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: MUTATIONAL SPECTRA OF SARS-COV-2 ISOLATED FROM ANIMALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript “Mutational spectra of SARS-COV-2 isolated from animals” is summarized the nucleotide and amino acid substitutions at different gene segments and proteins of SARS-COV-2 isolates. Authors tried to explain the comparison of the mutational effect of SARS-COV-2 in different animal species and human. However, the objective of the study and the effect of mutation should better explain which would benefit the reader in a greater extent. Similarly, the tables are full of content and difficult to understand. I suggest to revise the Tables and prioritize them in main tables and supplemental tables. I also recommend to use simple and fluent English language to easily understand the text.

There are some specific major issues to be addressed:
1.	Please consider doing a minor check across the manuscript for English usage, syntax and elaborate the abbreviations when comes first. For example: in line 72; the full name “pp”1ab is missing, in line 110; NCBI and GISAID, in line 258, 287; space is missing in between “animalisolate”.
2.	In line 100-101: the sentence (SARS COV-2 can infect …………………………chicken and duck) is a bit confusing. Make the statement clear with appropriate references. For eg., according to Shi et al., SARS COV 2 have poorly replicate in chicken and duck and also compare the statement with others observation eg. Schlottau et al., 2020
(https://www.thelancet.com/journals/lanmic/article/PIIS2666-5247(20)30089-6/fulltext)
3.	In the line 102: before mentioning the objectives, I suggest you to explain briefly the justification of the study.
4.	In line 184-186 (All SARS-CoV-2……………….at this position): not clear either animal virus substitutions are different from the human virus isolate. In the Table and Figure the substitutions are similar in animal viruses and human viruses.
5.	Line 233-235: rephrase the sentence to better understand.
6.	Line 287-290: Mentioned signature mutation for which species? What is the relation of this statement with the study?
7.	Line 298-299: The author stated animal have a role in the SARS-COV-2 outbreak but is not these animal viruses isolated after the pandemic? Whether the transmission is not the other way around? Please give enough statement and references to support the conclusions.
8.	Please explain the Figure 2 legend to understand better. The first paragraph of the legend is not completed (heptad rep….).
9.	Synchronization of the title and content of the Table 2 is missing. Proteolytic cleavage site is missing on the table 2. Please correct either the content or edit the title accordingly.
10.	Table 3 is unclear and difficult to understand. Please explain each headline and mention the effect of amino acid substitution (either bad or good) with appropriate references. What is the yellow mark stand for? If necessary, please use footnote to explain better. Similar observation for table 4.
Experimental design: Research questions are not well defined. Need further improvement.

Methods are well defined.
Validity of the findings: Results are explained well but discussion need to be specific and well written.

Speculation and conclusions need more supportive statement.
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: MUTATIONAL SPECTRA OF SARS-COV-2 ISOLATED FROM ANIMALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The current manuscript Ahmed Elsawad et al has tried to identify mutations within SARS-CoV-2 strains isolated from different animals. The idea is really good and worth exploring.

The introduction part is majorly written using content from Helmy et al., 2020. There are several places in the introduction which are approximately similar to Helmy et al., 2020. There are numerous publications available on the SARS-CoV-2 genome and COVID-19 disease. The introduction of any article is like a mini-review of our current knowledge procured from diverse publications, therefore my suggestion is to re-write the introduction section using that diverse information.

At several places, sentences should be rephrased such as line 65 should be "still the disease is spreading worldwide".
Experimental design: Considering the vastness of SARS-CoV-2 genome availability, it becomes tricky to select genome sequences. I do not understand on the basis of which criteria authors have selected those 38 human SARS-CoV-2. There are multiple clades diversifying the SARS-CoV-2 genomes based on diverse mutation sites, authors have not discussed any point in this regard.

Line 136-138: The authors should mention how many Ns are present in Cat and Dog CoV.
Validity of the findings: The phylogeny shown in Figure 1 is really vague and does not depict any real finding. As SARS-CoV-2 genomes from other animals (leaving bat and pangolin) are >99% identical, the SARS-CoV-2 clade is not showing any relationship amongst these strains. As the addition of bat CoV RaTG13, pangolin CoV and other CoVs will make the relative distance between SARS-CoV-2 genomes too high, It would be better to use only SARS-CoV-2 genomes and see how they are related with each other.

Wuhan-Hu-1 is the reference strain, however, now it is already known that there are more than 10 clades within SARS-CoV-2. The authors should explain the clade of each used strain. Later, they should use different representatives of diverse SARS-CoV-2 clades and classify these animal strains amongst any of the known clade or a diverse clade based on phylogeny and alignments.

Line 223-225: This is wrong speculation "This supports the speculation that animals have a role in the evolutionary pathway of SARS-CoV-2." It does not prove anything like this. These animals are just getting infected with the Human CoV (as they are >99.5% identical) and these are not in the line of SARS-CoV-2 common ancestors as in Pangolin and Bat-CoVs.

Authors should also explain synonymous or nonsynonymous mutations in each case. Simultaneously, They should explain that the identified mutation spectra belong to a transition or transversion event?

Line152-164: are these lines really required? You can combine this information in Table 2.

Line 258: B is missing from Bat.

The authors should provide more information in Figure 2. They should try to show the alignment for all descriptions in Line 178-189.

Table 2 is more like a Review figure.

Table 3: it is not correct to say alleles
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: MUTATIONAL SPECTRA OF SARS-COV-2 ISOLATED FROM ANIMALS
Review round: 1
Reviewer: 3

Output:
Basic reporting: THe overall representation of the manuscript is good and acceptable.
Experimental design: The bioinformatics analysis can be more rigorous.
Validity of the findings: The findings are genotypic and its impact on the structure of the proteins needs multiple analysis.
Additional comments: IN the manuscript entitled "Mutational spectra of SARS-COV-2 isolated from
animals", the authors  tried to correlate the evolutionary pattern of SARS-CoV-2 in animals. Unfortunately, the data set is very less (n=55) and out of which only 7 genomes for
are from animals (cat, dog, mink, mouse, and tiger) and the entire manuscript deals with the mutations around these animals.
I think the dataset should be increased, so that mutations pattern can be assured. Simultaneously, the authors should analyse the impact of these mutations.


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: MUTATIONAL SPECTRA OF SARS-COV-2 ISOLATED FROM ANIMALS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript “Mutational spectra of SARS-COV-2 isolated from animals” is summarized and compared the nucleotide and amino acid substitutions at different gene segments and proteins of SARS-COV-2 isolates from animals and human.” The manuscript has much improved and addressed most of the comments that has been previously raised.
Experimental design: Aims and objectives are clear in the revised version
Validity of the findings: The findings specially tables are much improved and understandable.
Data are well defined and controlled.
Additional comments: The author addresses most of the previous comments and improved the text and sequence analysis in a sound and scientific manner.


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: PSYCHOSOCIAL VARIABLES AND QUALITY OF LIFE DURING THE COVID-19 LOCKDOWN: A CORRELATIONAL STUDY ON A CONVENIENCE SAMPLE OF YOUNG ITALIANS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I found that clear and professional English has been used in the entire manuscript. The manuscript is remarkable in the extent of coverage of  previous literature. Article structure is very professional. The paper is self contained with relevant results to the hypotheses. This manuscript meets all the criteria set forth by PeerJ for basic reporting.
Experimental design: The article meets the aims and scope of the journal. Research questions are well defined and meaningful. I really liked the methods section in particular as it was very well defined and explained. I think authors have performed rigorous investigation.
Validity of the findings: The findings are valid and they corroborate the previous findings as well. This gives credence to their results. Authors also admit that the findings are culture specific and cannot be generalized to other age groups/nationalities.
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: PSYCHOSOCIAL VARIABLES AND QUALITY OF LIFE DURING THE COVID-19 LOCKDOWN: A CORRELATIONAL STUDY ON A CONVENIENCE SAMPLE OF YOUNG ITALIANS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is a basic study. There have been many papers investigating on the issue since the pandemic. English needs to be improved. Literatures should also include recent references including:
Add these to lines 69, 77 and 81-82.
1) Ma, Z. F., Zhang, Y., Luo, X., Li, X., Li, Y., Liu, S., & Zhang, Y. (2020). Increased stressful impact among general population in mainland China amid the COVID-19 pandemic: A nationwide cross-sectional study conducted after Wuhan city’s travel ban was lifted. International Journal of Social Psychiatry, https://doi.org/10.1177/0020764020935489

2) Zhang Y, Ma ZF. 2020. Psychological responses and lifestyle changes among pregnant women with respect to the early stages of COVID-19 pandemic. Int J Soc Psychiatry.doi:10.1177/0020764020952116.

3) Zhang, Y.; Ma, Z.F. Impact of the COVID-19 pandemic on mental health and quality of life among local residents in Liaoning Province, China: A cross-sectional study. International Journal of Environmental Research and Public Health 2020, 17, 2381, doi:10.3390/ijerph17072381.

