon signed-rank tests on the thresholded models (current and future) with an independent set of samples. These are conventional and acceptable methods.
Validity of the findings: With respect to the authors stated goals (listed in same order as Line 19):
1) this was done with four different parameterized approaches for current climate suitability. While the authors point out the general agreement of the models, there is variability in the southernmost extent in Florida. I wonder why all the current models show little/no suitability for the isolated northernmost portion of its range in Alabama? - the authors mentioned that in Alabama, the species is of high conservation concern. Are the climate models at this resolution failing to capture some local phenomena or is the occurrence of the snake there questionable? Or an issue with the SDMs? Perhaps it’s out of scope to delve into the factors thoroughly but should be worth noting in results or discussion since we are presented with the IUCN range map and then their distribution models, and it is relevant to their case that there is predicted expansion.
2) climate change models show much expansion and in some ways appear more like the IUCN range maps (which granted come with a large grain of salt) than the current climate models (also noted by authors). There is enough concordance between the two CMIP5 models that the overall future projections are convincing. However they do not address the fact that they are undoubtedly underestimating climate shifts with their RCP 4.5 even bracketing a pessimistic and more gentle models of change.
3) this is the most interesting (and speculative) part to me, and they conclude with caveats that the most recent localities which are predicted in the future climate models indicate that there are climate change responses already. I would prefer they visualized these data to be more convincing. (see note below)

Nice use of MESS analysis to identify no-analog/ novel climate space in the future climate models. It is valuable to see the differences based on model treatments and the CMIP5 models themselves.

Line 363 "The Full model type was presumed the most valid of the four models based on TSS and AUC” — but not by much as AUC values range from 0.828 to 0.8148 and had similar TSS. I think the Full model performed the best but I do not assume it’s the most “valid” which implies that it’s closest to the actual distribution of the species. As I mentioned before under Methods, weighing in the differences in extent of suitable habitat, the MESS, I favor the Reduced model but I am ok with a difference of opinion. I would prefer this Line reworded.

Tables and Figures, when checked how they were referenced in text, are in order and appropriate. Is there any figure or supplemental data that show testing vs. training data? Also, I would find it helpful, that is strengthen your case, to see the occurrence points on all the climate models, especially the future models since part of their conclusion that the species is already undergoing shifts in response to climate change is that the models better match the more recent localities. That is a big statement that would be best supported with a figure (either Fig 4 or 6, even if included in supplemental).

Figure 6: the color for Retraction is too similar to Expansion; please change (maybe red?)

Line 407: I appreciate this cautioning of interpreting modeling with no-analog climate; they reference appropriate papers here and discuss thermal optimum as a limitation. Does a largely fossorial species have special consideration here?

Line 448-449 I am glad they included this observation on dispersal limits as being also a limitation of this study. It would be worthwhile to include some more species specific details, such as habitat preference of forest types and understory/ leaf litter lifestyle and how these correlate with trends in forest loss or gain in the SE-US (an area which has seen much change in pine forest composition) and their models. (Tip: Global Forest Watch www.globalforestwatch.org has an interactive map that shows a lot of forest turnover in the SE.)
Additional comments: Title: Are not recognized common names of species capitalized? If so, it should be Eastern Coral Snake

Details to clean up:
Line 64 vs Line 67 - what are PeerJ guidelines? "Fig. 1 “ versus "Fig 1”? Or spelled out? Should be fixed to be consistent

Line 159: Typo in genus name “Mircrus” (sic)

Appendix S2 heading: please capitalize consistent with the organizations’ preference and your Methods section (ie., GBIF, VertNet, iDigBio)

Supplemental figures (all the bioclimatic maps) have two north arrows (likely unintentional) that should be cleaned up.
The response curves did not have headings and my review copy had a corrupted Word Doc so it was difficult to know what I was looking. Which models were these the response curves for?


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 TOBACCO SMOKE AND ELECTRONIC CIGARETTE VAPOR EXPOSURE ON THE ORAL AND GUT MICROBIOTA IN HUMANS: A PILOT STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	The use of EC and TS throughout the manuscript is quite confusing. Please spell out the words throughout the manuscript to make it easier to read. Abbreviations are ok in the figures as long as they are defined.
•	Lines 47-48: Redundant use of the words “including especially”. Please clarify.
•	Line 59: Please change “…community” to “communities”
•	Line 129: Please change “…have been” to “…had been”
•	Please change “a Prevotella” to “Prevotella” thoughout the manuscript.
•	The reason for doing the study is very clear and other studies that have done similar analyses on tobacco smokers are referenced and addressed in the manuscript.
•	Cannot locate raw data based on the project accession number provided by authors. Please provide exact URL that links to the page where the sequencing data can be found or searched for.
•	I commend the authors for generating visually pleasing figures and supplemental information.
•	Please address comments below regarding figures:
•	Figure 3 title: This states that these are the top 10 most significantly altered genera? But, these are not all significant. Please clarify or change title.
•	Please label boxplots (Figure 3 and Figure S3) with saliva, buccal, or feces so it’s easy to tell which location the graphs correspond to without referencing the text.
•	Supplemental figures are missing legends.
•	Figure S1: The boxplot colors in panel C are not defined. Consider changing the asterisk color so it isn’t so close in color to one of the boxplot groups.
•	Italicize genera in Table S1.
Experimental design: •	The research falls within the Aims and Scope of PeerJ (both biological and health sciences research.).
•	The research question is clearly well defined and the authors provide information as to why they chose to do this study. The new field of microbiome research needs data! I am happy to see that the authors present site-specific characterization of the microbiome prior to examining treatment differences.
•	Methods are described thoroughly and match those present in other epidemiology and microbiome manuscripts. IRB protocol is referenced.
•	Please address comments below regarding the Methods section:
•	Line 79: Please include information about the skewed sex-ratio of your participants (majority of study participants were male). This should be discussed, as sex-specific differences in microbiota have been shown in the literature.
•	Like 81: Please provide the form of informed consent (verbal or written) received. If verbal, please state “Verbal informed consent…”. If written, please provide an empty copy of the consent form as a Confidential Supplemental Information file. This is requested based on PeerJ’s requirements.
•	Line 84: Please define FTND. This is the first time it is mentioned in the manuscript.
•	Line 88: Please define CO.
•	Lines 93 & 94: Please include Catalog #’s for these DNA extraction kits in parentheses for easy reference.
•	Line 115: This sentence is a bit unclear. Is this referring to negative controls for each DNA extraction kit?
•	Line 120: Please define FDR. Also, please clarify this paragraph. Kruskal-Wallis p-values do not need to be adjusted for multiple comparisons. However, post-hoc test p-values (e.g. Mann-whitney) do need to be corrected.
Validity of the findings: •	Cannot locate raw data based on the project accession number provided by authors. Please provide exact URL that links to the page where the sequencing data can be found or searched for.
•	Line 40: This last sentence is too specific to microbiota. EC use may impact many other endpoints. Consider rewriting the sentence to include that other endpoints other than just microbiota composition and function should be assessed. There could be other detrimental impacts that are not captured by studying microbiota. Please also update the related sentence in the conclusion.
•	Line 136-138: This statistical analysis does not tell you if they are “significantly increased.” What can be said, however, is that there were statistical differences between the 3 groups. And that diversity was higher in x, vs. y. See general comment about doing post-hoc tests to see which groups are different.
•	Figure 1: See general comments on statistical analysis. Kruskal-wallis test should be used first and then, if that p-value is significant, a post-hoc test with multiple comparison adjustments can be conducted. This approach may help simplify your boxplots, as you can report a single p-value for the K-W test on the graph, and then use different letters to indicate pairwise differences if they exist.
•	Lines 160-161: These results would benefit from making pairwise comparisons to show that Prevotella and Bacteroides were significantly higher or lower in tobacco smokers feces.
•	Figure S1A and Figure 2: Nonparametric statistical analyses should not be used for beta-diversity metrics. A statistical test such as PERMANOVA or ANOSIM should be conducted to assess the global differences between the 3 groups instead.
•	Lines 162-164: For some races, diet groups, etc there was a n=0 or 1 person. Therefore, there are not enough individuals to make the claim that there are no statistical differences based on these endpoints. Please remove this statement or say it in another way.
•	Line 182: What alpha diversity endpoint was measured in the referenced study?
•	Lines 185-186: This “statistical difference” (e.g. increased OTUs in TS) may be eliminated if a Kruskal-Wallis test is done first to compare all 3 groups. Please see general comments about statistics.
•	Lines 202-204: Same comment as above. Based on the number of individuals, this claim should either not be made, or be more carefully made.
•	Line 204: Lim et al 2016 said sputum microbiota was influenced by tobacco smoke. In this study, you did not see any impacts on saliva. Please address why these differences are observed.
Additional comments: This is an original article that characterizes changes in microbial communities in different regions (oral and gut) in a pilot study of a small human cohort of controls, electronic cigarette users, and tobacco smokers. I am particularly elated that the authors characterized the overall differences in their sampling site communities prior to analyzing differences between treatment groups, as this is typically overlooked and provides a baseline understanding of the communities across sampling sites to help understand the inherent microbiome variability.

