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: DELIMITING THE BOUNDARIES OF SESAMOID IDENTITIES UNDER THE NETWORK THEORY FRAMEWORK
Review round: 2
Reviewer: 2

Output:
Basic reporting: My concerns about the introduction have been addressed, and it is clear and informative. The language has been improved throughout, and additional relevant comparisons with previous studies have been added. I have a few minor questions/suggestions: 1-4 are about the interpretation of results, while 5-10 are just about wording.

1.	Line 239: please make it even clearer here how your methods differ (e.g., which studies did not model muscles)
2.	Lines 81-82 “sesamoids reflect an antero-posterior differentiation pattern, being present mostly in the joints of the limbs and in the sacral vertebrae” – please clarify how presence in the limb joints and sacral vertebrae relates to the anterior-posterior axis.
3.	193-194: “The network modules reflect a functional clustering pattern, instead of a topological organization constrained to element's proximity.” Please justify this statement. It looks like most elements are grouped with other nearby elements, and it is stated later in Results that the patella and graciela sesamoids are the only elements in the network that do not follow a spatial neighborhood criterion.
4.	324-325 “connectivity similarities among paraphalanges and GSs could indicate that they may share a deep common origin.” I think this requires further justification. As I understand it, Saint-Hilaire was talking about connections to specific elements, while in this instance you simply have two types of elements that are loosely connected to the main skeleton.
5.	Abstract: “physical connections were considered links” – this is ambiguous. How about “muscles and bone to bone articulations were considered links” or similar?
6.	Lines 110-112 “and tendinous fascia (in case of not containing a sesamoid embedded)” Unclear what is meant here. Please rephrase.
7.	Lines 112-113 “The links are joints, muscles, tendo-muscular units, tendons, and aponeurosis (in case of containing a sesamoid embedded).” This is a bit confusing. An example might help. (this would also help to explain why you chose to model muscles as links!)
8.	Line 156: especially (not specially)
9.	265-266 “The above exposed is in tune” rephrase
10.	Line 316 “reminds those” Rephrase
Experimental design: The research questions have been condensed and clarified, and the authors have expanded on the reasoning behind their methodological choices. However, I still would like to see a fuller discussion of the latter issue. First, you might say explicitly in the manuscript what was stated in the rebuttal letter: that previous studies coded muscles differently, and why you chose to do it this way. Second, for the purposes of studying sesamoids, why is your approach better than coding muscles as nodes? You need muscles to study sesamoid connections because they don’t articulate with other bones, but why not model muscles as nodes, e.g. Diogo et al. 2015? (I’m not saying their approach was better, in fact yours makes a lot of intuitive sense, but when you deviate from previously published methods you should state your reasons.)
Validity of the findings: The conclusions are also more concise, and the links to the research questions are now clear. I think it would be even stronger if conclusion #2 were deleted (throughout the ms). Since embedded and glide sesamoids have very different network properties, it doesn’t really make sense to discuss them as a group, and I’m not even sure what it means. This is even more obvious when you get to conclusion #5, which states that embedded sesamoids are similar to canonical skeletal elements. You have a very clear story: embedded sesamoids have certain properties that are similar in some ways to canonical elements, whereas glide sesamoids have properties that are very different from either of the other two.
Additional comments: This is a very interesting study that raises important questions about the evolution and development of sesamoids and their role in the musculoskeletal network. In this revision, the authors have addressed many points raised by me and the other reviewers. I have some remaining minor questions and requests for clarification, which mainly center around the rationale for the study and the interpretation of results.


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

Input:
Title: FREQUENCY AND QUALITY OF FIRST AID OFFERED BY OLDER ADOLESCENTS: A CLUSTER RANDOMISED CROSSOVER TRIAL OF SCHOOL-BASED FIRST AID COURSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Thank you for this interesting assessment of the effectiveness of school based first aid for adolescents. As a parent of adolescents who is interested in my children participating in a similar program I found this to be of particular interest. Similarly, I feel this topic will be of interest to school and other adolescent based programs who offer such opportunities.
Over all the manuscript is very well written and relatively easy to follow. The references, background and context provided are appropriate. The structure of the article, figures and tables are professional. One note,Table 1 needs a minor formatting change - line up the information in the columns in the first section (session 1).

One piece not included that you may wish to consider is the inclusion of your hypotheses.
Experimental design: The experiment design appears appropriate -albeit uncommon as you mixed both quantitative and qualitative methodology without really synthesizing the findings together. Further in the study design section you note that a cluster randomised crossover trial was conducted. I like how well matched the subjects were given the need to utilize more than one school. In this section however there isn't a mention of the qualitative design... aspects of the study or a provided linkage of how the two approaches complement one another.  Also I think more detail regarding the coding framework used needs to be included. I would also like you to address the choice to quantify the qualitative data, as this approach is somewhat controversial among staunch qualitative researchers.

Measures: please speak to how the measures were created, who created them, and if validity or reliability analyses were carried out.
Validity of the findings: The validity of the findings are apparent. Consider adding some discussion as to the implications of the findings. Would schools / programs offer opportunities to refresh or practice skills at regular intervals? Should such first aid programs not be offered if findings bare out their ineffectiveness or does training offer something additional to teens such as feelings of confidence to be able to help in situations where a need arises? Perhaps a broader question needs to be answered. Other questions that come to mind are - 1. for CPR courses are the findings similar? What about life guard training or wilderness first aid? Are there regional or country differences noted. Does age of participant matter? Perhaps year 10 is the wrong target year.
Just a little fuel for thought as you seek to continue this work and widen the understanding of this topic.
Additional comments: Well done - nicely written piece. Enjoyable to read.


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: FREQUENCY AND QUALITY OF FIRST AID OFFERED BY OLDER ADOLESCENTS: A CLUSTER RANDOMISED CROSSOVER TRIAL OF SCHOOL-BASED FIRST AID COURSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a very professional piece of research exploring an important topic for adolescents. The paper is extremely well written and adequately covers the relevant literature. The structure conforms to the journal standards and all basic reporting criteria are met.
Experimental design: The experimental design is rigorous and responds to identified gaps in the field previously noted by He et al (2014) and Reveruzzi et al (2016). The methods are described in sufficient detail for replication and the supplementary files provide very clear guidance for other researchers. In passing, lines 68-70 of the Introduction have text that is replicated (printing error).

He Z, Wynn P & Kendrick D (2014) Non-resuscitative first-aid training for children and laypeople: A systematic review. Emergency Medicine Journal, 31: 763-768

Reveruzzi B, Buckley L & Sheehan M (2016) School-based first aid training programs: A systematic review. Journal of School Health, 86: 266-272
Validity of the findings: I especially enjoyed the rigorous methodology employed in this study. In the end the results were not surprising and in keeping with earlier studies – no differences between the groups on the frequency of appropriate first aid actions performed 12 months after training and the most common actions being sending for help and wound care.  Apart from the rigorous methodology employed the other real value of the paper is in the discussion where the authors have looked at possible limitations such as using open ended free text responses in an online survey . The authors identify where improvements can be made to investigate this topic and have indicated that a current study addresses these points. A particularly interesting topic would be to investigate willingness to offer first aid and CPR to family, friends and strangers and which barriers operate to reduce the likelihood of offering help to each group (fear of making a mistake and hurting the patient, legal disputes, fear of infection). These topics have previously been discussed in the literature, with greater willingness to assist family, then friends, then same age strangers and lastly adult strangers (Wilks et al., 2016). The distinction between assisting same age friends and same age strangers has important implications for real world adolescent bystander responses.

Overall, this is a very good paper that makes a very worthwhile contribution to the literature.

Wilks J, Kanasa H, Pendergast D & Clark K (2016) Emergency response readiness for primary school students. Australian Health Review, 40: 357–363
Additional comments: This is a very good paper that makes a very worthwhile contribution to the literature.