The authors need to be aware of the growing literature.
Experimental design: As I mentioned previously, many important references are missing. I would like to see proper references for any statements or points made. The sample size was too small, this can introduce bias. Also, I want to know how many people were approached and how many accepted the study, the reasons of rejection? The authors had omitted these details. How does the characteristics of the sample compared to the Italian general population? What samplings were used? the more I read the manuscript, I am asking for more details and these details are not included.
Validity of the findings: with such a small sample size and the details of study site, sampling etc. which were not incldued, it is hardly to recognise any meaningful impact or novelty from the study. The authors should take more time to polish the manuscript.
Additional comments: Please see above.
Also, more details on how the questionnaire was developed. Cronbach's alpha value? So many important details are omitted.

However, if these details can be included and stated explicitly, this might be a useful paper. But the title should be change to " in a sample of young Italians from XXX region", to better reflect the findings and does not mislead the readers.


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: PSYCHOSOCIAL VARIABLES AND QUALITY OF LIFE DURING THE COVID-19 LOCKDOWN: A CORRELATIONAL STUDY ON A CONVENIENCE SAMPLE OF YOUNG ITALIANS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comments
Experimental design: Young adult study is very limited especially based on how badly Italy has been hit due to COVID-19 pandemic. The experiment was performed with over 200 adults and the sample size seems small for any statistical modelling/analyses.
Validity of the findings: The conclusions seem relevant for a restricted data set from restricted regions. By using multiple measures, it demonstrates both robust evidence for a finding and/or the sensitivity of a finding to particular scale.
Additional comments: The figures have legends on the top and bottom. It is preferred to be at the bottom only.


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: PSYCHOSOCIAL VARIABLES AND QUALITY OF LIFE DURING THE COVID-19 LOCKDOWN: A CORRELATIONAL STUDY ON A CONVENIENCE SAMPLE OF YOUNG ITALIANS
Review round: 1
Reviewer: 4

Output:
Basic reporting: There are many English writing issues. I am not able to identify them all. The authors should consider getting writing/proofreading assistance

Abstract

"had a not significant effect on quality of life"

Did not have a significant effect

"young Italian people aged 21.2 (st.dev. =3.5) "

Missing a period after the paranthesis

"positive relationships with the put into action of specific behaviour"

Needs rephrased. With putting specific behaviors into action?

"to affirm one’s Country specific identity"

country not capitalized

"Finally, the COVID-19 fear "

drop the

"reflect on the importance of analyze the social context "

analyzing

Introduction

"Although the unicity of this pandemic, the world already faced off different epidemics."

Not clear what this means. Avoid uncommon terms like unicity

"The definition of quality of life comprise"

comprises

"social-related factors that impact on QoL"

social factors that impact QoL. (no social-related)

"Impact on" (a few places)

is not a correct phrasing, simply impact QoL

"Regards social support"

Regarding

"Noteworthy, the "

It is noteworthy that

"summarizing, "

Can't be used like that either

"situation could conceivable "

The end of the introduction has many meanwhiles, furthermores, in additions, etc. The narrative should be written more clearly with a varied sentence structure and make the predictions and connections to the literature clearer.

"had a not significant effect on QoL (H1b)"

Did not have a significant effect on is the correct wording (as per abstract)
I'm going to quit commenting on specific cases after the introduction. The whole paper needs to be carefully proofread.
Experimental design: The paper could use more clear justification for how the SEM model is structured and walk through the paths (particularly mediating ones) more clearly with examples and citations. Right now many of the predictions are simply mentioned without discussing much of the reasons for these predictions in a way that they make sense for readers not highly familiar with this area. It seems like many of the predictions are principled, but almost all of the rationale comes from a big long paragraph that is not well connected and does not make the importance of the current work clear.
Validity of the findings: Some of the alphas are below the criteria listed in the paper. It is not clear that, without additional validation, these scales can simply be translated from English to Italian, and this might have effected some of the psychometrics. This should be discussed as a limitation.

It's not clear the correlational nature of the results really suggest anything about interventions, despite the fact that this is stated as a goal.
Additional comments: The paper is difficult to read and thus difficult to assess in its current form. It appears to show some worthwhile correlations, but right now it is tough to read and understand how this fits into the broader literature. There are some technical limitations with how the surveys were translated and repurposed, which should be discussed.


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: OPTIMAL VOLUNTARY AND MANDATORY INSECT REPELLENT USAGE AND EMIGRATION STRATEGIES TO CONTROL THE CHIKUNGUNYA OUTBREAK ON REUNION ISLAND
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is professionally written in good English.

While the references are adequate,  the organization of the literature review is a bit confusing. [21] is a book that outlines the state of the art of the field of behavioral epidemiology at the time it was written; this should be made more explicit in line 66 of page 3, which right now sounds  a bit like if [21] had started it all. Line 83 at the same page mentions [39] in the context of statistical physics, but this is just part of a flashy tile of this article.  In reality, this is really an extensive and exhaustive survey of the parts (777 references in all) of behavioral epidemiology that most closely relate to vaccination games.

It is not clear whether Section 2  contains any novelty.  It should be made explicit whether and to what extent it goes beyond a condensed review of [40].  This should be made explicit.

Also, the results of subsections 3.1 and and 3.2 should be juxtaposed more clearly, perhaps by combining Figures 3 and 4 into one figure, so that it becomes visually obvious that the Nash equilibrium fall short of the societally optimal vaccination coverage.
Experimental design: The authors study meaningful questions and their methodology for analyzing the model  appear to  be sound and professionally executed.

However, some important information is missing, for example, it is not being explicitly said whether the graphs in Figure 4 rely on a numerical solver for finding the relevant equilibria, as I believe they would need to.
Validity of the findings: While my comments pertaining to points 1 and 2 can be addressed by minor modification, here I have a major issue:

Why is it that the two interventions considered by the authors lead to two qualitatively different predictions?  The authors appear to have completely overlooked this question, but it is an absolutely crucial one.

Let's take the point of view of a mosquito: In both cases it is faced with removal of potential blood meals.  Either because the human resource has become unapproachable due to repellent, or has been removed to another location.  In both cases the mosquito can either continue its search for another human provider of a blood meal until it succeeds, or content itself without a meal of human blood.  These scenarios would translate differently into differential equations. The authors have essentially shown (among other things and under certain assumptions about the costs) that in the second scenario the societally optimal strategy is to remove enough humans (by using insect repellent) to achieve herd immunity, while in the first scenario the cost of removing more humans will not lower the overall cost to society, as a higher proportion of the remaining susceptibles will eventually experience infection.  I find this quite interesting and it should be the major result of this paper.

But this issue is not addressed at all. Instead, the  authors set up their differential equations in such a way that insecticide repellent would apparently discourage mosquitoes from searching better-smelling humans that they might feed on [this is essentially what (12) translates into], while having fewer humans  around overall would still make them search until they succeed [translate (19) and the first line of (22)].  I find this highly implausible.  Insect repellent does not kill or disable mosquitoes, it only drives them away from particular humans.

Therefore I cannot recommend publication of the paper in its current form.
Additional comments: As you saw from point 3, I find some assumptions of your model implausible and did not recommend publication of this paper.

However, I believe that you have discovered, perhaps inadvertently, something that to the best of my knowledge has not yet been reported in the literature: The two different assumptions about mosquito behavior that I outlined above lead to two qualitatively different optimal strategies.  While I appreciate the care you took with using realistic parameters for a particular biological system to the extent possible,
I believe this is a more general finding about driving mechanisms and calls for a more general, abstract, treatment.  Such a paper might be a very valuable addition to the literature.

On the more empirical side: I don't know which assumption about mosquito behavior is (more) realistic. Perhaps the truth lies somewhere in between; perhaps there are empirical studies of this (in this case they should be cited); if not, such should be performed (in which case this should be recommended in your conclusions) .


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: OPTIMAL VOLUNTARY AND MANDATORY INSECT REPELLENT USAGE AND EMIGRATION STRATEGIES TO CONTROL THE CHIKUNGUNYA OUTBREAK ON REUNION ISLAND
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Optimal Voluntary and Mandatory Insect Repellent Usage and Emigration Strategies to Control the Chikungunya Outbreak on Reunion Island
 Sylvia R. M. Klein, Alex O. Foster, David A. Feagins, Jonathan T. Rowell, and Igor V. Erovenko

The authors investigated the potential for two intervention measures for under voluntary and mandatory protocols to control Chikungunya disease on Reunion Island when there is a risk of the disease becoming endemic.

General comment: I found the manuscript easy to read and follow and can easily be replicated.  I like the fact that the authors included their codes. I recommend it for publication subject to the revision below.

Specific comments
 Line 129: remove the extra “the”

Line 131: capitalize “table 1”

Equation 1: Give an explanation as to why disease-related dead is left out and not accounted for. The authors noted in the introduction 200 people dead from the disease on the Island.