The overall study design is limited, but this is clearly a pilot study and the authors do a nice job of addressing some of these limitations in the discussion. But, the lack of mention regarding the majority of the subjects being males was not addressed, and is particularly important given that other studies have shown sex-dependent differences in baseline microbiota and responses to toxicants.

The main findings of this article are 1) no impacts of electronic cigarette smoking were found on the microbial communities in the oral cavity (buccal and saliva) or gut (feces) and 2) tobacco smokers show significant changes in overall microbial community composition and increases or decreases in certain bacterial taxa, particularly in the feces. However, I have major concerns about the statistical analyses used to analyze the data in the manuscript. Beta-diversity metrics (PCA plots) should be analyzed using a PERMANOVA or ANOSIM to assess the global differences between the 3 groups. Alpha diversity metrics and individual taxa box plots can be analyzed with a nonparametric test as was done here, but the rule of thumb is that for 3 groups, a Kruskal-Wallis test should be done first and if the p-value is significant, then pairwise comparisons (Dunn or Mann-whitney) can be made with multiple comparison adjustments. In some cases, doing the analysis this way may benefit the authors and make the results less confusing (e.g. increased OTUs in tobacco smokers).

Other Specific General Comments
•	Please consider including a table of alpha diversity metrics for each group, and including richness and evenness.
•	Lines 124-132: This section is almost completely redundant with the materials and methods study cohort paragraph. Please consider combining these paragraphs so all information in both is retained. It seems reasonable to put it in the methods section as defining the study cohort.
•	Line 130: should “medium” be “median”? I also think this is an inaccurate measurement. Why did the authors choose to report the median and not the average and standard deviation instead?


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 TOBACCO SMOKE AND ELECTRONIC CIGARETTE VAPOR EXPOSURE ON THE ORAL AND GUT MICROBIOTA IN HUMANS: A PILOT STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study describes the effects of tobacco smoke and electronic cigarette vapour exposure in humans. This is an important study and it is well executed, although the presentation of the data and the statistical analysis can be improved. The writing style is clear.

The experimental design is very limited due to the low number of individuals included in this study. The results are promising but not likely to bring reliable conclusion. If this is really a pilot study, my understanding is that the authors will perform a bigger study. In this case, a publication as a preprint would be more suitable. However, this is only a recommendation. My role as a reviewer is not to comment on the publishing strategy.

I am not able to give a conclusion on whether or not I think that this study should be published because it needs to be extensively revised to be assessed at face value.

Bioinformatics pipelines for microbiome studies are relatively immature and several recent reviews emphasise the lack of consistency between the different pipelines. This is why the analysis should be fully reproducible. The authors should include their raw data (sequencing data posted on a data repository), their processed data (such as an OTU table), and the code used to process their data as a supplementary material.
Experimental design: The research question is very relevant and well addressed (for a pilot study). The statistics and the presentation of the data can nonetheless be improved. I believe that a minimal requirement would be that the authors present all the raw data, their processed data and provide their code in order to allow a full reproducibility of the study as stated above.

The microbiome is also influenced by alcohol consumption, and by the lifestyle. Where are these people living? Living in a polluted city could be a major confounding factor. Covariates should be more controlled. The authors report the measurement of CO levels. More details should be provided. It is very important to obtain a reliable biomarker of smoke ingestion since an EC user will be likely to go out with other individuals using conventional cigarette and thus be passively exposed. The authors should also run an association study to see if the CO levels correlate with relative abundance values in bacteria.
Validity of the findings: The authors should improve the presentation of their statistics. Is it not clear how the authors performed the adjustment for multiple comparisons. This is a general problem for microbiome studies since the data has a tree structure and it is thus difficult to understand how many independent tests are actually made.

Better statistical pipelines exist for pair-wise comparisons of gut microbiome compositions. A hierarchical multiple testing would be more suitable. Alternatively, the authors can adopt a multivariate strategy and use an sPLS-DA.

The presentation of the box plots could be improved by adding the actual data dots. It looks like the authors used R for the preparation of the figures. It is easy to add the data dots on the box plots.

The BMI should be presented; it is a better indication than the weight or the height of individuals. Is any of the covariate associated with relative abundance values?

More details on the overall structure of the microbiome can be provided. How many taxa were actually determined? The tree structure of the data is important to take into account. Thus, it would be important to present a tree of the different microbiome in order to understand the representation of the different taxonomic levels in this dataset. This can be a supplementary material.
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 TOBACCO SMOKE AND ELECTRONIC CIGARETTE VAPOR EXPOSURE ON THE ORAL AND GUT MICROBIOTA IN HUMANS: A PILOT STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am still not able to see supplemental figure legends. Please provide these with supplemental figures.

Figure 2 legend: “…reverse numerical order” is not clear.
Experimental design: Lines 90-93: Is this EC6 or 7 supposed to be the same EC#?

Lines 96-101 are still repetitive to lines 83-95 and can be condensed into that paragraph or removed.

Line 258: Thank you for addressing the male/female ratio. Consider replacing the words “are consistent in females” with “differ between males and females” to clarify what is meant here.

Line 136: Please add the acronym “FDR” after “…false discovery rate” since this acronym is used in subsequent figure legends.
Validity of the findings: Beta-diversity analysis: Please report overall PERMANOVA p-value for Figure 2. Were the pairwise p-values reported in Figure 2 adjusted for multiple comparisons (e.g. FDR)?

Lines 221-223: The following sentence needs to be removed from the discussion because the previous statement about significant effects of diet and lifestyle was removed due to the sample size being too low to make this claim: “Notably, diet and lifestyle was not found to drive the significance in this study, which is in accordance with Lim et al. (2016) who found that smoking was the most important lifestyle factor influencing the sputum microbiota (Lim et al., 2016)”
Additional comments: Thank you for addressing the issues raised previously and for providing the raw data. I commend the authors for their efforts. The CO data is also a great addition based on the other reviewer’s recommendation. I have a few other small recommendations that would, ideally, be addressed prior to publication:

Lines 93-95: The following sentence was moved from the results to methods, which is great. However, it still says “medium”. Should this be “median”?: “Electronic cigarette users… for a medium of 3 years”

Lines 176-178: “increased” should be “decreased” in the following: “Whereas Bacteroides showed significantly increased relative abundance in tobacco smokers compared to controls (P = 0.017) and electronic cigarette users (P = 0.003), but no difference between electronic cigarette users and controls (P = 0.684).” Please adjust in the figure legend too.

Line 266: Please correct spelling of “salvia”

Is the change in feces microbiota following tobacco smoke use some sort of stress response? The results presented here correspond with others in the literature, and are quite interesting.


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

Input:
Title: EFFECTS OF TOBACCO SMOKE AND ELECTRONIC CIGARETTE VAPOR EXPOSURE ON THE ORAL AND GUT MICROBIOTA IN HUMANS: A PILOT STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no further comment
Validity of the findings: no comment
Additional comments: The authors have taken into account all my comments. The manuscript is much better now. I am also glad to see that the authors have disclosed the totality of their raw data and their code. I wish every paper could be as transparent as this 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: ENDOPHYTIC FUNGAL COMMUNITIES ASSOCIATED WITH FIELD-GROWN SOYBEAN ROOTS AND SEEDS IN THE HUANG-HUAI REGION OF CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript reports a straight forward study of fungal diversity in soybean roots.  It is clear and unambiguous in describing the study and results.  Background literature is sufficient. The figures are well made and clear. It is self contained.
Experimental design: The experimental design is fine.  This was a survey of fungi in roots and seeds. The authors do indicate how this work will contribute to further studies on use or role of endophytes in agriculture. The study seems well done to assess diversity of fungi in soybean. The described methods were adequate.
Validity of the findings: These data are robust and valid--and conclusions support by results.
Additional comments: This study of diversity adds to our knowledge of diversity of fungal endophytes in plants. A next step would be to use the isolated endophytes to assess affects on soybean.  This is really where this research would go in the future.  This is a nice foundation.


