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: RISK OF PHOSPHORUS LOSSES IN SURFACE RUNOFF FROM AGRICULTURAL LAND IN THE BALTIC COMMUNE OF PUCK IN THE LIGHT OF ASSESSMENT PERFORMED ON THE BASIS OF DPS INDICATOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript evaluates the performance of the universal DPS-index method for assessing the risk of phosphorus loss in surface runoff from agricultural land at the Baltic Commune of Puck. The manuscript is generally written is acceptable English, but there are many sentences where the writing style is not appropriate for scientific publication. The authors need to put significant effort into using the correct terms and avoid sentences like “(HCL solution is an aggressive extract, water – not)”.

The manuscript is very convoluted, particularly the “Results and Discussion” section, which is longwinded and difficult to read and extract the key findings and contributions. I suggest the authors to consider splitting this section into two sections, one for “Results” and one for “Discussion”. In the “Results” section, please simply describe and highlight the important results (plots and tables) and move the description of the case study to “Materials and Methods”. In the “Discussion” section, please examine your results in greater detail and compare your findings with the literature. This is the most important section of your paper and it should clearly identify the main contributions of your work (based on your results) to the scientific community – please build concise sentences and be direct to the point avoiding vague statements such as “, in environmental terms,”.

The tile does not reflect the actual contribution of the work presented. As said in the introduction “The aim of this work is to test the usefulness of the Pothig et al. (2010) method to assess the risk of P loss in…” I suggest the authors change the tile to something more informative, such as “Evaluating the performance of the universal DPS index method for assessing the risk of phosphorus loss in surface runoff from agricultural land: the case of the Baltic Commune of Puck”. This suggestion is a bit long, but this is just a suggestion for the authors to work on.

Finally, I also find that the scientific contribution of the paper is poor. The authors seem to have a substantial dataset in their hands – pH and P content for 61 farms. However, there is no map showing the location of these plots nor, for instance, an analysis of the spatial distribution of pH and P content - this is just an example of the type of work that could potentially be included to make the paper more interesting. Also, the measurements presented should be better described (e.g., when did these measurements took place) and should be related to hydrological processes - is there a temporal dependency worth to explore. Nothing is mentioned about the hydrology of the region, although it is well known that it can have a determinant effect on soil P concentrations. Also, to improve the quality of the manuscript, the authors could focus on improving the “universal” DSP-index method and propose a way forward – this would be a much stronger contribution.

For all the reasons above, I think that this paper requires profound improvements and, therefore, I recommend REJECTION, but I urge the authors to re-submit it after the issues identified have been addressed. I provide below many comments and suggestions that will help the authors to improve the quality of the paper.
Experimental design: The authors do not describe the experimental design in enough detail. I provide below examples of how this could be improved.
Validity of the findings: As said before, the "Results and Discussion" section is convoluted and needs major revisions. I provide below examples of how this could be improved.
Additional comments: GENERAL NOTES:
Line 39: I suggest changing slightly the portion “it was considered that the assessment based on the DPS index may be unreliable” to something more constructive or adding some more detail. For example: “…may be unsuitable for… (specify)”
Line 49: the citation (National…, 2016) is not properly handled. Probably use an acronym based on the what is in the reference listing, NWMB (standing for “National Water Management Board”).
Lines 51 to 58: In my opinion these sentences are unsuitable for a scientific paper. I would rephrase and remove for instance “made at the Conference of Ministers for the Environment of the HELCOM countries”
Line 63: Eutrophication parameters? What are these parameters? The authors should be more specific
Line 67: What “component”? Very vague; please make the sentence clearer
Lines 120-133: This is an article, not a report. In my opinion, the authors should adopt in-line numbering or simply integrate the information in “normal” sentences.
Line 136-150: This listing is unnecessary.
Line 155: I don’t think that “the results of laboratory analysis were also developed statistically”? Do you mean that statistical tests were performed on the lab results? Please clarify
Line 157: “Statistica 6”? The reader might not know what software this is. Please add a reference and maybe some details about what it does and what functionalities did you use for this particular study
Line 158-164: This whole paragraph is very confusing. Are you trying to say that you complemented your DSP-index analysis with a land use classification assessment? Please rephrase the sentence to be more direct to the point.
Line 167-169: This whole paragraph is not part of the results. It should be in the Materials & Methods section if not included there already. In this section (Results) focus only on the results.
Line 172-178: please remove this list and integrated the information in a proper sentence.
Line 179; This sentence is cumbersome. Please rephrase. And this is not really the results of this study. This is a general description of the study site and should me move to “Materials and Methods”
Line 195-196: what is P_ER and P_HCL? This seems to be the first time these are mentioned in the text so they need to be described.
Line 194-200: The whole paragraph is very cumbersome. E.g. “The available P content in the discussed layer of analysed soils…” and “P content in these in relation to 62.3% of plots was…”. Please revise profoundly.
Line 206: The authors need to improve the English significantly. What do you mean by “the P content in the shallower of the analyses”? are you referring to the topsoil?
Line 227: What do you mean by “The P content was most strongly correlated with 0-5 and 0-30 cm soil profiles,…”? This makes no sense. Do you mean correlated (or varied) with the soil depth?
Line 254: What do you mean by “in environmental terms”? Please just remove this type of statements all along as they don’t add anything and just make the text convoluted.

TECHNICAL NOTES AND SUGGESTIONS:
Line 21-22: suggest changing to: “… is a basic measure that should be included in action plans to counteract eutrophication in…”
Line 23: used either “, which” or “that”
Line 25: add “the” after “Based on”
Line 44-45: suggest changing to “… excessive river inflow of phosphorous (P).”
Line 45: add “estimated” after “… Baltic basin is”
Line 47: add “About” before “12.7 tons of…”
Line 61: replace “reasons” by “factors”
Line 63-64: replace “in” by “by” before “42%” AND add “by” before “66%”
Line 88: I suggest rephrasing this sentence to something like “This method is attractive because it is simpler and has the potential of being applicable to different soil types.” It is important to write this sentence carefully because it will create a certain expectation in the reader, and as said in the abstract “… the DPS index may be unreliable”. The message and objective of the paper should be clearly articulated throughout the paper.
Line 89-92: In this sentence “In the MONERIS model,… , it is particularly used…” it is unclear what “it” is referring to.
Line 93: I suggest replacing the work “usefulness” by “ability” AND replace “the Pothig et al method (2010)” by “the method proposed Pothig et al. (2010)”
Line 93: “method” should go after “(2010).
Line 94-95: Rephrase to something like: “This was accomplished by…”
Line 101: replace “The” by “This”
Line 101-107: Please rephrase this paragraph. Some suggestions: remove “by nature” AND replace “danger” by “risk” AND link better the different sentences
Line 108-114: this information is not relevant for the purpose of this paper. The purpose of this paper is to inform other scientists about the performance of a method, so please remove everything that isn’t important for this goal - just say that the study presented in this paper is part of a larger project/study and add a reference (and remove everything else)
Line 134: Replace by “… according to the following criteria: (1)…”
Line 203: add “the” before “P content”
Lines 187-188: no need to capitalized “manganese”, “iron” and “aluminium”