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: FREQUENCY AND QUALITY OF FIRST AID OFFERED BY OLDER ADOLESCENTS: A CLUSTER RANDOMISED CROSSOVER TRIAL OF SCHOOL-BASED FIRST AID COURSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Trial Registration- The ethical trial registration lists a study of performing first aid versus intervening during a mental health crisis based on the training provided to the cohorts. Whereas the abstract discusses the impacts of physical first aid training versus teen mental health first aid in the frequency of performing these skills on their peers. Was ANZCTR advised of the change?
From Abstract-“This study aimed to establish whether the training programs produced differences in the frequency with which students offered and provided first aid to peers experiencing an illness or injury and the amount of first aid provided that was in line with current first aid guidelines.”
From Ethical Applications- “Cluster randomised controlled trial of secondary school student training in teen Mental Health First Aid versus physical first aid on ability to assist peers with a mental health problem.”

In general the article is well written with professional English and structure.

Some clarity would be helpful in regards to the original hypotheses whether the subjects are responding to physical or emotional emergencies.
Experimental design: As listed above, it was difficult to correlate the results based on the difference between the initial goals and the results.
Validity of the findings: Unable to validate until clarification is given on the research question and hypothesis, as I feel that these may change.
Additional comments: I don't feel that I am able to get to the point of verifying the accuracy and references and so on until I receive clarification on the desired results. I feel that the manuscript is very hard to evaluate as I am continuously having to question the true aim of the study.


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

Input:
Title: SPATIAL MODELING OF LONG-TERM AIR TEMPERATURES FOR SUSTAINABILITY: EVOLUTIONARY FUZZY APPROACH AND NEURO-FUZZY METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is generally well written, and easy to follow. The methods are reliable, and sound. The results are well presented.
Experimental design: Original primary research within Scope of the journal.
Methods described with sufficient detail and information to replicate.
Validity of the findings: All underlying data have been provided; they are robust, statistically sound, & controlled.
Additional comments: (1) Introduction:
Summary of existing comparisons of the ANN and the three ANFIS methods is not adequate enough. There are many references available:
Prediction of hourly solar radiation in Abu Musa Island using machine learning algorithms;
Modeling daily water temperature for rivers: comparison between adaptive neuro-fuzzy inference systems and artificial neural networks models
….
(2) Four statistics used in the study should be moved to the Methods section rather than in Section 4.
(3) Discussion of the results is not enough. I suggest adding more in-depth analysis in the Section 4 or adding a new section “Discussion”.
(4) Figures:
Figure formats and quality need improvement, such as Fig. 5. Where are the points and lines? Before the final submission, the authors should check carefully about the pdf file, this is the basic requirement. Figs. 6 and 7, can you adjust to make them clear and precise? The same problem for Figs. 9 and 10.
(5) Tables:
The contents in Table 1 didn’t show up completely, please check.
(6) Formulas:
Formulas (7) and (8), please revise the positions of (7) and (8).
(7) Language
The English should be improved. I suggest proof-reading by a native English speaker.


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: SPATIAL MODELING OF LONG-TERM AIR TEMPERATURES FOR SUSTAINABILITY: EVOLUTIONARY FUZZY APPROACH AND NEURO-FUZZY METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: - In general, the article is well written with an appropriate use of scientific terminology.

- However, authors should increase the number of references in the introduction. Between lines 59-67, only old references are cited. It is suggested to add more recent publications when contextualizing the subject of GIS interpolations. For example, Brunetti et al. (2014); Gonzalez-Hidalgo et al. (2015); Serrano-Notivoli (2019); among others.

Brunetti, M. , Maugeri, M. , Nanni, T. , Simolo, C. and Spinoni, J. (2014), High‐resolution temperature climatology for Italy: interpolation method intercomparison. Int. J. Climatol., 34: 1278-1296. doi:10.1002/joc.3764

Gonzalez‐Hidalgo, J. C., Peña‐Angulo, D. , Brunetti, M. and Cortesi, N. (2015), MOTEDAS: a new monthly temperature database for mainland Spain and the trend in temperature (1951–2010). Int. J. Climatol., 35: 4444-4463. doi:10.1002/joc.4298

Serrano-Notivoli, R., Beguería, S., and De Luis, M.: STEAD: A high-resolution daily gridded temperature dataset for Spain, Earth Syst. Sci. Data Discuss., https://doi.org/10.5194/essd-2019-52, in review, 2019.

- Please, in Figure 1, review the source of the image. Who is the provider of the map? What is the meaning of DSI initials in the bottom-left corner?

- Please check the labels of figures 5,6 and 7 because they are misspelling the degrees Celsius symbol: (◦C), not (oC).
Experimental design: The Experimental design has practically no loopholes since the methods used are widely described which allow the readers to understand the whole process.

However, the question arises whether the authors have reviewed the quality of the temperature data. Perhaps they should provide some homogeneity test to guarantee the validity of the data used. For example, a simple method to apply would be the Von Neumann test.
Validity of the findings: the authors provided different metrics to evaluate the interpolation error, thus giving robustness to the results presented.

However, the authors did not offer any kind of discussion that would allow their results to be compared with other similar studies. It is mandatory to add a discussion section to discuss the results obtained.
Additional comments: The authors satisfactorily interpolated the temperature in Turkey, using an advanced and complex methodology.

The article deserves to be published in the journal if the authors introduce the changes and improvements suggested in the review.


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: SPATIAL MODELING OF LONG-TERM AIR TEMPERATURES FOR SUSTAINABILITY: EVOLUTIONARY FUZZY APPROACH AND NEURO-FUZZY METHODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper is generally well written, and easy to follow. The methods are reliable, and sound. The results are well presented. The authors have addressed most of the review comments.
Experimental design: Original primary research within Scope of the journal.
Methods described with sufficient detail and information to replicate.
Validity of the findings: All underlying data have been provided; they are robust, statistically sound, &
controlled.
Additional comments: (1) The authors added more references in the Introduction section as suggested. However, I still have some concerns. In lines 77-83, the authors have already stated the goal and the contents of this study. I am quite puzzled why the last paragraph (Lines 84-91) were placed there. The authors should carefully read the Introduction section again to make it more clear.
(2) Still, figure quality (Figs. 1, 8-10) is not good enough, which needs further improvement.


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

Input:
Title: EFFECTS OF ORIGANUM VULGARE ESSENTIAL OIL AND ITS TWO MAIN COMPONENTS, CARVACROL AND THYMOL, ON THE PLANT PATHOGEN BOTRYTIS CINEREA
Review round: 1
Reviewer: 1

Output:
Basic reporting: ok
Experimental design: need some corrections as revised.
Validity of the findings: need some modifictaion in the discussion part
Additional comments: The revision has been made as track revision in the attached word file.


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

Input:
Title: EFFECTS OF ORIGANUM VULGARE ESSENTIAL OIL AND ITS TWO MAIN COMPONENTS, CARVACROL AND THYMOL, ON THE PLANT PATHOGEN BOTRYTIS CINEREA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see all my comments under section 3
Experimental design: Please see all my comments under section 3
Validity of the findings: Manuscript # 45399
Title: Effects of Origanum vulgare essential oil and its two main components, carvacrol and thymol, on the plant pathogen Botrytis cinerea
Authors: Huiyu Hou, Lin Zhou, Xueying Zhang, Te Zhao

In this study, the authors examined the impact of Origanum vulgare essential oil and its components on the plant pathogen Botrytis cinerea. The study, overall, is well designed and has strong potential in expanding our understanding of how these essential oil components may be used to control the pathogen and its spread. The manuscript is well written - clear, professional and articulate English has been used throughout. Authors have used good literature citations, and sufficient background and rationale for the work has been included in the manuscript.

I have the following comments about the manuscript, please see below.