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: ENDOPHYTIC FUNGAL COMMUNITIES ASSOCIATED WITH FIELD-GROWN SOYBEAN ROOTS AND SEEDS IN THE HUANG-HUAI REGION OF CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript was generally well written. The study was well designed. Specifically, using both conventional (cultural isolation followed by conventional Sanger sequencing) and innovative (Illumina MiSeq sequencing using eDNA) to study fungal diversity is the best merit of this study. The results were properly analyzed. Comparisons among results obtained from three methods plus previous reports have important implications. Here are some comments in order to help the authors to further improve the manuscript.

Abstract
The Abstract reads fine, while I encourage the authors to further improve it. First, there are already many numbers in the Abstract, which is somehow confusing. Are the numbers “18” in line 22 and 23 necessary? Second, is “based on internal transcribed spacer (ITS) sequencing” in line 23-24 necessary? Third, the sentence in line 25-26 is confusing and can be improved. Fourth, in line 27-29, although the analyses indicated the difference in population and diversity, it is confusing in stating “significantly different” after indicating “Fusarium……in EVERY sample”.  Please improve the logical flow, for example, add “even though”, or “except for” before the “Fusarium in every sample” statement. Fifth, the statement in line 29 is not true. Phyllosphere fungal endophytes were not investigated in this study.

Introduction
The Introduction generally reads fine. To better serve the purpose of literature review and correlate with the later findings, I suggest the authors add literature reviews on the major fungal genera found in this study. Specifically, previous reports of Fusarium as endophytes should be reviewed, since it was the dominant genus found in this study.

Line 70-77: T-RFLP and DGGE are not “new” or “emerging”.

Materials and Methods
Line 95: the authors may want to add a sentence indicating the popularity of the ZhongHuang 13 cultivar in the Huang-Huai region to justify this selection.
Line 100: “in the field” or “in each field”?
Line 124: “Amended” to replace “treated”
Line 126: delete “kinds of”; replace “3 cities x 2” with “6”
Line 130: delete “carefully”
Line 133 to 141: shorten this section if the methods were the same as those in cited articles.
Line 144: What method was used to quantify DNAs if “equal amounts of DNA…were mixed”?
Line 153: Revise the section title to specify that this section was about sequencing and sequence analyses of the MiSeq or CI.
Line 173-176: indicate where and how the diversity analyses and subsequent graphics plotting were conducted.

Results
The Results section is generally clean and well written. The findings are clearly reported. The figures are nicely plotted/made.  Minor mistakes are still present. The major issue is that many discussions should be moved to the next section.
Line 189: The term “ITS sequencing method” is misleading. ITS sequencing was also used in the CD-iso method following cultural isolation.  The authors may want to find another way to describe these methods.
Line 191-192: “…indicating that…..of low abundance” belongs to the Discussion. Please only report findings in the Results, while move all related discussions to the Discussion.  Also, this statement of indication is contradictory to that in Line 270-272.
Line 194-196: “Thus, …..” belongs to the Discussion.
Line 199-200: “indicating……” belongs to the Discussion.
Line 206-208: “These difference might……” belongs to the Discussion.
Line 210-221: Please only report findings from this study. This whole section should be moved to the Discussion.
Line 251-257: “These results suggested that……seed germination” and “further indicating that…..” should be moved to the Discussion. Please simply report the findings.

Discussion
Compared to the above sections, the Discussion section feels weak. It should be strengthened after moving many discussions in the Results to the Discussion.

Line 266-274: The best merit of the methodology in this study was using a variety of isolation/identification methods. The results from different methods were well illustrated in the Results. However, they deserve better comparison and discussion here. There is a lot that can and should be discussed. For example, obtaining fungal cultures provides fundamental materials for studying the biological and ecological roles in subsequent research. This is also related to the following discussion paragraph on the determination of endophytisms and pathogenicity of the isolated fungi. Second, CD-iso recovered the lowest quantity of fungal population, but its identification had the best quality. Most fungal isolates were identified to species-level with >98% identity (Table S2). On the other hand, the other methods amplified shorter DNA fragment and their identifications were subsequently compromised to only genus-level. Third, it is interesting that some fungi were only found by isolation. Was the less abundance the only reason, or were there other potential reasons related to the isolation and sequencing methods?

Line 292: The dominance and potential role of Fusarium deserve a more detailed discussion.
Line 293-308: More in-depth discussions are warranted in this paragraph. Was there any difference in the soil types and weather conditions at the time samples were collected among these fields? The discussion here can be more valuable if such potential correlation between fungal community and environmental conditions are discussed.
Also, this may be a good place to indicate that phyllosphere fungal endophytes were not studied and warrant future 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: MOVEMENT VELOCITY IN THE CHAIR SQUAT IS ASSOCIATED WITH MEASURES OF FUNCTIONAL CAPACITY AND COGNITION IN ELDERLY PEOPLE AT LOW RISK OF FALL
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors should be commended for presenting a concise and very well-written manuscript. Below I provide specific comments of the different sections of the manuscript that I hope they could contribute to improve the presented manuscript.

ABSTRACT
Line 84. Change “consisting on” by “consisting of”.
Line 87. Delete N=40 because it is redundant.
Line 88. It would be interesting to include the load used in the CSQ test.
Line 89. Delete “Additionally”
Line 92. Use the abbreviation for “chair squat exercise” or remove this abbreviation from the abstract.
Line 93. … correlations “were obtained” between…
Line 95-96. If possible, include the value of the ES after each variable instead of using the range.
Line 97-99. I think that the conclusion would be more informative if the author indicates in what consisted the “muscular performance test” instead of the device that was used for the measurement.

INTRODUCTION
Line 118-119. Introduce the abbreviations of these variables if the abbreviations are going to be used later in the manuscript.
Line 122. Change “this” by “these”.
Line 124-127. Consider to change this sentence for clarity to: “Finally, it has been shown that the velocity recorded against a submaximal load can be used to accurately quantify the maximal strength capacity in a large number of resistance training exercises”. Add at the end of this sentence several references that support this statement.
Line 129. Change “correlations” by “relationship” or “association”.
Line 132. Consisting “of”.
Line 135-136. I miss a couple of sentences in the introduction to justify the hypothesis of the authors.

MATERIAL AND METHODS
General comment: I suggest to describe first the chair squat test and then all the remaining variables that were correlated with the performance in the chair squat test.
Line 138. “Matherial” should be changed to “Material”.
Line 143. Consider to change to: “Participants were also divided…”
Line 149. Change “participant´s” to “participants´”
Line 159. If authors decide to use abbreviations (e.g., HGS), they should use these abbreviations consistently through the manuscript.
Line 164-165. I miss one parenthesis after 0-20.
Line 179. “ms” should be changed to “s”?
Line 186. Change “calculations” by “statistical analyses”.
Line 190-192. This sentence would fit better when describing the chair squat test.
Line 196. Why using 95%CI for correlations and 90%CI for standardized mean differences.

RESULTS
The results section is concise and very easy to follow.

DISCUSSION
Line 222-224. It could be interesting to highlight in the discussion the benefits of the test used in the present study compared to the 30-s chair test (e.g., faster assessment).
Line 234. The word “thus” could be omitted.

CONCLUSIONS
Consider to change to: “This study showed that the mean velocity recorded by a smartphone app during the chair squat exercise is positively related to…”.


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: MOVEMENT VELOCITY IN THE CHAIR SQUAT IS ASSOCIATED WITH MEASURES OF FUNCTIONAL CAPACITY AND COGNITION IN ELDERLY PEOPLE AT LOW RISK OF FALL
Review round: 1
Reviewer: 2

Output:
Basic reporting: I want to thank the author for write this interesting manuscript. However:

1) I suggest count with a professional English servirse to improve the re-phrasing, use of punctuations and minor mistakes with grammar.

2) The reference need to be expanded to give support some topics as methods. Please see attached comments.