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: RISK OF PHOSPHORUS LOSSES IN SURFACE RUNOFF FROM AGRICULTURAL LAND IN THE BALTIC COMMUNE OF PUCK IN THE LIGHT OF ASSESSMENT PERFORMED ON THE BASIS OF DPS INDICATOR
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors made a significant effort to improve the paper based on the comments provided by the two previous reviewers. However, I still think that the paper doesn't meet the minimum requirements for publication as grammar errors can still be found throughout the paper, as well as unclear sentences. Furthermore, I think that the paper is long-winded, which makes it very difficult to follow, particularly the Discussion section. I tried to provide suggestions on how to improve the overall quality, but it took me a long time because there are really too many English problems, as there were in the first version.
I also have concerns regarding the scientific contributions of this paper. It is said that "It should be recognized that the risk level of P losses in surface runoff from the analyzed plots determined by the DPS indicator is uncertain. This uncertainty is increased by the lack of linking the DPS index with other significant factors in P runoff losses such as type of crop and area inclination." However, the title of the paper is "Risk of phosphorus losses in surface runoff from agricultural land in the Baltic Commune of Puck in the light of assessment performed on the basis of DPS indicator”, which seems to be in conflict. If the method used is uncertain for the main objective of the paper, what is the contribution of the paper “risk of phosphorus loses?
The authors may still be able to publish the paper, but they’ll need to identify the really valuable contribution of their research work, which seems to be more on the applicability of the DPS indicator across different regions? One possibility could be to change the title to “Use of the DPS indicator for risk assessment of phosphorus losses in surface runoff: evaluation of the method and application to the Baltic Commune of Puck”, and the authors would need to make a significant effort to really identify the key contributions (take away messages) from this research, and maybe create sub-sections in the Discussion section with titles that summarise those key points and, then, only bring the critical information necessary to support that the conclusions in that sub-section inside “Discussion”.
Experimental design: No major comments here.
Validity of the findings: No major comments here.
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: RISK OF PHOSPHORUS LOSSES IN SURFACE RUNOFF FROM AGRICULTURAL LAND IN THE BALTIC COMMUNE OF PUCK IN THE LIGHT OF ASSESSMENT PERFORMED ON THE BASIS OF DPS INDICATOR
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors made a great effort to improve the overall quality of the manuscript. The manuscript is now well written and much more readable. I continue to disagree with the authors about the Discussion section, which I continue to think that it should be split into sub-sections. Presently, this section is 7 pages long, with the authors discussing different aspects of the results; one after the other. I provide again below some suggestions for ways in which that section could be split into sub-sections. This would also help to highlight the key messages as the authors go through their data, as well as make the whole section more digestible. This is my honest opinion, but I will leave the final decision on this matter to the authors. I provide below also a few minor suggestions, but I recommend "minor changes" to this manuscript as I think that the paper is now of good quality and ready for publication pending the authors addressing the few points listed below.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: 1) Line 100: remove “in the”
2) Line 231-233: I would elaborate on the spatial heterogeneity a bit more instead of saying only that it’s very heterogeneous. For example, it seems that the soil in the northwest areas tends to be more acidic and have a lower P content when compared to the southeast areas. Maybe you can elaborate on why that could be happening.
3) Discussion section: The authors do not agree with me in that the Discussion section should be divided into sub-sections. I respect the decision of the authors, although I utterly disagree.  The discussion section is seven pages long; isn’t there a way to break this into smaller sub-section that would help to highlight the key messages? I can clearly identify a few:
3a)	DPS index assessment suggests a high risk of P loss (lines 346-359)
3b)	Fertilizer use to protect plant yields (lines 219-233)
3c)	Comparing the DPS indicator to agronomic test methods
3d)	….


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: ASSESSMENT OF A TAKAGI–SUGENO-KANG FUZZY MODEL ASSEMBLY FOR EXAMINATION OF POLYPHASIC LOGLINEAR ALLOMETRY
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have formulated a rather complex protocol for converting problems of “complex allometry” into ones of “polyphasic, loglinear allometry.”  Inasmuch as the new routine is based on logarithmic transformations, I see no real improvement over existing methods, all of which are flawed at some level or another by the transformation itself.  If the authors are interested in the scaling relationship between, say, surface area vs. mass of eelgrass leaves, then these are the data that need to be examined graphically and then studied analytically --- not their logarithms.  In both “complex allometry” and “polyphasic allometry,” the departure from linearity in log domain reflects on failure to identify the proper functional form for the allometric equation on the arithmetic scale.  The present study does not address that problem.


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: ASSESSMENT OF A TAKAGI–SUGENO-KANG FUZZY MODEL ASSEMBLY FOR EXAMINATION OF POLYPHASIC LOGLINEAR ALLOMETRY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper describes an interesting modeling approach. The topic is attractive. The theory is presented at the minimum understandable level and the application is trying to support the theory. The paper has potential to be appreciated by the readers and I suggest its improvement in terms of the general comments to the authors.
Experimental design: Please read the general comments to the authors.
Validity of the findings: Please read the general comments to the authors.
Additional comments: 1) The main contribution must be specified.
2) The motivation of the approach must also be specified in the context of other similar modeling approaches. Authors' strong well-acknowledged results in the field should also be included in this discussion.
3) Discussion of related work on other nonlinear modeling approaches including fuzzy ones should be extended with the following papers, which recently came into my attention because they proved to be successful in various applications:
- A review on information accessing systems based on fuzzy linguistic modelling, International Journal of Computational Intelligence Systems, vol. 3, no. 4, pp. 420-437, 2010.
- New results in modelling derived from Bayesian filtering, Knowledge-Based Systems, vol. 23, no. 2, pp. 182-194, 2010.
- Predicting tDCS treatment outcomes of patients with major depressive disorder using automated EEG classification, Journal of Affective Disorders, vol. 208, pp. 597-603, 2017.
- Model-free sliding mode and fuzzy controllers for reverse osmosis desalination plants, International Journal of Artificial Intelligence, vol. 16, no. 2, pp. 208-222, 2018.
4) The paper contains some grammar problems. Their correction is needed.
5) Please arrange the modeling approach in terms of an identification algorithm with clear steps.
6) What about the convergence of the identification algorithm? Please discuss.
7) This is an application paper. It is not clear how the theory from the previous sections is applied in the application one. More details are necessary. The comment 5) helps.
8) Transitions from section to section should be smoother. The comments 3) and 7) help.
9) More details on the models are needed. How did you train them?


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: ASSESSMENT OF A TAKAGI–SUGENO-KANG FUZZY MODEL ASSEMBLY FOR EXAMINATION OF POLYPHASIC LOGLINEAR ALLOMETRY
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment. Some really minor issues are described in my comments to authors.
Experimental design: Some statistical tests are used without explanations. The "SC" method also does not have clear explanations. These methods should be explained in more detail prior to the manuscript being considered further.
Validity of the findings: no comment
Additional comments: Dear authors,

Here I completed reviewing a manuscript entitled "A Takagi-Sugeno-Kang fuzzy model assembly for examination of weighted polyphasic loglinear allometry", submitted for PeerJ. This study looks at a highly controversial topic of whether the original arithmetic scale or the geometric scale should be used in studies of allometry. Authors proposes the Takagi-Sugeno-Kang fuzzy model (hereafter referred to as TSK) as a universal model that includes both the fit of non-linear model on arithmetic scale and the fit of a linear model on geometric scale as special cases. Authors apply TSK for empirical data to demonstrate that the method identifies and estimates parameters of polyphasic loglinear allometry only when breakpoints are clearly visible. Based on these results, authors claim that TSK is a useful model in allometric studies that reconciles the heated debate on the use of log scale in allometry.

I like the general idea of this manuscript. Especially I agree with authors' view that a method that inclusively treats issues of scaling should eventually resolve the current division of opinions in the field. The presented method appears to be generally credible, and the validation of the method are conducted with full transparency (original data and source code are available). Results suggest that TSK performs well in all three data examined.

Overall, the presented manuscript would fill an important knowledge gap in studies of allometry and its relevant fields. However, there are a couple of shortcomings in the current manuscript. They should be given further treatments before the manuscript will be considered further.

---------------------------------- start

1. The biological context in studies of allometry

In the introduction, authors provides a concise overview of the issue of scaling in allometry. This has generally a good coverage of relevant papers and well written, but one important aspect is missing, which is about the biological context in studies of allometry. One of the most fundamental discrepancies between the proponents of multiple parameter complex allometry on arithmetic scale, namely Gary Packard and his collaborators, and supporters of traditional allometry (TAMA, in your manuscript) lies in the degree of appreciation of biological theory in place of statistical correctness (e.g. Lemaître et al. 2015 Biol. Lett. 11:20150144, Pélabon et al. 2018 Biol. J. Linn. Soc. 125:664-671). As I describe below in comments on the use of visual inspection, this study, being a totally legitimate and scientifically correct endeavor, still has a great possibility to nourish the "useless" divide between two schools of allometry researchers. The fundamental issue is that this study focuses on the argument that TSK provides a good statistical fit to the data, based on AIC, r2 etc. We can easily find models based on multi-parameter complex models of allometry on arithmetic scales that fits data equally well, or even better than TSK. Thus, if we are to judge the validity of models based on statistical fit alone, TSK may well not be the best model of allometry.

I thus suggest authors to be explicit that the appreciation of biological theory is one major axis of polarization between the two schools. Then, briefly argue that TSK could be a highly biologically meaningful model of allometry, because it can model the breakpoints while keeping the meanings of allometric exponents as Huxley's original formulation. This is the key part, in my view, that is lacking in the current manuscript. If authors are unfamiliar with these arguments, I suggest reading Lemaître et al. (2015), Pélabon et al. (2018) as a starting point. Also, pp. 19-20 of Houle et al. (2011, Q. Rev. Biol. 86:3-34) is an excellent material to read.

2. Similar existing models

Authors introduces TSK as a novel model in studies of allometry, which I certainly agree, but the current manuscript does not give credit to existing approaches to fit polyphasic loglinear allometry. For instance, various methods for "the broken-stick" or "the broken-line" regression are available (e.g. Muggeo, 2003 Statistics in Medicine 22:3055-3071) and applied in allometry studies (e.g. Tsuboi et al. Nat. Ecol. Evol. 2:1492-1500). These methods are trying to solve the very same issue that TSK incorporates. Authors therefore should 1) briefly review existing broken-stick models, and 2) explain what is the uniqueness of TSK, and what is specific benefit(s) of using TSK over existing models.

