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 NEUROPSYCHOLOGICAL PROFILE OF PROFESSIONAL ACTION VIDEO GAME PLAYERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is generally written clearly and professional English is used throughout. In the annotated document I have highlighted some instances where the language was not clear or was ambiguous.

For example, in the abstract the phraseology 'do not make unanimity' is a bit awkward. I believe Statista (2018; on line 42) is using 'AVG' in different sense from the video game literature which makes the opening paragraph a bit difficult to follow. And so on. These should not take long to correct.

The article is well referenced however, there are two pockets of literature which need to be included. First, its acknowledged at the start of the paper that researchers have found null results in the video game literature but none of these studies are cited. Second, the authors emphasise the relation between video game play and cognitive abilities. And given that I think the authors think that the direction of the relation runs from video game play to cognitive abilities it would have been good to cite some targeted training studies with healthy populations.

I have included some examples of authors in both groups in the annotated document.
Experimental design: There are some missing details in the methods section. For example, the reader is not told what the selection criteria the casual video game group had to respect was. Nor are we told how many were excluded for failing those criteria (or the BDI-II inventory for that matter). Was handedness self-reported or assessed using something like the EHI? And so on.

There are a number of other minor queries of this nature in the annotated document.
Validity of the findings: It would have been better to report exact p-values when possible, and report standard deviations rather than standard errors (unless the authors have some reason for doing otherwise). There are a number of other minor queries in the annotated document. For the most part this section is fine.

Currently, the discussion really only acknowledges that the current results are consistent with elite video game players being self selected at the very end. Up to that point the authors describe video game play being 'related' to certain capacities but 'related' here is ambiguous. While there are training studies which show that no video game players who play video games show abilities in the direction of causal video game players, there is currently no training studies that show that causal video game players matching elite video game players. This is no problem but it does raise interesting questions about the use and testing of elite players (supposedly it would be to identify target capacities for enhancement) and what the possible limits of training might be. Given that this is a cross sectional study there are limitations on what can be concluded about the relationship between video gaming demographics and cognitive capacities.

Issues surrounding this point come up a number of times in the discussion and there are number of comments and suggestions in the annotated document that I hope will help.
Additional comments: This was an enjoyable paper which makes a novel contribution to the literature through presenting findings of actual professional video game players (Houston Outlaws). Very few research groups have access to such demonstrable experts video game players and so this research will be on interest to a wide readership.

I have attached an annotated manuscript with questions, queries, concerns and suggestions which I had while reading through the manuscript. I hope this document assist the authors with making revisions to the manuscript. The changes are not substantial but should hopefully make the paper much stronger.


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 NEUROPSYCHOLOGICAL PROFILE OF PROFESSIONAL ACTION VIDEO GAME PLAYERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the present article the Authors investigated the cognitive differences in two groups of video game players, i.e. elite n=14 vs. casual n=16. The findings indicate that elite video game players, in comparison to casual video game players, show higher performance in visual attention and working memory, as well as in the learning of 3D multiple-object-tracking task, suggesting a better functioning of dorsal ("When" and Where") "Action" pathway.

The article is interesting and it is a very good paper for Peer J.

Here some constructive (major and minor) comments:

Main comments

1. The relationship between selective attention and working memory could be better describe considering also the relevant reviews such as:
a. Edward Awh, John Jonides (2001). Overlapping mechanisms of attention and spatial working memory. Trends in Cognitive Sciences, Vol. 5, Issue 3, p119–126
b. Adam Gazzaley, Anna C. Nobre (2011). Top-down modulation: bridging selective attention and working memory. Trends in Cognitive Sciences, Vol. 16, Issue 2, p129–135
c. Nicholas E. Myers, Mark G. Stokes, Anna C. Nobre (2017). Prioritizing Information during Working Memory: Beyond Sustained Internal Attention. Trends in Cognitive Sciences, Vol. 21, Issue 6, p449–461

2. Only group analysis are reported. I suggest to explore also individual data. How many EVGPs perform better than the control group? For example, the Authors could use a cut off of 1 DS.
In addition, in order to explore better the possible relationship between selective attention and working memory in visual domain (main comment 1), a simple bivariate correlation analysis could be used. The same for the possible relation between working memory in visual and auditory domains (see main point 4).

3. A multivariate analysis between the two groups could be use to investigate the global results of the complex neuropsychological evaluation.

4. The multi-sensory effects of FPS action video games (i.e., WAIS Digit Span result) could be better considered also reporting recent results from action video game training in children with neurodevelopmental disorders such as developmental dyslexia (see Franceschini et al., 2013 Curr Biol, 2017 Sci Rep; Franceschini & Bertoni, 2019 NeuroPsychologia).

5. The interesting result of learning during 3D-MOT task could suggest the relevant role of FPS action video games mainly on dorsal-action pathway (but see the null results on visuo-motor skills also reported in the present article).

Minor comments
-pg. 8/32 line 66 "spatial cognition" is reported both as "robust impact" (line 65) as well as "less substantial" (line 68).
-pg 16/32 line 251 and 252 please check and report complete statistical results (reported in Table 3).
-Table 1 is equal to Table 2. Probably the Table 1 should report Neuropsychological tests.


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 NEUROPSYCHOLOGICAL PROFILE OF PROFESSIONAL ACTION VIDEO GAME PLAYERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Minor Comments:

Line 58
"between focused and distributed state of attention" missing 'a' before focused?

Line 61
FPS games are introduced as a sub-category of AVGs. However, all the previous examples of AVGs on Line 49-50 are FPS games. If AVGs are meant to be a more general category, then it might be worth giving some examples earlier of AVGs which are not FPS games.

Line 65
"must" is meant to be 'most'.

Line 90
"The concept of elite experts is useful when considering difference in performance amongst experts."? Unclear sentence.

Paragraph beginning Line 119
While I get the comparison being drawn elite athletes I think that it would be worth making clear that this is just being used as a paradigm case of expertise and its associated wide-ranging benefits. After all, the same point being made here with elite athletes could also be made using elite musicians (for example) who also show similar wide-ranging benefits (including to the visuospatial domain).

Line 138
Not sure what cognitive expertise means in this context. Is is something over and above the previously listed enhanced cognitive abilities?

Line 141
Is "distinct" the right word? 'Enhanced'?

Line 146
Change "extraordinary". Perhaps just use 'enhanced' due to previous usage.

Line 156
"Will" should be 'would'

Line 158
"were" should be 'have been found'

Section beginning Line 191
For clarity start a new paragraph with each measure.

Line 214
Incomplete brackets

Line 231
The Grooved pegboard test description could be clearer.

Line 479
Missing 'a'

Line 491
Sentence beginning "Those studies..." is unclear.

Line 496
It might be worth making absolutely clear that while it might not be a characteristic of expertise it is open that it might be a characteristic of video game experience (or exposure of some sorts).

Line 508
Could be stated more clearly. The behavioural results observed here are not evidence that action video games impact on the dorsal pathway. They are simply consistent with the hypothesis that they do impact on the dorsal pathway.

Line 517
"athletes" is repeated

Line 519
Any thoughts why there might be such a difference between athletes and video game players on the 3D-MOT?

Line 552
"planification" better as 'planning'?

Line 561
What was the question raised by Dye et al. (2009)?

Line 569
Delete "Matthew WG"

Line 576
Beginning of the sentence could be stated more clearly.
Experimental design: Minor Comments:

Line 179
What was the reason for excluding participants who played more than 20 hours per week? Given the focus on expertise, then so long as these participants were not competitive (the other exclusion criteria) I am not seeing why they should be excluded. It would be good to say a little more about why this exclusion criteria was used.
Validity of the findings: No comment
Additional comments: The authors have addressed all my major concerns and I am happy to recommend the paper for publication.

I have made a few minor comments. However, most of my comments are language queries as I had small difficulties in certain points when reading through. I would recommend the authors give the paper one last proof-reading pass as I am sure there might be further minor things which I might have missed.


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: PREDICTING DISEASE OCCURRENCE OF CABBAGE VERTICILLIUM WILT IN MONOCULTURE USING SPECIES DISTRIBUTION MODELING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is clear and unambiguous. In general, well written with some exceptions.

Some references are missing in the text but in general they are sufficient.

Figures are well presented, although Figure 4 needs correction.

Hypothesis is relevant and well presented.
Experimental design: The research fits well within Aims and Scope of the journal.

Research question well defined, relevant & meaningful.

Investigation was performed rigorously.

Some details are missing in Methods and require to be included. As it is presented, there's no enough information to replicate the study.
Validity of the findings: Novelty state.

Findings and method are meaningful and relevant.
Additional comments: Dear authors,

I have reviewed the manuscript “Predicting in disease occurrence of cabbage Verticillium wilt in monoculture using species distribution modeling” for possible publication in PeerJ. In this study, the authors used species distribution modeling to predict the potential occurrence Verticillium wilt using survey data in central Japan. The study has great value as it is a pioneer at using SDM to predict soil-borne plant disease, a topic of great relevance related to food security and sustainable agriculture. The manuscript is in general well written, with some grammatical issues noted in the attached document, although balance among the different sections is not good, especially in the abstract. In its current form is not ready for acceptance, as there are some issues that need to be addressed before publishing.