3) The manuscript fulfils the scientific structure. But i suggest to add a universal format of data .csv o .txt. Minor changes are attached in comments.

4) The hypothesis is relevant for clinical context, but needs to improve their previous knowledge (introduction), discussion and conclusion with major changes. Please see attached comments.
Experimental design: 1) the manuscript fulfils the aims of the journal.

2) the aims of the study requiered minor changes. Please see the comments attached.

3) the manuscript fulfils ethical standard.

4) Method needs major revision. Please see the comments attached.
Validity of the findings: 1) If the authors could support the methodological deficits, the manuscript will show an interesting tool for clinician in world-wide.

2) Data needs revision. Please see the comments attached.

3) Conclusion need major revision. Please see the comments attached.

4) Speculation need major revision. Please see the comments attached.
Additional comments: Dear Editor:

1) the manuscript is na original idea but need to major revision in methods, if the article do not present in their next version eligibility criterias' need to be rejected. However could be a mistake of authors.

2) The manuscript is interesting and original. I invite to authors to perform the revision contained in the manuscript to improve the quality of this 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: MOVEMENT VELOCITY IN THE CHAIR SQUAT IS ASSOCIATED WITH MEASURES OF FUNCTIONAL CAPACITY AND COGNITION IN ELDERLY PEOPLE AT LOW RISK OF FALL
Review round: 2
Reviewer: 1

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


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

Input:
Title: MOVEMENT VELOCITY IN THE CHAIR SQUAT IS ASSOCIATED WITH MEASURES OF FUNCTIONAL CAPACITY AND COGNITION IN ELDERLY PEOPLE AT LOW RISK OF FALL
Review round: 2
Reviewer: 2

Output:
Basic reporting: 'No comment'
Experimental design: 'No comment'
Validity of the findings: 'No comment'
Additional comments: The authors accomplished a well scientist manuscript revision. I appreciate the scientific improvement of the manuscript and i think now could be published. Only some minor details must be checked before to publish the manuscript as punctuation in line 119, space of parenthesis in line 140, the correction of 'nor' in line 146 if was a mistake and the parenthesis use in line 157-158.


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: VALIDITY AND RELIABILITY OF THE WIMU INERTIAL DEVICE FOR THE ASSESSMENT OF THE VERTICAL JUMP
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study is well designed and the manuscript is generally well structured. The results indicated the validity and reliability of the inertial device WIMU for two different type of vertical jumps. The paper focuses on a topic which should be of interest to coaches, trainers, physical educators, and sports scientists, and has relevance to exercise training. However, I have some reservation that the practical impact of the work may be limited and that the work has limited novelty despite the limited research in this area. Overall, I recognize the strong scientific merit of the research, which uses a good research design and robust methods of measurement and analysis.
Experimental design: The purpose of the study is investigate validity and reliability of the inertial device WIMU for two different type of vertical jumps. The authors used test and retest method to evaluate reliability of the data. At the same time, authors also used the force platform as a reference to investigate the validity of WIMU.
Validity of the findings: The statistical analysis was also support the purpose of the study. Intra class correlation, coefficient of variation, standard error measurement were used to evaluate the reliability and validity of WIMU device. The statistical analysis support the purpose of the study. However, there are few improvement need to be made that listed below in the comments section.
Additional comments: The study is well designed. There are a few areas need to be improved. The specific comments below highlight areas where improvement is recommended although these are relatively few because of the good quality of design and analysis. Overall, this paper presents work of good quality but there remains a significant question on how much new knowledge the work brings to the field. Evaluation of potential impact is a difficult challenge and it can be difficult to predict the impact (in terms citations) of a paper before it reaches the public domain. Ultimately this should be a decision for the editor.

Abstract: Please provide the full name of CMJ and SJ as they introduced at the first time.
ln 136: how did you define flight time from force platform? What is threshold? Since using the force platform why not calculate jump height from integration of impulse?  Any filter being used for signal processing from the two instruments?
ln 146: please check typo CCI.
ln 151: please explain what is mean power spectrum of the two repetitions? Power spectrum analysis?
References: please provide completed page number of articles (reference 6 and 27)


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: VALIDITY AND RELIABILITY OF THE WIMU INERTIAL DEVICE FOR THE ASSESSMENT OF THE VERTICAL JUMP
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, this is a well-designed study that aims to evaluate the validity and reliability of the WIMU device. Despite the effective design of the study, the manuscript has a fatal flaw, in that all of the sections about the WIMU or QUIKO reads as though it is an advertisement for RealTrack systems. There are numerous sections that don't really serve to give information relevant to the study, rather it serves to inform the reader of the neat features and the advantages that WIMU and QUIKO has over other technologies.

In fact, after finishing the methods section, I had to go back into the text to make sure that none of the authors had a conflict of interest with RealTrack. I did some Googling, and it turns out the lead author lists himself as being employed by RealTrack systems on Linkedin. While I cannot see the acknowledgments section in my version of the manuscript, if the first authors' LinkedIn profile lists this as his employment, it really should be included as an affiliation in a manuscript. This conflict of interest (that should have been disclosed as an affiliation), has obviously bled into the authors' writing, which is a significant flaw with this manuscript.

I have chosen not to address the rest of the paper until the "advertorial" writing issues have been cleared up.
Experimental design: Did not assess.
Validity of the findings: Did not assess
Additional comments: Here are the major issues that I came across in the writing. I chose not to read any farther than the methods section.

Line 64-71 This section could have been accomplished in a single sentence. Instead, the authors chose to list off neat features of the device, and how much better it is than other types of technologies. This particular section reads like an advertisement.

L117-130 Only about half of this information is actually useful to the reader for the purpose of informing or allowing for replication. The other half of the information reads like a bullet-pointed list in a product pamphlet of all of the customization options for the Quiko software. We do not need to know, as a reader, about the adjustment parameters ("features"), unless you are providing this information so that we can replicate or better understand your methods.


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: VALIDITY AND RELIABILITY OF THE WIMU INERTIAL DEVICE FOR THE ASSESSMENT OF THE VERTICAL JUMP
Review round: 1
Reviewer: 3

Output:
Basic reporting: The research havea real interest and deser to be published. However, the author should disus about parameters and device potential  that has not been investigated inn the present study. Jumping performance needs to be introduced in the results and needs to be discussed.
Experimental design: The research is original and within scope of the journal. The research question is fairly well defined and relevant. However, the analysis of the results only focus on flight time, while jumping performance (in meters) is never considered. Methods used to analyse the validity and the reliability seems to be appropriated and  complete.
Validity of the findings: Findings should be implemented with data on jumping performance. Moreover, in many cases the authors discus about parameters of the device that have not been investigated. These parameter are out of subject and caution is really needed as they haven’t been investigated.

Moreover, the authors have to be aware that they can compare their flight time results with only the studies that investigated that particular parameter.

Conclusion is too long and should focus on the research hypothesis.
Additional comments: Abstract :
Why don’t you gather all reliability analysis at the same time ? You introduce ICC L25 then you introduce CV and SEM L28-29.
L34. Remove “on any surface” as it  hasn’t been investigated.

Introduction :
L40-L43. Out of subject and not necessary.
L60 The authors should explain in more detail why and how jumping performance can be estimated through the measurements of flight time. References are also needed here.
L65-L70. This is the place where authors could explain in more details all potential possibilities of the WIMU system. After the general presentation the authors should exclusively focus  on the jumping measurement.
L68-69. Why the fact that WIMU system receive data during the jump could be  an  advantage ass we only take into account the flight time ? Moreover, with accelerometer, it is much more difficult to detect accurately the moment where the subject leave the floor and when he lands. The argumentation of the authors need to be changed. If the data measured during the flight phase with the WIMU system are used to improve the estimation of flight time and  jumping performance, please explain how !

Material and methods
L89. Wimu installation  on the back need more details.
L105. Was it the same examiner ?
L113 : what was the instruction for landing ?
L122 : does the WIMU contain any Gyroscope ?
L140 and L144. Why do the authors separate the two reliability analysis ?
L151 and 152. What does mean “_” in the formulas ? Not clear.

Results
Jumping performance results also need to be presented.
L177 and L178 : could you check your data ? Why in CMJ  a superior  average systematic tends leads to a lower % ?