3. Visual inspection as part of the proof

Authors' argument on the validity of TSK in fitting empirical data is based on a combination of statistical support (AIC, r2, etc.) and visual inspection of data in log-log plot. Proponents of non-linear models in arithmetic scales would immediately claim that the visual inspection should be made on the original arithmetic scales, and in this scale, the breakpoints seen in the log scale will most likely disappear. As such, the argument will fall back to an unproductive loop that authors courageously try to solve.

My suggestion is to examine and discuss whether the breakpoints estimated by TSK could be understood in biologically meaningful manners. An important difference between complex multi-parameter allometry model and polyphasic loglinear allometry is the existence of breakpoints in the latter. Because the goal of allometry study is to understand the biological processes that generate covariance between traits, the polyphasic loglinear approach, including TSK, should be favored regardless of statistical fit if the breakpoint has important biological meanings.

----------------------------------

Other comments

line 69: "In" spite of

Line 72-73: What is the exact unreliability of TAMA in this example? Please be specific.

Line 85-97: Is this a problem? If so, why?

Line 152: "Analyses"

Line 267 onwards: What is SC? This is not introduced anywhere earlier, but it seems to be very important step that analytically determines the number of breakpoints (q). This must be explained in details, either in Appendix or in the method section.

Line 272: "established"

Lines 352-374: in this paragraph, several statistical tests are presented without explanations of what they are. Let's take one example, "an Anderson-Darling test", in line 363-365. The value of A, as well as its associated p-value is absolutely meaningless without readers knowing what the test is, what the A is, and what the null hypothesis is. Thus, all of these tests here and elsewhere should be briefly explained in the method section.

Line 378: I guess you mean "b" not "d"

Line 382-383: the sentence ", while... heteroscedasticity" is redundant, so should be removed.

Line 488: Akaike's information "criterion"

Line 497: remove ", of Akaike's AIC index (Akaike, 1974)" to avoid redundancy

Line 594: "Kerkhoff"

Line 671: The term "glowing" seems to be incorrect here, but I am not sure what authors want to deliver in this sentence. Please read it again, and rewrite it accordingly.

---------------------------------- end

I hope my comments will help improving the manuscript further.

Sincerely yours,


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: ASSESSMENT OF A TAKAGI–SUGENO-KANG FUZZY MODEL ASSEMBLY FOR EXAMINATION OF POLYPHASIC LOGLINEAR ALLOMETRY
Review round: 2
Reviewer: 1

Output:
Basic reporting: I agree with the modifications done and the responses to my comments. The paper is improved and meets the level of publication in this journal. Therefore, I suggest again the publication of this paper.
Experimental design: I agree with the modifications done and the responses to my comments. The paper is improved and meets the level of publication in this journal. Therefore, I suggest again the publication of this paper.
Validity of the findings: I agree with the modifications done and the responses to my comments. The paper is improved and meets the level of publication in this journal. Therefore, I suggest again the publication of this paper.
Additional comments: I agree with the modifications done and the responses to my comments. The paper is improved and meets the level of publication in this journal. Therefore, I suggest again the publication of this paper.


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

Input:
Title: ASSESSMENT OF A TAKAGI–SUGENO-KANG FUZZY MODEL ASSEMBLY FOR EXAMINATION OF POLYPHASIC LOGLINEAR ALLOMETRY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear authors,

I deeply impressed by authors' serious effort on revising this manuscript in response to my and other reviewers comment. All my comments are satisfactorily responded. I only have a few minor suggestions that may further clarify your point. Please see attached file for detail of my suggestions.

Otherwise, I just want to leave one final comment on your manuscript. Although I fully see the value of your model and I agree with you that TSK model would be preferred over MPCA in many cases of biological research, there are some occasions in which MPCA can be biologically reasonable model. I can think of at least two.

First, MPCA can model a situation in which the initial timing of development of the trait itself and overall size are very different. This situation will generate negative intercept that cannot be modeled in the log-domain. Second, the error structure can be additive while the biological process underlying allometry is multiplicative. This situation also calls for analyses on the arithmetic scale or modeling of heterozchedastic errors in the log scale. These are two situations that I know of, but there might be potentially more of these. Empirically, these situations may not be very common in biology but they "can" exist. If I were to add anything to the manuscript further, I would make it explicit that MPCA can be biologically insightful.

Congratulations for your fine work. I am looking forward to seeing this study published.

Sincerely yours,


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: SV-CALLERS: A HIGHLY PORTABLE PARALLEL WORKFLOW FOR STRUCTURAL VARIANT DETECTION IN WHOLE-GENOME SEQUENCE DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors propose the workflow for structural variant detection in whole-genome sequence data. It is necessary and useful, however, there are some concerns that need to be addressed:
- The system can detect general variants or specific variants for cancer? Because as shown in the title, it maybe detect the general variants, but in the text, the authors mentioned a lot in cancer.
- Can the system classify the structural variants (i.e., deletion, insertion, ...)?
- The system can be extended to be used in the other operating system apart from Linux. It is necessary to have broader users.
- As many similar systems can support the use of GPU in running, can we config the "sv-caller" for using GPU?
- The most important thing is that the authors have to provide more comparisons and discussions on the previous workflows. There are a lot of published systems for variant calling, what are the advantages and disadvantages of this workflow?
- I have checked that I cannot access the link of Research Software Directory (https://research-software.nl/software/sv-callers), please check that it is always active.


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: SV-CALLERS: A HIGHLY PORTABLE PARALLEL WORKFLOW FOR STRUCTURAL VARIANT DETECTION IN WHOLE-GENOME SEQUENCE DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors has implemented an SV discovery workflow that integrated multiple publicly accessible SV algorithms for easy application. This tool is reliable and extendable, making it easier for users to generate SV calls. However, assessment of the tool, as represented in the manuscript, were mostly focused on the computational efficiency but lack discussion about quality of the SV callset generated from this tool. The authors should have provided a comprehensive evaluation of the tool, with comparison against individual algorithms, based on the two samples discussed in the manuscript to show superiority. Moreover, the content is not well organized in this manuscript as is confusing to read. Serious re-format is necessary here.
Experimental design: Whole design of this manuscript focused on improving computational efficiency of SV calling algorithms, but didn't provide enough details about important aspects such as  1. SV comparison criteria adopted   2. benchmark the proposed method to other existing ones
Validity of the findings: Kuzniar et al aimed at building a reliable tool for SV discovery in germline and cancer genomes. They proved reliability of their tool, however, failed to provide enough evidence that the performance of their workflow are of high sensitivity and low false discovery rate.
Additional comments: Major point:

Kuzniar et al. mostly focused on building a user-friendly, high efficient workflow for SV discovery via integration of different SV calling methods, however, little results were provided about the quality of output SV callset. These following points should have been addressed in both method and result :
1.	explain the criteria adopted for the comparison of SVs from different algorithms;
2.	provide evaluation of SVs from each algorithms, and explain their strategy of filtering SVs.
3.	Most importantly, the authors should provide enough evaluation of the callset from their workflow, show the increase in sensitivity and specificity of their workflow compared against others.

The method part includes information that should have been moved out to other sections. Eg. The ‘Workflow implementation and deployment’ section under method need serious re-writing, the introduction and discussion of snakemake would fit better under ‘Introduction’, while the discussions about figure 1 should be under ‘methods’.

Minor points:
1.	In line 176, author mentioned that time cost of the tool on different system differ substantially, and this difference were contributed by data size and load of the computing system. This reads confusing,  as for a fair comparison the same dataset should have processed then why is there a difference in data size? And how many jobs were processed in parallel on each system?
2.	Which computing system were number in table 2 concluded from?


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: SV-CALLERS: A HIGHLY PORTABLE PARALLEL WORKFLOW FOR STRUCTURAL VARIANT DETECTION IN WHOLE-GENOME SEQUENCE DATA
Review round: 2
Reviewer: 1

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


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

Input:
Title: SV-CALLERS: A HIGHLY PORTABLE PARALLEL WORKFLOW FOR STRUCTURAL VARIANT DETECTION IN WHOLE-GENOME SEQUENCE DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors present sv-callers, a computational workflow managing execution of multiple SV-detection tools for ensemble analysis of a given NGS dataset. The code is made publicly available and free to use without limitations. This manuscript includes links to example output from the software, along with a Jupyter Notebook for others to reproduce their results. The software is readily configurable for additional SV-callers and runs to completion on multiple HPC infrastructures. The manuscript's narrative is clear and easy to read. An attentive reader of this manuscript will come away with awareness of a new tool available for use in their research.
Experimental design: There is a need in the community for workflows like sv-callers. The authors cite competing tools by English, et al. (2015) and Fang, et al. (2018), The authors claim that sv-callers "enables analyses that would otherwise be difficult and/or slow to perform." While this may be true, the manuscript provides no evidence supporting this conclusion. Otherwise, the questions answered by the authors address runtime, resource utilization, and whether the output is usable in downstream applications (i.e. R), none of which qualify as meaningful and relevant research questions in the fields of Biological, Environmental, Medical, or Health Sciences Sciences.
Validity of the findings: no comment
Additional comments: Reviewer 2 gave the authors an opportunity to augment their findings with an interpretation of their results in light of a research question, i.e. "how accurate are the merged calls?" In response, the authors demurred, suggesting an effort of massive proportions (e.g. Kosugi et al.) would be required. Even if the authors manually review 20 deletions contained in a NA12878 SV truth set (e.g. Parikh H et al. BMC Genomics. 2016 Jan 16;17:64) some semblance of an answer would emerge with allowances for uncertainty based on the small sample size.