Introduction, Lines 55-56 – Please elaborate on “and the point mutations have been identified”.

Introduction, paragraph 3, Line 72 – Please elaborate of what “bioactivities”. Please also cite some articles on this.

Materials and methods, Section 1, Inhibitory activity of different plant essential oils on mycelium growth of B. cinerea – Please spell out PDA (I am assuming this is Potato Dextrose Agar) on first use. On paragraph 2 of this section, can you please put a citation here for this method?
Materials and methods, Section 3, In vitro antifungal activities of O. vulgare essential oil, carvacrol and thymol against B. cinerea – With ‘mother liquor”, do you mean a combined solution? Please change this term with a more scientific usage.

Materials and methods, Section 4, Antifugal activity of O. vulgare essential oil, carvacrol and thymol on spore germination – “The spore concentration was adjusted to..” – how did you adjust spore concentration? Please elaborate and include a citation to the method here.

Materials and methods, Section 5, Effects of carvacrol and thymol on fresh and dry mycelial weight of B. cinerea – Please spell our PDB on first use (I am assuming you mean Potato Dextrose Broth?)

Results, Inhibitory activities of 17 plant essential oils on mycelial growth of B. cinerea – I suggest putting the percent inhibition rates on a plot for better understanding. The table is useful, if needed it could be put into a supplement.

Results, GC-MS analysis of O. vulgare essential oil – Is this table 2? Do you have the GC-MS peaks images/plots from this analysis? I suggest using a figure with the peaks rather than a table here.
Results, Antifungal activities of the O. vulgare essential oil, carvacrol and thymol against mycelial growth of B cinerea in vitro – I suggest the same here, please include a figure here instead of a table.

Results, Antifugal activities of O. vulgare essential oil, carvacrol and thymol on spore germination - I suggest the same here, this data is better depicted in a plot to include as a figure here instead of a table.

Results, Antifungal activities of O. vulgare essential oil, carvacrol and thymol on spore germination of B. cinerea - I suggest the same here, this data is better depicted in a plot to include as a figure here instead of a table.

Results, Effects of carvacrol and thymol on the mycelial morphology of B. cinerea - I suggest the same here, this data is better depicted in a plot to include as a figure here instead of a table.

Results, Effects of carvacrol and thymol on wet and dry mycelium weight of B. cinerea – I suggest you also include a plot with inhibition rates here (this could potentially be a supplemental figure but including a plot lays out your findings well)

Figures 2 and 3: On the legend, please explain your X and Y axes.

There is not Figure 7 cited within the submitted manuscript, although there is one included at the end of it.

Discussions, Line “a hot-topic with great application potential in controlling important fungal diseases of crops – this needs more elaboration with a few citations.
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF ORIGANUM VULGARE ESSENTIAL OIL AND ITS TWO MAIN COMPONENTS, CARVACROL AND THYMOL, ON THE PLANT PATHOGEN BOTRYTIS CINEREA
Review round: 1
Reviewer: 3

Output:
Basic reporting: I have recommended some language corrections in the attached file
I have some comments in the from of queries for the authors which  I wish they answer
There are two refrences missing form the list of refrences
Experimental design: no comments
Validity of the findings: no comments
Additional comments: All corrections and comments are embded to the attached file


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

Input:
Title: EFFECTS OF ORIGANUM VULGARE ESSENTIAL OIL AND ITS TWO MAIN COMPONENTS, CARVACROL AND THYMOL, ON THE PLANT PATHOGEN BOTRYTIS CINEREA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors made necessary changes to the manuscript based on my previous review suggestions.
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: EFFICACY OF A NEW DIETARY SUPPLEMENT IN DOGS WITH ADVANCED CHRONIC KIDNEY DISEASE
Review round: 1
Reviewer: 1

Output:
Basic reporting: - In its current form the are a number of English grammatical errors and sentences that do not read correctly/make complete sense. I would advise getting a native English speaker to proof read the manuscript before any future submissions. Examples include lines 39-40, line 46, line 55, etc.

- The literature references often include review articles, but these are not sufficient to back up specific findings from individual studies. A reader should be able to go straight to the source of the data backing up any points you are making, without having to look up the actual reference in a review article. An example is lines 195-197; the references here should be the actual studies that present this data (ie Elliott 2000, Plantinga 2005, Ross 2006) and not the Polzin review paper.
There are also times when the referencing is not correct, e.g. referencing the de Brito-Ashurst 2009 and Polzin 2013 papers in a sentence about bicarbonate in human medicine (lines 69-71).

- The article has been structured well and includes a number of figures and tables. However, the figures need revising as they have been "cut off" so I can't review the whole figure. The tables also include a very large amount of information, and I don't find it easy to interpret the system of a,b,c indicating statistically significant differences. Please add additional information to explain this.

- The article is self-contained with relevant results to the hypotheses.
Experimental design: - This study does fit within the original primary research Aims and Scope of this journal, although it is not a typical journal for veterinary research,

- The research question is well defined, relevant and meaningful. There is a need for data to support the use of nutritional supplements to address hyperphosphataemia and metabolic acidosis in later stages of CKD.

- The study has been performed rigorously with attention to detail and a large amount of data collected.

- Some additional information needs adding to the methods:
1) Please state the laboratory used for the CBC, biochemistry, urinalysis etc. Please state the machine make used for the venous blood gas analysis. Was ionised calcium not measured on the venous blood gas machine?
2) Please be clear whether dogs were assigned to one specific renal diet, or whether an individual dog could eat any of the renal diets you mention during the study.
3) Does the supplement being tested have a name? If so, please include.
4) Were hypertensive dogs excluded? Please state this. Also, as you were not starting treatment for proteinuria, were dogs with a UPC above a certain number excluded? How do you justify not treating the proteinuria in dogs with a UPC >0.5?

- I would strongly suggest reviewing your statistical methods, because ideally this analysis should have been done using linear mixed models. This would have allowed you to test time vs treatment group more easily, without the need for lots of multiple comparisons and would be easier for you to summarise the data (in figures, rather than huge tables of lots of numbers). If you are not prepared to do this, then we need more information to make it clear where you have applied your Bonferroni correction (and other multiple comparison corrections) - usually, with Bonferroni, the new P value for significance is stated. So where you 7 time points, the P value of 0.05 becomes 0.008: I can't see that you have applied this because you say your superscript letters indicate significant differences at <0.05 between timepoints, but shouldn't these only indicate differences at <0.008?
Validity of the findings: - You have supplied lots of data, but it is difficult to confirm that the data is statistically sound at present (see above).

- Your conclusions are fairly sound, although I am not convinced that changes noted could be due to an improvement in GFR: creatinine that changes <25% is essentially stable given the biological variability of its measurement.

- Please state clearly which IRIS stage the dogs in the study were in. Were they all stage 3, or were some stage 4? If they are all stage 3, then you can say throughout the paper that the supplement was assessed in dogs in IRIS stage 3 (rather than saying "in dogs in advanced stages").
Additional comments: Well done for performing a prospective, randomised, controlled study. Did you consider having a placebo for the control group? You have provided a large amount of data and generally the paper is logically ordered. However, there are a number of points that I would advise you consider (as detailed in the other 3 sections).


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: EFFICACY OF A NEW DIETARY SUPPLEMENT IN DOGS WITH ADVANCED CHRONIC KIDNEY DISEASE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This prospective, blindly randomized work (treatment group vs. placebo) aims to describe the efficacy of a feeding stuff in treating metabolic acidosis and hyperphosphatemia in dogs with advanced chronic kidney disease.