A major shortcoming of this study is that it is a case study. While this is not a major problem, it is not mentioned anywhere in the manuscript, and all analyses proceeded as if the study could be replicated. Nor is this issue much discussed in the Discussion. While lack of replication is often unavoidable in SDMs studies at a low spatial scale particularly using survey data, this has to be reported as a limitation of the study.

The abstract is not well balanced. The authors present too much background and just a few details on the results, mentioning mainly how the model was evaluated. Here the authors need to highlight their findings and the relevance of their study.

The introduction reads well and has all the important topics. Although, I suggest adding more information regarding the uses and importance of SDMs. Here, it is important that the authors differentiate between MaxEnt the algorithm and the software.

Material and Methods need more information. For example, authors need to include a justification of the selected variables. A simple description is given for TWI in L157 (A high TWI indicates that a site is moist) but it is not enough. Why the authors selected those variables? How do they influence Verticillium wilt occurrence? Because there is no justification for variable selection, the information on variable contribution feels weak later in the discussion. Also, I am curious to know why no climate predictors were used to model potential occurrence.

In L190, the authors provide the percent contribution of each explanatory variable. I suggest to report instead, or at least include also in the manuscript, the permutation importance of each variable, as this parameter is more relevant and informative within MaxEnt outputs.

L192-193. The authors must justify why they used Maxent's default setting. This point is very important since the authors are basing their results with this configuration without trying to optimize the model. There are many articles that have explored this problem (see Anderson and Gonzalez 2012; Warren and Seifert 2011; Merow et al. 2013; Radosavljevic and Anderson 2013; Syfert et al. 2013; Halvorsen et al. 2016). I would have expected that at the very least and to increase the quality of the models, the ‘hinge’ and ‘threshold’ features would have been deactivated to avoid overfitting response curves.

Regarding the evaluation of model performance, the authors only used the AUC value, which although it is high, does not show much, only that the model is not bad. Getting high values of AUC is very common for models based on presence-only data such as the one developed in this work. If the model is evaluated with cross-validation (for example, using spatial blocks, as in Roberts et al. 2017, http://onlinelibrary.wiley.com/doi/10.1111/ecog.02881/full), a high value of AUC would be more convincing. AUC levels for good performance are only relevant for presence/absence data. Since the authors base all the performance of the model on this statistic, it is necessary to justify it, especially since problems with AUC are well documented (see Lobo et al. 2007). Another option would be to calculate an additional statistic, such as ‘true skill statistics (TSS)’, to corroborate model performance.

How was the 'background' selected for the model? There is no mention of this anywhere in Methods and it is important to describe it.

As the results are just one paragraph, I suggest combining these with the Discussion. Although, I am not sure if the journal accepts a section combining “Results & Discussion”.

In the results, I don’t understand why the authors did not project their model to the whole agricultural area in Tsumagoi village (i.e. all grid cells in Figure 3A, including outside the agricultural area). This would be easy to do and informative considering agricultural expansion. This will provide information on which areas are more vulnerable to the disease in potential new locations.

In L223-224 the authors say "Some grid cells where Verticillium wilt of cabbage had never been recorded had a high probability of disease occurrence"; here, the authors need to discuss these findings. This is very important, is it because these grid cells have the optimal conditions for the occurrence of the disease? This is the place to speculate and discuss in more detail the response of the disease to the selected variables and the conditions across the landscape.

The authors also need to provide a better discussion on the contribution to the prediction for the variables. Specifically, including why TWI (7.7%) and slope (7.1%) had the lowest values. This has to be connected to the justification of the variable selection (see comment above)

There’s no need to have two panels in Figure 4. Just present Figure 4B.

There’s no need to include in figure 3 and 4 legends "These maps in this figure were arranged by ArcMap (ver. 10.2.2, ESRI, Redlands, CA, USA)" and "The occurrence probability calculated in each grid was arranged by ArcMap", respectively.

See more specific comments in the document attached.


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: PREDICTING DISEASE OCCURRENCE OF CABBAGE VERTICILLIUM WILT IN MONOCULTURE USING SPECIES DISTRIBUTION MODELING
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript describes a substantial research effort to assess potential distribution of  cabbage Verticillium wilt in a specific area in Japan. Literature reference is not sufficient, because different sections such as Introduction and Discussion are without a background of the disease, niche theory and on species distribution models. All results are inconsistent, since they were obtained of a weak and unclear modelling approach and a limited data set. A manuscript should show a relevant objective. Species distributions models are not only maps. You need to improve this manuscript a lot with all suggestions included in this document.
Experimental design: I included in a file with commented details of the experimental design.
Validity of the findings: I included in a file with commented details of the validity of the findings.
Additional comments: No additional comments


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

Input:
Title: PREDICTING DISEASE OCCURRENCE OF CABBAGE VERTICILLIUM WILT IN MONOCULTURE USING SPECIES DISTRIBUTION MODELING
Review round: 1
Reviewer: 3

Output:
Basic reporting: Lines 23-24: Please review the use of infected (the host) vs. infested (soil, inanimate objects) here and throughout (Lines 79, 80, . I would tend to write this sentence as “…IPM methods are sometimes inadequate to bring about control in severely infested fields.” since the field is infested, unless you are talking about the plants in the field.
Line 26: do you mean disinfestation?
Line 29: “…to a soilborne…”
Lines 66 and 72: change disinfection to disinfestation
Lines 122-124: I think more information on the pathogens could be provided here (e.g. how long it survives in soil, host range – wide in V .dahliae vs. brassicas in V. longisporum. What is there relative abundance in relation to each other? This can be important depending on what other crops (hosts vs non-hosts) are rotated in the fields.
Experimental design: Lines 112-119: what other crops are grown here? Are other hosts grown in rotation? Does infected seed play a role in the introduction of the pathogen into fields?
Line 121: are certain cultivars of cabbage grown that are resistant or susceptible? Verticillium may go undetected in fields planted to resistant cultivars or detected more frequently in fields planted more often to susceptible cultivars.
Line 125: Were fields sampled more or less equally, or is there any potential bias for certain cells?
Line 135: Do some fields or areas have longer histories of cabbage production than others? Since Verticillium can survive in the soil and buildup over time, would fields with longer histories of cabbage production be identified as infected more instances over time than newer fields?
Lines 157-158: what was the sample size (n)?
Validity of the findings: Lines 77-78: what about exclusion and prevention?
Lines 80-82: it could also be mentioned that knowledge of infested and non-infested fields could help growers prevent infesting new fields or re-introducing the pathogen to fields that have been fumigated.
Additional comments: The manuscript "Predicting disease occurrence of cabbage Verticillium wilt in monoculture using species distribution modeling" by Ikeda and Osawa describes research to predict this important disease in a major production area using historical survey data and novel, ecological-based approaches. Their analyses showed that road density was a major predictor of Verticillium occurrence in this agricultural area. These findings make perfect sense, as the pathogen is soilborne (seedborne in some hosts) and primarily moved with soil or agricultural products via human activities. I think this is a very important and interesting work and recommend publication after the authors address/respond to my 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: PREDICTING DISEASE OCCURRENCE OF CABBAGE VERTICILLIUM WILT IN MONOCULTURE USING SPECIES DISTRIBUTION MODELING
Review round: 1
Reviewer: 4

Output:
Basic reporting: The manuscript is clearly written. There are some relevant references missing.
Please note the following references when you state that there is no previous studies utilizing SDMs in plant pathogen epidemics.
Modeling and mapping the current and future distribution of Pseudomonas syringae pv. actinidiae under climate change in China Wang R, Li Q, He S, Liu Y, Wang M, et al. (2018) Modeling and mapping the current and future distribution of Pseudomonas syringae pv. actinidiae under climate change in China. PLOS ONE 13(2): e0192153. https://doi.org/10.1371/journal.pone.0192153

Narouei-Khandan, H.A., Harmon, C.L., Harmon, P. et al. Potential global and regional geographic distribution of Phomopsis vaccinii on Vaccinium species projected by two species distribution models. Eur J Plant Pathol 148, 919–930 (2017). https://doi.org/10.1007/s10658-017-1146-4

When, where, and whether to manage a plant epidemic
Nik J. Cunniffe, Richard C. Cobb, Ross K. Meentemeyer, David M. Rizzo, Christopher A. Gilligan
Proceedings of the National Academy of Sciences May 2016, 113 (20) 5640-5645; DOI: 10.1073/pnas.1602153113

I also suggest you discuss more on the road network in disease occurrence in the light of other studies eg.
The spread of a wild plant pathogen is driven by the road network
Numminen E, Laine AL (2020) The spread of a wild plant pathogen is driven by the road network. PLOS Computational Biology 16(3): e1007703. https://doi.org/10.1371/journal.pcbi.1007703
Experimental design: The experimental is adequately designed.
Validity of the findings: The authors should discuss how their findings could be extended to wider areas. Is it possible that this model can be used in other Japanese or global areas and why.
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: PREDICTING DISEASE OCCURRENCE OF CABBAGE VERTICILLIUM WILT IN MONOCULTURE USING SPECIES DISTRIBUTION MODELING
Review round: 2
Reviewer: 1

Output:
Basic reporting: I have reviewed the revised manuscript "Predicting disease occurrence of cabbage Verticillium wilt in monoculture using species distribution modeling" for publication in PeerJ.

The authors have made a good effort addressing all my previous comments but the manuscript still has issues that require attention before acceptance.
Experimental design: Some details are required. See comments in the file attached.
Validity of the findings: Some details are required. See comments in the file attached.
Additional comments: Dear authors,
I appreciate your effort addressing all my previous comments; however, the new version of your manuscript has some issues that need to be clarify and corrected before acceptance.