This reviewer hopes the authors will make the suggested (or equivalent) effort, and increase visibility of their software via publication in PeerJ.


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

Input:
Title: DIAGNOSIS AND TREATMENT OF EPIDIDYMAL TUBERCULOSIS: A REVIEW OF 47 CASES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article entitled "Diagnosis and treatment of epididymal tuberculosis：A review of 47 cases" is interesting and usefull.
English should be inproved.
List of references is sufficient.
Information about patients is incomplete. Authors missed some important data. I have done my comments and recommendations in the text of this article.
The quality of figures is poor.
Experimental design: This study is a simple retrospective non-comparative. Authors analysed 47 cases of histologically-confirmed epididymal tuberculosis in patients treated in the hospital for four years - and it is rich experience.  They have got their purpose - but result ic incomlete, as some important data were missed.
Validity of the findings: The study was descriptive, and in Conclusion there are no clear specific recommendations, common well-known words only. The data were good - but analysis and conclusion poor.
Additional comments: I support this study and wish success to authors, but this article need major revision. Sure, this may be brilliant.


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: DIAGNOSIS AND TREATMENT OF EPIDIDYMAL TUBERCULOSIS: A REVIEW OF 47 CASES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The Manuscript is clear and unambiguous, professional English used throughout except little mistakes
Literature references, sufficient field background/context provided except sourse marked in RED
Professional article structure, figures, tables. Raw data shared
Self-contained with relevant results to hypotheses.
Experimental design: Well done.
No comment
Validity of the findings: No comment
Additional comments: Well done interesting work. However please correct the text accordind changes marked RED. Please, add reference Banyra O, and Shulyak A. Acute epididymo-orchitis: staging and treatment. Cent European J Urol. 2012; 65(3): 139–143


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: DIAGNOSIS AND TREATMENT OF EPIDIDYMAL TUBERCULOSIS: A REVIEW OF 47 CASES
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This peice of Journal has education purposed, therefore it's agreed upon to be published.


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: DIAGNOSIS AND TREATMENT OF EPIDIDYMAL TUBERCULOSIS: A REVIEW OF 47 CASES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Congratulation, now it is perfect!


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: DIAGNOSIS AND TREATMENT OF EPIDIDYMAL TUBERCULOSIS: A REVIEW OF 47 CASES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment'
Experimental design: no comment'
Validity of the findings: no comment'
Additional comments: WELL DONE!
CONGRATULATIONS


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: DIAGNOSIS AND TREATMENT OF EPIDIDYMAL TUBERCULOSIS: A REVIEW OF 47 CASES
Review round: 2
Reviewer: 3

Output:
Basic reporting: Clear and unambiguous, professional English used throughout. Also have enough reference.
Experimental design: no comment
Validity of the findings: Conclusions are cleared and acceptable.
Additional comments: This piece of journal has education purposes, therefore it is agreed upon to be published


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: CHANGES IN LONG NON-CODING RNA TRANSCRIPTOMIC PROFILES AFTER ISCHEMIA-REPERFUSION INJURY IN RAT SPINAL CORD
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. English is used appropriately.
2. Literature references and background are provided sufficiently.
3. The quality of several figures should be greatly improved, such as Fiugure 4 and Figure 5.
4. The results positively support the scientific hypotheses. More lncRNA should be validated using qPCR.
Experimental design: The experimental desigh is reasonable.
Validity of the findings: More lncRNAs and mRNAs should be validated using qPCR.
Additional comments: This author Zhibin Zhou et al have provided the lncRNA and mRNA expression profiles after SCII and validated the differential expression lncRNA and mRNA. They further used bioinformatics tools to analyze the DElncRNAs and DEmRNAs. Generally, the article have some referenced value for developing novel diagnostic or therapeutic biomarkers for SCII. However, there are still some issues to be solved before publication.

Major revision
1. In Materials & Methods section, the number of the rats that were assigned into SCII group and sham group should be noted. What’s the L4 and L6 mean?
2. The primer sequences of the lncRNA and mRNA that were validated in qPCR assay should be provided.
3. The quality of several figures should be greatly improved, such as GO and KEGG figures.
4. More lncRNAs and mRNAs should be validated using qPCR.


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: CHANGES IN LONG NON-CODING RNA TRANSCRIPTOMIC PROFILES AFTER ISCHEMIA-REPERFUSION INJURY IN RAT SPINAL CORD
Review round: 1
Reviewer: 2

Output:
Basic reporting: Zhou et al. screened genome-wide expression patterns of lncRNAs and mRNAs in spinal cords from three SCII groups and three control tissues of the sham-operated group rats via RNA sequencing. They did gene ontology (GO) and KEGG pathway analyses as well as built co-expression networks to explore the functions of DElncRNAs and DEmRNAs.
RNA-seq is a powerful tool that has been used to identify novel protein-coding and non-coding RNA transcripts involved in the regulation of gene expression. Noncoding RNAs (ncRNAs) comprise a majority of the genome and play important developmental and physiological roles, as well as roles in disease mainly by regulating gene expression. Long noncoding RNAs (lncRNAs) are traditionally defined as having greater than 200 nucleotides. Accumulating evidence has shown that lncRNAs have various biological functions in RNA processing and translation and act as important gene expression regulators, however, few studies have explored the important role of the neuroprotective effect LncRNA of the spinal cord during ischemia-reperfusion injury.
Zhou et al findings could provide basic functional research on the lncRNAs involved in SCII and might be critical for providing new insights into the identification of potential targets for SCII therapy.

I have several major comments about this work as follows:

1.	Your introduction needs more detail. I suggest that you provide more justification for your study and mention other similar studies. Specifically, you should expand upon the knowledge gap being filled. For example, there is similar study “https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488167/” and “https://www.ncbi.nlm.nih.gov/pubmed/29998804“ I suggest the authors compare the result with previous similar studies.
2.	The full bioformatics pipeline, the commands used and the parameters need to be provided. I would like to be able to see and verify the main analysis of the paper.
3.	Also, the raw data should be uploaded in a public repository and a link provided in the paper
4.	 The authors need to be much more clear in the exact steps taken to do the statistical calculation. For example, the approach used for the prediction of cis- and trans- regulated genes of LncRNAs is not written. It should be written in detail.
5.	The authors claimed to have seen 1455 DELncRNAs and 6707 DEmRNAs genes, while previous studies had a much smaller number of DE genes. How do the authors explain this discrepancy?
6.	The MDS or PCA of the samples are needed for both LncRNAs and mRNA.
7.     I would like to see additional figures for the figures 5A, 5B and 7B to have the over-represented p-value adjustment instead of p-value and enrichment score.
Experimental design: I have several major comments about this work as follows:
1.	Various cytokines peak in the serum within the first 24 hr of ischemia-reperfusion injury. It needs justification of why the authors decided to study 48 hours after the reperfusion to harvest L4 to L6 spinal cords?
2.	The number of samples (n=6) is not enough for making a strong claim. I do understand that it might be not feasible to have additional samples, authors at least need to discuss this issue in the paper.
3.	SCII rat models have a bigger spinal cord infarct zone than the control rat spinal cord. I was wondering if the authors provide this factor during their experiment.
4.	Which neurological function assessment they have done during their experiment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: CHANGES IN LONG NON-CODING RNA TRANSCRIPTOMIC PROFILES AFTER ISCHEMIA-REPERFUSION INJURY IN RAT SPINAL CORD
Review round: 1
Reviewer: 3

Output:
Basic reporting: I think the figures were not of sufficient resolution, which should be improved.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Zhou et al investigated the differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) in rat spinal cords in the SCII model by high-throughput RNA sequencing method. They found that SCII could reshape the expression patterns of LncRNAs and mRNAs in the rat spinal cord after SCII. These results provide a lot of potential molecules that may play some role in post-SCII pathophysiological processes. This is a well-performed study with correct bioinformatics approaches and correct statistical analysis. I only have some minor comments that need to be considered by the authors before accepted for publication.

1 Although English is of a very good standard, some minor errors remain. A thorough check should be performed by the authors
2 Clear, and Consistent font type and size should be used in the author's figures.
3 Figure 1.  C and D should have the same size.
3 The resolution of Figures 4, 5 and 6 should be improved.