Discussion
L197-198. Give an example. (comparison betwwen Myotest and Optojump ?).
L222 : ”this device is enable to… ” which device ? Th WIMU ? It is not clear.
The authors should discuss the importance of the landing with all device measuring the jumping performance through flight time !
The discussion should include the results on the jumping performance. Moreover, the authors have to be aware that they can compare their flight time results with only the studies that investigated that particular parameter.
L226. The authors discuss about the ability of the WIMU system to measure other parameters like force. They need to be more prudent and to use conditional as they didn’t investigate this parameter. Moreover, a short overview on the figure 1 seems to show that values are not the same from WIMU to the force plate.

Conclusion
Conclusion is too long and should focus on the research hypothesis.
L250. The authors could present other potential advantages of the system but have to mention that investigations are needed to verify their reliability.

References
Please check all references format

Tables
As ICC is a reliability parameter. Why don’t you introduce it in the table 2 ?
Table should also include Jumping performance (in meters)

Figures
Figure 1 : what is the measured parameter ? Present the unit.
Figures 3 and 4 : Please introduce the linear regression and the R². Please introduce the unit.
Figures 5 ans 6 : please introduce the unit.


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: VALIDITY AND RELIABILITY OF THE WIMU INERTIAL DEVICE FOR THE ASSESSMENT OF THE VERTICAL JUMP
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors revised the previous errors. The results indicated the validity and reliability of the inertial device WIMU for counter movement and squat vertical jumps.  Overall, I recognize this research, which uses a good research design and robust methods of measurement and validation analysis.
Experimental design: The purpose of the study is investigate validity and reliability of the inertial device WIMU for two different type of vertical jumps. The authors revised the errors and typos. Test and retest method to evaluate reliability is appropriate for the study.
Validity of the findings: The statistical analysis was also support the purpose of the study. However, in the conclusion part. the authors indicated that the new device is valuable tool for controlling the training and competition. This may not be answered by this study. There are numerous factors that would influence training and competition outcomes other than vertical jumps. Long term study may be needed. Therefore,  the authors should consider to rewrite this senescence.
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: VALIDITY AND RELIABILITY OF THE WIMU INERTIAL DEVICE FOR THE ASSESSMENT OF THE VERTICAL JUMP
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall, this has been a big improvement over previous. There are still numerous areas to be improved, and another advertorial section to tone down in the discussion. The authors need to be careful of over-stretching the conclusions they draw from this study. Flight time measured by the WIMU is valid and reliable, but we have no data to suggest anything about other measures that might come from the WIMU, thus we cannot conclude anything about the device quality  overall, just about its measurement of flight time.

Finally, having a native English speaker would be helpful for dealing with some if the awkward and confusing language throughout. I have tried to point out the worst of them, but I certainly didn’t get them all.
Experimental design: Design is organized well.
Validity of the findings: The findings are logical and fine. The conclusions are not- the authors found that flight time, and only flight time, is valid and reliable, yet write about the device as though it has been validated for many other variables. They seem to be really stretching the scope of their findings.
Additional comments: Abstract
Which CV did you use? Hopkins’ spreadsheet?
Do you mean that you used standard error of the measurement,
T, rather than standard error of the means? These are not the same thing.

L39 please check this for grammar

L46 please change ‘measured’ to “estimated”.
L49 “jump height is an indirect method to jump height”...? Please check this sentence for accuracy.
L50 different methods for calculating what exactly?
This error of 2.2 cm- under what conditions and when using which devices does this error occur?
L75 what do you mean by early stages?
L77 what kind of training? Sport practice?
L90 with HANDS on their hips
L98 how much rest did each participant get between trials?
L116 can you explain more about the specific statistic you’re referring to when you say you calculated CV
L143-146 ICC only tells you about the reliability of the device- not the relationship between devices- the Pearson correlation does. Please modify accordingly.
L159 are you sure that you mean misreported? This rd doesn’t make sense in the context of the rest of the sentence.
L186-188- I don’t think you are referring to contact mats still- please add a phrase in this sentence that your focus is on the accelerometer.
L199-210 And here we are again with the advertorial- in this study you ONLY tested flight time, this you cannot talk about how great the other features are. You specifically suggest that the WIMU can make the same measurements as a force plate, then state in the next sentence that these measurements are valid. You don’t have multiple measurements/variables that are valid- you have ONE. A sentence or two about how this type of technology has advantages over force plates is fine, but you have here another entire paragraph about hoe great the WIMU is...
L217 Neither of these two studies support your statement- these two studies measured barbell kinetics/kinematics, not flight time as was measured in this study.


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

Input:
Title: VALIDITY AND RELIABILITY OF THE WIMU INERTIAL DEVICE FOR THE ASSESSMENT OF THE VERTICAL JUMP
Review round: 2
Reviewer: 3

Output:
Basic reporting: L43 : a sentence explaining that accelerometer coul be sucessfull to measure jumping performance is missing here (maybe with a reference to another system)
L47 : replace "the more" by "an"
L49 : I suppose you mean "Also, the Fight time is an indirect technique to measure jump height or muscle power ...
L50 : "suggested"(?)
L54 : You need here to describe that one  function of the WIMU is to assess jumping performace throught the measurement of flight time.
L58 : replace inthe by in the
L90 : "Once in position, the subjects were instructed (approximately a 90° angle) as quickly as possible and then jump as high as possible." What are they instructed to do AQAP ? Not clear.
Table 1 : Insert standart deviation  for jumping performance  + units
L199-206 : As you did'nt compared force signals of both systems in the present study, you cannot affirm that WIMU allows providing a similar analysis of force plate. In this paragraph, you need to be more prudent in your assertions. Conditional should be used in the otentioal of the WIMU. However you can write that it need to be verufy by further investigations.
L204 "These measurements are valid for both the CMJ and the DJ." => The only measurement that has been demonstrated to be valid in your study is "flight time"
Conclusion : You research is on  flight time validity and reliability  and not on training control. I invite the authors to  keep the conclusion around the research hopothesis. They can of course suggest the need of additional researches in order to investigate te complete potential of WIMU system.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript has been improved but a few change are still needed before acceptance. All the text need to be carefully ready to remove all typo errors.The potential of the WIMU could be in the discussion, but conditional tense should be used and authors have to be aware that they can not assert things that has not already been investigted. But they can suggest a need for further studies. The conclusion still needs improvements


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: VALIDITY AND RELIABILITY OF THE WIMU INERTIAL DEVICE FOR THE ASSESSMENT OF THE VERTICAL JUMP
Review round: 3
Reviewer: 1

Output:
Basic reporting: Authors made corrections based on the previous review. However, there is one part still need to improve.
From Ln 182 to Ln 186. The variable from study was flight time based and only one variable was investigated. The rest of the variables were not being analyzed (compare to other reference), so you can only indicate the reliability of flight time compare to force plate. The reliability of the other variables were not being evaluated in this study. So, you may consider to change the statement.
Experimental design: The design of the study was good
Validity of the findings: Validity of the study was confirmed based on the comparison of flight times from
 two different instruments.
Additional comments: From Ln 182 to Ln 186. The variable from study was flight time based and only one variable was investigated. The rest of the variables were not being analyzed (compare to other reference), so you can only indicate the reliability of flight time compare to force plate. The reliability of the other variables were not being evaluated in this study. So, you may consider to change the statement.


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: VALIDITY AND RELIABILITY OF THE WIMU INERTIAL DEVICE FOR THE ASSESSMENT OF THE VERTICAL JUMP
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors have made a substantial effort toward improving this manuscript. They should be commended for their immense improvements. Only a few comments related to writing.
Experimental design: Still good.
Validity of the findings: The authors have limited the scope of their conclusions enough so that they appropriately reflect the findings of the study.
Additional comments: L175 Please change "really" to "very"
L233 Please change "thought" to "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: VALIDITY AND RELIABILITY OF THE WIMU INERTIAL DEVICE FOR THE ASSESSMENT OF THE VERTICAL JUMP
Review round: 3
Reviewer: 3

Output:
Basic reporting: The authors answered successfully to the reviewer's comments. From my point  of view, the article is now acceptable.
Experimental design: The authors answered successfully to the reviewer's comments. From my point  of view, the article is now acceptable.
Validity of the findings: The authors answered successfully to the reviewer's comments. From my point  of view, the article is now acceptable.
Additional comments: The authors answered successfully to the reviewer's comments. From my point  of view, the article is now acceptable.