English requires a review, as there are sections that are not easy to follow or understand the ideas.


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: PREDICTING DISEASE OCCURRENCE OF CABBAGE VERTICILLIUM WILT IN MONOCULTURE USING SPECIES DISTRIBUTION MODELING
Review round: 2
Reviewer: 2

Output:
Basic reporting: line 89 - change to "given"
line 98 - change to "approaches"
line 145 - change to "In recent years, cultivars..."
line 147-148 - change to "...times in total by official and private.....capable of distinguishing..."
line 149 - change to "The surveys and field collections were approved..."
line 173 - change to "roads"
line 258-259 - this sentence is not clear to me.
line 272- change to "uninfested"
line 276 - should "becoming" be changed to "detecting"? This sentence is not clear.
line 278-280 - This sentence is not clear. Do the authors mean that the disease was present but not detected due to some unknown control measures that were being used?
line 285 - delete "more"
Experimental design: Is there a(n) (auto)correlation between road density and agricultural production that would confound these results (i.e. there are more roads in agricultural areas, and more Verticillium in agricultural areas, hence road density and Verticillium appear to be correlated)?
Validity of the findings: No comment
Additional comments: The authors addressed my comments on the first draft. I just have a few more comments/corrections to note.


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

Input:
Title: ANALYSIS OF GENES AND UNDERLYING MECHANISMS INVOLVED IN FOAM CELLS FORMATION AND ATHEROSCLEROSIS DEVELOPMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is overall easy to understand. The introduction is clear, focusing on foam cells in the context of atherosclerosis. Flow chart of the analysis helps to follow the different comparisons.
Major comments:
-	The papers corresponding to the published datasets used in this paper have not been referenced and discussed. It would be important to explain how different this new analysis is compared to the ones done in the original papers.
-	The discussion section does not correspond to a discussion of the results but instead to an extended introduction. For example, no mention of the results is done between lines 255 and 275. This needs to be modified and should include the following points. In particular, the authors should include a comparison to the previous analysis of the microarray/RNAseq and stating the novelty/difference of their approach. The detection of only a small overlap between SMC-FCs ad M-FCs should be discussed (due to cell type difference? experimental design?). The authors analysed data from in-vitro experiments as well as in-vivo/tissue. These analyses come with different limitations. Indeed, tissue heterogeneity will limit the interpretation of the result and this needs to be mentioned.
Minor comments:
-	A few typos and unclear sentences can be found along the manuscript such as Line 82 “plague”; Line 206 “GSE5466 was expressed”; Line 244 “corelated”; Line 333 “compared its expressing in”
-	Figures: Gene names in Heatmap should be removed if they are too small to be read. A title for the x-axis of the bubble diagram should be added and the size of the text should be increased.
-	The manuscript contains many abbreviations that make the reading of the manuscript difficult. For clarity, some abbreviations could be removed (“atherosclerosis” shouldn’t be abbreviated to AS).
-	In Figure 6B, all significant comparisons are shown while only a few comparisons are described in the text. It might be better to show only the statistics for the relevant comparison.
Experimental design: Overall, relevant questions and objectives are well defined with the flow chart helping to understand the analysis. Methods section is comprehensive.
Major comments:
-	The choice of dataset to include in this analysis has not been explained clearly. Does it correspond to all publicly available data on foam cells and atherosclerosis? If only some dataset were included, can the authors justify their selection criteria.
-	The choice and order of comparisons in this analysis are not completely claer for me. Indeed, the authors started by analysing 2 in-vivo dataset then 2 in-vitro datasets. Finally, they finish by analysing another in-vivo and in-vitro dataset, for validation. I do not understand why the 3 in-vivo datasets where not analysed and integrated together. What is the justification for choosing the dataset for target identification versus the dataset for validation?
Minor comment: The threshold (fold change and pvalue) to identify DEGs should be included in the Methods.
Validity of the findings: While the comparison of in-vitro and in-vivo dataset to identify genes involved in foam cell formation in atherosclerosis is interesting, the interpretation of results and discussion is sometimes missing.
Major comment: As mentioned above, the Discussion of the manuscript should include the limitations of this type of analysis
Additional comments: In summary, this manuscript provides an interesting analysis by integrating different datasets to find relevant candidates involved in foam cell formation. The different steps of the analysis and results are clearly explained. However, the choice of data comparison is not justified and does not seem pertinent. The authors only find 4 candidates, probably due to the limitations of this kind of approach. Discussion of the results and limitation should be incorporated to this manuscript.


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

Input:
Title: ANALYSIS OF GENES AND UNDERLYING MECHANISMS INVOLVED IN FOAM CELLS FORMATION AND ATHEROSCLEROSIS DEVELOPMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: Figures need to be remaking.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript provided a list of genes that are functional related to the development of atherosclerosis. The idea to utilize multiple existing datasets is valuable, though I am not fully convinced by the technical details of the proposed approach. My comments are list as follow.

Major comments:
1. For the DEGs associated with AS, the first gene sets come from DE analysis of early versus late atherosclerosis samples, and the second gene sets come from DE analysis of athrosclerotic versus nonathrosclerotic samples. It is not clear to me why you need to take the intersection of the intersection of the DEGs of the two comparisons. The first experiment design is comparing between patients with AS not health control. Theoretically, the second set of gene is enough. Please explain the specific purpose of including first gene sets.
2. To generate SMC-FCs related genes and M-FCs related genes. The authors simply take the overlap genes between studies. This approach ignores the difference of sample size across study designs and the effect size of each gene in DE analysis. It would be better to use a meta-analysis which adjusted for these effects.
3. To validate the four coregulated genes associated with FCs, the authors used vulnerable AS plaques dataset. It seems to be a logic gap need to be filled. It is not clear to me why the vulnerability of AS can validate the role of FCs in AS. Please explain it in more details.

Minor comment:
The gene names (row names) of Figure 2 and Figure 4’s heatmaps are unreadable. Please make a clearer figure or completely remove the gene name and put them in supplementary tables.


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

Input:
Title: ANALYSIS OF GENES AND UNDERLYING MECHANISMS INVOLVED IN FOAM CELLS FORMATION AND ATHEROSCLEROSIS DEVELOPMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: My first review of the manuscript contained several major and minor comments. While a few of these comments have been addressed, I still have some issues.

The remaining comments are:

-	While the authors justified the validation of their candidates in 2 additional in-vivo datasets looking at vulnerable plaque (in response to comment (9)), the authors do not explain why they choose GSE9874 to validate their candidates. I do not understand why this dataset was not analysed in combination with GSE54666. Can you justify this choice?
GSE9874 corresponds to -/+ oxLDL from 15 healthy/athero. The Figure 6 includes -/+ oxLDL conditions but there is no mention of healthy vs athero.  Can you explain if both healthy and athero samples were used?

-	The discussion still needs improvement. In comment (1) and (2), I mentioned that previous studies, that generated these datasets, have not been referenced and discussed. The revised discussion only comments on 2 out of the 7 studies. For this reason, I consider that my comment has not been fully addressed.  To answer comments (3) and (8), the authors included a new section to the discussion corresponding to the limitation of these studies. The 2 new sections of the discussion (discussion of the datasets and limitations of the study) are vague and poorly written. The authors need to explain what they mean by “interexperiment variability”, “cell heterogeneity”. The authors should point out which datasets is affected by the limitation of the sample size. The “sequencing related technical errors” shouldn’t be mentioned within the “external clinical traits”.


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

Input:
Title: ANALYSIS OF GENES AND UNDERLYING MECHANISMS INVOLVED IN FOAM CELLS FORMATION AND ATHEROSCLEROSIS DEVELOPMENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors addressed all my comments in the revised version. I have no further questions or 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: THIRD MOLAR AGENESIS IN MODERN HUMANS WITH AND WITHOUT AGENESIS OF OTHER TEETH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clearly written and meets these criteria. Comments on improving the Tables are included in the general comments.
Experimental design: See general comments
Validity of the findings: Issues with the sample composition, level of analysis and limitations are integrated with the general comments.
Additional comments: This study of tooth agenesis compares third molar agenesis in individuals with agenesis of other teeth (cases) and control individuals with no agenesis of other teeth. The authors are to be commended for their improved study design over many earlier studies on the subject.

The study has problems with aspects of the data collection and samples, analytical methods, presentation of tabular data and inconsistency with dental terminology.
In terms of the data collection, it may be that the case and control selection is not clearly described, but it appears that the individuals were selected from orthodontic patients who had records from the last 12-year period. This is confusing as the sample demographics include individuals up to 40 years of age. Does this mean individuals of age 40 who were orthodontic patients? It seems unlikely that individuals of the older ages would still be regular orthodontic patients or that their records would include information on extractions performed long ago and perhaps by another practitioner, such as premolar removal to facilitate corrective procedures.
Secondly, the authors state that individuals with syndromes were excluded. Yet they included 27 oligodontia cases (almost 9% of the cases) with up to 20 missing teeth (Suppl. Table 1). With 20 missing teeth, the most extreme case had only 8/28 teeth occurring. How is this extreme level of oligodontia justified for inclusion, as non-syndrome oligodontia is extremely rare? The use of these extreme cases seriously compromises the integrity of the results.