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

Input:
Title: CHANGES IN LONG NON-CODING RNA TRANSCRIPTOMIC PROFILES AFTER ISCHEMIA-REPERFUSION INJURY IN RAT SPINAL CORD
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The author has revised the manuscript according to my comments. In my opinions, the manuscript could be accepted just before some minor revisons are made.
1. The author forgot to describe the validation results of DElncRNA and DEmRNA by qPCR. Please add the corresponding content.
2. The criteria for screening the DElncRNA and DEmRNA is contradictory in Abstract, Materials & Methods and Results. FC ≥1.5 or FC ≥2.0? Why the author choose this criteria for screening the DElncRNA?
3. In the discussion, it is better to provide more directions about how to study the DElncRNAs in this research in the future. The author could tell us which lncRNA is the potential diagnostic or therapeutic target for ischemia-reperfusion injury.


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: CHANGES IN LONG NON-CODING RNA TRANSCRIPTOMIC PROFILES AFTER ISCHEMIA-REPERFUSION INJURY IN RAT SPINAL CORD
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have adequately responded to my comments. I have no further comments.
Experimental design: No further comments.
Validity of the findings: No further comments.
Additional comments: No further comments.


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

Input:
Title: CHANGES IN LONG NON-CODING RNA TRANSCRIPTOMIC PROFILES AFTER ISCHEMIA-REPERFUSION INJURY IN RAT SPINAL CORD
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed all of the criticisms. I have no additional comments and suggest acceptance.


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

Input:
Title: THE EFFECT ON TWINNING RATE OF TRANSFERRING DOUBLE VITRIFIED-WARMED EMBRYOS IN WOMEN OF ADVANCED REPRODUCTIVE AGE: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written and clear, with a good languange property.

The background and literature review is sufficient, but due to the year of the data, the 2017 ASRM guidelines for the number of embryos to transfer were not cited.
At the same time, the policy of double embryo transfer in the population is not acceptable for every institution all over the world and this can be a big problem when you try to make your research interesting to different public.

The first table is not cited in the text, and it could be substitute in part the description of the prognosis criteria in the Subjects part.
Experimental design: the research question is clearly defined and interesting: reducing the twinning rate is a goal of every ART center.
This is a retrospective study with all the known problems.
Methods are clearly presented.
Validity of the findings: At the other hand the statistical approach is quite poor: the stratification process and the Chisquare analysis does not permit to fully considered the conclusions as statistically robust.
A review with a professional statistic is much needed.
Additional comments: The article is interesting and very clear, even with a quite poor statistical approach.
The only limitation is that the embryotransfer policy is based on criteria that are not accepted in other countries so the findings are not completely exportable in other contexts.


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

Input:
Title: THE EFFECT ON TWINNING RATE OF TRANSFERRING DOUBLE VITRIFIED-WARMED EMBRYOS IN WOMEN OF ADVANCED REPRODUCTIVE AGE: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are minor grammatical mistakes in some places.

     1. In the last sentence in the Introduction section, "...the number of previous cycle ..." should be replaced by "...the number of previous cycles ..." and so on.

     2. Line 138, "continuous normal distributed" should be replaced by "continuous normally distributed."  Please go over the article to correct all such mistakes.

     3. Line 208: "previously studies" should be "previous studies."
Experimental design: No comments.
Validity of the findings: 1. There are several entries in the P-value column in Table 2 and Table 3  designated as "NS." This acronym should be explained in the statistical analysis section. Also, why were these p-values not computed?

2. You should report standard errors with the estimates of proportion (e.g., the clinical and twin pregnancy rates in Table 3).

3. The authors claim (line 235) that "From these results, it was concluded that women with a favorable or average prognosis who are 35-40 years have a high risk of twin pregnancies." The corresponding p-values in Table 3  are still reported as "NS."
Additional comments: No additional comments


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

Input:
Title: THE EFFECT ON TWINNING RATE OF TRANSFERRING DOUBLE VITRIFIED-WARMED EMBRYOS IN WOMEN OF ADVANCED REPRODUCTIVE AGE: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Manuscript Review: The effect on twinning rate of transferring double vitrified warmed embryos in women of advanced reproductive age.

Summary

The study is a retrospective analysis of advanced reproductive aged women who underwent vitrified-warmed double embryo transfer (DET). The purpose of the study was to investigate the twin pregnancy rate in women of advanced reproductive age undergoing DET in order to determine which population would be better served for elective single embryo transfer (eSET). The patients were subdivided into age (35-37, 38-40, and >40), as well as prognosis based on embryo transfer history and grade of embryos to be transferred. All embryos transferred were at the Day 3 stage, and each patient only contributed one cycle. Clinical pregnancy rates were defined by the observation of a gestational sac with our without fetal heartbeat on ultrasound evaluation 35 days after embryo transfer. The number of sacs was taken as the number of successful implantations. The results showed that both patients over 40 and those patients with poor prognosis had < 20% probability of twin pregnancy rate which can be used as a guide for practices in determining who to recommend eSET vs DET.
Experimental design: Appropriate
Validity of the findings: Appears appropriate
Additional comments: In your methods, line 81-82, you mention all embryos transferred were Day 3. Is this standard for your practice? Do you culture any embryos to day 5? If you culture embryos to day 5, do you have any results for blastocyst DET in advanced reproductive age women? ASRM committee opinion Blastocyst Culture in clinically assisted reproduction recommends blastocyst transfer in good prognosis patients due to higher likelihood of live birth.

The aim of the manuscript is to assist practitioners in knowing which patients of advanced reproductive age are at risk for twin pregnancy and who to recommend eSET. Do you have any data showing the live birth rates for these patients? That would be another helpful statistic to enhance the data and help with stronger evidence to support the recommendation.

I commend the authors on the retrospective study with a large patient volume and extensive data set that was compiled over many years. The manuscript is written clearly and is unambiguous with professional English language used throughout. It is a well-defined research question, and the methods are described with sufficient detail and information to replicate. The tables have sufficient information and are easy to read. I also commend the authors on the novel aspect of stratifying the patient by prognosis as well as age. The author has listed many weaknesses, including transfer of two cleavage stage embryos. Given the increased nature of blastocyst being the preferred embryo stage of transfer (ASRM committee opinion Blastocyst Culture in clinically assisted reproduction), I feel the data would be more helpful to have information on clinical and twin pregnancy rates of the subset of patients in embryos transferred in blastocyst stage. If there is data on this information, it should be included.


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

Input:
Title: THE EFFECT ON TWINNING RATE OF TRANSFERRING DOUBLE VITRIFIED-WARMED EMBRYOS IN WOMEN OF ADVANCED REPRODUCTIVE AGE: A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: In the new form, it is well improved.
Validity of the findings: no comment
Additional comments: I am really pleased to note all the efforts of the authors in order make the paper clear and understandable.
In my opinion, it should be accept in the actual form


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

Input:
Title: THE EFFECT ON TWINNING RATE OF TRANSFERRING DOUBLE VITRIFIED-WARMED EMBRYOS IN WOMEN OF ADVANCED REPRODUCTIVE AGE: A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Minor comment:
In lines 143-144,  "... way analysis of variance (ANOVA) test and the nonparametric Kruskal-Wallis test depending on normal or abnormal distribution, respectively. ..."

The line " ...  abnormal distribution ..." should be changed to "...  non-normal distribution ..."


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

Input:
Title: THE EFFECT ON TWINNING RATE OF TRANSFERRING DOUBLE VITRIFIED-WARMED EMBRYOS IN WOMEN OF ADVANCED REPRODUCTIVE AGE: A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 3

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


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

Input:
Title: GENE CO-EXPRESSION NETWORK FOR ANALYSIS OF PLASMA EXOSOMAL MIRNAS IN THE ELDERLY AS MARKERS OF AGING AND COGNITIVE DECLINE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Authors have applied weighted gene co-expression network analysis (WGCNA) to investigated miRNAs within plasma exosomes of elder people. Using hub node and pathway enrichment analysis they further narrow down four microRNAs (hsa-mir-376a-3p, has- miR-10a-5p, has-miR-125-5p, has-miR-15a-5p) that have regulatory roles in the development of ageing and cognitive dysfunction. Considering ageing is a complex phenomenon, the prediction of biomarker only focusing on exosomal miRNAs is overstated.
Experimental design: The analysis was done using the datasets available on the website. The tools they have used for miRNAs target prediction are also web-based tool. Details parameter for analysis is lacking in each step. Statistical analysis details are also missing.
Validity of the findings: The results are solely bioinformatics based, require experimental validation in a large number of patients before making use as a biomarker for ageing.
Additional comments: Authors have applied weighted gene co-expression network analysis (WGCNA) to investigated miRNAs within plasma exosomes of elder people. Using hub node and pathway enrichment analysis they further narrow down four microRNAs (hsa-mir-376a-3p, has- miR-10a-5p, has-miR-125-5p, has-miR-15a-5p) that have regulatory roles in the development of ageing and cognitive dysfunction. Overall, this is a good effort to identify biomarkers using bioinformatics tools. However, the manuscript lacks in-depth details of the methods. The inclusion and exclusion criterion choosing the dataset is missing. A brief of Pipeline describing the steps and criteria followed will give a more clear idea about data analysis. More importantly, considering the complex nature of the ageing, how did the authors deal with the false positivity? What cut off values they have used choosing miRNAs from different modules.