Line 139: Do the authors expect to get a different result if this simplification is no made?

Line 153: Which of the two equilibria are the authors referring to?

Line 159: What is the justification for the use of repellant as a control measure?

Line 160: Revise the order of the papers cited.

Equation 10: The authors should give an explanation of this equation. It is not immediately clear from the description given. The authors should give explanation as given in one of Erovenko’s previous papers.

Equation 13: The derivative of equation 12 is not this simple expression. Are Z^* and N^*constant? Are these not functions of r? They contain beta_1 = beta_1(r). Or am I missing something?

Line 206: replace “stopping” with “stop”.

Equation 22: It is not clear how this equilibrium is greatly different from the first equilibrium in equation 6, the model with emigration is the same as equation 1 except for the addition omega from the S class.

Line 296: “resulting in a greater proportion”. Am I missing something here? How is this so? The magnitude of I/N is 10^(-4).

Line 327: “Are shown in Figure 8”. Discuss the graphs as was done for Figure 9. There are four panels in the figure, what do they say individually?


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: OPTIMAL VOLUNTARY AND MANDATORY INSECT REPELLENT USAGE AND EMIGRATION STRATEGIES TO CONTROL THE CHIKUNGUNYA OUTBREAK ON REUNION ISLAND
Review round: 2
Reviewer: 1

Output:
Basic reporting: The writing and has been done professionally.
Experimental design: The modeling itself appears to have been competently executed.
Validity of the findings: As the authors now acknowledge in the new paragraph at the end of their Discussion (Section 4),  their findings crucially depend on certain modeling assumptions that are now spelled out in that paragraph. I see two problems with these assumptions and the way they are presented.

1. The assumptions do not seem particularly plausible to me.  While my guess here is no better than the author's, the main problem is that these assumptions have not been substantiated by references to the literature on actual mosquito behavior.  Therefore the findings of this paper are only valid as an implication: "If we can assume that mosquito biting satisfies to a reasonable approximation the assumption spelled out in this paragraph,  then we can deduce the following policy recommendations."  As far as I can see, this implication has been validated by the paper, but the assumptions have not been experimentally confirmed.   The implication itself would still be a valid finding for a mathematical modeling paper, but it needs to be clearly presented as such.

2.  There is a mismatch between the categorical announcement of policy recommendation in the abstract and earlier parts of the paper, and the nature of the findings, that are only an implication, with experimentally unsubstantiated premises. This is misleading.  The new paragraph is buried near the end of the paper.  While the authors claim in their response that the last paragraph of the introduction now addresses the modeling assumptions, it does no such thing.  It simply restates the model's prediction.

I must, unfortunately, conclude that the current presentation of the findings is outright misleading.  If this publication is to have value as a contribution to the scientific literature, it needs to be clearly stated, in the abstract, that these conclusions were reached under certain assumptions about mosquito biting that await experimental verification, and these assumptions need to be clearly spelled out in a prominent place early in the paper.
Additional comments: The minor points that I had made in my previous review have been adequately addressed, but as you see from the previous item, my major objection still stands.


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: OPTIMAL VOLUNTARY AND MANDATORY INSECT REPELLENT USAGE AND EMIGRATION STRATEGIES TO CONTROL THE CHIKUNGUNYA OUTBREAK ON REUNION ISLAND
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have answer all my concerns.


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: OPTIMAL VOLUNTARY AND MANDATORY INSECT REPELLENT USAGE AND EMIGRATION STRATEGIES TO CONTROL THE CHIKUNGUNYA OUTBREAK ON REUNION ISLAND
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: I had concerns with some of the phrases that were used in the previous version to describe the meaning of the findings.  They have been addressed.
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: THE EFFECT OF DECAY AND LEXICAL UNCERTAINTY ON PROCESSING LONG-DISTANCE DEPENDENCIES IN READING
Review round: 1
Reviewer: 1

Output:
Basic reporting: Excellent reporting, although minor improvements are possible (see General Comments)
Experimental design: The only thing that remains unclear is if/how spillover was taken into account (see General Comments)
Validity of the findings: No comment
Additional comments: This paper deals with a topic that is very timely and relevant to the study of human sentence processing. The experiments are well designed, the analyses are state of the art, and the writing is very clear. Nevertheless, there are two issues need to be resolved before I can recommend publication.
(1) Does the verb in every stimuli sentence require a particle? If not, how was the “no particle” option incorporated in the cloze test and data analyses?
(2) There is no mention of how spillover was taken into account, even though this phenomenon is prevalent in reading, in particular self-paced reading. Were reading times on words directly following the particle also taken considered? If not, could this be why the expected effects were not found?

Minor comments:
- line 47: what does it mean for something to be “anecdotally assumed”?
- When introduction German particle verbs, it would be good to mention that moving the particle to after the object NP is required in German.
- line 115: the Dutch prefix “ver” in “verdelen” is not a particle (i.e., it is not split: “hij deelt het ver” is not possible)
- line 127-128: “self-paced reading and eye tracking modalities” and “reading modalities” -> shouldn’t this be “paradigm” instead of “modality”? In both cases, the modality is written/visual.
- Table 1 shows 95% CI instead of the standard error mentioned on line 221. Also, the caption is not quite accurate because the table presents cloze statistics but not the cloze test results.
- It would be helpful if the goal of the cloze test data analysis were explained before the technical details (starting on line 226)
- line 232-234: “the probability of the target particle was lower … for the interaction” -> for which combination of factor levels was the probability lower?
- The violin plots of Fig. 4 shows probability mass for negative values of entropy, even though entropy is by definition non-negative
- line 414: what did the preprocessing of eye-tracking data entail?
- line 417: the citation to R is “R Core Team”, not just “Team”.
- line 448-449: the problem of evaluating multiple dependent measures is not a “limitation of the BF analysis” in particular, is it?
- line 474: “The statistical analysis” should probably be  “The outcome of the statistical analysis”


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

Input:
Title: THE EFFECT OF DECAY AND LEXICAL UNCERTAINTY ON PROCESSING LONG-DISTANCE DEPENDENCIES IN READING
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment (see section 4 below for all comments).
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The paper reports the results of one self-paced reading and one eyetracking-while-reading experiments, aimed to investigate the effect of decay, and its interaction with predictability, on the processing of verb particles which are dependent on the verb but appear downstream from it. This is an interesting question, the experiments are overall well thought-out, the analysis is rigorous (with data and code provided) and the discussion is careful and responsible.
In my view, the main weakness of the manuscript is the presentation the hypotheses of the two frameworks, and in particular that of the LV05 model. Personally, the reasons for the predictions of the LV05 model were not clear to me, which made it hard to understand certain aspects of the experiment and interpret the results. Below I elaborate some more on this concern, and offer some other, more minor, comments.

Clarity of opposing hypotheses, particularly the predictions of the LV05 model:

To me, the Introduction (particularly the first page) was very confusing.
If I understand correctly, the experiments set out to test the predictions of Surprisal vs. LV05. First, I think this should be stated clearly and consistently throughout the paper, starting from the abstract (where now only Surprisal is mentioned, in contrast to "other theories") and then explicitly in the Introduction. (And also in the discussions - the Discussion of the SPR results now starts with "we hypothesized" – is "we" = LV05? and the hypothesis of Surprisal is not spelled out at all there; the authors only state that the results were not consistent with it).
Once this is established, I think the authors should take MUCH more time to introduce these frameworks. Currently, both Surprisal and the LV05 model are simply mentioned (along with their relevant predictions for the current research, but with no other explanation). I think the authors should present these theories for readers who are unfamiliar with them. What is Surprisal theory, what are its main tenets? What is the LV05 model? What is it modelling, what are its assumptions?
Then, the authors should explain both frameworks' hypotheses about decay and its interaction with predictability. For Surprisal, line 40 "interference and working memory constraints may negatively impact…": in "working memory constraints", do the authors mean "the limited capacity of working memory"? Why would interference and limited capacity interact with predictability in this way, according to Surprisal? More explanation is needed, along with the relevant results (from German? Hindi? Persian?)
Even more so, for the LV05 model, it was hard to understand the proposed reason for the interaction between predictability and decay. The crucial sentence is this: "If an upcoming lexical item is highly predictable, it can be pre-integrated into the pursued parse, facilitating its retrieval once encountered. However, if there is uncertainty about the lexical identity of a word, this will increase the likelihood that the parser either pursues a parse with a different lexical item to the one yet to be encountered, or makes no lexical prediction at all".
This raised a lot of questions for me:
-	Line 149 onwards "in the absence of interference, decay over distance … will make the long condition more sensitive to predictability". Why? I understand that these are the results of a simulation, but can the authors provide an intuition as to why this is so? Do the authors claim that when a lexical item is highly predictable, it is integrated (prior to its occurrence in the input) and it is therefore amenable to decay? If so, it should be stated clearly.
-	What's "highly predictable"? Consider for example a verb from the small set size group which takes five possible particles. If one of them appears in 80% of cases, and each of the other four – in 5% of cases, is the most probable one highly predictable, therefore integrated and amenable to decay? If this is the case, shouldn't that also happen for a hypothetical verb in the large set size group which takes 15 particles, with the most probable one appearing in 80% of cases, and each of the other ones in ~1.5% of cases? And what about a "small set" verb with 60%-10%-10%-10%-10% distribution of particles and a "large set" verb with 60%-4%-4%... distribution? Would the most probable particle be integrated?
-	What happens when there's no one highly predictable completion? What's the role of decay in these cases? What's the predicted difference between a small set verb with 5 possible completions each appearing in 20% of the time, and a large set verb with 15 completions, one appearing in 20% of the time and each of the others in ~6%?
-	The upshot from the last two questions is: shouldn't we look at constraint (cloze probabilities) *at the verb* in order to know what was preactivated/integrated there? Or perhaps at entropy, if it is assumed to modulate preactivation/integration (e.g. integration only happens when there are no strong competitors, i.e. low entropy), but again, *at the verb*? As the authors say in line 263, the study wanted to test "whether the number of potential particles pre-activated at the verb would affect reading times". But to know whether they are preactivated, don't we need cloze data from that point? (even though possibly subsequent material, i.e. the object, can prove our prediction wrong, leading to reanalysis? Since I'm not sure what the assumptions of LV05 are, I don't know what it will predict).
-	The manuscript does discuss entropy, but measured right before the particle. In the pre-test, it turns out that there's no difference between the two groups, but this is only discussed in the Results section, before carrying out the alternative analysis. I think it would be much better to acknowledge the potential problem, namely that the two verb types have similar entropies (before the particle), and why this may undermine the verb type manipulation, when the pretest is presented. Otherwise the reader is left very confused.
-	This is related to another minor point that was not clear to me: how were the verbs selected? Based on the cloze pretest, namely based on their preference after the object, before the particle? Or based on their particle selection options regardless of the specific object?