The simple investigation of associations among the agenesis patterns is not sufficient for documenting the complexity of agenesis in this study. What are the odds associated with these combinations?

Tables should be simple and easy to understand. They need to be ‘stand alone’, meaning that they can be understood without reference to the text, which should only elaborate upon the data presented in the table. Several in this manuscript are not.  Varying use of tooth numbering terminology and descriptors, in the text and tables should be corrected. For example, in Tables 1 and 2 the heading ‘Tooth number’ is not clear and is not consistent with a subsequent table that uses the FDI system (Table 3). The non-standard table terms should be explained as a footnote below the table.
Tables 3 and S2, S3, S4: What does the header “Index’ mean?
Table 4: The header ‘Pattern Symmetry’ seems to be over the wrong column.

Finally, the authors state that their findings are consistent with other studies so the clinical nature of the study is not a true limitation. Unfortunately this point is moot, as agreement with other studies that are also clinical is not relevant to the limitation issues.


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: THIRD MOLAR AGENESIS IN MODERN HUMANS WITH AND WITHOUT AGENESIS OF OTHER TEETH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The purpose of this study was, to explore the patterns of third molar agenesis in modern European subjects with and without agenesis of other teeth. The topic fits within the scope of PeerJ. The Intro section ends with the null-hypothesis that there is no difference in third molar agenesis patterns between individuals who have agenesis in teeth other than third molars, and those who do not.

1.	This is a well-written manuscript that follows the guidelines of the STROBE reporting guideline.

2.	The Introduction section is adequate concerning the quoted literature. However, I invite the authors to better explain in the Intro section why a new study was needed – what is the knowledge gap - considering the high-quality systematic review and meta-regression of Carter and Worthington in 2015. Their analysis included 24 studies on Europeans, so this constituted a large sample size  for meta-analysis.

3.	Figures and tables are relevant but there is some improvement possible.
Experimental design: 4.	This is a well-conducted study, but the authors do not clearly state the design of the study. Is this a case-control study? Please add.

5.	The inclusion criteria for the controls could be described better. It is only stated that a control group of 303 individuals without agenesis of teeth other than third molars, were matched for age and sex, but what about the other criteria as for the cases group?

6.	It is not completely clear how the study size was arrived at. The authors state that they took a sample of n=303 with agenesis from about  10.000 cases. But it then means that the incidence for agenesis in this sample is about 3%, which is much lower than reported in literature. This does not comply with statements in the discussion section that the sample has external validity. Could you explain, please?

7.	Table 1 only gives numbers and not the percentages, please add.

8.	Figure 1, legend in the table. ‘’agenesis’’ It is confusing, please make it ‘’agenesis group’’ and ‘’controls’’
Validity of the findings: 9.	When considering the percentages of the number of missing third molars per individual in the control and the agenesis group, a tendency was found towards more missing third molars in the agenesis group. Thus is the key finding of the study. The authors only mention ‘’genetic factors’’ which is a very vague term. The discussion could be enhanced by trying to explain this finding from the embryological viewpoint, f.e. the studies of Inger Kjaer in Copenhagen.

10.	About the external validity, see my question above.

11.	The discussion section could also be broadened by elaborating on the clinical significance of the findings. As a reader I am left with question ‘’OK, so what …?”

12.	The raw data are in a text file and rather useless for a check. It is up to the editor to decide if this is sufficient.
Additional comments: No additional comments


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

Input:
Title: THIRD MOLAR AGENESIS IN MODERN HUMANS WITH AND WITHOUT AGENESIS OF OTHER TEETH
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article is satisfactory in this regard, but some small corrections of the English is needed.
Experimental design: Some shortcomings with the analysis are discussed in the comments.
Validity of the findings: See comments.
Additional comments: In terms of the data collection, it may be that the case and control selection is not clearly described, but it appears that the individuals were selected from orthodontic patients who had records from the last 12-year period. This is confusing as the sample demographics include individuals up to 40 years of age. Does this mean individuals of age 40 who were orthodontic patients? It seems unlikely that individuals of the older ages would still be regular orthodontic patients or that their records would include information on extractions performed long ago and perhaps by another practitioner, such as premolar removal to facilitate corrective procedures.
Authors’ Response: The reviewer is right that the individuals were selected from orthodontic patients. Various archives were searched to identify pre-treatment records of eligible patients that were obtained the defined 12-year period. It is true that certain patients were also up to 40 years old and this is indeed a clinical occurrence. Of course, this is not the majority of orthodontic patients, but it is only a small part. With a thorough anamnesis and medical history kept in all searched archives, it was possible in most cases to determine the reason of any tooth absence. When the treating doctors judged that the cause for missing teeth was unclear, the case was excluded, according to our eligibility criteria. Through this approach, misdiagnosis might not be fully excluded, but it was limited considerably, in order not to critically affect the outcomes. This consideration has now been added to the limitations of our paper and the issues mentioned just above have been clarified in the methods section.
--The authors have dealt with this issue in a satisfactory manner.

Secondly, the authors state that individuals with syndromes were excluded. Yet they included 27 oligodontia cases (almost 9% of the cases) with up to 20 missing teeth (Suppl. Table 1). With 20 missing teeth, the most extreme case had only 8/28 teeth occurring. How is this extreme level of oligodontia justified for inclusion, as non-syndrome oligodontia is extremely rare? The use of these extreme cases seriously compromises the integrity of the results.
Authors’ Response: As mentioned, our sample consisted orthodontic patients, including two university centers. Thus, the prevalence of oligodontia could be different from the general population, since such severe cases are usually referred at the university and they usually need orthodontic treatment. Regarding syndromes, we based our decisions on the thorough review of the medical and dental history of the patients. Thus, there is always a risk of including a non-diagnosed syndromic patient in our sample. This risk cannot be totally eliminated and this is mentioned in the limitations of our study. The fact that we might have in our sample more oligodontia patients as it might be expected for the general population does not compromise our findings, since the case-control study design, for the hypotheses tested, does not require or did not aim to represent the general population. Thus, for the specific hypothesis tested in the study, including the effect of the number of missing teeth on the third molar formation, it is actually an advantage to have adequate numbers of cases on the different agenesis severity levels. Relevant information is now provided in the limitations section of our manuscript.
--The authors have not dealt with this major bias and design flaw. The problem is not whether oligodontia occurs with greater or lesser frequency in their cases vs. the general population, but their failure to address how these extreme cases (most probably due to syndromes that were supposed to be excluded) may actually bias their results. They should have done an analysis treating them separately to evaluate the impact of their inclusion.

The simple investigation of associations among the agenesis patterns is not sufficient for documenting the complexity of agenesis in this study. What are the odds associated with these combinations?
Authors’ Response: Table 3 and Supplemental Tables 2 and 3 describe the most common tooth agenesis patterns present at the studied sample along with the associated frequencies. These descriptive data provide an overview of the agenesis patterns observed in the sample and along with the other data presented in the study provide a thorough description of the current sample. A more detailed tooth agenesis pattern assessment similar to that previously published by our team is beyond the scope of the current study (Gkantidis N, Katib H, Oeschger E, Karamolegkou M, Topouzelis N, Kanavakis G. Patterns of non-syndromic permanent tooth agenesis in a large orthodontic population. Arch Oral Biol. 2017;79:42-47. doi:10.1016/j.archoralbio.2017.02.020). Furthermore, since the raw data are provided with the submission, future researchers that might be interested in specific patterns can use our data for their purposes.
 --This is not a satisfactory response; it is their responsibility to present their findings in an informative, analytical manner and not to leave it up to the reader. Secondly, the article they cite here as containing or pertaining to the requested analysis, is not even referenced with regard to the agenesis patterns in the results.

Finally, the authors state that their findings are consistent with other studies so the clinical nature of the study is not a true limitation. Unfortunately, this point is moot, as agreement with other studies that are also clinical is not relevant to the limitation issues.
Authors’ Response: To clarify this issue, that was also noted by the reviewer in a comment above, we rephrased this paragraph and transferred this information to the limitations section. We hope that following the clarifications provided here and in the revised versions of our manuscript, the reviewer’s concerns on this issue will be adequately addressed.
--Elsewhere the authors have argued that their study is not designed specifically for clinical purposes but ‘ it is more relevant to basic research than to clinical research’, yet they use the clinical basis of their samples to argue for the strength of their results. I think they do not demonstrate an understanding of the underlying biological premises of conducting this type of research.

Reviewer 2 (Anonymous)
2. The Introduction section is adequate concerning the quoted literature. However, I invite the authors to better explain in the Intro section why a new study was needed – what is the knowledge gap - considering the high-quality systematic review and meta-regression of Carter and Worthington in 2015. Their analysis included 24 studies on Europeans, so this constituted a large sample size for meta-analysis.
Authors’ Response: The study of Carter and Worthington had a large sample size and a valid methodology, and so we used the reported value for third molar agenesis as a reference value for our sample. However, Carter and Worthington (2015) looked at the prevalence of third molar agenesis in the population. This meta-analysis did not assess whether other teeth were missing or not. In our study, we investigated the chances of a missing third molar when other teeth are also missing. The last part of the introduction has been revised to clarify this issue.
This partially addresses this concern raised.