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: ARE LOWER LEVELS OF PHYSICAL ACTIVITY AND SELF-RATED FITNESS ASSOCIATED WITH HIGHER LEVELS OF PSYCHOLOGICAL DISTRESS IN CROATIAN YOUNG ADULTS? A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is generally well written and good to read. Some observations were made directly into the file.

There are some citations lacking and some parts of the Methods were not presented, which was pointed out in the text presenting Tables.

Other problems, information and questions were indicated in the pdf.
Experimental design: Observational study.
Validity of the findings: See the pdf.
Additional comments: Review needed.


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: ARE LOWER LEVELS OF PHYSICAL ACTIVITY AND SELF-RATED FITNESS ASSOCIATED WITH HIGHER LEVELS OF PSYCHOLOGICAL DISTRESS IN CROATIAN YOUNG ADULTS? A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text should be revised throughout as there are some English Language issues.

The text is sufficiently well referenced but there the authors should consider elaborating more on physical fitness and physical activity

The raw data is clear and well described

The objective and hypothesis is clear
Experimental design: This is a good research involving a population-based study on a very specific topic.

The investigation was performed to a highly professional and ethical standard

There is room for improving the description of the methods used (for example the questionnaires...)
Validity of the findings: The data is statistically sound and robust

The conclusion section answers the hypothesis question well.
Additional comments: First of all, it is a pleasure to read this article built on a population-based study investigating the relationship between physical activity and fitness and psychological distress. The following points should be considered to make this research article more robust.

1. The English language used in this manuscript should be improved so as the readers can understand clearly your text. Some examples where language could be improved are lines 83 to 86; line 96, line 119 (65000 to 70000 students...), line 145 (...ranging from 0 to 24). The text should be thoroughly revised to check for grammatical errors such as line 260(possibility..)

2. There is a big chunk of repetition of the last sentence in discussion (lines 264 to 266) and conclusions (272 to 274)..Consider rather to elaborate this sentence further in discussion and explain more the ALPHA battery fitness objective method, how it is better than the previously used method(s).

3. Authors posited that there was no significant difference in line 132 but does not explain clearly differences among which factor(s)...same need to explain the statistics and which variable when they mentioned the two measures in line 164 to 165.

4. The authors should consider elaborating what is physical activity and physical fitness as well as psychological stress further in the text (introduction) and distinguish between physical activity and physical fitness.

5. Overall the text seem fine with clear objectives and clear conclusions as well as good presentation of statistical result in tabular forms.

6. I thank the authors for the providing the raw data with clear variable names for each column of data.


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: ARE LOWER LEVELS OF PHYSICAL ACTIVITY AND SELF-RATED FITNESS ASSOCIATED WITH HIGHER LEVELS OF PSYCHOLOGICAL DISTRESS IN CROATIAN YOUNG ADULTS? A CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The text is clear and the English issues are addressed.

The text is sufficiently well referenced.

The raw data is clear and well described.

The objective and hypothesis is clear.
Experimental design: This is a good research involving a population-based study on a very specific topic.

The investigation was performed to a highly professional and ethical standard.
Validity of the findings: The data is statistically sound and robust as well as the conclusion section answers the hypothesis question well.
Additional comments: First of all, it is a pleasure to read this article built on a population-based study investigating the relationship between physical activity and fitness and psychological distress. Overall the text seem fine with clear objectives and clear conclusions as well as good presentation of statistical result in tabular forms. I thank the authors for the providing the raw data with clear variable names for each column of data.


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: SIDERITIS SCARDICA EXTRACTS INHIBIT AGGREGATION AND TOXICITY OF AMYLOID-Β IN CAENORHABDITIS ELEGANS USED AS A MODEL FOR ALZHEIMER’S DISEASE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: The methods employed are generally described sufficiently and to an extent that allows for replication. However, a few minor questions came up at some points, which I think could improve the replicability:
•	Could you please add a little bit more information on how the solid-liquid separation was performed? I.e., what solvent was used to dissolve the extract in the beginning? Were both solvents, ethanol and water used subsequently to elute the respective compounds from the resin?
•	How did you identify the specific polyphenolic compounds by HPLC? E.g. how were flavonoids distinguished from the other polyphenols in the mixture? Please add this information very briefly or provide a reference, if the method has been established before.
•	Thank you for providing the results of the thin layer chromatography! I think this is a very valuable method (however, often underestimated) to obtain fast and good overviews to easily compare the composition of extracts and fractions. The picture of the plate in figure 2 was given in grayscale; would it be possible to provide a coloured picture? This would enable the reader to better distinguish between the compound classes.
•	Could you please add the amount of time the worms were treated in hypochlorite solution? As this is the standard procedure for age-synchronized cultures, I don’t think it has to be described in detail, but since the concentration of NaOCl and NaOH may vary among protocols, the duration of the treatment would be necessary for replication.
•	Also, it would be helpful for the reader to add the information about the respective mutant C. elegans strains somewhere to the Materials and Methods section. All necessary information is given within the figures or results, but I would suggest to additionally present it at the beginning.
•	In line 142, it reads “…to induce the expression”; probably the expression of the Aβ peptide? Please add this information.
Validity of the findings: no comment
Additional comments: Results and discussion are conclusive and cover all of the research questions presented in the beginning. The data presented are statistically sound with a sufficient number of replicates and individuals treated, negative and positive controls are included in every experiment. However, a few questions / remarks remain:
•	As you stated in line 243/244, acteoside might play a major role for the bioactivity of the extracts. I think it would have been interesting to include the isolated compound (which I think is commercially available) in your bioassays to see whether it exerts an activity itself, and if yes, to what extent compared to the whole extracts. Why wasn’t the compound included?
•	You mentioned the presence of tannins (line 168). As their Rf values are rather high (0.7 – 0.8) using the mobile phase described, the respective compounds are presumably rather small and therefore lipophilic. If this information is available, could you specify on the type of tannins? Also, EGCG is used as the positive control, which could be regarded as a monomeric unit of a condensed tannin, and you mentioned tannins to be present in the lipophilic / active fractions; couldn’t this compound class contribute to the bioactivity as well?
•	Regarding figure 3: Is the example shown in picture A the 0.5 % EtOH control or a treated worm? Please indicate the treatment in the legend. An additional picture showing the 0.5 % EtOH control versus a treated worm (e.g. EtOH40) could visualize the effect more clearly.
•	Figure 4: I suggest to specifiy “control” in the legend within the graph a bit more in detail (e.g. “control EtOH 0.5 %) and to indicate that “No Aβ” means that a different strain was used (e.g. “No Aβ (strain CL…)”). Although the information is given in the legend, reading only the figures, “control” could be confused with EGCG.
•	Line 189/190: I suggest to write “negative control”, instead of just “control”, because you previously mention the “positive control EGCG” for the plaque reduction assay and the “control strain CL802”, so the term “control” is not immediately clear from the text.
•	Line 168: “Rf 0,3… “: please replace “,” by “.”


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: SIDERITIS SCARDICA EXTRACTS INHIBIT AGGREGATION AND TOXICITY OF AMYLOID-Β IN CAENORHABDITIS ELEGANS USED AS A MODEL FOR ALZHEIMER’S DISEASE
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.    Please include relevant information for strains NL5901 and  CL802 that would make them appropriate controls respectively for the aggregation formation and paralysis  experiments, respectively.

2.	Please include the deviation in the percent (%) reduction of aggregates observed per treatment group. For example, there was 21% ± (deviation)%, and not the mean alone.

3.     Even though the experimental design involved using 20, 40, 50, and 70% ethanol extractions, and used 200, 400, 600, 800, and up to 1000 ug/mL  concentrations for treatment, there  appears to be only a select number of data presented in the paper. If certain data were presented, while others were  not presented, can you please provide a rationale for this decision?
Experimental design: 1.	The C. elegans strains used (CL2006 & CL4176) express amyloid-beta in muscle cells, and are therefore expressing these aggregatory proteins in the whole body. Is there any reason why only the head region was observed?

2.     Why was the analysis limited to the number of amyloid-beta aggregates, and the size of amyloid-beta aggregates ignored? Were all the amyloid-beta aggregates formed (and observed) the same size? Instead of counting aggregates, would it be possible to quantify the total amount of amyloid-beta aggregates by summing up the fluorescence of the aggregates?