Other comments:
Line 36, after "there are accounts modeling the effect of intervening material…": I think it is natural to start the Introduction with the discussion of decay (which now appears in the second paragraph), as these are the more traditional approaches to distance effects. Then, Surprisal and anti-locality can be presented.
The manipulation of decay was introduced by adding a very short constituent – a two-word phrase. Could that be the reason why no effect of decay was found? Does the LV05 predict an effect of decay with such a minimal manipulation? Related to this, line 526, "it would have been difficult to construct longer sentences without reintroducing these factors (interference), which supports the idea that they are the source of processing difficulty": why does it support this idea? I think it only means that it's very hard (perhaps impossible?) to test the influence of decay by itself.
When entropy is first discussed, the concept should be explained – not only with a mathematical formula, but also with the intuition as to what it means.
Minor comments
Line 47 "activation decay is anecdotally assumed…": another relevant reference here is Chow & Zhou (2019), which is a replication of Wagers & Phillips (2014) (though the original authors do not frame their study as investigating decay).
Line 52 "decay is not a useful predictor": perhaps also cite Van Dyke and Johns' (2012) review which argues against a role for decay in sentence processing.
Materials section: Do all the experimental verbs necessarily take particles at all? I assume this is the case, but I think this should be stated explicitly
Line 217 "24 items that suited the experimental design" – meaning what? That they selected 6 or less, or 15 or more, particles?
Online norming study (line 249 onwards): Why is this pretest necessary? In the experiment, the verb is several words upstream from the particle, so why are reading times of the verb+particle relevant?
Line 382, "a second possibility is that locality and antilocality effects simply cancelled each other out": how is this relevant to the effect of predictability, which is the topic of discussion? I would think that it is relevant to the (lack of) effect of decay, not predictability.
Line 484 "speed up at the verb": this sounded to me like the authors were referring to a speed up at the verb relative to preceding material; it took me some time to understand that it means lower reading times in the large set verbs compared to the small set verbs.
Line 544, "a potential explanation for the lack of speed-up… more preactivated particles may have led to slower reading". I'm not sure I would predict this. I would think activations are not usually viewed as costly. Perhaps the source of increased reading times here is that the verbs are more ambiguous/vague, i.e. have more possible meanings?
Typos etc.
line 34: length > amount
line 166: items > item
line 128: delete second 'also'
line 319: delete second 'the'
line 341 and caption for Figure 5: I initially thought the RTs in the table are reading times for the particle (and wondered why they were so high). The text and caption should say that these are RTs for answering the comprehension questions. Same for line 440 and table 9.
Line 389: the number "1" is missing
Line 457, "the results of the statistical analysis": in all the reading time measures? If so, maybe "analyses"?

References:
Chow, W. Y., & Zhou, Y. (2019). Eye-tracking evidence for active gap-filling regardless of dependency length. Quarterly Journal of Experimental Psychology, 72(6), 1297-1307.
Van Dyke, J. A., & Johns, C. L. (2012). Memory interference as a determinant of language comprehension. Language and linguistics compass, 6(4), 193-211.
Wagers, M. W., & Phillips, C. (2014). Going the distance: Memory and control processes in active dependency construction. The Quarterly Journal of Experimental Psychology, 67(7), 1274-1304.


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

Input:
Title: THE EFFECT OF DECAY AND LEXICAL UNCERTAINTY ON PROCESSING LONG-DISTANCE DEPENDENCIES IN READING
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is well-written and clear. References and contextualization are appropriate, and the paper is self-contained. Raw data is available.

Comments:
Supplemental material is referenced (e.g., Page 8, last sentence (in Analysis section), states “...can be found in the supplementary material”. Similarly page 13, in Data analysis.), but I couldn’t find supplementary material, either at the end of the PDF, in the PeerJ review materials, nor in the OSF repository. This was not a big problem for me because all information is contained in the source code, but this should be fixed (if it was not my oversight).
Experimental design: no comment (everything is satisfying)
Validity of the findings: The statistical analysis methods are appropriate (e.g., using a full random-effects structures and an appropriately transformed dependent variable). While I did not check all the details in the source code, the code ensures reproducibility of reported results. Planned and exploratory analysis are cleanly separated. Conclusions are well stated and supported by the analysis results.

Minor comment (change encouraged but not mandatory):
The authors note that surprisal generally predicts that long dependencies reduce processing effort. Whether this actually applies to the verb-particle dependencies studied here is, however, not easy to establish due to data sparsity (as the authors explain convincingly). Intuitions about surprisal can be misleading, and simulations with a probabilistic model are needed to really understand what the predictions are. Given that the stated surprisal predictions are not supported by simulations, I suggest the authors temper their claim about the predictions of surprisal. This is already acknowledged in the "Predictions" section, but I'd suggest the authors also acknowledge it in the conclusion. E.g., in the conclusion: “the surprisal account would predict…” is an overstatement given that there are no simulation results or explicit claims about these particles in the cited surprisal studies to support this. After all, it might well be that the absence of evidence for a distance effect is exactly what surprisal would predict. The authors already acknowledge this possibility in the Conclusion, where they state that previous observed antilocality effects might have been due to stronger lexical constraints created by intervening material than in the stimuli here.
For what it’s worth, I ran five replicates of a high-quality neural language models (trained on 800M words of German Wikipedia text, about at the level of the models in Gulordava et al. 2018 (https://www.aclweb.org/anthology/N18-1108/)) on the stimuli to obtain surprisal estimates on the particle, predicting model surprisal from distance (long/short) and the entropy from the cloze study, with full random effects for items and model replicates. There was a clear effect of entropy (beta=1.2, t=3.1), at most marginal evidence for an effect of distance (beta=-0.17, t=-1.6), and no evidence for an interaction (t=-0.8). (Also, no evidence for any effects was observed when predicting from the discrete 2x2 contrasts of the experiment). So, while these surprisal models predict an effect of entropy, they predict at most a small effect of distance.
Given that there are no established neural language models for German that have been used in prior psycholinguistic research, there is no need at all to run such models for the purposes of this paper. However, these simulation results suggest being careful with evaluating surprisal predictions and emphasizing that the predictions described here are intuitive, not based on an actual probabilistic model. Alternatively, the authors could also acknowledge that it is simply not clear whether or not surprisal predicts an antilocality effect for these data.
Additional comments: No additional comments


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

Input:
Title: THE EFFECT OF DECAY AND LEXICAL UNCERTAINTY ON PROCESSING LONG-DISTANCE DEPENDENCIES IN READING
Review round: 2
Reviewer: 1

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


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

Input:
Title: THE EFFECT OF DECAY AND LEXICAL UNCERTAINTY ON PROCESSING LONG-DISTANCE DEPENDENCIES IN READING
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I thank the authors for their serious and thorough consideration of my comments from the previous round of review. I find the manuscript much improved, clearer, more focused and easier to read and understand.
I still have some remaining relatively minor comments, mostly with regard to the presentation in the Introduction. If these concerns are addressed, the manuscript can be published in PeerJ.
Specific comments:
Abstract:
"Locality effects induced by interference and working memory have been…": this would probably be clearer if it said "induced by interference and working memory load have been…" (as in the previous sentence).
Also - and this is not crucial at all for the paper, I'm just wondering about it – I'm not sure what the authors mean by "effects of working memory load". The way I see it, interference can come about as an effect of high working memory load (more items that are similar to one another); decay can also come about as an effect of high working memory load (not enough resources to keep the item active while keeping other items active too). So I wonder if in saying "working memory load" the authors mean some other effect, possibly displacement, i.e. forgetting some material to make room for other material?