Few additional issues:
1.	Line 92 : Which correlation calculation method you have applied?

2.	Line 195 : …… data came from 79 elderly patients.  In Line 137 …. 97 samples. Which one is true?

3.	Line 245 : ….. identified the highest number of genes targeted by hub miRNAs
How did you perform this?


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

Input:
Title: GENE CO-EXPRESSION NETWORK FOR ANALYSIS OF PLASMA EXOSOMAL MIRNAS IN THE ELDERLY AS MARKERS OF AGING AND COGNITIVE DECLINE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Paper refers to plasma exosome -derived miRNAs changes and its relation with cognitive function in older people. In general, the paper is well-written, however, and english proofreader will improve importantly the reading. There are some miscues in the references since some of them have DOI and others don´t please try to make it more homogeneous. Some figures have a title and others do not, please correct, additionally add more explanation to figures ( #1, #3, #4, #5 ) so they could be more easy to understand. In cases of Figure 6 and 7, please put the most representative images and add the others to supplementary figures, otherwise, they are quite difficult to follow. In the case of Table 1, perhaps it could be added somehow to Table 2 since it´s quite small and has very few information.  In the introduction, it is not stated why the cognitive decline is related to miRNA´s profiles? please add more information on the subject.
Experimental design: Please, add more information on the WGCNA package. It´s not clear what was the experimental procedure performed in the papers, since authors state that they investigated miRNAs within plasma exosomes of older people, but it is not stated in methodology the blood extraction or the exosome isolation, otherwise please clarify.
Validity of the findings: In general, it is not clear the experimental analysis of exosomes and miRNAs, since this study is a bioinformatic analysis, authors should specify such relation. Discussion is quite big please try to diminish information and discuss the differences of your findings with other papers and clinical importance of such.
Additional comments: In general, this paper seems to be a complete bioinformatic analysis, however, experimental relation between exosomes and miRNA´s is not clear. Additionally, there are miscues that difficult reading, so perhaps proofreader could be useful. Figures need more information so they could be easy to understand. Reference needs to be more homogeneous.


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: GENE CO-EXPRESSION NETWORK FOR ANALYSIS OF PLASMA EXOSOMAL MIRNAS IN THE ELDERLY AS MARKERS OF AGING AND COGNITIVE DECLINE
Review round: 1
Reviewer: 3

Output:
Basic reporting: In general it is well raised, even with the suggested corrections.

But I have some suggestions:

In the introduction the relationship between miRNA and aging is poorly established.

Line 195: It says:

In this study, data came from 79 elderly patients

Was it mentioned before?
Experimental design: The methodology part is well raised
Validity of the findings: the findings are conclusive, it would have been important that trials are designed to validate the miRNA found as prevalent: hsa-mir-376a-3p, miR-10a-5p, miR-125-5p, miR-15a-5p
Additional comments: They are mentioned 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: GENE CO-EXPRESSION NETWORK FOR ANALYSIS OF PLASMA EXOSOMAL MIRNAS IN THE ELDERLY AS MARKERS OF AGING AND COGNITIVE DECLINE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper addresses a very current theme.

It is very well written, with references that provide what they point out

only comments that are less than:

The introduction has different font sizes.

There is no consistency of how they write miRNA or even how they cite references
Experimental design: they used the right tools
Validity of the findings: each result is analyzed very well also serves to build the following result
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: BLOOD LACTATE DYNAMICS IN AWAKE AND ANAESTHETIZED MICE AFTER INTRAPERITONEAL AND SUBCUTANEOUS INJECTIONS OF LACTATE—SEX MATTERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an extremely important finding, especially for rodent studies intended for mimicking humans.  The effect of animal handling and the adrenergic response in rodent was very well documented in the 1970 through 1990 literature. This was time when IUCAC would permit decapitation without anesthesia so a true comparison could be determined. I think a more thorough review of the literature would permit the authors to add in the Abstract that "the difference in awake and anesthetized mice is due to the adrenergic response caused by mouse handling.  A fairly thorough review of this early literature was performed in a section from a recent review (Tikunov et al, Current Metabolomics, 2013). They should know that most of the hepatic glucose is derived from glycogen in those initial minutes during handling. Without 13C-labeled lactate given SC or IP,  it is not possible distinguish glucose from glycogenolysis or gluconeogenesis.  In fact, early In vivo 13C MRS glycogen studies infused beta-blockers to lab animals to inhibit glycogenolysis (in Tikunov et al., 2013). The finding that male mice have a significantly elevated response to handling and this could be due to their inherent aggressiveness could be novel but a more thorough literature review is required. They should include some of these earlier references to help substantiate their results, which are taken from the Tikunov et al, 2013 review. Especially important for thier study are the first 4 references and the last 4 books in this list:

Gariépy, J. L.; Rodriguiz, R. M.; Jones, B. C. Handling, genetic and housing effects on the mouse stress system, dopamine function, and behavior. Pharmacol. Biochem. Behav., 2002, 73, 7-17.

Tabata, H.; Kitamura, T.; Nagamatsu, N. Comparison of effects of restraint, cage transportation, anaesthesia and repeated bleeding on plasma glucose levels between mice and rats. Lab. Anim., 1998, 32, 143-148.

Zethof, T. J.; Van der Heyden, J. A.; Tolboom, J. T.; Olivier, B. Stress-induced hyperthermia in mice: A methodological study. Physiol. Behav., 1994, 55, 109-115.

Annamunthodo, H.; Keating, V. J.; Patrick, S. J. Liver glycogen alterations in anaesthesia and surgery. Anaesthesia, 1958, 13, 429- 433.

Besch, E. L.; Chou, B. J. Physiological responses to blood collection methods in rats. PSERM., 1971, 138, 1019-1022.

Arola, L. L.; Palou, A.; Remesar, X.; Herrara, E.; Alemany, L. Effect of stress and sampling site on metabolite concentration in rat plasma. Arch. Internat. Physiol. Biochim., 1980, 88, 99-105.

Healing, G.; Smith, D. Handbook of pre-clinical continuous intra- venous infusion Taylor & Francis: New York, 2000.

Reilly, R. Variables in animal base research: Part 2. Variability associated with experimental conditions and techniques. ANZCCART News, 1998, Vol.11.

Balcombe, J. P.; Barnard, N. D.; Sandusky, C. Laboratory routines cause animal stress. Contemporary Topics, 2004, 43, 42-51.

Hau, J. S. Handbook of Laboratory Animal Science, Third ed.; CRC Press: Boca Raton, FL, 2010.
Experimental design: No comments
Validity of the findings: no comment
Additional comments: I think a more thorough review of the effect of anesthesia and animal handling is important.  In doing so, they may discover their gender are in fact in an earlier report on sex difference in mouse handling.


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: BLOOD LACTATE DYNAMICS IN AWAKE AND ANAESTHETIZED MICE AFTER INTRAPERITONEAL AND SUBCUTANEOUS INJECTIONS OF LACTATE—SEX MATTERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper presents an interesting and useful evaluation of the differential effects of lactate injection in mice.  The therapeutic utility of lactate treatment may not be familiar to the general readership of PeerJ and the introduction would benefit from an expanded discussion of the background of this application.  In particular, on line 40, the authors mention that lactate administration has therapeutic potential for several neurological diseases.  This could lead to an expanded discussion with appropriate references.
A brief discussion of some of the potential side-effects & toxicity potential of lactate therapy would be interesting as well.
One significant issue with the paper is in the presentation of the data in figures 2 and 3.  The histogram format is rather cumbersome to review.  To present the time course data, it might be more readily interpreted to show a trajectory plot for each treatment.  Furthermore, the data could be split into separate figures presented as a series of tiles.  For example, Figure 2 could be presented as shown in the attached file.

A simple line diagram or better yet, boxplots of the data could be presented with connecting lines to highlight the trajectory.  Separating the PBS and lactate injections, while maintaining a consistent axis range will make the data interpretation much easier.   Statistical significance for each plot could be readily represented and differences between plots could be presented in a table format.
The authors could consider the potential effects of differences in total lean muscle mass between the sexes on lactate metabolism.
Experimental design: The experimental design is well constructed and executed.  The use of linear mixed effect regression models is appropriate for the studies.
Should be clarified how the 2g/kg body weight dose was determined. It appears to be designed to mimic the effects of high intensity exercise, but this should be stated.
Validity of the findings: Findings are of significant interest and utility to the scientific community involved in research involving lactate metabolism.
Additional comments: Overall a very valuable investigation of lactate metabolism and kinetics using mouse models.