It is this reviewer’s opinion the paper reveals numerous lacks concerning either the study setting and compliance with currently ethical standards. Not treating dogs with metabolic acidosis may lead to a sudden worsening of their clinical condition, with complications often leading to death. Contrary to what the authors report, clinical trials of this type require, as long as this type of request  is accepted, a specific authorization of an Ethical Committee, a fundamental and missing element in this study. Moreover, the authors do not actually demonstrate a real efficacy of the treatment, rather preferring to repeatedly refer to statistical evaluations between the treatment group and the control one, almost forgetting that the therapeutic purposes are represented by the achievement of endpoints capable of modifying the patient's prognosis. Might the authors state that raising blood bicarbonates from 16.3 to 17.4 mmol / l can actually change the patient's clinical picture and prognosis?
According to the IRIS treatment recommendations, the aim of the treatment should be to maintain serum bicarbonates between 18 and 24 mmol / l, a condition that has not even been achieved in the T group. This reviewer is also curious to know the reasons why the authors decided to treat patients with a dosage of alkalizing of approximately 50% of what suggested in literature. In fact, the product used contains 5 g / 100g of sodium bicarbonate and it was administered at the rate of 0.2g / kg bw orally divided into two daily administrations; assuming the sodium bicarbonate used was 100% pure, each patient was treated with a dose equal to 10mg / kg bw orally divided into two daily administrations (5 mg/kg twice a day) compared to approximately 8-12mg / kg every 8-12 hours which are usually suggested
Experimental design: SEE COMMENTS FOR THE AUTHORS
Validity of the findings: SEE COMMENTS FOR THE AUTHORS
Additional comments: Revision
Line 79-81 When diet alone is insufficient, administration of alkalinizing agents such as potassium citrate, aluminum hydroxide, aluminium carbonate, calcium carbonate, calcium acetate and lanthanum carbonate is indicated…
•	The list of raw materials doesn’t include alkalizing agents (eg. “aluminum hydroxide, aluminium carbonate, …. and lanthanum carbonate”). It’s advisable the authors amend the list, reporting just the alkalizing agents.

Line 90 A case-control study on dogs affected by CKD was performed in 2015.,
•	Authors should better clarify which criteria were used in classifying patients as affected from CKD;
•	Study was performed in 2015, then did the Authors use the IRIS staging system available at that time or the current one (2019)?
•	“2015.,” cancel the fullstop “.”

Line 92-94 Dogs affected by other concomitant diseases (acute kidney injury, pre-renal or post-renal azotemia, genitourinary tract inflammation or infection, urinary tract obstruction, chronic heart failure, neoplasia, hypothyroidism, diabetes) were excluded from the study.
•	How have the other diseases been diagnosed (laboratory evaluations, instrumental ones, both)?
•	Were patients suspected of being affected  from other disease excluded too?

Line 96 They provided a written informed consent before the enrollment of their animal.
•	Was the study approved from an Ethical Committee which also endorsed the informed consent?

Line 97-98 …dogs were randomly assigned to two groups: the control group (C group, n=10) and the treated group (T group, n=10).
•	Which software or system of randomization was used?

Line 103 indirect Doppler
Line 104 method using the radial pulse.
•	Join the sentences on the same line

109 …Canine k/d, Hill’s Pet Nutrition)”.” from…
•	Cancel “.”

Line 119 Blood Sampling and Assay.
•	CBC: neither sampling technique nor the instrument used to process the blood samples undergoing CBC are reported.

Line 120-121 Venous blood gas analysis was also performed in all cases with a standard analytical device.
•	It is this reviewer’s opinion there is no analytical device defined as “standard“ to perform a blood gas analysis. As well as they did for the determination of other parameters, Authors should report  the device used.

Line 122-126 The laboratory performed CBC and serum biochemical analysis recording the values of: ionized calcium (iCa), serum bicarbonate (HCO3), blood urea nitrogen (BUN), creatinine (CREA), phosphorus (P), total protein (TP), albumin (ALB), albumin/globulin (A/G), glucose (GLU), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin (BIL), and cholesterol (CHOL).
•	This reviewer is a bit puzzled. Were either iCa and HCO3 determined through another method than hemogasanalysis? Determining iCa without a blood gas analysis, requires strictly anaerobic methods and if these had been used they should then be described, together with a list of both the devices and methods used. Even the determination of HCO3, if not done with blood gas analysis, should be better described, also considering in the discussion section all the factors potentially influencing this determination. Also determination of HCO3, if not performed through hemogasanalysis, should be better described, considering all the factors potentially influencing this determination in the discussion section.

Line 156-158 Values of haematocrit (HCT), haemoglobin (HG), red blood cells (RBC), white blood cells
(WBC), neutrophil (N), eosinophil (EO), lymphocytes (LYM) (Table 3) were recorded during the entire study.
•	These data should be included in M&M rather than in Results. Also timing of sampling and evaluation of samples should be described.

Line 171 No difference in the serum iCa level between groups was reported at day 0.
•	Do the Author mean “no statistically significant difference”?

Line 180-182 At the baseline) all dogs (n=10) of the C group, were proteinuric (UPC, 0.5), while 8 dogs in the T group were proteinuric (UPC, 0.5) and 2 were borderline proteinuric (UPC, 0.2 and 0.5) (Table 7).
•	Was a statistical evaluation at T0 performed in order to evaluate the difference between groups? If this kind of assessment was carried out, authors should clarify it and report it in the paper; if that was not done, this is a required data.

Line 192 …observation was longer (180 vs 28 days) than other similar studies (Zatelli et al. 2012).
•	This reviewer cannot understand this statement. The study of Zatelli et al. 2012, which authors refer to, had a much longer duration (44 weeks) than the study presented (180 days). What do the “28 days” refer to?

 Line 201-202 In this study, the values were recorded at 4–8 weeks following the supplement
administration (Zatelli et al. 2012).
•	As indicated above, the paper the authors refer to was carried out during a 44 wks period; it needs the concept to be better explained.

Line 212 – 214 The current IRIS guidelines ((IRIS)  2019) for phosphorus control suggests to start phosphate binding therapy if serum phosphorus remains above 5 mg/dL after the initiation of dietary restriction.
•	Recommendation above refers to patients in IRIS stage 3. Authors should better explain this aspect.

Line 235-237 …of inclusion in the trial, had a blood bicarbonate concentration below 18 mmol/l and at the end of the study (T=180), blood bicarbonate concentration increased significantly (p<0.01) in the Tgroup compared with the C group (Figure 1, Table 2).
•	As it is correctly reported by the Authors, in the case of a patient with metabolic acidosis, the therapeutic aim is to raise bicarbonates above 18 mmol / L and not just to increase them still remaining in an acidotic range (we have no idea if such a result can modify the patient’s prognosis). In order to correctly evaluate the usefulness of the therapy administered, it is necessary to insert a table with values of the single patients as well as offer a statistical determination of the intra-group significance before and after treatment.

Line 247-248 UPC had a significantly decrease at days 150 and 180 in the T group compared with C (p<0.01).
•	Based on tables and data provided, it would seem more correct to state that in group C there was an increase in proteinuria, rather than saying in group T this decreased during treatment. The increase of proteinuria in group C could be justified by the higher level of intra-group proteinuria at the time of inclusion compared to group T. A statistical evaluation of the difference between groups at T0 as well as an intra-group one is missing.

Line 252- The pathogenethic mechanism is controversial, but anyway may reflect a possible improvement in glomerular filtration rate (GFR), as also confirmed by the significant reduction in CREA values in the T group compared to the C group (Table 5).
•	It is hardly understandable to this reviewer the link between improved GFR and reduction of proteinuria, considering that the product administered is based exclusively on alkalizing (sodium bicarbonate) and intestinal phosphorus binders. It is the opinion of this reviewer that the Authors should better express what is reported.

Line 275-277 All procedures, treatments and animal care were in compliance with the guidelines of the Italian Minister of Health for the care and use of animals (D.L. 4 March 2014 n. 26 and D.L. 27 January 1992 n.116) and UE (Directive 86/609/CEE).
•	A clinical trial involving randomization and division into two groups of dogs with advanced chronic kidney disease and exclude
•	ing the control group from therapy of metabolic acidosis and hyperphosphataemia (both causes often leading to clinical worsening and death), cannot be carried out without the ethical committee approval.