3.     Some researches have mentioned that for strain CL4176, there is already a considerable expression of amyloid-beta and formation of aggregates even at 16 degrees Celsius before the upshift in temperature. In your hands, did you also observe this?

4.     Is there a reason why there were no data points plotted for EtOH50 beyond 600 ug/mL in Figure 3-C?
Validity of the findings: 1.	Can you give an explanation as to why the total phenols was ‘surprisingly high’ in the 40% ethanol fraction?
Additional comments: No comment.


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

Input:
Title: SIDERITIS SCARDICA EXTRACTS INHIBIT AGGREGATION AND TOXICITY OF AMYLOID-Β IN CAENORHABDITIS ELEGANS USED AS A MODEL FOR ALZHEIMER’S DISEASE
Review round: 2
Reviewer: 1

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


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

Input:
Title: BEHAVIOR AND BIOCOMPATIBILITY OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS WITH BACTERIAL CELLULOSE MEMBRANE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Style of reporting is inconsistent.
Language needs to be improved.
e.g. 56 Researchers have been studying bone marrow mesenchymal stem cells, the cells
57 applicability in regenerative medicine and improving methodologies presented a greater
58 effectiveness (Dimarino, Caplan & Bonfield, 2013; Wei et al., 2013; Li et al., 2016).
59 The mesenchymal stem cells use appears promising in the regenerative medicine field.
60 On the other hands, these studies developed,
66 The tissue engineering comprises a promising multidisciplinary field that involves
67 materials development or devices capable of specific interactions with biological tissues (Langer
68 & Vacanti, 2016). The researches expansion in this area has accentuated the search for subsidies
69 to suport the stem cells biocompatibility in biopolymers to attend in vitro tissues develops to
70 replace injured areas.
71 In the last years
83 In tissue engineering the bacterial cellulose supported the researches interest because it is
84 an abundant biopolymer in nature with biodegradability and able to be synthesized by several
85 bacteria adding the low cost in its manufacture. The nanofibrils formed between 20-100nm.
86 Their which intertwine forming a network and allied to its molecular structure, giving its high
87 hydrophilicity. Many biomedical researches have
101 A one year old male New Zealand rabbit considered clinically healthy, was used for the
102 isolation procedure of BMMSC. A mouse Mus muscles was used as a peritoneal machophages
103 source. For the cellular viability determination trypan blue and subsequent graphical
104 representation in the growth curve were done. For the fibrolastoid colony forming units (CFU-F)
105 assay were used cells collected from the bone marrow cultured in petri dish in the sixth passage.
106 For the differentiation potential in mesenchymal lineages study were used the means of
107 chondrogenic and osteogenic inductionic. In the verification of BMMSC biointegration to BCM
108 confocal microscopy and Scanning Electron Microscopy (SEM) were used and to analyze the
109 BCM phagocytic capacity, toxicity and cytotoxicity were used peritoneal macrophages
sowing the
164 concentration of 1 x 104 cells/mL in five six-well plates counting two wells every 24 hours in the.

Recent references on in vivo studies  using bacterial cellulose based scaffolds need to be included.
The results are relevant to hypothesis
Experimental design: Methods have been described in sufficient detail.
Validity of the findings: The data on differentiation of the stem cells needs to be supported by atleast one more independent assay/technique
Additional comments: Major inconsistencies are observed with reference to citing the references.
Several typographic errors are observed.
Improvement in the language of presentation of paper is suggested.
Image quality (light microscopy images) need to be better.
An annotated PDF is being provided. Most of the suggested corrections have been marked.


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: BEHAVIOR AND BIOCOMPATIBILITY OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS WITH BACTERIAL CELLULOSE MEMBRANE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper would be significantly improved by careful editing of the English language and grammer. It is hard to understand in places. Some phrases are misleading (eg homogenised instead of mixed, peal instead of passage in differentiation instead of undifferentiated-line 269). There are also typographical errors which should be fixed
The way the paper is structured leaves me unclear about the author’s aims.  The initial focus is on the isolation of rabbit-derived bone marrow stromal cells, these are then cultured on a bacterial cellulosic membrane. The paper then switches to some characterisation of the membrane. It might be clearer to describe the membrane properties then to present the cell isolation data then to assess the cell and membrane combination.
In the discussion light and shading are included as physical conditions which influence mammalian cell culture, (lines 341 and 343) can the authors support this with references? I don’t believe this to be a factor for stromal cell culture.
The references cited are very heavily biased towards the last 4-5 years. The methods section should include citations to relevant papers, particularly around the cell isolation and characterisation used.
Some figures are not very clear. In figure 4 it is hard to see any cells. Can the stated magnification for figures 8 C and D be checked?
Experimental design: The aims and objectives of the work could be more clearly stated in the introduction.
Citations should be added to the methods.
The experimental  methods are not clear in several places
-Was only 1 rabbit used to isolate MSCs?  How reproducible was the cell isolation and characterisation-including the behaviour of the cells on the bacterial cellulosic membrane?
-Was the chondrogenesis assay conducted on a monolayer of cells? (Normally these assays are done in a pellet or micromass culture). Can the authors comment on which protocol was used and cite the appropriate paper?
-At what stage was the CFU-F assay performed? On cells directly isolated from marrow or after the MSCs had been in culture for some time? If the former do the authors have data on the number of CFUFs isolated as a function of the number of cells plated?
-Can the detail about the number of peritoneal macrophages added to the bacterial cellulosic membrane be added?
-How was the BCM isolated and prepared? How were MSCs seeded onto this?
Validity of the findings: Data are presented showing isolation of MSCs from rabbit marrow. This seems to be from 1 animal.  How reproducible is this work-in particular in relation to the behaviour of the cells on BCM?
Chondrogenesis data are shown based on what appears to be a monolayer assay which is unconventional. The staining shown is not of high quality-the sample appears to have peeled and folded onto itself. What do the authors mean by “cytoplasmic integration” in line 287?
There is a confusing jump in the data from rabbit MSCs on BCM to characterisation of BCM which should be better explained in the results.
Figure 9 shows that the BCM causes a significant increase in macrophage activity- what does this mean? Some explanation would be helpful.
The authors describe the levels of NO production as non-cytotoxic (line 310) can they put this into context?

Part of the discussion is a validation of the rabbit MSCs by comparison to definitions of MSCs from different groups, the authors should compare their cells to others who have isolated MSCs from rabbit bone marrow. There are publications from over a decade ago which isolated these cells.
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: THE EFFECT OF USING GAMES IN TEACHING CONSERVATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript lacks clarity in many respects, and in particular on the methods and experimental design. The manuscript also needs additional review by an English speaker for clarity of language.
Experimental design: This study was designed to investigate the effect of using different types of games in conservation training courses for early-career professionals. The topic and questions explored in this study are interesting and worthy of attention, and the effort to collect data on instructional effectiveness and learning outcomes is commendable. The authors are clearly thoughtful educators and raise important questions in the discussion that are worthy of further investigation. However, the study seems to have a number of weaknesses in terms of the experimental design and data collection methods, and the manuscript lacks clarity in many respects. The latter would require significant re-writing, but the former are harder to address.
Validity of the findings: I was unable to fully evaluate the statistical analyses in detail because of my lack of familiarity with the methods used, but have important concerns about the methods.

Concerns with the experimental design and justification of methods:
- The overall design is not clearly described or justified. Table 1 supplies some information, but it is that comprehensive and leaves many open questions. How long was each game played for? Can they be considered analogous? How was the data binned or combined for analyses?
What would be required is a table or figure that clearly lays out: a global picture of how many students were included in the data collection in each course and each year, with each set of analogous questionnaires (since these changed over time), that is, which sets of student experienced the same questionnaires and design (repeated measures versus counterbalanced), and what the sample size was for each set.

- Why were two different designs used (repeated measures and counterbalanced)? There is no explanation for the choice of these approaches, nor sources cited, or why both were used in this study, nor how they affected the results. The potential for order effects is raised in the methods section (Line 178) but not furher discussed.

- The application of three types of instruction (supplememntal games, experiential games, and didactic instruction) is evaluated in terms of its effect on perception (through questionnaires), learning (through content quizzes), and behavior (through observations). Unsufficient information is provided on how these data collection tools were designed and validated and the questionnaires and quizzes themselves do not seem to be available. How were the questions formulated? Where they tested in any way? For example, in the observations of behavior, the asking/answering of questions was quantified - were the type of questions relevant? When observing and recording behaviors, was this done for the whole group playing the game, or were these recorded per individual? If a single individual asked most of the questions this could impact the data.