Introduction:
I think the presentation of surprisal and related ideas is still somewhat confusing, mainly because, I believe, there are two distinct, important predictions made by surprisal, but the presentation sort of mixes the two:
1.	First, surprisal says "more predictable is easier". This is by no means something that was first claimed by surprisal theory. It was shown in ERP studies since Kutas & Hilliard (1980) and in reading times since Ehrlich & Rayner (1981) (and maybe before?). Surprisal is just one way to model this observation. This generalization is the basic tenet in the predictions of both theories contrasted in Figure 1 (in both, reading times on the left are shorter than on the right).
So I think in the subsection discussing word predictability, the discussion shouldn't really start with or focus exclusively on surprisal. In fact, on the next page the authors offer an explanation for the effect of predictability based on decay (lines 116-118). The discussion in the subsection discussing word predictability can therefore outline the main observation (predictable is easier) and findings, and then mention the surprisal account, and also the decay account.

2.	The second thing, which is more specific to surprisal, is the prediction for antilocality effects. Antilocality is briefly explained in the abstract and then is sort of assumed, but never really presented methodically.
So I would suggest including an explanation of this effect in a dedicated subsection. So the order will be: the "word predictability" section (the prediction of which are identical for the two hypotheses); the "antilocality" section (suprisal); and then the "decay" section.
Also, the two paragraphs on the interaction of predictability with distance (p. 2 line 65 onward) are very confusing. They sort of go back and forth between discussing the interaction of predictability with distance and discussing the interaction of predictability with working memory load without explicitly explaining why or whether the two (distance/working memory load) are interchangeable.
Finally, the bottom line of these two paragraphs is "facilitation in the reading times of a distant word .. may only occur when that word is highly predictable" (this is also stated in the predictions section, and in the conclusion) – but this interaction is not represented in Figure 1, where the effect of distance is identical for more predictable and less predictable words.
One general suggestion: it would perhaps be helpful to have one example sentence in the introduction (perhaps even with a verb-particle dependency) to accompany the discussion, so the different predictions can be exemplified with regard to that sentence, to make them concrete and easier to understand.
Experiment 1 Methods:
I think it would be helpful to state explicitly, around line 307, that the set size manipulation therefore did not result in a difference in the predictability of the particle.
The entropy formula on line 320 should be explained.
One last thing – just a thought, no need to do this – I wonder whether in the eyetracking there would be effects on rates of skipping the particle altogether (since we know that more predictable words are skipped more often). The authors say that the particle was not always fixated – I wonder, for future studies, if there could be something interesting there.

Typos and very minor comments:
Abstract, 8th line from bottom: should be "decay, predictability or their interaction".
Abstract, 5th line from bottom: perhaps instead of "facilitate or hinder reading times", change to "facilitate or hinder processing"?
Line 210: parentheses missing around Lewis and Vasishth, 2005.


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

Input:
Title: THE EFFECT OF DECAY AND LEXICAL UNCERTAINTY ON PROCESSING LONG-DISTANCE DEPENDENCIES IN READING
Review round: 3
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: I thank the authors for the very thorough revision of the Introduction! I find the Introduction extremely clear now. The discussion around example (1) in particular is very helpful and accessible (and I believe it will benefit other readers too). I'm looking forward to teaching this paper in my classes!

One tiny comment, on line 119: please clarify what PCFGs stands for.


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

Input:
Title: BMD: AN R PACKAGE FOR BENCHMARK DOSE ESTIMATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: The scientific community should be encouraged to develop novel tools for BMD modeling, considering the drawbacks of the current software such as US EPA’s BMDS and Netherland’s PROAST. This "bmd" R package developed by Jensen et al. has some advantages over the BMDS/PROAST. Notably, one of the major claimed novelty of "bmd" is on the model averaging. However, as far as I know, BMDS has already incorporated the function of Bayesian model averaging for dichotomous data, and the development of model averaging for continuous data is ongoing and proposed to be done this year. In addition, the BBMD system (Shao et al. 2018) have already developed model-averaging BMD estimations for both dichotomous and continuous data.

I do appreciate the authors efforts of developing alternatives to the current BMD tools. However, when the authors stated that this R package “can be seen as a flexible alternative to BMDS and PROAST”, they need to make clear arguments on when or under what circumstances the risk assessors should use their “alternative”. After all, the two commonly used tools have been extensively validated in the past decade and have good credibility. This brings out another issue – only several datasets were analysis and there were no parallel comparisons between the results from this R package and those from BMDS/PROAST. Is there any difference on the value of BMD/BMDL estimation? If so, how would you explain the differences? These questions cannot be simply ignored especially for a methodology paper. Besides, the use of “bmd” R package had already been mentioned in the previous paper five years ago (Ritz et al. 2015), which makes me wonder the purpose of the current manuscript. I thought there would be a relatively large-scale comparison between BMD estimates using “bmd” vs BMDS/PROAST, basically to show the validity of “bmd”. Otherwise, how would you justify the validity of these results?