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: EFFICACY OF A NEW DIETARY SUPPLEMENT IN DOGS WITH ADVANCED CHRONIC KIDNEY DISEASE
Review round: 1
Reviewer: 3

Output:
Basic reporting: English language revision would be beneficial, considering the amount of grammatical errors. This does not only make the reading less smooth, but also alters the information that is providing due to the confusing message that is often given.

The structure of the discussion needs to be altered. It should only comment on own results (providing explanations for them and commenting on their relevance, and comparing them to what was already known in literature on that specific matter) and not provide a literature overview again that belongs in the introduction section.
Experimental design: The research is well defined, relevant and meaningful. It should however be more clear which substances of this dietary supplement have been tested before and which have not, so why this is a 'new' dietary supplement. Also, it should be made clear what the goal of each substance is (phosphate binder and/or alkalinizing agent and/or other effect?), this is now not specified anywhere.

It is also not clear whether this is a double-blinded placebo-controlled study, and if so, why this is not mentioned in the title. In the materials and methods section, the authors claim the following: 'Both investigator and owner were not aware of the group assignment'. However, the word 'placebo' is not mentioned anywhere in the article, which is confusing.

Possibly due to language issues, it is often not clear which data have been compared to which date (e.g. are T180-results compared with T0-results within one treatment group, or are the results of the treatment group compared with those of the control group at a certain time point). This is often confusing and hinders clear interpretation of the results.

Lastly, it is not clear whether ethical approval has really been given for this study (see my comment on this in the manuscript)!
Validity of the findings: The discussion is now more a review of literature than a critical approach to the own results, so this part will need adjustments.

Where the author speculates, this was mentioned in the comments in the revision.
Additional comments: Potentially valuable article (especially considering the results 'phosphate binding effect' and 'palatability'), but major revisions are necessary to make it acceptable for publication.

I have not uploaded all tables again, only the manuscript, but it would be preferable to write all laboratory values in SI units.


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: IDENTIFYING DISEASE-ASSOCIATED SIGNALING PATHWAYS THROUGH A NOVEL EFFECTOR GENE ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article can be refined by improving few specific portions (mentioned in my detailed comments in general comments section) with regards to english and literature references.
Context and background was adequately provided.
Experimental design: This article introduces a new approach to signal pathway analysis named "Signaling Pathway Functional Attributes Analysis" (SPFA). It addresses the shortcomings of the previous analysis methods by incorporating functional attribute finding and dissecting which parts of the pathway are abnormal.
Research questions, aims and scope were well defined. Methods had adequate details and the code was also provided via Github. The approach investigated here was compared with 7 other approaches in the literature.
Validity of the findings: Conclusions are well stated and can be further enhanced by addressing the following questions in the general comments section.
Additional comments: Must address:
1. What are the reasons to choose the 3 mentioned rules to select the 33 data sets from KEGGdzPathwaysGEO and KEGGandMetacoreDzPathwaysGEO R-package. The authors should elaborate and justify the reasons for their 3 rules.
2. Follow up to the same, what would the impact on the analysis if the related target pathways of these datasets are not KEGG pathways?
3. How is the p-value combined to acquire the significance of each signaling pathway?
4. What was the rationale for choosing the 3 measures to compare all the signaling pathways analysis methods? Are there any other measures in literature which are used?

Good to address/incorporate (this should be treated as suggestions to enhance/improve this study or follow-up study):
1. Line 282:  “signal variations received by these effector genes were also in the top ranking.” What was the exact rankings?
2. What factors contribute to the low sensitivity of SPFA compared to other signaling pathways compared in this manuscript? Is it the case where the statistical model presented here as high accuracy (prioritization) but low sensitivity?
3. Can one create a hybrid metric that would optimize all 3: sensitivity, specificity and prioritization instead of just summing the ranks from individual ones?
4. Are there certain target pathways for which SPFA performs better/worse than others? The performance distribution of SPFA within different target pathways would be nice to look at?

Minor english/grammatical modifications:
1.  “contribute to disease cell biological process” is repetitive in line 23 and 24. The sentence should be re-written.
2. The line 138 should be removed: Add your materials and methods here.
3. Equation 1 dse is not defined, should be defined right after the equation.
4. Line 170: the year of the referenced article not mentioned: Jung & Berger, as the format of the reference followed in this manuscript is name and year. The same thing should be checked for all the references.
5. Equation 2: ASD acronym full form not mentioned in text. Does it stands for Accumulated signal deviation?
6. Equation 6 and 7: all the variables should be defined properly


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: IDENTIFYING DISEASE-ASSOCIATED SIGNALING PATHWAYS THROUGH A NOVEL EFFECTOR GENE ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing is extremely poor, almost unreadable.  Nearly every sentence requires revision. This includes the abstract. Only after reading the paper am I able to understand the abstract.

Some background is provided however very little about recent developments. Most references are pre-2014.  Beyond that, there is one from 2019, two from 2016 and one from 2015.  A quick pubmed search uncovers several more recent publications that describe new methods. This paper should at least mention them and state why they are not evaluated in the current publication. In fact, the 2016 publication that is cited proposes and evaluates a new method LEGO, however this method is not mentioned in the current manuscript. Their new method is compared to several other methods, but not rationale is provided for how they were selected for comparison or why others were omitted.

Structure is acceptable, however the authors appear to misunderstand the purpose of each section.

The manuscript is self-contained.
Experimental design: The article is in the Biological and Health Sciences.

The research aims to describe and evaluate a new method for identifying biological pathways whose activity differs between cases and controls.  The new method attempts to incorporate signalling pathway structure into its search for affected pathways.  Most previous methods ignore structure.  There are other methods that do consider structure but these are not considered or discussed in much detail.

Evaluation of the new method is based on the approach of a previous publication (Tarca et al. 2013) although the authors mistakenly credit Bayerlová et al. (2015). Some 'cherry picking' is evident when they compare methods according to three measures, sensitivity, prioritization, and specificity.  They correctly note that no method is likely to be best under all measures and choose to sum performance ranks for sensitivity and prioritization but not specificity.  Under this ranking scheme, SPFA performs best.  They fail to note in the text, however, that it is actually tied with PADOG (Table 2). Furthermore, if performance ranks are summed across all three measures, then PADOG actually turns out to be the overall winner (rank sum=7 compared to SPFA rank sum = 10).  This is not mentioned in the text.

SPFA identifies dysregulation in pathways where correlation between pairs of genes with signaling relationships differs between cases and controls. Given this, it seems odd that the authors chose to use such small datasets to showcase their method (one 15v8 and the other 4v4).  Does it even make sense to compare correlations calculated for n=4 samples and compare them to correlations calculated for another n=4 samples?  Surely much larger datasets are available from GEO or certainly through TCGA.

The statistical significances of correlation differences in SPFA are evaluated by gene label permutation.  Unfortunately this approach is likely to inflate statistical significance because genes within the same pathway tend to be correlated much more highly than genes in different pathways.  Consequently, under random gene label permutation, correlations will tend to be much lower.  It would be much better to compute a null distribution, as in Tarca et al. 2013, by permuting group labels instead.

Methods are sufficiently described. The authors provide code (https://github.com/ZhenshenBao/SPFA) however this lacks documentation and provides incomplete description of an example.  Repeating the example requires the user to write additional code.  The authors have clearly performed the complete analysis so it's not clear why the complete code is not provided. The R code relies on a few R packages: KEGGgraph, igraph, and ggplot. These dependencies are not documented.
Validity of the findings: The manuscript, including the abstract, claims that SPFA, unlike previous methods, is alone able "to help to find which functional attribute is in case of an exception between two conditions (normal vs. disease) and which parts of a pathway are abnormal in the disease biological process."  This is simply not true.  It is possible for example for any other method to evaluate enrichment in a sub-pathway.