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

Input:
Title: A NEW SPECIES OF OLIGODON FITZINGER, 1826 FROM THE LANGBIAN PLATEAU, SOUTHERN VIETNAM, WITH ADDITIONAL INFORMATION ON OLIGODON ANNAMENSIS LEVITON, 1953 (SQUAMATA: COLUBRIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is well written manuscript based on extensive morphological and molecular comparisons and fieldwork to distinguish the new species in question. There is no doubt that this is a valid species and the authors have made a detailed description. There is some minor weakness in this paper, e.g using body ratio based on one sample to distinguish it from other congeners. This can be modified as per the suggestions I have made. There are other suggested minor changes which i have pointed out in the MS.


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

Input:
Title: A NEW SPECIES OF OLIGODON FITZINGER, 1826 FROM THE LANGBIAN PLATEAU, SOUTHERN VIETNAM, WITH ADDITIONAL INFORMATION ON OLIGODON ANNAMENSIS LEVITON, 1953 (SQUAMATA: COLUBRIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is very interesting
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I made my fe corrections with track back in the text, so I can sent the word file, I cannot upload it as DOCX.
The tables and figures are fine, with one mistake inside:
Table 5:
Oligodon eberharti
Change to Oligodon eberhardti


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

Input:
Title: A NEW SPECIES OF OLIGODON FITZINGER, 1826 FROM THE LANGBIAN PLATEAU, SOUTHERN VIETNAM, WITH ADDITIONAL INFORMATION ON OLIGODON ANNAMENSIS LEVITON, 1953 (SQUAMATA: COLUBRIDAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The english in the manuscript requires revision, some phrasing and grammar needs to be corrected, key points are addressed in my comments. Literature references are fine, a few sources need to be checked for consistent formatting. Raw data is missing, mainly the raw tree used in the study. Photographs are in poor resolution or are mislabelled and need to be corrected.
Experimental design: Description of the new species and comparisons is straightforward. I do not like the way the account of new data on Oligodon annamensis is structured. I prefer that the authors re-write this section as a redescription. The specimen descriptions can be lumped together more and this will help organize the manuscript better. The species Oligodon lacroixi is not talked about at all in the paper despite its presentation a new DNA sequence. This needs to be addressed more or removed from the manuscript because it is not a close phylogenetic relative anyways.
Validity of the findings: The new species is based off a single male specimen, but given its phylogenetic position based on multiple loci and the morphological differences, I do not have any objections to its validity or diagnosis.
Additional comments: The article "A new species of Oligodon Fitzinger, 1826 from Langbian Plateau, southern Vietnam, with additional information on Oligodon annamensis Leviton, 1953 (Squamata: Colubridae)" needs important revisions before being accepted into this journal. The following issues need to be resolved in the next version of the manuscript.

1. Reformat the account on Oligodon annamensis as a redescription or a seperate species account. Discussion section for this species looks unorganized and would be streamlined if redone as a formal description of variation.
2. Remove or add more information on Oligodon lacroixi since it is a novel sequence you are reporting in the study.
3. Consistent use and clarification of hemipenial terminology used (for example: papillae as an in-situ appendage vs. papillae as everted hemipenis ornamentation; "forked and "unforked" vs. bilobed and unilobed; use of bifurcation to describe hemipenis shape vs. use of bifurcation to describe the sulcus spermaticus). Also the pictures of the hemipenis need to be redone to show ornamentation/structure better.

Additional comments and suggestions are outlined in the attached PDF and should be addressed.

– Justin L. Lee


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

Input:
Title: A NEW SPECIES OF OLIGODON FITZINGER, 1826 FROM THE LANGBIAN PLATEAU, SOUTHERN VIETNAM, WITH ADDITIONAL INFORMATION ON OLIGODON ANNAMENSIS LEVITON, 1953 (SQUAMATA: COLUBRIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: See previous review
Experimental design: See previous review
Validity of the findings: See previous review
Additional comments: The authors of this paper have drastically improved their manuscript according to comments made by the reviewers and myself. The revisions made are adequate, the paper reads well and mistakes in earlier drafts were addressed.

There are a few instances in the tables, the species diagnosis for Oligodon rostralis and in the discussion where the hemipenial term "forked" is used when it should be "bilobed", and in line 774 in the discussion, "unilobed" is used when it should be "unforked". This may need to be revised for consistency. Other than that, I believe the manuscript is ready for acceptance pending comments from other reviewers.

– Justin Lee


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: CAS9/GRNA-MEDIATED GENOME EDITING OF YEAST MITOCHONDRIA AND CHLAMYDOMONAS CHLOROPLASTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Manuscript reports on DNA engineering in Chlamydomonas chloroplasts and yeast mitochondria using an independently replicating episomal element as an expression platform. Authors selected replicons that have been described in the literature. The so called Edit Plasmids carry the expected genes: Cas9, sgRNAs processed by tRNAs, the selectable plastid or mitochondrial marker, and a GFP flanked by short target sequences for insertion at the target site.  The chloroplast data are more comprehensive, and with the addition of relatively small amount of data can make a convincing case for CRISPR/Cas action. The mitochondrial data are sketchy.
Experimental design: Problems with the experimental design and suggestion to improve the text  are included in general comments.
Validity of the findings: See in general comments.
Additional comments: 1. Introduction: Lines 69-71: “Examples include DNA transformation in yeast mitochondria (Fox, Sanford & McMullin, 1988) and Chlamydomonas chloroplasts (Boynton et al., 1988), and high protein expression in tobacco chloroplasts (McBride et al., 1995).” To be equitable, first transformation should be cited for higher plant chloroplasts also.

2. What authors refer to as chloroplast DNA replacement is GFP insertion at the target site facilitated by homologous recombination. DNA replacement would be replacing one gene coding region (or sequence) with an altered coding sequence, such as one psaA coding sequence with a different psaA coding sequence. Make changes throughout.

3. Data presentation should be improved in the text. On Lines 399-400 authors state: “Twenty eight days post transformation, cell lysates from 20 independent colonies of all constructs except for YP23 and YP24 with 16 colonies were prepared in pools.” In reality, the pool sizes were different. When accounting for lanes in Figure 2B, the caption states “Numbers of transgenic colonies were 8 for Lane 1 & 2, 4 for Lane 3, 5”. Thus, for YP13 three pools were analyzed;  two pools of 8 and one pool of 4. The pool size could be included in Figure 2B, and the lengthy explanation deleted from the caption.

4. Ultimately, two GFP carrying colonies were found in 20 transplastomic events, and none in 69 “controls” where CRISPR/Cas9 was ineffective or absent, suggesting that the two homologous recombination events were triggered by cleavage of the target site by CRISPR/Cas9. Apparently, when the target site is cleaved, shorter homology is sufficient to facilitate homologous recombination. These are the only data supporting the claim that  insertion is due to CRSPR/Cas action, and are not the products of homologous recombination mediated via the short targeting sequences.  Authors argue that the sequences are too short to yield transplastomic events. However, Dauville et al. (2004) report on average 2 and 3 events per 2x107 cells using 75 bp/52 bp and 51 bp/52 bp homologies, respectively (Table 2 in reference). The homologous regions in Dauville et al. (2004) are shorter than the 74 bp and 76 bp used in present study. One would like to see shorter homologies and larger sample sizes tested to be comfortable with the conclusion that insertion is due to CRISPR/Cas action.

5. Insertion was into the psaA gene coding region is generating a fusion protein. Are the Chlamydomonas cells expressing the fusion protein photoautotrophic? Was the medium set up to accommodate photoheterotrophic cells? Why were the GFP insertion colonies not screened by fluorescence under UV light?

6. Line 433 Section title: “Cas9/gRNA-induced NHEJ DNA repair in Chlamydomonas chloroplasts”. The title should be changed, since there is no evidence for DNA repair by NHEJ. The section title should be “On target mutations at the Cas9 target site”.  It appears that there was positive selection against recovering products of NHEJ. The target sites are within two photosynthetic genes, psaA and psbD. CRISPR/Cas9 most often causes single nucleotide insertions/deletions generating an early stop codon. If the Chlamydomonas cells were grown on photoautotrophic medium, only nucleotide substitutions would be compatible with photoautotrophic growth because during photoautotrophic grown there is strong selection to maintain functional psaA and psbD copies. Indeed, only nucleotide conversions were obtained, that would suggest functional constrains. Discuss the impact of photoautotrophic / photoheterotrophic growth on the outcome of the DNA repair experiment.

6. There are extensive data on chloroplast DNA repair after cleavage by the I-CreII homing endonuclease (Kwon T et al. PNAS 2010;107:13954-13959). Should be cited and discussed. In Arabidopsis, psbA defective sectors survived because were supported by wild-type sectors enabling detection of NHEJ.

7. The statistics of CRISPR/Cas9 mutations is debatable: 4 events in 100 clones with CRISPR/Cas9 and 2 events (based on 1 event in 50 clones) in the absence of CRISPR/Cas9.