When compared to the BMDS, the best argument of this R package could be the flexibility of handling the count data, the method of which has been mentioned in the parent R package (Ritz et al. 2015).  In the current manuscript, using just one dataset is apparently not enough to justify the validity of BMD estimation for count data using this new package. Besides, how does the results compare to those from BMDS, when the count data were treated continuous? The authors were correct that this package may have potentials in the field of ecotoxicology. As far as I know, the count data in other fields of toxicology is not common – most of the endpoints are dichotomous or continuous. Therefore, focusing on “selling” the package using much more datasets (especially count data) on ecotoxicology can be helpful.
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: BMD: AN R PACKAGE FOR BENCHMARK DOSE ESTIMATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors don’t seem to have a clear motivation or audience for this manuscript, which I think has led to its disorganization and lack of clarity. What is the authors’ motivation for writing this package and manuscript? How is the package novel, useful, and important? Further, to whom are the authors targeting this manuscript – is it to toxicologists who would use bmd to estimate BMRs and BMDs, to statisticians/mathematicians interested in the mathematical basis of this package, or to some other group altogether? Selfishly as a toxicologist, I would think this paper would be most widely read by toxicologists wanting to use this package for BMD estimation. If so, the authors need to rewrite the manuscript with that lens. The authors assume too much background understanding of BMDs, dose-response, risk assessment, and other toxicological ideas that is not appropriate for a non-tox focused journal like PeerJ. The authors also assume that the reader understands the different statistical structures outlined in Section 2 and why they are different for different data structures. The authors also are not careful enough with their word choice, especially in regard to chemical concentrations vs organismal response, which is crucial for a discussion of a metric that describes organismal response to different chemical concentrations (doses). The writing needs a lot of reworking with this consideration – I’ve pointed out a few specific places under “general comments” below in which I thought it was unclear, but this needs to be considered throughout the manuscript.
Further, I am not overly familiar with PeerJ’s “Bioinformatics and genomics” section, but I do not completely agree that this is relevant to that section. It is description of an R package for the estimation of a statistical metric specific to toxicology describing a biological response, but this isn’t the type of analysis when I think of when I think about “bioinformatics,” which usually is in relation to large data sets or high-throughput processing.
Experimental design: The authors do not clearly define their research question or the knowledge gap they seek to fill. I do think the examples portion of this manuscript is the strongest part of it, although the authors need to be more explicit in how they chose certain analytical steps (e.g. choice of risk definition and BMR).
Validity of the findings: The authors do not clearly define the impact or novelty of this work. The authors’ conclusions in the “Conclusion” are not supported by the manuscript (see my specific note below).
I think this is the section to report on the provided data and code – overall, the data and R code are provided in a way that is very clear and easy to use. I particularly appreciate the Word doc which carefully goes through the R code for all of the examples – this is excellent. The only part of the code that didn’t work for me is the sandwich estimation of the CopperTemp.m2 model – the block of code on the top of pg 5 gave me this error:
Error in UseMethod("estfun") :
  no applicable method for 'estfun' applied to an object of class "drc"
I have two more questions about the R code/analysis: first, for the bootstrapped estimates for example 3.3 on pages 11 and 12 of the word doc, I got the exact same BMD and BMDL estimates as the example code. If these are bootstrap techniques, shouldn’t they be stochastic? I’m probably missing something here because I’m not a statistician, but I wasn’t expecting to get the exact same results, down to four decimal places, as the example code.
Second, what is the significance/meaning of a negative BMDL estimate (pg 13 the BMDL estimate for alpha.m2.bmd)? How would one use this negative BMDL in practice?
Additional comments: 1.	The first sentence of the abstract makes some assumptions that the reader knows what the authors mean by “dose”, “hazard characterization”, and “risk assessment”. Nowhere in the abstract do the authors make it clear that this package is designed for use in toxicology and chemical risk assessment (or however the authors wish to describe it). Suggest revision to make that clear since PeerJ is not a specialized tox journal.
2.	Introduction, L16-23: I’m a toxicologist myself and I don’t understand this introduction to BMD. How is this different from more commonly calculated tox statistics such as NOEC or LOEC (this seems almost identical to a NOEC to me)? More specifically, what “predefined small change” is used? Are there established, normal changes (5%? 50%?) that are usually used for BMD estimation? By “background level”, do the authors mean control response or the background concentration of the contaminant? How is “experimental design” directly included by calculating a confidence interval (L22-23)? And the BMD refers to the contaminant concentration, but this is not clear when the authors talk about “reference values” and “limit values”. Overall, this important introductory section needs to be rewritten with careful word choices to make it very clear what BMDs are, what they estimate, and what they’re used for. Be explicit if the changes or limits or values are in regard to organismal response or chemical concentration. Figure 1 is not at all helpful in this introductory section (e.g. what is p0?).
3.	Introduction, L44-45: The authors do a good job of reviewing the software available for BMD estimation but only state that the package they’re describing here is a “flexible alternative”. What specifically was the goal of developing this package, if there are already software packages that are freely available (I think?) online? How is bmd different from what’s already available?
4.	L51-59: Again, this section is very confusing to me, specifically L52-53 – how could there be absolute and relative changes when you’re comparing the response to the “background level”? Should the responses always be considered in comparison to the control (so relative)? Do the authors mean that comparisons of the response to prior knowledge are “absolute” (as they describe in L66-67)? Further, the authors use the parameter p0 for the first time in L59 as an example of the opposite to the standard case before they’ve ever defined p0 or adequately described how a standard case (BMD for an increasing dose-response relationship) is estimated. To me, the authors are making too many assumptions on the background knowledge of the reader and should rewrite this section and the introduction to be clearer.
5.	L66: “mean background level/risk of an adverse effect” – does p0 refer to the chemical concentration or the organismal response? Looking at Figure 1, I think it refers to organismal response but this explanation is confusing.
6.	L71: what do the authors mean by “risk definition”? It should be explained at the beginning of the methods section. And I don’t understand what the reader is supposed to glean from section 2.1 – how would I, as a toxicologist wanting to use this package, use this information to decide how to use bmd? Outside of the different data types, how do I decide which “definition” to use?
7.	L78-86: I don’t understand this – why isn’t p0 just the control response and is instead estimated assuming a normal distribution? The parameter x0 is the “level for which the response is considered adverse” – is this the level of the chemical (concentration) or organismal response?
8.	L205: define BIC
9.	The examples are overall very helpful. The authors chose very different examples of various exposure types (soil vs aquatic), organisms, endpoints, and issues with the data (e.g. overdispersion in example 3.2) that strengthen this paper and the reader’s ability to use these examples for his or her own work. It would be helpful if the authors explain how they chose the “definition” for each example (e.g. L252 the authors chose the “additional risk definition” for this example – why?). Similarly, how do the authors chose the BMR? It is 0.05 for example 3.1 (L256) and 0.1 for example 3.2 (L272) – why?
10.	L252-260: Similar to my comment for Figure 1, what do the units mg/kg refer to in example 3.1? I think it’s mg chloroacetamide/kg soil, assuming the earthworms were exposed through the soil, but this should be explicitly defined.
11.	L285-6: what is the evidence for overdispersion? How would one know that there is “more variation than can be explained by the model”? The authors explain an indication of overdispersion in L294 but that’s only after it has been corrected for.
12.	L328: “do” should be “due”
13.	L349: “analyzes” should be “analyses”
14.	L352-363: a summary of this should be included in the introduction to describe the differences between bmd and existing programs (see my earlier note). The authors need to include in this section and others the significance of these differences, however. From my reading, the main difference between bmd and BMDS and PROAST is that it’s connected to drc, includes fractional polynomials, semi-parametric alternatives, and standard error estimations. The authors do a good job explaining the significance and utility of standard error estimations in L359-360, but the significance of the other differences are not stated.
15.	L386-388: the authors conclude by saying that the “BMD methodology has great potential in ecotoxicology” and it is applicable to “various analytical issues” but they have not described that explicitly in any other part of this paper. I cannot make those conclusions having read this paper, and the authors need to more clearly lay out their evidence for these claims throughout the paper.
16.	Figure 1 legend: define BMD, BMDL, BMR, and p0 in the figure legend, along with what the units of mg and kg refer to (I assume mg is of the contaminant and kg is soil because it was a soil exposure?).


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: BMD: AN R PACKAGE FOR BENCHMARK DOSE ESTIMATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: - Many of the math expressions in the manuscript are improperly formatted. For example, I have no idea what is going on in equations (7) (8) and (9).   Equations/expressions need to be cleaned up throughout the manuscript.

- Line 52 - 55.  This text needs to be rewritten and clarified. In reality, some fo the definitions are only applicable to certain data types.  That is the meaning is relative to the type of data.  For count, the meaning is usually based upon the rate etc. This needs to be clarified.

-Their Hybrid response is based upon the normal distribution with constant variance.  It would nice to know if their approach extends to non-constant variance or Log-normal models.  The latter two are usually used in Europe and North-America.

- It is unclear what the author's mean by 'after-fitting.' on line 120.  This is the first time I have encountered this term in the literature (statistics or otherwise).  Some discription should be given to the reader.
Experimental design: -  There is no comparison to the other software packages (e.g. BMDS and PROAST).  As many of the models are shared between all three packages some attempt at comparison should be made.

-  Additionally timings of the algorithms should be given. Choice between the use of R-BMD bootstrap model averaging and PROAST bootstrap model averaging may come down to computational efficiency.  Currently the online EFSA/ PROAST tool takes 10s of minutes for several hundred bootstraps. It would be nice to know the difference.

-  The Author's do not compare their estimates using the variety of methods they propose.  For example, table 3 gives bootstrap BMDL estimates.  They should give a table that gives the results for every method presented.
Validity of the findings: - The findings are sound.
Additional comments: The paper is very well written and should be ready for publication given correction of the above concerns.


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: BMD: AN R PACKAGE FOR BENCHMARK DOSE ESTIMATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No more comments
Experimental design: No more comments
Validity of the findings: No more comments
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: BMD: AN R PACKAGE FOR BENCHMARK DOSE ESTIMATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: I am re-reviewing this manuscript after submitting reviews on the initial submission. Overall, the authors did a good and thorough job responding to my comments and addressing them through changes in the manuscript. I appreciate the authors hard work and time on this. There are a few points that are still unresolved or unclear which I’ve listed under ”General comments”, but overall I think this manuscript is very close to submission with minor changes (mostly focused on the clarity and structure of significantly rewritten sections such as the Introduction and Validation of the R Package).
Experimental design: The authors responded to my comments sufficiently and added a new section comparing the bmd package to the existing programs (Section 4) which is quite good (although needs editing and restructuring, see General Comments below). Overall, the experimental design is strong.
Validity of the findings: The authors responded to my comments sufficiently, however a new paragraph in the discussion is concerning (L627-633) and seems to potentially undermine their conclusions and the utility of this package. The authors need to address this.
Additional comments: L15-29: I appreciate the authors taking time to rewrite this section for clarity and it is improved but is still a bit confusing. What do the authors mean by ”agent” in L16? Why not use the word chemical or stressor? And the authors need to define the many acronyms they use in L24-25. This whole paragraph needs to be closely re-read for mistakes and edited for clarity (e.g the last sentence doesn’t make sense: ”also when exposure is a concentration or a contaminant level”?)
Overall, the introduction is much improved and does a good job explaining the background and motivation for the paper. However, there are typos, confusing language, fragmented or run-on sentences, and awkward or non-existent transitions between paragraphs throughout that will lead to a lack of clarity (e.g. L51 – what do the authors mean by ”compare e.g. Bresica, 2020”? Is that reference an example of BMD or NOAEL or the differences between them?). The introduction needs to be closely re-read by all the authors.
L157: the authors did not resolve my comment below – they literally rearranged the words that were already there and it is still unclear.
Previous comment and authors’ response:
Reviewer: The parameter x0 is the “level for which the response is considered adverse” – is this the level of the chemical (concentration) or organismal response?
Authors: Thank you for the comment. This has been changed to: x_0 is the response level considered to be adverse.
L257-274: I again appreciate the hard work the authors have put into this revision, including this new section which I think does help the reader substantially. However, like the introduction, there are small typos and errors throughout that need to be addressed with careful re-reading by all co-authors (e.g. L263 ”cause” should be ”course” and the sentence that runs L270-272 doesn’t make sense).
”Validation of the R Package”: I really like this idea behind this section and I think it does a great job of comparing bmd to the existing packages! However, again, this section needs to be closely re-read by all authors (the sentence that runs L512-513 does not make sense in professional English). Also, the structure of this section is all over the place – the authors present results (L536) and then return to methods (L537-561) and then present more results (L562) before discussing the results (starting in L563). I think this section needs to be reorganized.
L627-633: this paragraph is concerning – the authors do a good job discussing differences in results between the packages in L566-581 but here the differences sound much more significant. I also don’t understand what the authors mean that ”none of the packages can be considered the ”truth”” (L631). The authors need to be much more clear on this important point, since the implications of differences between bmd and BMDS and PROAST that have been ”validated extensively” (L627) are potentially a big problem for users interested in bmd.