That said, SPFA does appear to be different than previous methods in that it takes into account correlations between genes with signaling relationships in pathways. Because of this, it can potentially identify dysregulated pathways missed by methods focused on gene set enrichment.
Additional comments: Lacking from the manuscript is a more in-depth discussion that attempts to explain why the different methods perform the way they do. For example, PADOG likely has improved specificity because it down-weights the contribution of genes involved in multiple pathways. Given the superior performance of PADOG, perhaps a similar approach could be applied to improve SPFA specificity.

SPFA attempts to combine two different approaches based on enrichment expression differences and correlation differences.  I'm curious why the correlation difference approach was not evaluated as an approach on its own since it identifies a different type of dysregulation.  In fact, it might be more useful as part of a 2-stage approach to pathway analysis where enrichment is used to identify pathways and then correlation-differences are used to identify the most dysregulated relationships in that pathway.

Throughout it is assumed that the enrichment approach is entirely independent from correlation differences approach (hence the method for combining p-values on Line 210).  However, this is not entirely obvious and should be more formally investigated.  This outcome of this investigation is critical for a thorough understanding of SPFA.

It should be mentioned in the discussion that a weakness of the approach is its dependence on manually curated signaling pathways.  The level of signaling knowledge varies significantly between and even within pathways.

Minor comments:
Line 175 ‘Euclidean distance’ should be ‘Network distance’.
Line 226 ‘Absolute difference’ implies that differences are always non-negative. However, the formulas given earlier suggest actual difference.
Line 246  Who is Michaela?


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: IDENTIFYING DISEASE-ASSOCIATED SIGNALING PATHWAYS THROUGH A NOVEL EFFECTOR GENE ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors significantly improved the manuscript in terms of english. The manuscript is much more understandable from its previous version.
In the present version ,the authors also did a good job in reviewing the current landscape in the introduction section compared to the previous version.
Experimental design: The authors also improved the method section and listed the reasons for collecting the data and using the comparison metrics. The authors also addresses the concerns I raised in the previous review.
Validity of the findings: Conclusions are well stated, and the authors also compared their results to the other 7 techniques and quoted those studies.
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: PHYTOTOXIN SYNTHESIS GENES AND TYPE III EFFECTOR GENES OF PSEUDOMONAS SYRINGAE PV. ACTINIDIAE BIOVAR 6 ARE REGULATED BY CULTURE CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: I would like to see a statistical test applied to the results of Figure 1 to make its findings more robust.
Validity of the findings: well done, only further clarification needed.
Additional comments: line 124-128: Please provide version #s for all bioinformatic processing software
line 174: I presume size screening was also part of the confirmation of PCR products.

Line 237: For figure 1, I anticipated a figure that showed the degree of fit in the correlation between the RNA-seq and RT-PCR analysis. I would encourage you to change the way this is plotted to increase the confidence of fit.

Line 259 - "most of them were thought to be hrpL-dependent effector genes" needs a citation.

Paragraph starting line 266: This section is a bit difficult to follow. I recommend you pull parts out it and incorporate that into the earlier paragraph. Tie the lack of Hrp box sequences to the longer-expressed phytotoxin synthesis genes

I appreciate the many supplemental figures the authors provide. I do wonder if perhaps a final main figure for the text could be a cartoon that shows the expression pathway and comparative expression they discuss towards the end of the paper.


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

Input:
Title: PHYTOTOXIN SYNTHESIS GENES AND TYPE III EFFECTOR GENES OF PSEUDOMONAS SYRINGAE PV. ACTINIDIAE BIOVAR 6 ARE REGULATED BY CULTURE CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is acceptable although usage of "it is thought" as a default statement for well verified results could be substituted with stronger language

Raw RNAseq reads do not appear to be archived in a public database.

This claim that Psa6 is the only P syringae strain that produces two phytotoxins is not true.
Most Psy pv. syringae strains produce three toxins syringomycin,syringopeptin, and syringolin. Some P syringae pv syringae strains produce phaseolotoxin in addition to the toxin triad and some also produce mangotoxin
https://doi.org/10.1371/journal.pone.0036709.
Pto DC3000 produces both coronatine and phevamine A.

Considering the importance of culture medium to this study, the composition per liter for each medium should be included in the methods at least. A table comparing the four media would be preferable.

Be more specific about "using R software", which packages?

Consider converting red/green heatmaps to blue/yellow to accommodate the color blind.

In Figure 1, include the R^2 value.
Experimental design: There are several problems in the design of the RT-qPCR experiment

The PCR efficiency of test genes hasn't been conducted.

In addition you should calculate NRQ using empirically determined efficiencies rather than just assuming the efficiency to be perfect (=2).

Melt curve analysis doesn't seem to have been conducted to verify the amplification of a single product under actual testing conditions.

It isn't clear to me if the primers designed for testing expression of Psa6 genes were verified as valid for the other strains. Different reference genes were used for each strain?

Ideally reference genes should be validated for use and several levels of expression so that test genes can be compared to a references with similar levels of expression. Using all high expression reference genes could result in artifacts and bias. I recognize that there are different opinions on use of reference gene.  There should be some editorial guidance on whether this is an issue worth addressing.
Validity of the findings: Other Psy strains were included to verify regulation differences from Psa6 but I really would have been better to use a well validated strain like Pto DC3000 known to respond well to HMM. This would provide a good internal control for the study.

 Line 273, The claim the late induced effectors lack hrp box promoters needs a deeper analysis. Are theses genes in operons downstream of other hrp regulated genes?

Line 353- citation that salicylic acid is antimicrobial? chitnases are not likely to exert an effect on bacteria.

The discussion of specific effectors doesn't bring much value. There is not obvious trend identified in either group and each effector is likely to exert these roles only in the context of specific hosts.
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: PHYTOTOXIN SYNTHESIS GENES AND TYPE III EFFECTOR GENES OF PSEUDOMONAS SYRINGAE PV. ACTINIDIAE BIOVAR 6 ARE REGULATED BY CULTURE CONDITIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: all corrections were made
Experimental design: thank you for adding the R2
Validity of the findings: All corrections were made
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: PHYTOTOXIN SYNTHESIS GENES AND TYPE III EFFECTOR GENES OF PSEUDOMONAS SYRINGAE PV. ACTINIDIAE BIOVAR 6 ARE REGULATED BY CULTURE CONDITIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: My previous concerns have been adequately addressed. 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: METHYLENE BLUE INHIBITS NUCLEATION AND ELONGATION OF SOD1 AMYLOID FIBRILS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article reporting the inhibitory action of Methylene Blue on nucleation and elongation of SOD1 amyloid fibrils. However, there are major problems in this manuscript which are as follows:
1. There is recent work on effect of this same dye on fibrillation in lysozyme which has not been cited.
2. The results of Congo Red, Nile Red Red assay should be discussed in detail in the main manuscript and not in the Supplementary section.
Experimental design: There are some major flaws in the experimental design which need to be addressed. e.g.
ANS assay should be performed in fluorescence.
The CD experiments should be performed to ascertain the conformational changes and the CD curves should be deconvoluted to decipher the changes in the secondary structure of the protein.
Intrinsic fluorescence experiments should also be performed.
 The interaction of the dye with the protein should have been done at first using UV-Vis, steady-state, and time-dependent fluorescence as well as Isothermal titration calorimetry.
Molecular docking should also be performed.
Above all, the effect of methylene blue should be investigated on proper neurological cell lines if it is to have any potential anti-amyloidogenic activity.
Validity of the findings: In the absence of the experiments suggested above the paper appears to be flawed fundamentally and the results reported are too thin and premature to jump to any conclusions.
Additional comments: The work reported is not sufficient to validate the anti-amyloidogenic property of the dye and the authors should attempt to study the effect of methylene blue on a more realistic model preferably some apt cell line or animal model before they jump to any conclusion regarding the potential inhibitory effect of the dye on amyloid fibrillation.