Experimental design
5. The inclusion criteria for the controls could be described better. It is only stated that a control group of 303 individuals without agenesis of teeth other than third molars, were matched for age and sex, but what about the other criteria as for the cases group?
Authors’ Response: Except from tooth agenesis, the other inclusion criteria of the control group were identical to those of the agenesis group. This is now clearly stated in the Methods section.
--This is now adequately addressed.

Validity of the findings
9. When considering the percentages of the number of missing third molars per individual in the control and the agenesis group, a tendency was found towards more missing third molars in the agenesis group. This is the key finding of the study. The authors only mention ‘’genetic factors’’ which is a very vague term. The discussion could be enhanced by trying to explain this finding from the embryological viewpoint, f.e. the studies of Inger Kjaer in Copenhagen.
Authors’ Response: We thank the reviewer for the suggestion. In terms of developmental timing, the third molar is the last tooth in the molar series and also the last tooth to develop in the dentition. It is known that the last tooth in each tooth series shows more often developmental disturbances, including agenesis, thus being more vulnerable to genetic or environmental factors that might be present (Townsend et al. Morphogenetic fields within the human dentition: A new: Clinically relevant synthesis of an old concept. Arch Oral Biol. 2009;54:S34–S44. & Gkantidis et al. Patterns of non-syndromic permanent tooth agenesis in a large orthodontic population. Arch Oral Biol. 2017;79:42-47.) This might be another contributing factor relevant to the present results. It has also been shown that overall dental development is delayed in patients with tooth agenesis, compared to controls, with a weak correlation between dental developmental stage and number of missing teeth (Lebbe et al. 2017). This information has been now added in the discussion section.
--This is a much needed improvement to the discussion.


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

Input:
Title: THIRD MOLAR AGENESIS IN MODERN HUMANS WITH AND WITHOUT AGENESIS OF OTHER TEETH
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript (revision v2) is clearly written in professional English and has been improved according to the referees’ comments.
The Intro introduces the reader to the topic and literature cited is adequate.
Table legends have been improved.
Experimental design: Based on the comments of the referees, in the revised version the authors have adequately addressed issues that were raised.
Validity of the findings: I would not characterize this study as novel, but data is valid and could be useful in further studies. I have nothing to add here.
Additional comments: The authors have answered the comments of the referees satisfactorily and revised the manuscript accordingly. I have no further comments or suggestions for the authors.


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: INCREASED COPY NUMBER OF SYNCYTIN-1 IN THE TROPHECTODERM IS ASSOCIATED WITH IMPLANTATION OF THE BLASTOCYST
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is the review report for the manuscript titled ‘Increased copy number of syncytin-1 in the trophectoderm is associated with implantation of the blastocyst’ by Guo et al. Human trophoblast cells express syncytin-1 and syncytin-2 that are derived from HERV-W and HERV-FRD envelopes, respectively, and known to be expressed to form syncytiotrophoblasts in placenta. However, it is still unclear whether syncytin-1 and syncytin-2 have important roles during implantation. The authors showed syncytin-1 but not syncytin-2 has increased genomic copy number in the pregnant group when compared with the non-pregnant group even though there were no significant differences in the clinical conditions. The authors also conducted in vitro experiments using H1 embryonic stem cells to suggest that the increase of syncytin-1 but not syncytin-2 genomic copy number is associated with differentiation into trophoblast cells. The manuscript is concluded with these data that syncytin-1 could be the indicator for the successful implantation after frozen embryo transfer.
This study is quite interesting and important for the development of methods for accurate selection of embryos to be transferred. In particular, the clinical data of syncytin-1 copy number is valuable to gain insights into the strategy for embryo transfer. However, the manuscript needs to be improved before going to be published in the journal PeerJ.

Major comments
1. The English needs to be improved to make your text more clearly understandable. For examples, ‘We want to know… (lines 69-70)’, ‘The results indicated that… (lines 156-157)’, ‘As implantation of the … (lines 174-176)’, ‘The transcription factor CDX2…(lines 262-263)’can be proofread.

2. The Introduction is not effective to understand the background of this study. It is hard to understand how these syncytins are expressed and how these syncytins contribute to placental development. The authors should include more of what are known already about the roles and molecular characteristics of syncytin-1 and syncytin-2 during placentation to improve the Introduction.
Experimental design: 3. The authors described that the aim of this study is to examine the association of genomic copy numbers and expression of syncytins with the outcome of the pregnancy (lines 69-70). This, especially the genomic copy number, gives off an erratic impression, the authors therefore should explain more details why they focused on the factor genomic copy number in this study.

4. The authors need to explain the purpose of the experiments using H1 cells before going ahead (line 178-).
Validity of the findings: 5. The discussion about the differentiation from embryonic stem cells to trophoblast cells (lines 256-269) should be incorporated in the subsection ‘BMP4 induced differentiation of H1 stem cell line into trophoblast cells’ of Results to make it easy to understand the meanings of RT-qPCR data. This part (lines 256-269) also can be shrunk by excluding the redundant descriptions such as ‘We used BMP4…(lines259-260)’, ‘This indicated that the expression of NANOG… (lines 261-262).’, and ‘A previous study found that the trophectoderm… (lines 263-266)’.

6. Increasing the genomic copy number of syncytin-1 is the important finding of this study. Hence discussion about the phenomenon is necessary to increase the impact of this manuscript. I would suggest the authors to incorporate the explanation that how the genomic copy number affects gene expression as well as protein function (could be general). The authors should also discuss about why only syncytin-1 but not syncytin-2 altered genomic copy numbers.
Additional comments: 1. Please explain what the A, B, C and D indicate in the figure legends of Figures 3 and 4.

2. In line 211, please delete ‘the’ before ‘the H1 cell line’

3. Delete ‘the’ before HERV (lines 58, 59, 62, 239)

4. Authors’ first and last names should not be separated by commas in the References.


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: INCREASED COPY NUMBER OF SYNCYTIN-1 IN THE TROPHECTODERM IS ASSOCIATED WITH IMPLANTATION OF THE BLASTOCYST
Review round: 1
Reviewer: 2

Output:
Basic reporting: In overall, this manuscript is clear and data are interesting.
Concerning the scientific writing style, some small changes are required such as using adequate academic wording instead “so, we wanted” (line-19) and when necessary introducing passive voice sentences instead of the pronoun “we” (i.e. lines 196/200/213).
The abbreviation hCG for human chorionic gonadotropin is not the same throughout the manuscript and figures (i.e. lines 104/194 and table 2).
All abbreviations should be defined at their first use (line  63 – HERV-FRD, line 96- BMI) and redefined in each figure.
The flow in the material and methods is broken at a few places and requires some brief changes:
•	line 7, “patients and sample collection” section is introducing DNA storage. I would suggest splitting into two sections the patient information and the clinical samples.
•	lines 108- 111, “When a couple lived together and had a normal sex-life, without the use of contraception, if after 1 year the wife did not fall pregnant, the patient was diagnosed as infertile. If the patient had never been pregnant, the patient was diagnosed as having primary infertility” could be placed in “Patients and sample collection” section
Experimental design: The title “Increased copy number of syncytin-1 in the trophectoderm is associated with implantation of the blastocyst” suggests you looked at Implantation whereas you investigated until day 33. Could you develop in the discussion section your choice of date (biochemical vs clinical pregnancy in relation to Implantation)?
In your study, have you included couples suffering from unknown infertility or only male fertility factors?
The use of an in vitro model of stem cell differentiation to mimic in vivo embryonic trophoblast cell differentiation needs to be incorporated into the study aims section to contain the hypothesis.
The BMP4 induction protocol should be stated as a common practice and referenced.
The choice of statistical analysis t-tests for multiple comparisons seemed inappropriate and ANOVA / Mann Whitney tests are recommended. More details are required in the Methods section.
Lines 147 – 148: “Spearman’s Rank Correlation Coefficient (rs) was used to compare the relative odds of clinical factors associated with blastocyst implantation”. Could you develop how you measured odd for each embryo using Spearman’s rank? Could you demonstrate by using a correlation on a binary outcome that the Point-Biserial Correlation Coefficient is equal to Spearman’s rank in this study? How is the figure 1C different from the figure 1A?
The details about the number of technical and experimental replicates should be enumerated in each figure and present in the method section.
Validity of the findings: In your hypothesis, “whether the envelope proteins of HERV-W have an effect on the development of the trophectoderm”: it is either a cause or causality, but the data are limited and the mechanism is not fully established in your results. Therefore, the hypothesis should be rephrased.
Lines 178 – 180: “The small undifferentiated cells had obvious nuclei and gathered together like a clone. After differentiation, the morphology of the H1 cell line changed significantly. The cell volume was bigger and the cell had increased protrusions and branches (Figure 2)”. The research standards are not achieved with the wording “obvious” in the absence of a nuclear marker and the absence of quantification for the cell volume. In order to conclude these findings, a phase contrast-nuclei marker staining and measures of cell volume (using Phalloidin staining?) with statistical analysis are expected.
In Table 3, data for a few parameters are incomplete/missing such as the incidence of primary infertility (18 out of 29) or embryo quality (24 out of 29). However, it was stated in the line 104 “The number of quality embryos in all of the blastocysts was collected”.
The clinical collection finished in 2018. Could you update the manuscript and add an analysis of live birth/late miscarriages and syncytin DNA copies?
The discussion section requires to be developed on the study limitations (numbers, infertility, use of stem cells model, in vivo/in vitro…).
After identifying an increase in mRNA, the dataset would be strengthened by the identification of an increase at the protein level (i.e. syncytin-1) in the in-vitro model overtime. Therefore, additional work to investigate the protein levels are highly recommended.
In the conclusion section, line-282 “Syncytin-1 may be an important marker in detecting embryos”. Would you not conclude on the DNA copy number of Syncytin-1 as a potential marker instead of the functional protein?
Additional comments: The authors have identified an increase in DNA copy of syncytin-1 in differentiating trophectoderm in vitro and observed that a significantly higher number of copies was found in trophectoderm from embryos that implanted and are responsible for successful pregnancy until 33rd day compared to embryos in the “non-pregnant” group. It is a very interesting finding.
However, the manuscript requires some improvements: re-structuring and adding more details. Moreover, the key findings would be strengthened with some additional work.