8. Mitochondrial replicons (HS8, HS6, etc)  should be listed in a Table, as the chloroplast vectors in Table 1.

9. Data for mitochondrial replicons should be included in a Table, similar to Table 3A for chloroplasts.

10. Line 512: DNA replacement is a misnomer – should be DNA insertion. See Point 2 for chloroplasts above.

11. Since GFP was flanked by homologous targeting sequences, the role of CRISPR/Cas in the insertion is questionable. See point 4 for chloroplasts above.

12. Supplemental Figure S1 is apparently missing. To avoid the problem, all  Supplemental Figures and Tables should be included as a single pdf file. Raw data should be in separate folder with a list of all documents systematically labelled.


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: CAS9/GRNA-MEDIATED GENOME EDITING OF YEAST MITOCHONDRIA AND CHLAMYDOMONAS CHLOROPLASTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript demonstrates chloroplast genome editing in Chlamydomonas reinhardtii and mitochondrial genome editing in Saccharomyces cerevisiae, mediated by CRISPR-Cas9 system with the Edit plasmids. This is the first report of CRISPR-based organelle genome editing; therefore, this study harbors potentially sufficient impact and importance for publication in PeerJ. However, there are a lot of points that need to be clarified, improved, and discussed before publication.
Experimental design: See general comments.
Validity of the findings: See general comments.
Additional comments: 1) Two key experiments are missing in this study. The first point that needs to be addressed is the percentages of edited organelles in the selected clones. If there was only a small portion of edited organelles in the whole population of clonal cells, the authors' approach is not practically useful. This point should be examined by out-out PCR (i.e., primers C1 and C4 in psaA targeting, and primers C and F in COX1 targeting). The second critical point is stability of the edited organelles. The authors have to conduct long-term monitoring of the edited clones, and the proportion of edited organelles should be assessed over time.

2) Related to the above point, the clonal analysis of yeast mitochondrial genome editing is definitely needed.

3) The manuscript title should include organism names for clarification. The following title should be more appropriate: "Cas9/gRNA-mediated Genome Editing of Yeast Mitochondria and Chlamydomonas Chloroplasts."

4) The authors should explain why they selected the exon 3 of psaA gene as a target site for chloroplast genome editing and what the function of psaA gene is.

5) Scale bars should be added to Figures 2A and 5A.

6) Fluorescence images should be shown, because GFP genes were knocked-in in chloroplast and mitochondrial genomes.

7) Full sequences of the Edit plasmids with the annotations should be provided to secure the reproducibility.

8) Ideally, various lengths of homology arms should be tested and their comparative analyses should be performed. This point should at least be discussed as a future study.

9) Appropriate reference for the previous studies reporting mitochondrial genome editing in humans using ZFNs and TALENs should be added and discussed (Bacman et al., Nature Medicine, 2013; Bacman et al., Nature Medicine, 2018; Gammage et al., Nature Medicine, 2018).


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: CAS9/GRNA-MEDIATED GENOME EDITING OF YEAST MITOCHONDRIA AND CHLAMYDOMONAS CHLOROPLASTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a strong and convincing report on successful editing of the organellar (both chloroplast and mitochondrial) DNAs.  The research was completed using plasmids introduced into the organelles using biolistics, and the plasmids carry the coding regions for the guide RNAs and cas9 protein.  The experiments were carried out in yeast for the mitochondrion, which makes sense as amodel system because of the existence of mitochondrial mutants in this fungus, and the algae Chamdymonas, which has one big chloroplast and is therefore a good model system.

The manuscript is extremely well written and clear.

One potentially important point is that the introduced plasmids could be maintained in the organelles indefinitely, even though they could not detect the plasmids after 30-40 generations.  Would it be possible to remove the origin of replication from the plasmids so they could not replicate in the organelles, and therefore disappear quickly from progenies?

There will be many challenges to editing of the organellar DNAs in other eukaryotes, given that there will be many mitochondria and/or chloroplasts in cells, requiring the introduction of a selectable marker such as antibiotic resistance. This will have regulatory issues over the long term.  This point may need to be addressed in the manuscript.  Nevertheless the technology is robust for basic research.

The evaluation of edited sites by restriction enzyme analyses (for example lines 446-451) is not very robust approach, it would be much better to sequence the regions and report any unanticipated sequence changes or rearrangements.

Overall, the observation that unintended indels and substitution events were extremely rare is a very positive indication that editing of the organellar DNAs will become an important tool!!
Experimental design: Robust and appropriate
Validity of the findings: Very convincing results
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: CAS9/GRNA-MEDIATED GENOME EDITING OF YEAST MITOCHONDRIA AND CHLAMYDOMONAS CHLOROPLASTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Well written and understandable. Lt of unnecessary on line supplemental information.
Experimental design: Byung-Chun Yoo and colleagues report on DNA engineering in Chlamydomonas chloroplasts and yeast mitochondria using an independently replicating episomal element as an expression platform. The “Edit Plasmids” carry the Cas9 endonuclease gene, sgRNAs processed by tRNAs, the selectable plastid or mitochondrial marker, and a GFP flanked by short target sequences for insertion at the target site.  Having short, homologous sequences on the donor DNA was a fatal flow in the experimental design. However short, the homology could direct integration by homologous recombination. To provide irrefutable evidence  for CRISPR/Cas directed integration, authors should repeat the experiment without any targeting sequence on the donor DNA. They should then document that the integration of the cassette occurs at a different site, site dependent of the choice of sgRNA cutting the genome at a different location. NHEJ typically results in the loss or gain of at least a few nucleotides. Perfect homology at the target site is a red flag, the hallmark of homologous recombination.
Validity of the findings: see above.
Additional comments: Please, repeat experiment without targeting sequences on donor DNA.


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: CAS9/GRNA-MEDIATED GENOME EDITING OF YEAST MITOCHONDRIA AND CHLAMYDOMONAS CHLOROPLASTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Since the authors avoided clarifying the points raised in the comment #1 of my initial review, the actual applicability of the authors' method is still questionable. However, as the authors claimed, the phenomenon itself (i.e. CRISPR/Cas9-mediated organelle editing) was validated in this study regardless of its efficiency. Therefore, I basically accept the authors' claims.
Experimental design: See general comments.
Validity of the findings: See general comments.
Additional comments: 1) According to the authors' response, "the goal presented in this manuscript was to test whether or not CRISPR/Cas9 works in organelles at all," and the editing efficiency is still unclear. This point should be clearly described in the main text. The actual applicability cannot be guaranteed without showing the data covering this reviewer's requests in the initial review.

2) The reference information should be added in line 662.


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: CAS9/GRNA-MEDIATED GENOME EDITING OF YEAST MITOCHONDRIA AND CHLAMYDOMONAS CHLOROPLASTS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The Topical Review by Thurtle-Schmidt and Lo entitled “Molecular Biology at the Cutting Edge: A Review on CRISPR/CAS9 Gene Editing for Undergraduates” cited by Corresponding Author is based on engineering the nuclear genome. Integration of foreign DNA in the nucleus in the absence of a double-strand break, by homologous recombination alone, is very rare, observed in 1 out of 100,000 integration events (or less frequent). If a double-strand DNA break is introduced, this frequency is boosted to 1/5. The role of double-strand DNA break in facilitating insertion of sequences at a specific location in the nuclear genome is well accepted. For discussion see for example Wright et al. (2005) High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant Journal 44(4) 693-705.

In contrast, in chloroplasts and mitochondria there is no need for double-stranded breaks to observe homologous recombination, because homologous recombination is the rule. The question is what role, if any, double-strand DNA break plays when homology is provided for the insertion of heterologous DNA. This reviewer does not doubt that precise homologous recombination can be obtained using CRISPR/Cas9. This reviewer doubts whether or not the CRISPR/Cas9 construct reported here is active at all in either chloroplasts or mitochondria, because identical outcomes can be obtained in chloroplasts and mitochondria without CRISPR/Cas9, by homologous recombination.

The lack of evidence for non-homologous end joining (Analyses for on-target mutations ..) reported here could be the consequence having non-functional CRISPR/Cas9 vectors.  Mutations inactivating chloroplast genes could provide a simple and convincing outcome regarding the functionality of the CRISPR/Cas9 vectors. Two chloroplast photosynthetic genes could be targeted in two different cultures and defective (photoheterotrophic) mutants collected from each. In cultures where the first gene is targeted, most phototrophic mutants should have mutations in the first gene at the predicted target site. If the second gene is targeted, the mutations should be in the second gene. An elegant, recent example for non-homologous end joining in mitochondria was provided by Kazama et al. in Nature Plants 5: 722-730, 2019, using TALEN-mediated gene inactivation. Authors could design similar experiments using their mitochondrial system.

Independent confirmation of CRISPR/Cas9 activity of the reported constructs should be the prerequisite of publication. The possibility of alternative explanation, integration by homologous recombination, should be pointed out.
Experimental design: see above
Validity of the findings: see above
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: CAS9/GRNA-MEDIATED GENOME EDITING OF YEAST MITOCHONDRIA AND CHLAMYDOMONAS CHLOROPLASTS
Review round: 3
Reviewer: 2

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