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: METHYLENE BLUE INHIBITS NUCLEATION AND ELONGATION OF SOD1 AMYLOID FIBRILS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the present manuscript, Musteikyte and co-workers perform an in vitro study on the activity of the dye Methylene blue as an inhibitor of SOD1 amyloid formation under denaturing conditions. The text is clear; the English is mostly correct. The introduction and associated literature are adequate. Raw data is shared, and the Figures in the main text and supplementary files are of good quality.
Experimental design: There are two main problems with the methodology in the present manuscript.

1)The aggregation conditions are far from physiological conditions and question the nativeness of the protein at the beginning of the reaction. First, the protein is incubated in 5 mM DTT, a very high concentration of reducing agent that should reduce the intrasubunit disulfide. Besides, the protein is demetallated, which also destabilizes it. On top of that, they add 0,5 GuHCl, and the protein solutions are incubated at 60 oC with intense agitation. The authors rationalize that it is necessary for a reproducible on-pathway aggregation. Still, I am afraid that under these extreme conditions, the protein is aggregating from a  significantly unfolded state, questioning the relevance of the acquired data in a biological context and the applicability of MB as a drug for SOD1 related diseases. The authors should demonstrate that, under the assay conditions, the protein is initially significantly folded and, ideally, active.

2) The aggregation kinetics are followed with Th-T, whereas it is known that MB interferes significantly with the amyloid dye's signal. This is also the case for CR; MB has been reported to compete for the binding of Nile Red to fibrils and aggregates. This lets us only with the AFM data, which are convincing but should be complemented with at least an alternative biophysical technique. Orthogonal light scattering kinetics or DLS measurements at selected time points might help.
Validity of the findings: Apart from the methodological aspects mentioned above, which I think are important:

1-It is said that MB acts at the nucleation stage, but if I am not wrong, this is deduced from the direct observation of the curves. Calculation of the respective nucleation and elongation rate constants would provide support to this assumption.

2- In the seeding assays, the amount of MB equal or even is several-fold higher than this of Th-T, raising question on whether the quenching effect mask any effect of the dye on seeded aggregation. This might explain the strange curves obtained at concentrations higher than 100 uM of MB. One of the explanations the authors provide to this phenomenon is: "An alternative explanation would be that MB changes the surface or structure of the formed aggregates, thus impeding their ability to bind amyloid-specific dyes." If this is the case, this phenomenon would have important implications for the kinetic assays recorded in the manuscript.

3- In the "Seeding properties of non-fibrillar SOD1 aggregates formed with MB" section, as long I understand, there is not any step to purify the aggregates formed in the presence of 400 uM of MB. If this is the case and the solution is directly used as a seed in the lower 1% proportion, the seeded reaction would have 4 uM of MB and in the 10% seeded reaction 40 uM, this makes difficult to elucidate if the dye contributes at least in part to abrogate the seeding. In any case, controls should be included.

4- I am not sure that the differences in the FTIR spectra of fibrils formed in the presence and absence of MB are enough to derive the structural conclusions the authors extract. The deconvolution of the absorbance spectra on their components might help to support such findings.

Overall I am not convinced that from the experiments in the present form of the manuscript, it can be concluded that MB might be a therapeutic lead for the treatment of SOD1 associated amyloid disorders.
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: CORRELATION BETWEEN FRUIT WEIGHT AND NUTRITIONAL METABOLISM DURING DEVELOPMENT IN CPPU-TREATED ACTINIDIA CHINENSIS ‘HONGYANG’
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please add more relevant recent papers.
Experimental design: Please add more relevant recent papers.
Validity of the findings: Please add more relevant recent papers.
Additional comments: Please add more relevant recent papers.


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: CORRELATION BETWEEN FRUIT WEIGHT AND NUTRITIONAL METABOLISM DURING DEVELOPMENT IN CPPU-TREATED ACTINIDIA CHINENSIS ‘HONGYANG’
Review round: 1
Reviewer: 2

Output:
Basic reporting: Although this research aims an original topic trying to relate the content of sugars and organic acids with the weight of kiwi fruit by the effect of the preharvest application of a growth regulator, the main research question is not well supported.
Experimental design: Even when the introduction makes clear that the CPPU regulator influences the development and metabolism of kiwifruit, it is not clear why it is important to study the relationship between fruit weight and the content of a particular component.

The implemented techniques are not the more advanced but they are adequate for the proposed response variables.
Validity of the findings: The manuscript is well written, the results seem sound, discussion and interpretation are sober and supported by their results, however to my respect the generated knowledge may not be relevant enough to the field and there is not a clear meaningful contribution to be considered for publication in PeerJ.
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: CORRELATION BETWEEN FRUIT WEIGHT AND NUTRITIONAL METABOLISM DURING DEVELOPMENT IN CPPU-TREATED ACTINIDIA CHINENSIS ‘HONGYANG’
Review round: 1
Reviewer: 3