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

Input:
Title: INCREASED COPY NUMBER OF SYNCYTIN-1 IN THE TROPHECTODERM IS ASSOCIATED WITH IMPLANTATION OF THE BLASTOCYST
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this well written paper the authors investigate whether the fusogenic envelope proteins of HERV-W (human endogenous retrovirus-W), syncytin-1 and syncytin-2, are expressed by the trophoectoderm of the blastocyst and if they have any association with the implantation of the blastocyst.

The HERV-W proteins are mainly distributed in the placenta and play a key role in the fusion of the progenitor cytotrophoblast to the fused multinucleated syncytiotrophoblast. A key step in embryo implantation is the adhesion to and invasion of the endometrium by the blastocyst trophectoderm.

This is a paper with good novelty and with clinical significance as good markers of preimplantation success for a healthy pregnancy outcome are lacking in the IVF field.  The patient groups are large and the ethics approval of the study and the statistical tests used are appropriate.
Experimental design: In this study the authors utilise whole-genome amplification of 6-8 cells of the human blastocyst trophectoderm to measure the copy number of syncytin-1 and syncytin-2 using real time qPCR. In addition, clinical data associated with the outcome of pregnancies was collected, including
age, BMI, bFSH, rate of primary infertility, the thickness of the endometrium on the day of
endometrial transformation, the levels of estrogen and progestin on the transfer day, the days and the morphological scores of the blastocysts. The authors also examine the expression of mRNA and the copy numbers of syncytin-1 and syncytin-2 in H1 stem cells, and in differentiated H1 cells, induced by BMP4, were measured using real time qPCR.

They show that the relative copy number of syncytin-1 in the pregnant group of patients was significantly higher than in the non-pregnant group following IVF with a single blastocyst. This was shown to significantly correlate (rs = 0.681, p < 0.001) with successful blastocyst implantation after embryo transfer. Syncytin 2 did not reach significance.

As expected as the H1 ES cells differentiated under BMP4 stimulation, shown by the expression of NANOG mRNA decreasing, and the expression of CDX2 and β-HCG mRNAs increasing asses by qRTPCR. The copy number of syncytin-1 increased significantly during differentiation.

The authors conclude that : Preceding the transfer of frozen embryos, the increased copy number of syncytin-1 in the blastocyst trophectoderm was associated with good outcomes of pregnancies.
Validity of the findings: Q1) The adhesion, attachment and infiltration of the blastocyst is mediated by the first syncytiotrophoblast originating from the fusion of the trophoectoderm layer.  The authors should state this as it is directly relevant to the function of syncytin 1 and 2 in trophoblast fusion. It could be mentioned in the discussion in the context of increased syncytin 1 and a successful IVF outcome being dependant on the ability of the blastocyst to implant.
Additional comments: Q2) the photos of the H1 cells don’t really add anything, H1 are known to default to syncytiotrophoblast under BMP4 stimulation. The paper would be strengthened by doing IHC for syncytiotrophoblast markers such as CK7, AlkP, E –Cad.

Q3) Suggest the clinical significance is addressed in the conclusion.


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: INCREASED COPY NUMBER OF SYNCYTIN-1 IN THE TROPHECTODERM IS ASSOCIATED WITH IMPLANTATION OF THE BLASTOCYST
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is the review report for the revised manuscript titled ‘Increased copy number of syncytin-1 in the trophectoderm is associated with implantation of the blastocyst’ by Guo et al. The manuscript is carefully revised and English was improved.
Experimental design: no comment
Validity of the findings: Major comments
The genomic copy number of syncytin-1 in differentiated H1 cells was only 1.5-fold (Figure 5), which was quite lower than that observed in pregnant embryos (4-5-fold higher than non-pregnant embryos) (Figure 1). Moreover, the copy number in H1 cells was seemed to be already plateau on D1 after differentiation even though the expression was continuously increased until D7 (70-fold). These data support the authors’ hypothesis that increase of syncytin-1 copy number is associated with the successful pregnant, however, it seems to be difficult to find the association between the increase of copy number and expression amount. The manuscript may get improved by discussing the other possible meanings of syncytin-1 transposition. e.g. affecting the relevant genes expression through epigenetic modification.

Minor comments
1. Lines 16-18 must be revised. Syncytin-2 is not the envelope protein of HERV-W but HERV-FRD. Please reconsider and modify the sentence.

2. ‘It did not increase the transcripts by changing…’ (Lines 333-334) needs to be modified to such as ‘Syncytin-2 transcription was not increased by changing…’.
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: INCREASED COPY NUMBER OF SYNCYTIN-1 IN THE TROPHECTODERM IS ASSOCIATED WITH IMPLANTATION OF THE BLASTOCYST
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is well-referenced and unambiguous with a clear hypothesis.
Experimental design: Matching the hypothesis, the experiment design has been refined to achieve rigorous scientific standards.
Validity of the findings: In this version of the manuscript, the impact and limitations of the results have been clearly stated and discussed.
Additional comments: A well-written resubmitted manuscript which incorporated all answers to previous reviewers' comments. Your findings set the scene for further scientific projects to improve clinical ART rates.


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: INCREASED COPY NUMBER OF SYNCYTIN-1 IN THE TROPHECTODERM IS ASSOCIATED WITH IMPLANTATION OF THE BLASTOCYST
Review round: 2
Reviewer: 3

Output:
Basic reporting: Thankyou to the authors for their extensive revisions to the paper.  The changes have significantly improved the readability of the manuscript.  All reviewers concerns have been fully addressed.
Experimental design: No concerns
Validity of the findings: No concerns
Additional comments: See above


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

Input:
Title: MIR-26A-5P INHIBITS GSK3Β EXPRESSION AND PROMOTES CARDIAC HYPERTROPHY IN VITRO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article includes adequate introduction and background to evaluate the work into the specific context of interest.
Experimental design: Methods were described with sufficient details. Supplementary data should be better described.
Validity of the findings: More experimental results are required to elucidate the correlation between autophagy and cardiac hypertrophy.
Additional comments: In this manuscript, Liqun Tang et al. propose to investigate the role of miR-26a-5p expression in the regulation of autophagy and cardiac hypertrophy.  This paper contains some interesting observations about the role of miR-26a-5p in cardiac hypertrophy, however there are a consistent number of concerns that need to be addressed by the authors to adequately validate these observations.
Major concerns:
1)	The correlation between the induction of autophagy by miR-26a-5p upon phenylephrine (PE) treatment and its ability to promote cardiac hypertrophy by reducing GSK3β expression is not discussed by the authors and further work is needed to better characterize the role of autophagy induced by miR-26a-5p upon phenylephrine (PE)-induced cardiac hypertrophy. It would be important to provide additional experimental evidences in order to assess whether the up-regulation of autophagy by miR-26a is effectively responsible for the cardiac hypertrophy stimulation. To this aim, the authors should analyze if the decreased expression of GSK3β observed during (PE)-induced cardiac hypertrophy in presence of miR-26a is affected by treating cells with the autophagy inhibitor 3-methyladenine or by silencing specific autophagic genes, such as Beclin1 or ATGs. (Fig. 2)

2)	The authors analysed the levels of lipidated LC3 after PE treatment in combination with miR-26a agomir or miR-26a inhibitor. In Fig. 1B it is shown that LC3-II/LC3-I ratio (please do not indicated this as LC3b/LC3a because these names refer to different LC3 genes) in PE-induced cardiac hypertrophy treated with miR-26a agomir group was higher than control groups. However, according to the published guidelines for monitoring autophagy (Klionsky DJ et al., 2016), the authors should analyse LC3 lipidation by measuring LC3II/GAPDH ratio.  In addition, according to the published guidelines for monitoring autophagy (Klionsky DJ et al., 2016), authors should analyse autophagy flux by comparing LC3 levels in the presence or absence of lysosomal inhibitors to rule out that the increase of LC3-II levels is due to a block in autophagosome-lysosome fusion. Moreover, authors should analyse autophagic cargo degradation by analyzing p62 protein levels, in the presence or absence of lysosomal inhibitors. (Fig. 1)

3)	The authors confirmed by in vivo experiments the ability of miR-26a to inhibit GSK3β expression and promote cardiac hypertrophy, but they did not perform any in vivo experiments on the autophagy regulation by miR-26a. To this aim, authors should analyse LC3 and p62 protein levels in this in vivo system. (Fig. 4)

4)	The authors showed that GSK3β expression is inhibited by miR-26a at transcriptional level. To validate this finding, authors should analyse also GSK3β protein levels in H9C2 cells and in SHR rats. (Fig. 2-4)