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: MUSCLE STRENGTH AND INFLAMMATORY RESPONSE TO THE TRAINING LOAD IN ROWERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This research article presents original data about assessing the biomechanical and biochemical profile changes in training forms were introduced. Therefore, the study counted with seventeen male sweep-oar rowers, members of the Polish national rowing team. The authors examined anthropometric, biomechanical, and biochemical factors related to skeletal-muscle function and performance at two moments: 1) at the beginning of the preparatory period; and 2) at the end of preparatory period.  In summary, the training program of approximately five months was sufficient to lead a significant (P<0.05) decrease in some biomechanical factors: global strength of lower limb extensors (right), torques – (torso, flexors), and torque (torso rotator, right); and some biochemical factors: CK, TT, and TT/C. Nevertheless, total phenolics increases significantly after the intervention. For this reason, the authors stated that the amount of strength might be significantly affected by the antioxidant defense promoted by excessive muscle damage.

The manuscript is well written and can improve physiologists and exercise sciences/health sciences professionals' understanding.

The article included a sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge. Moreover, the relevant prior literature was appropriately referenced. However, there are some aspects regarding the validity of the finding that should be clarified better.

1) The central point that needs more clarification is why the authors stated in the conclusion that the antioxidant significantly affects the amount of force? The reason I am asking is that there is no relation of causality between those conditions. The authors indeed bring a contextualization that points to this affirmation, however, the increase of total phenolics presented in this research does not allow the affirmation that antioxidant defense is playing a role on muscle strength, it can be  "Randomness." Therefore, I suggest rewriting the sentence suggesting the antioxidant profile as a possible variable that might be playing a role in muscle strength. Moreover, further studies should also investigate if there is a causality effect.
Experimental design: The study design is appropriate to address the primary aim of the study.
Validity of the findings: The validity of the findings agrees with the experimental design. The only correction needed is at the conclusion (mentioned right above).
Additional comments: There are a few minor comments:

1) add the sample size at the caption of all tables;
2) State the statistical test performed to find the P-value at the table legends.
3) Why did the authors not present a table or a figure with the correlation? This illustration may facilitate the reader.


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: MUSCLE STRENGTH AND INFLAMMATORY RESPONSE TO THE TRAINING LOAD IN ROWERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: English is not my primary language. However, I find that the text presented for evaluation is fully understandable and the information contained in the chapters describes well the problems and methods presented in the article.
The authors used sufficiently extensive literature on the impact of intense exercise on biomechanical and biochemical changes in the body of highly qualified athletes. There are many publications on this subject, while this publication refers to the most important items in the literature on rowers.
The structure of the paper corresponds to the general principles of scientific publications, i.e. division into sections, references, tables and figures.
Raw data for biomechanical measurements and biochemical analyzes were provided in a form that could be reproduced and recalculated.
The experimental group of players, measurement methods and biochemical analyzes as well as the results and their adequate statistical analysis were well selected in order to verify the research hypotheses.
Some of the cited references indicate a tendency to "strengthen" the scientific argument carried out by the authors of this article. I did not notice the glaring "clustering" of similar thematically similar publications.
Experimental design: The article clearly presents two aims: i) research (scientific) and ii) practical (training). The first was to obtain information on the relationship between training in the preparatory period and changes in strength in selected skeletal muscle groups and the biochemical basis of these changes. The practical goal is to link the training loads with the value of strength gains and overtraining rates in the preparation period.
The applied measurement methodology meets all the highest standards in biomechanical and biochemical research. Stands for measuring force in isometric conditions are widely used in sports research and the implemented metrological solutions are patented. The scheme of the experiment is very simple, it consists in verifying the effects "before-after". The factor that initiates changes in the body of the studied persons is the training load in the preparation period.
The consent for the experiment with the participation of humans was issued by the Regional Bioethics Committee at Poznań University of Medical Sciences.
Standard biochemical methods were used, citing the references in which they were described. Similarly with the methodology of measuring muscle strength. The description in the text is supplemented with a view of the subject on the stand, illustrating the construction of the stand, positions during measurement and the method of stabilizing the adjacent body segments. In the description under Figures 1-3, it would be advisable to add the information "under static condition".
Validity of the findings: The value added to literature is the association / correlation of representative biomechanical and biochemical parameters with the content of rowing training. It should be emphasized that they were rowers of the highest sports level, often the best at world. Inducing an increase in physical fitness features in such highly qualified athletes with long-term training experience is a great challenge for training methods. This article provides the content and forms of training during the 24 weeks of the preparatory period. There are few publications presenting so precisely the training methodology not only in rowing but also in other sports.
Raw data is available and can be further processed. The applied statistical analysis is simple and accurately selected to verify the purpose of the work. It would be advisable to give in tab. 3 and tab. 4 information on which test (t-Student or Wilcoxon) detect significant differences in the relevant parameters. An additional comment to the statistical analysis is provided in the attached file.
The work is completed with short and careful conclusions regarding recommendations for improving the training process for this group of athletes. The conclusions follow directly from the results of the experiment. The few correlations in this work seem to indicate that the antioxidant potential of the organism has an influence on the value of the skeletal muscle force. However, confirmation of this conclusion requires a larger group of voluntary subjects, representatives of various sports disciplines.
Additional comments: The experiment is very interesting and scientifically valuable and brings new information to sports practice. It is necessary to run and check the statistical analysis of the results again.

SELECTED PROBLEMS RELATED TO THE STATISTICAL ANALYSIS OF THE RESULTS.
Table 4 shows the biochemical parameters that changed after training in the preparation period. These include: CK (U / L), TT (ng / ML), TT / C, Total phenolics (g GAE / L). Using Statistica v.13.3, the analyze has been re-calculated.
	                                          Shapiro-Wilk test	                    t-Student	Wilcoxon test
	                                            Examination
	                                         First	     Second
CK(U/L)	                              p=0,0181	       p=0,0010		                                   p=0,0245
TT (ng/ML	                       p=0,9366	       p=0,0430	                           (-)	                 (-)
TT/C	                               p=0,3382  	p=0,2305                 p=0,0062	           p=0,0615
Total phenolics (g GAE/L)	p=0,4632 	p=0,4610	                p=0,0123

1)	According to the raw data provided by the authors, the mean and standard deviation values for TT_1 and TT_2 are respectively:
TT_1 = 5,74±1,13, while in tab.4 = 5,81±1,13)
TT_2 = 5,21±1,27, while in tab.4 = 5,28±1,27)
Therefore, it is not known for what values the difference between the tests for this parameter was calculated.
2)	It has to be decided which of the t-Student test or Wilcoxon test is to be used to analyze the differences in the TT/C parameter. The basis is the assessment of the normality distribution of the differences between the paired values of the first and second measurements (delta). The Shapiro-Wilk test shows that the distribution of empirical data (delta) is not close to the theoretical one because W = 0.9156 (p = 0.1251). A borderline value of p = 0.3 is assumed to assess the compliance of these distributions. Thus, for the TT / C parameter, the Wilcoxon test to evaluate the differences is adequate. As shown in the table above, it cannot be concluded that there are differences in the "before-after" measurements of TT / C.


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: MUSCLE STRENGTH AND INFLAMMATORY RESPONSE TO THE TRAINING LOAD IN ROWERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors successfully addressed all the reviewers comments, increasing the manuscript quality.
Thank you.


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

Input:
Title: MUSCLE STRENGTH AND INFLAMMATORY RESPONSE TO THE TRAINING LOAD IN ROWERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is prepared in the form of good standard scientific articles. Literature selected adequately to the subject and appropriately used in the chapter "discussion". There are no objections to the structure of the tables, which well illustrate the measurement results and their analysis. After specifying the purpose of the work as part of the self-correction, the final conclusions are clear.
Experimental design: The problem undertaken at work corresponds to the profile of the journal. The work is well grounded in the research on the impact of maximum physical effort on the human body. The original research methodology and properly selected statistical analysis of the results should be emphasized.
Validity of the findings: The results of this work cover a certain gap between the basic physiological knowledge and the training theory in competitive sport. The conclusions enrich the basic knowledge on the border of physiology and biochemistry and constitute the premises for the methodology of sports training.
Additional comments: Interesting publication. A well-planned experiment. It is worth emphasizing that measurements were carried out on such a large group of competitors of the highest sports level.