- Why were changes made to the questionnaires over time (as per lines 194-195) and how did these potentially affect the results? This is not explained or discussed.

- There is no discussion of the possible limitations of self-assessment questions.

A suggestion: A summary of good practices for validation can be found in Dirks, Clarissa, Mary Pat Wenderoth, and Michelle Withers. Assessment in the college science classroom. WH Freeman, 2014. Chapter 7.
Additional comments: Additional / specific comments:
Line 97 - I would disagree with the authors that conservation biology focuses on cause-and-effect systems. In my view, they are dynamic and complex systems with feedbacks and time-delays, not cause-and-effect. The authors should include citations to support this characterization.

- In Line 186 the authors state that additional assessment was done for a portion of the students after one week to assess "long-term” knowledge acquisition. I would disagree that one week can be considered long-term, or would like to see references supporting this characterization.

Line 205-207 is unclear.
Line 222 - it is not clear what is meant by “statement" here.

Lines 342-344 are unclear

Line 372 Sentence starting with "Second, instead of regurgitating..” is poorly written and unclear. It is unclear, who is regurgitating the knowledge here and what is
being proposed instead and on what grounds.

Line 391 - what is meant by “first-hand”?

Lines 354-356 The authors interpret the lack of interaction between personality scores and lesson type on questionnaire scores (behavior) as evidence that the preference for EG over DI is universal. I would argue that this but one interpretation. This could also be interpreted as limited sensitivity of these tools or the experimental design to changes in preferences. These other interpretations should also be discussed as opposed to making this strong claim.

Minor errors:
Line 366 centre or central?
Line 325 uleast = least
There is a typing error in Question 3 in Supplemental material “Learning_style_survey"

The manuscript needs additional review by an English speaker for clarity of language. For example, the second sentence in the abstract is confusing:
Line 22 - "Games are an increasingly popular approach for conservation teaching. However, we know little about the effectiveness of the games on conservation teaching.” Do the authors intend to mean effectivess of the teaching? That is not what is being measured in the study, but rather the effects of the games on student experiences and knowledge acquisition.

Other examples of sentences that would benefit from a review of language for clarity and precision are:
Line 24-25 - I think was is meant is: goal-oriented tasks found in real-life situations
Line 60 - both or all?
Line 95 emphasizes


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

Input:
Title: THE EFFECT OF USING GAMES IN TEACHING CONSERVATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript can be improved by making the writing clearer and more concise. Because there are many elements to the study, it is difficult to keep track of the different components, so making sure that the same terms are used, clarifying definitions, and putting details into table form can do much in helping the reader navigate. There also repetitions of statements and extraneous language, particularly in the introduction.
Experimental design: The authors have collected data on many aspects of the students' experience and performance over several groups and years, which makes it difficult to organize in a coherent manner. More clarity about some aspects of the methodology are needed, particularly why certain parts of the data collection only applied to certain groups and especially how biases were eliminated (example, the person conducting the sessions giving equal effort to all treatments; how the concepts and content of different modes of instruction were made comparable). The behavioural data collected on 'joyful behavior' seems particularly biased towards showing positive outcomes for the games versus the didactic instruction, so I am not sure if this should even be included. The presentation of the results may benefit from being summarized both in text and figures.
Validity of the findings: I do think the findings are interesting, but am concerned that there are statements that try to explicitly link what happens in the classroom setting will also result in behaviors and attitudes with positive outcomes for conservation are an overreach.  I would suggest really being careful and specific about where the findings/conclusions of this study, especially outside of a learning environment, would apply.  The conclusion section should probably be rewritten with this in mind.
Additional comments: I do think that this study has merit and interesting results, but how the manuscript was put together was confusing at times. I think that it can be made more coherent and cohesive - links are needed between the perception of the students about the experience of games in the classroom and the benefits this provides for learning or social outcomes. I provide more comments in the pdf which I hope will improve it.


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

Input:
Title: THE EFFECT OF USING GAMES IN TEACHING CONSERVATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: High standard of academic English used that was clear and easy to understand to someone versed in the discipline.
Good and correct use of referencing.
Some of the data could perhaps be more easily conveyed to the reader through the used of tables or charts.
Overall well structured and presented.
Experimental design: Overall a very good experimental design that used multiple approaches to triangulate th results of the data.
Some more description of the observational aspects of the research (e.g. observing the behaviour students) might be required within the main text of the article.
Validity of the findings: Findings appear valid and well constructed.
The use of multiple avenues of data collection as well as more than one research design (i.e. both counterbalanced and repeated-measures) provided reliability to the data.
Additional comments: Overall an excellent paper. Some minor adjustments in terms of the presentation of the results and the data could just improve the overall quality of the paper.


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

Input:
Title: FLUXPYT: A PYTHON-BASED FREE AND OPEN-SOURCE SOFTWARE FOR 13C-METABOLIC FLUX ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The technical details on "How-To" use the FluxPyt is clear in general (Suppl File 2), however there is still a bit more description required of the output files and figures the program generates.

Another technical detail is the need of the folder name (where the models are stored) is needed - the authors mentioned that there is no need to enter this folder name in the path to run FluxPyt. However, at least, I needed to add the model directory as well to the path to run FluxPyt.

Some text in the draft needs to be re-written, i.e., lines 33-36, 44-46, 79-81, 187-188 etc to make it a bit more clear.
Experimental design: There are lot of .pckl files generated along with some standard txt, png and svg file formats. An additional supplementary file describing at least the most useful files will be helpful, for example, flux map is definitely useful for the end user and are there any other file formats and descriptions that needs to be mentioned about?  The authors should walk through the order of the output files generated as the program runs through different phases.

Either there should be a bit more introduction to EMU (elementary metabolite units) method or at least the authors should direct the reader(s) to Antoniewicz et. al's paper [PMC1994654] that describes EMU in detail. I see the reference citing the above article, but there should be a bit more stress, as Antoniewicz et. al's paper gives a very comprehensive description of the method. Also, did the authors try to compare FluxPyt to Metran (as Metran is from Antoniewicz/Stephanopoulos lab)?

Flux standard deviation calculations are a bit unclear (lines 124-128) and might need a bit more explanation of the need and use of this method.

Would this program be useful to run on non-stationary metabolic fluxes datasets too? How about the experiments with multiple tracers? Did the authors compare these datasets (i.e., multiple tracer experimental data) to OpenFlux as well? Since EMU framework seems to reduce the computational time, compared to traditional isotopomer methods, the authors should at least benchmark datasets with multiple tracers.
Validity of the findings: Most of the programs like OpenFlux and Metran that are based on EMU framework, even though are free for academia and not-for-profit institutions, should still be run on MATLAB which is not free. FluxPyt is built on python framework and makes this MFA software, a truly open source. The program is relatively easy to use, however the authors did not make any comments on use with other operating systems, like *nix or macOS. They should clearly mention that it is not compatible for other OS, or if the work is in progress to accommodate other OS.
Additional comments: The figures generated, ease of program will help other labs to use this program. However if there is a bit more description on how the output files can be used, it will be even more helpful.


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: FLUXPYT: A PYTHON-BASED FREE AND OPEN-SOURCE SOFTWARE FOR 13C-METABOLIC FLUX ANALYSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper presents a python based software for 13C metabolic flux analysis. Through two case studies and comparison with other software, the authors demonstrate their software is able to generate valid results quantifying cell metabolism. The language is in general clear, and the figures and tables are supportive to the conclusions.
Experimental design: Since this is a software paper, there is no experiment for this study.
Validity of the findings: The authors have confirmed their software can reproduce MFA results that have been published previously.
Additional comments: I have some questions for this software, and hope the authors can address these questions in their manuscript if necessary.
1. As a matter of fact, many researchers use python 2.7 for programming, do you know if your software can work well in both 2.7 and 3.4 releases.
2. If someone wants to use FluxPyt to study a cyanobacterium, will the flux map automatically create a Calvin cycle? Or the flux map template is only for heterotrophs?
3. It will be better if you can give a full list of metabolites and their abbreviations somewhere in the manuscript.