Minor concerns:
1)	All the supplementary data should be better organized and classified. In particular, the authors should provide more details on the supplementary tables where quantification data are present. It is not clear to which figures they refer and the meaning of the numeric values.
2)	Figure 2 title should be corrected in “MiR-26a inhibits GSK3β expression and promotes cardiac hypertrophy in vitro”

3)	The abbreviations should be explained with the full name the first time they appear in the main text:
-	PE    line 75
-	CK    line 134
-	SHR  line 179

4)	Line 163: mark should be corrected with marker

5)	Line 138 - 140 – 183 – 238: obviously should be removed


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

Input:
Title: MIR-26A-5P INHIBITS GSK3Β EXPRESSION AND PROMOTES CARDIAC HYPERTROPHY IN VITRO
Review round: 1
Reviewer: 2

Output:
Basic reporting: (1) Page 8 line 134: Please add the full name of short-form “CK”.
(2) Page 8 line 144-145: Through cell experiments, the authors concluded that "miR-26a promoted myocardial cell autophagy activation in PE-induced cardiac hypertrophy." However, as shown in Figure 1B-E, I find no significant changes of LC3b/LC3a levels in H9C2 cells transfected with miRNA-26a inhibitor after PE stimulation compared to cells stimulated with PE alone. In my opinion, this means that reducing miRNA-26a expression in cardiac hypertrophy has no effect on autophagy activation. I feel sorry that I cannot understand the statement, and I am worried that the figure may contradict the conclusion authors have drawn in this section. Please check the results carefully and, when necessary, supplement relevant description and analysis in this part.
(3) Page 16 Figure 1C-E: Please add error bars to the histograms and clearly mark the compared groups and P values.
(4) Page 17-18 Figure 2C: In figure legends, the authors stated that “the cardiac hypertrophy marker protein α-actinin (red)”. However, the actual immunofluorescence staining of α-actinin was green. Please correct this mistake.
(5) Figure 2C, Figure 3A, and Figure 4C: Please add appropriate scale bars to these figures.
(6) Page 19 Figure 3A: I couldn’t see what is in this figure. Please pay attention to this issue and provide clearer images.
(7) Page 21 Figure 4C: I find it difficult to clearly distinguish the difference of cardiomyocyte size between these two groups. Please provide a higher magnification image or quantitative analysis of cardiomyocyte size.
(8) Page 23 Table 1: Please explain why the listed sequence of primers for qRT-PCR contained homo primers such as Homo U6, Homo GSK3β, and Homo GAPDH since the experimental subjects were rat cardiomyocyte cell line (H9c2 cells) and rats.
Experimental design: (1) Page 7 line 114-116：It could be better to describe the animal experiments in more detail, including the age of the spontaneously hypertensive rats, the husbandry condition, and whether it complied with the animal experiment ethics. Furthermore, the results section mentioned the observation of PE-induced cardiac hypertrophy (Page 9 line 183), but the methods section did not mention the intervention of PE in rats. Please revise to ensure the uniformity of the experiment.
(2) Page 8 line 135-145: The primary antibody anti-LC3 (1:1000, 12741S, CST) involved in this study was used to recognize endogenous levels of total LC3A and LC3B proteins according to the manufacturer's instructions. In Western blot analysis, two bands with molecular weights of 16 kDa and 14 KDa appeared, which represent LC3A/B-I and LC3A/B-II, respectively. I couldn’t understand why the authors analyzed relative LC3a level, LC3b level, and LC3b/LC3a instead of LC3A/B-II/LC3A/B-I to observe autophagy levels.
In addition, it could be better to supplement the protein level of p62/SQSTM1, which is another commonly used autophagic marker.
(3) Page 8 line 152 and Page 9 line 177: The authors observed that MiR-26a inhibits GSK3β expression in cardiac hypertrophy, both in vivo and in vitro. So, does miR-26a directly target GSK3β? It could be better to explore and verify the downstream target genes of miR-26a.
Validity of the findings: Page 6 line 69-74：This section described the research background associated with miR-26a, including its protection of vascular smooth muscle cells from H2O2-induced injury, mediating allergic inflammation, and inhibition of neuropathic pain and neuroinflammation. However, I think these previous studies are not enough to be the reason for choosing miR-26a. Please investigate and supplement the progress of research on the role of miR-26a in cardiovascular diseases.
Additional comments: This is a good original primary research which sought to explore the role of miR-26a-5p in cardiac hypertrophy and its specific mechanisms. After detecting the high expression of miR-26a-5p in PE-induced hypertrophic cardiomyocytes, the authors further explored the mechanism by using miR-26a agomir and inhibitor to transfect H9c2 cells. Finally, miR-26a-5p was found to significantly inhibit GSK3β expression in vitro and in vivo, and also promote the autophagy activation of cardiomyocytes. The topic of this manuscript is timely and interesting, which provides new insights into the pathogenesis of cardiac hypertrophy. However, substantial efforts will be needed to improve the logic of the article, experimental design, and professionalism of figures. In conclusion, this manuscript cannot be accepted in the current situation. Perhaps, after major revisions, it can be accepted.


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: MIR-26A-5P INHIBITS GSK3Β EXPRESSION AND PROMOTES CARDIAC HYPERTROPHY IN VITRO
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In this manuscript Tang and colleagues studied the effects of miR-26a on cardiac hypertrophy in both in vitro, phenylephrine (PE) treated H9C2 cells, and in vivo, spontaneously hypertensive rats (SHR) models. They found that miR-26a-5p was highly expressed in PE-induced cardiac hypertrophy as well as the LC3 levels. Moreover, miR-26a agomir induced LC3 accumulation and the cardiac hypertrophy related protein α-actinin expression, while inhibiting the expression of GSK3β, a negative regulator of cardiac hypertrophy. Finally, the PE and the miR-26a agomir treatments increased the cellular hypertrophy both in vitro and in vivo.

Although the work is potentially interesting one of the main conclusion drawn by the authors (i.e.  miR-26a-5p activates myocardial cell autophagy) is not supported by the data presented.

In fact, the authors stated:

- Line 138. “protein expression levels of LC3-a and LC3-b were obviously elevated in H9C2 cells treated by 200 μM PE, suggesting that the autophagy was activated in PE-induced cardiac hypertrophy compared to controls”.
- Line 144. “Therefore, miR-26a promoted myocardial cell autophagy activation in PE-induced cardiac hypertrophy”.

The authors reported increased levels of LC3 protein upon PE treatment (+/- miR-26a agomir); this is not sufficient to state that these treatments induced the autophagic flux, in fact an increased LC3 levels could be due to either an increased autophagosomes formation or an inhibition of autophagosomes degradation. LC3 levels should be measured in presence of an autophagy inhibitor (NH4Cl; Bafilomycin etc.; See Rubinsztein DC et al. Autophagy. 2009 Jul;5(5):585-9).

Other main points:

-ABSTRACT. The authors stated “MiR-26a-5p was highly expressed in PE-induced cardiac hypertrophy. MiR-26a-5p promoted LC3b/LC3a ratio in PE-induced cardiac hypertrophy”.
Generally, the authors confused LC3-a and LC3-b (genes) with LC3-I and LC3-II (proteins) forms. Authors showed results only about LC3-I and LC3-II forms.

- Line 129. The authors stated “Firstly, we assessed whether the cardiac hypertrophy model was successfully built in vitro”. However, no data were presented about the suitability of the hypertrophy cellular model. Moreover, in figure 3B it seems that the PE treatment does not induce cellular hypertrophy.

-Figures 1C, 1D and 1E. How many experiments were performed? Standard deviations and statistic should be performed.

-The quality of figure 3A is too poor.

-Figure 4. The enlargement of cells of miRNA-26 agomir treated SHR rats is not clear, an enlargement of the picture is required.

-Figure 4. It would be very interesting to compare the levels of GSK-3 and miR-26a of SHR to those of healthy rats.

-Figure 4. It would be very interesting to measure LC3 levels in the SHR rats, treated with either miR26 agomir or NC agomir, and comparing them to those of healthy rats..

-Figure 4. It would be important to show, as a control, the levels of known miRNA-26 target genes, in both H9C2 cells and SHR rats’ heart

- In the Discussion section would be important for the readers to have hypothesis on how miR-26a can affect GSK3 and α-actinin.

-Text should be checked carefully by a native english speaker

Minor points

- Line 157. The author stated “To further assess the function of miR-26a in cardiac hypertrophy, we tested cardiac hypertrophy related mark protein GSK3β expression”. It should be stated clearly that GSK-3β is a negative regulator of cardiac hypertrophy.

- Line 182. The authors indicated “…in PE-induced cardiac hypertrophy treated……”, however SHR rats is a spontaneously Hypertensive Rats (SHR) model as stated by the authors in the Material and methods section.

-Figure 1A. What CK stand for? No indication is present in figures legend.
-Figure 2 “….. hypertrophy marker protein α-actinin (red)” should be “….. hypertrophy marker protein α-actinin (green)”
-Figure 4C. “miR-26a NC” should be “NC agomir”

- Lines 58/59. The authors stated “Hence, LC3-II/LC3-I ratio has been used to affect autophagy activity”; Actually, LC3-II/LC3-I ratio is used to measure autophagy levels and/or autophagy flux.