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: LONG-TERM ADMINISTRATION OF VITAMIN B12 AND ADENOSINE TRIPHOSPHATE FOR IDIOPATHIC SUDDEN SENSORINEURAL HEARING LOSS: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comments
Experimental design: no comments
Validity of the findings: no comments
Additional comments: This manuscript described that long-term administration of vit B12 & ATP helped prevent the progression of hearing loss after idiopathic sudden sensorineural hearing loss (ISSNHL).
Figure 2 revealed that hearing outcome was better during 4 – 6 months after the onset of ISSNHL in the group taking vit B12 & ATP than that not taking vit B12 & ATP. Although the difference was small, it was significant statistically.

Because many otologists consider that hearing level became stable after three months from the onset of ISSNHL, more detailed description is required.

#1
My understanding is that hearing level moves longer in low-tone frequencies that in high-tone frequencies after the onset of ISSNHL. What was the frequency area of the slowly progressing hearing loss during 4 – 6 months after the onset of ISSNHL?

#2
It is generally considered that hearing recovery can be expected longer in acute low-tone SNHL than in ordinary ISSNHL.  I expect some comments about the initial hearing type of ISSNHL in association with the delayed hearing change after the onset of ISSNHL.

#3
Furuhashi’s paper (Clinical Otolaryngol, 2002) described recurrence of ISSNHL for a very long period like ten years.
I would like to recommend a paper investigating for more than three months after the onset of ISSNHL for one of the references.
Yeo, SW, Lee DH et al.: Hearing outcome of sudden sensorineural hearing loss: Long-term follow-up. Otolaryngol Head Neck Surg 136;221-224. 2007.


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: LONG-TERM ADMINISTRATION OF VITAMIN B12 AND ADENOSINE TRIPHOSPHATE FOR IDIOPATHIC SUDDEN SENSORINEURAL HEARING LOSS: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Vitamin (vit) B12 and adenosine triphosphate (ATP) are usually used for the treatment of idiopathic sudden sensorineural hearing loss. However, we don’t know how long these medicines should be used. This paper clearly answers this question. In this paper, authors clarified that vit B12 and ATP did not influence the overall prognosis of idiopathic sudden sensorineural hearing loss; however, the long-term administration can prevent the progression of hearing loss after idiopathic sudden sensorineural hearing loss. I think that this must provide clinically useful information to the readers of PeerJ. Actually, the number of enrolled subjects is not many (n=117), but this issue is mentioned as a limitation of this study. Therefore, I would like to recommend this paper for the publication.


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

Input:
Title: CHARACTERIZATION OF DNA METHYLATION IN MALAWIAN MYCOBACTERIUM TUBERCULOSIS CLINICAL ISOLATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Characterization of DNA methylation in Malawian Mycobacterium tuberculosis clinical isolates

Ndhlovu et al. report data from an interesting study examining potential mechanisms for MTB phenotypic heterogeneity, despite >99% genetic homology.  Epigenetic factors play an important role in regulating an extensive number of processes and influencing phenotype. Since there is sparse data on the effects of epigenetic modifications on the phenotype of MTB this study is important to field.

Methods

1)	Choice of isolates to study – lines 100-101 – “Out of a total of 133 MTB positive isolates, 18 were selected based on which isolates were the first to be successfully revived from frozen state”

Since this is an observational study the data is valid but please can the authors comment in the discussion on this strategy for selecting isolates – might speed of growth be related to epigenetic modifications. A comment should be made in the discussion as to how selection of isolates may have influenced the methylation data.

Results

2)	Figure 1: Is the reference here from H37Rv? Please mention in the legend.

3)	Figure 2: Please can you provide X and Y axis legends. Fig 2A and B particularly their Y axis is given as ‘0’.  Please can you increase the font size of all text on Figure 2 – it is very hard to read the legends and labels. I don’t find this figure very easy to interpret.

You are reporting ‘Distribution of methylated samples in each lineage for the motifs. It might help if there were numbers stated in the legend or on the bars. How many samples of each lineage do you have here? Maybe it is because the Y axis doesn’t have a legend that I cannot quite follow this.

Fig 2 C and D – You state that there is high variability in methylated motifs in modern strains (L2/L4) while largely uniform in ancient strains (L1/L5/L6).

Please can you state how ‘modern’ and ‘ancient’ are defined.

In Fig2D – L5 and L6 indeed appear to have little variation – the points are all around 1.0 in the violin plot – however L1 looks to have the largest variation of all. Please can you explain why you consider this variation less than L2/L4.

4)	You state in line  266 that ‘in some cases, low methylation could possibly be attributed to sequencing errors”. This is important and you should give a numerical range or expected inaccuracy of the results based on system used.

5)	Line 294 “ as a result of sample ERS2711943 being lowly methylated at 56%”

Please change to “ as a result of sample ERS2711943 having low levels of methylation (56%) as compared to the other samples at >95%.

6)	Line 46-47 “We report previously unreported mutations…”
Please change to “We report previously unknown mutations”

Discussion

7)	‘Studies of Mtb DNA methylation using SMRT sequencing have previously focused on strains originating from the USA, Asia and…’ please can you give approx. numbers studied and lineage to put your study better into context.


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: CHARACTERIZATION OF DNA METHYLATION IN MALAWIAN MYCOBACTERIUM TUBERCULOSIS CLINICAL ISOLATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Ndhlovu et al provides insights into the methylation profile of clinical strains of Mycobacterium tuberculosis from Malawi. This study provides evidence of methylation events and lineage specific comparisons of methylation activities of a total of 18 strains. As methylation profiling of bacteria with clinical importance such as M. tb is a new field made possible with the current advances in whole genome sequencing this study paves the way for future more advanced studies to follow where specific methylation signatures could be mapped to specific phenotypic traits.
Experimental design: This manuscript is clearly written, provide enough background and referencing relevant papers in the field. Hypothesis and experimental approaches are well documented with all ethical standards.
Validity of the findings: The data analysis is robust, and the authors highlight the importance of novel mutations found in methyltransferases in M. tuberculosis. The authors present their data clearly in figures 1 to 3. However, figure 4 would have been greatly improved by presenting the different strains in a circular representation as it is common when you compared variability of DNA.
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: COMBINATION OF CIRCULATING MIR-145-5P/MIR-191-5P AS BIOMARKER FOR BREAST CANCER DETECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is well written regarding language and data structuring.
Sentence in Line 38 is not clear (until recently? Meaning like 10 years ago).
There isn’t enough literature in the introduction or the discussion to justify the gap in knowledge. The main reason for the need of liquid biopsy biomarker for early BC detection is the decrease in sensitivity of mammography in dense breasts especially in young females who are not recommended to do the test.
Figures require more clarity and labelling regarding acronyms. Figure 1 is hard to follow. What is SV? What does the x and y axis represent in Fig1A?
Experimental design: -unclear gap in knowledge: The choice of microRNA studied is not well explained. In the line 69, it was described that 6 microRNA were investigated in circulation without mentioning them but three of them were later considered as endogenous controls. It is not enough to study only 3 miRNA in circulation without any explanation about the choice of miRNA.
-Unclear results: At the beginning of the article, GeNorm was used to show that the optimal number of control miRNA is three but only one control miRNA was used. Furthermore, when NormFinder was used. why were miR-222-3p and miR-29c-3p as endogenous control? miR-16 and RNU6B are widely used as endogenous controls. In your data, miR-16 was not significantly dysregulated.  Which level of stability value that determines whether it is considered as an endogenous control?
-It is said that the normal women were selected by ethnicity and age: were they matched?
Validity of the findings: Data analysis is not sound. When analyzing the real time PCR data, it was concluded that miR-145 and miR-191 were upregulated. We can’t compare delta CT values but either fold change which is 2^(-delta delta CT) or negative delta CT. miR-145 has delta delta CT=2.36	 So Fold change= 0.195 meaning it is downregulated in BC compared to control or you can say that negative delta ct of control is higher than negative delta CT of BC. Data are not well analyzed and require modification. For more guidance on analysis, refer to Si et al., 2013 or  Nassar et al., 2014.
Regarding the association with clinic-pathological parameter, the results should be represented in a figure or table. Also, it is not clear what was compared between the 2 groups. Levels of miRNA meaning fold change? Delta CT? Reanalyze the data according to what was discussed above.
The ROC curve should be rechecked after reanalyzing the data.
Discussion should be remodified accordingly.
Additional comments: The article requires reanalysis to ensure solid results. The choice of endogenous control could be based on the literature if the cel-miR-39 is not valid. Furthermore, the choice of microRNA should be further discussed. The article focused alot on the choice of endogenous control more than the dysregulated miRNA.