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: MIR-26A-5P INHIBITS GSK3Β EXPRESSION AND PROMOTES CARDIAC HYPERTROPHY IN VITRO
Review round: 2
Reviewer: 1

Output:
Basic reporting: no further comments
Experimental design: no further comments
Validity of the findings: no further comments
Additional comments: The authors should review the text and figures in order to replace the LC3II/LC3I ratio analysis with the LC3II/GAPDH ratio as requested.

Figure 1C - 1G – 6D: Did the authors analyze the levels of LC3II/GAPDH ratio? Do the new graphs refer to the measurement of the LC3II/GAPDH ratio? If so, the authors should modify the legend of the graphs in the figure and in the text that still report LC3II/LC3I ratio.

Manuscript Text that should be modified if LC3II/GAPDH results were reported:
Line 200, 203, 206, 207: LC3II/LC3I ratio should be corrected with LC3II/GAPDH or replaced by LC3II or lipidated LC3.
Line 210, 211: LC3 should be corrected with LC3II or lipidated LC3.
Line 216: LC3II/LC3I ratio should be corrected with LC3II/GAPDH.
Line 292: LC3II/LC3I ratio should be corrected with LC3II/GAPDH or replaced by LC3II or lipidated LC3.
Line 302: LC3II/LC3I ratio should be corrected with LC3II/GAPDH.
Line 335: LC3II/LC3I should be corrected with LC3II or lipidated LC3.
Figure 1F: Bar-A1 should be corrected with Baf-A1


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

Input:
Title: DIFFERENT ANALYSIS STRATEGIES OF 16S RRNA GENE DATA FROM RODENT STUDIES GENERATE CONTRASTING VIEWS OF GUT BACTERIAL COMMUNITIES ASSOCIATED WITH DIET, HEALTH AND OBESITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is written in rather clear and technically correct language. The introduction and background revealed the problem of reproducibility in microbiome studies and main factors that may have impact on the result of such investigations. However, please try to keep the same format of  in-text citations, preferably with comma before year (please compare 51-52 and 72 lines). Also I believe that not several but numerous studies have investigated the relationship of microbiome contents and diet, health and obesity (line 79) especially if we would search not only for studies done in humans but also in rodents.

I suggest to improve some phrases to ensure that an international audience can clearly understand the text. Some examples where the language could be improved include lines 23, 50 – the current phrasing makes comprehension difficult.

The shown results are sufficient to support the conclusions made by the authors.

The links to the raw data are supplied in acceptable form.
Experimental design: The research question is well-defined, relevant and very important. I suggest adding to the description of the studies which region of the 16S rRNA gene was sequenced and with which primers set.

Also used DNA extraction technique may be very relevant factor to study across the samples too. (Have you tested this factor? It could be added to the supplementary materials.) As it is known that method of DNA extraction may affect the reported microbial community structure it may be interesting to test this hypothesis.

Can you please, provide explanation why in some cases you have used QIIME which is not supported for several years instead of QIIME2?

Also you have tested influence of the factor ‘sequencing technique’, with two levels: pyrosequencing and MiSeq across the samples. As you wrote that there was only one study utilizing pyrosequencing and a set of primers that differs from all other studies it is worth to emphasize that the effect of the factor ‘sequencing technique’ can be observed for several reasons (different  sequencing technique, different set of primers, the study, or the combination of several of them).

Moreover, I suggest to test whether the quantitative levels of microbial taxa differed by sample source with more approaches then only PCoA. Especially, pay attention to the UMAP (Uniform Manifold Approximation and Projection) algorithm for dimensional reduction of diversity within large amounts of data by non-linear multidimensional clustering (McInnes et al., 2018) which is appropriate for such type of data and started widely used for such type of analysis.

Furthermore, on the PCoA are two the most definable clusters that are species-specific (mice and rats), it would be interesting to inspect only the mouse samples in order to find some more relevant clustering factors as they can be masked with the impact of the species specificity. It could be added to the supplements.

References:
McInnes L, Healy J, Saul N, Grossberger L. 2018. UMAP: Uniform Manifold Approximation and Projection. Journal of Open Source Software. 3:861. DOI:10.21105/joss.00861.
Validity of the findings: All the underlying data have been provided but I suggest to use not only PCoA but also UMAP for the patterns’ search.The aim of the study to assess the impact of different analysis strategies and several other factors on the resulting microbiome composition is achieved. The conclusions are well stated, and they are connected with the investigated question.
Additional comments: 1. Please pay attention that it is commonly suggested to test whether the quantitative levels of microbial taxa differed by sample source with multiple approaches including UMAP.
2. Please consider correcting the in-text citations and some hard for the comprehension phrases.
3. Add a little bit more relevant data about the studies (which region of the 16S rRNA gene was sequenced and with which primers set; the DNA extraction method in order to have an ability to test if there is batch-effect due to this factor).
4. It would be valuable to look for influence of the factor of DNA extraction method on the microbial community structure.
5. Please, provide in the text explanation why in some cases you have used QIIME which is not supported for several years instead of QIIME2.
6.I suggest to inspect additionally only the mouse samples in order to search for some more relevant clustering factors as they can be masked with the impact of the species specificity.


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

Input:
Title: DIFFERENT ANALYSIS STRATEGIES OF 16S RRNA GENE DATA FROM RODENT STUDIES GENERATE CONTRASTING VIEWS OF GUT BACTERIAL COMMUNITIES ASSOCIATED WITH DIET, HEALTH AND OBESITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clearly written and good structured. Raw data is available.
Experimental design: Research question is well defined, all analysis methods are described in sufficient detail.
Validity of the findings: This is very interesting and useful study about the influence of analysis parameters on the results. I do not have any major questions, only some minor:
1) For close-reference OTU-picking strategies does it have sense to add the number (or percent) of unused reads in each analysis? Could it appears, that only the minor fraction of reads was considered by these approaches? Also could the difference of the number of used reads between Closed97 and Closed99 strategies produce such changes in predicted bacteria phyla proportions shown in Table2? Because De novo and Open-reference strategies that use all reads give much more consistent phyla proportions.
2) It could be advantageous to add figures with PCoA using weighted UniFrac, because authors mentioned that it gives results that differs from obtained with unweighted one.
3) The number of observed OTU can be highly inflated because of chimeras, did authors consider to use any chimera filtering methods, especially for De novo and open-reference strategies?
One additional moment, that potentially is out of the planned scope of the manuscript. It could be very interesting to extend the analyses of the “study effect” to consider not only close-reference strategies, but also open-reference and especially DADA2 (of course for studies that use same 16s rRNA region).
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: DIFFERENT ANALYSIS STRATEGIES OF 16S RRNA GENE DATA FROM RODENT STUDIES GENERATE CONTRASTING VIEWS OF GUT BACTERIAL COMMUNITIES ASSOCIATED WITH DIET, HEALTH AND OBESITY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is written in a good English with the sufficient amount of background information. It has the professional structure and self-contained.
Experimental design: The research question is well defined, although it is very broad and requires much more data to be analyzed to be answered in full. The title is somewhat misleading as only rodent gut microbiomes were studied and it is not clear what health conditions other than obesity were implied. The current manuscript is an exploratory analysis of effects of various clustering strategies on OTU assignment to 16S sequences and factors relevant for explanation of variations in the data.
Validity of the findings: Authors used several strategies on binning 16S sequences to OTU with QIIME1 software and GreenGenes reference database and showed that the results are highly unstable. This is consistent with other reports (like Prodan et al. 2020 PLOS ONE, Alimeida et al. 2018 Gigascience) and general knowledge in microbiome community. Although the authors provided the links to 16S reads in the SRA it would be highly beneficial to attach processed OTU abundance tables as the direct foundation of the conclusions.

The factor analysis is consistent with the current knowledge, but with only 7 studies it is limited for generalization. Especially for factors “sequencing technique” and “anatomical site” because of the strong confounding effect with the "study" factor.
Additional comments: 1)	Although 16S sequencing data is provided through NCBI SRA it would be good to add full OTU tables in the supplement.
2)	I suggest changing title to better represent narrow scope of the research as only rodent gut microbiome was studied and the only health condition was obesity.
3)	On figures among treatments levels indicated “obese” and “lean”. Are they control groups treatments? If this is true – are control obese and lean mice had different treatment? In the table S1 indicated several diets for control groups, but in all studies lean and obese mice had the same diet. Please check your factor levels assignment.
4)	To put more weight for the factor analysis I suggest trying it only on control groups as it should show the study effect clearer.
5)	On line 186 indicated n=164 samples in the study although on line 191 used n=162 and n=163 samples. Why 1-2 samples were omitted?
6)	The line 346 stated that Cyanobacteria “showed the lowest SD among the OTU picking strategies”, however it has very low abundance and therefore low SD. It seems that this phrase could be deleted as do not lead to any conclusion.
7)	The line 360 states that explained variation could be found in the Table 4. However it does not contain any information on the explained variation.
8)	Line 447 “More importantly, the fact that each bacterial cell displays a high level of individuality raises doubts about the relevance of nucleotide similarities to estimate or predict similarities in nature.” It is not clear what does it mean exactly. Does it mean that bacteria with identical 16S can have substantial variance in genomes or bacteria with identical genome could exhibit different phenotype? Although it is a discussion it should be stated clearer.
9)	 In the Table 2 phrase “This data was obtained from the analysis of sequences using a 97% similarity against the reference OTU files.” is unclear as this table includes data from different approaches.
10)	In Table 4 it is not clear what percents are shown.
11)	Line 421 “Brüssow 2019” should be 2020.