Output:
Basic reporting: English needs substantial editing.
some references missing, some not appropriate (See details below).
Figures need improvement (some suggestions below)
Raw data shared.
Experimental design: The statistical analysis is not very clear and the experimental design dubious. Not sure if what it is called a biological replicate is a true biological replicate or more of a technical replicate given that the experimental unit was not really defined. Some graphs, where stars show statistically significant differences the error bars between the treatment and the control overlap, raising concerns about how the statistical analysis was performed.
Methods need more information (see detailed suggestions).
Research questions at the end of the introduction not very clear (see detailed suggestions)
Validity of the findings: The authors have assessed the effect of low dosage CPPU (5 ppm) on ‘Hongyang’ kiwifruit applied at 25 days after anthesis. Fruit were 14% larger. SSC was not affected by CPPU but TA decreased in treated fruit. Analysis of soluble sugars revealed that CPPU increased hexoses (fructose and glucose) but lowered sucrose. This is in disagreement with the SSC data, but the authors do not discuss it in the manuscript. TA and acids are in agreement.
Discussion does not consider factors such as CPPU concentration applied and timing of application. this should be added when drawing conclusions as it has been clearly shown how different timing of application in 'Hayward' have different outcomes.
Additional comments: Some specific comments:
Line 22: …to influence post-harvest quality. Not sure this is the reason why it is used. it is more a consequence of its use.
Lines 44-45: Guo et al 2017 reports the figure of 1.06 million tons per year, but they do not have a citation for it. What is the source of this production figure?
Line 46: kiwifruit species have been recently reclassified. Actindia chinensis var. deliciosa (A.Chev.) A.Chev. is the species for ‘Hayward’ and A. chinensis Planch. var. chinensis is the species for ‘Hort16A’ and ‘Hongyang’. Please check manuscript and edit accordingly. Actinidia can be abbreviated after first mention. Authority to be added to materials and methods section only.  Check spelling for species name Actinidia arguta throughout the manuscript.
Line 54: red fleshed core? ‘Hongyang’ core is white. It is the locules tissue where the seeds are (inner pericarp) that accumulates anthocyanin. The outer pericarp is green or yellow according the level of degreening that occurred and the maturity/ripening stage. Please amend accordingly.
Line 54: check text to make sure ‘Hongyang’ (and any other cultivar name) is between single quotes and not between double quotes or single quotes are missing.
Line 55: the kiwifruit in Henwood et al 2018 is not ‘Hongyang’ (star-red fleshed) but is a block-red fleshed fruited genotype. therefore this reference is not appropriate here.
Lines 62-65: references missing, please add.
Line 67: nutritional qualities? Please change to fruit quality attributes, here and for the rest of the manuscript as this terminology is more appropriate.
Lines 80-82: what’s your hypothesis? It is not really clear from this paragraph. add some more rather than only have we have tried this product on a different cultivar and checked the effects.
Line 87: were the kiwifruit vines grafted or were they on their own roots? Please specify. Also this sentence needs English editing.
Line 90: unsure why you cite this paper here (Cruz-Castillo 2014) as they did pre-flowering CPPU treatments. Were just the CPPU exposure and the concentration used that were similar?
Line 90: half of the fruit were dipped. This is unclear. I think the author, reading the text that follows, meant that they treated half of the vines with CPPU and half of the vines were control.
Line 91: kiwifruit plants are called vines and not trees, as they are botanically vines and not trees. Please check the manuscript for consistency in terminology making sure you do not use the word tree.
Line 94: the experimental design is vague and I am not satisfied the ‘biological replicates’ are really independent. The author state that 30 fruit were randomly collected and then subdivided into 3 biological replicates. To me you have one biological replicate and 3 technical replicates. The vines should have been subdivided into 3 groups and fruit from each group treated as a biological replicate.
Line 102: please make sure that all equipment cited in the paper has maker’s name, city and country.
Line 104: there are no details on how sugars and starch were extracted (only details on how they were determined). What’s the method used? please describe. It is also important to describe how samples were handled (how were they ground, were they kept frozen during grinding?)
Lines 112 and 114: is the pH of the NH4H2PO4 solution 2.5 or 2.54? please check you have the correct figure there.
Line 117: please delete Toussant as it is Barboni’s first name.
Line 123: I do not think the three replicates were true biological replicates and independent from the description given in the materials and methods given that they were randomly harvested and the grouped later.
Line 124: you need more details about the ANOVA. Did you consider you had repeated measures into your statistical analysis? You are analysing a time course. Add more details. What software did you use for the anova? Which are the factors? Is time a factor in your analysis? How did you treat the replicates?
Line 220: kiwifruit protein influencing fruit quality? The only protein that really affects kiwifruit quality is actinidin as it can trigger allergies in some individuals. Either add references in this part or remove the protein discussion point as it is not relevant for this paper.
Line 224: the statement about starch digestibility is not appropriate here. in kiwifruit starch is accumulated during development as a strategy to store carbon without affecting the osmotic pressure of the fruit and maximise its sink capacity. When eating ripe, starch is not there as it has been converted in soluble sugars. Please remove sentence.
Figure 3 shows that CPPU treated fruit have higher soluble sugars (%). This is because CPPU advances fruit maturity. How do you relate this to unchanged SSC in Figure 2? How would the author explain no differences in SSC, but higher soluble sugars and no change in starch? Starch is the ‘carbohydrate’ long term storage in kiwifruit fruit. A comment about this point in difference in ‘Hongyang’ might be needed in the discussion. Also, timing of application (After anthesis) and concentration of CPPU applied should be factored in. Also, what was the crop load like? If the crop load was quite low, then it is likely that vines were not carbohydrate limited and tolerated the moderate increase in fruit size (only 14%) better than other conditions when the increase in size was far more dramatic.
Figure 1: what is DAA? I assume days after anthesis. what is CK? I assume it is untreated control, but it is not stated in the figure legend of any of the figures (this comments apply to all of the figures). The figure legends should allow figure interpretation without the rest of the manuscript (Stand alone). Please revise all of the figure legends to make sure that sufficient details are provided. X-axes: please add a title for it. example: Developmental stage (DAA). This comment also applies to all of the graphs in the manuscript.
Figure 2 is deformed and needs fixing. Also, comments that applies to other graphs. Please have the X-axes and the Y-axes starting from zero. Interruptions of axes are useful when you have a large increase in a particular parameter you are measuring, not for a few units. Please remove when not required to improve clarity.
All figures and text: when talking about metabolites, you have to refer to it as concentration not content. E.g. Sucrose concentration (mg/100g).
Are the concentration in mg/100g of fresh weight? If so specifiy.
References: please check them accurately for spelling, capitalisation, italic etc…


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: CORRELATION BETWEEN FRUIT WEIGHT AND NUTRITIONAL METABOLISM DURING DEVELOPMENT IN CPPU-TREATED ACTINIDIA CHINENSIS ‘HONGYANG’
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am satisfied with the changes
Experimental design: I am satisfied with the changes
Validity of the findings: I am satisfied with the changes
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: CORRELATION BETWEEN FRUIT WEIGHT AND NUTRITIONAL METABOLISM DURING DEVELOPMENT IN CPPU-TREATED ACTINIDIA CHINENSIS ‘HONGYANG’
Review round: 2
Reviewer: 2

Output:
Basic reporting: A professional English is used in most of the manuscript, some details that need edition are suggested and marked at the reviewed pdf file. The title could be edited to say: "Correlation between fruit weight and nutritional metabolism during development in CPPU-treated Actinidia chinensis ‘Hongyang’".
References were added by the authors according to the suggestion previously made. However, two references are misplaced at the materials and methods section, they are marked at the pdf file of the manuscript reviewed.
Figures are professional and a few suggestions are given at the pdf reviewed to make figure legend shorter at Y axis.
Results are well presented and some suggestions are made at the pdf manuscript to make them clearer and a few questions are made to the authors.
Experimental design: The manuscript presents an original research to determine the effects on physiological and nutritional contents of Actinidia chinensis fruit during development, due to the application of a fruit growth promoter. The addressed question is stated in the manuscript. Methods are referenced to published papers. Three biological replicates of 10 fruit each were used to perform the experiments. Comparisons with other Actinidia chinensis that used the same growth promoter are made in the research presented. A correlation analysis of fruit weight with quality parameters is presented. Besides effects on sugars and organic acids after the treatment are smaller when compared with other cultivars.
Validity of the findings: The benefit of assaying different concentrations of the fruit growth promoter is stated in the paper and compared to other kiwi cultivars. The statistics are sound and discussed in the paper. Conclusions are based on the data presented and discussed.
Additional comments: The paper presents a sound work and some suggestions are made on the pdf to make it clearer. Two references were switched with respect to the methods used to determinate TA and soluble protein. The Y axis at figures could be shortened as suggested. Some suggestions are made throughout the manuscript and also for the title. I hope those suggestions are helpful to 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: CONFIDENCE IN THE DYNAMIC SPREAD OF EPIDEMICS UNDER BIASED SAMPLING CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, a stochastic model to simulate the sampling of a population is developed based on a class of hypergeometric distribution with population rBT+(1-r)T and infected rBT is provided, where B is the same biased used in our sampling model below, r is the proportion of the population that is infected, and T is the total population size. Such model simulates testing for a disease in an epidemic with known dynamics, allowing us to use Monte-Carlo sampling to assess metric confidence. Also, the SIR epidemic model and SAIR epidemic model are tested. The model provides an essential tool in designing an effective response to the outbreak of an infectious disease. The obtained results are interesting. The effect of precautionary measures on the behavior of the model was confirmed that the quarantine period should be long enough to achieve the desired result.

The authors should do the following to improve the quality of the manuscript:
I. The paper is well written, and it is written in a truly sporty manner. English is generally good, I think it needs to be polished further and some typos need to be revised. Further punctuation marks should be checking through the paper, especially after the equations and at the end of the statements.
II. The original papers should be much better presented.
III. References need to be arranged properly according to the journal format.
IV. The literature should be updated with the relevant bibliography "Solution of Fractional SIR Epidemic Model Using Residual Power Series Method, Applied Mathematics and Information Sciences 13 (2), 153-161 (2019)", "On the homotopy analysis method for fractional SEIR epidemic model, Research Journal of Applied Sciences, Engineering and Technology, 7 (18) 3809-3820 (2014)", "Analytical Study of Fractional-Order Multiple Chaotic FitzHugh-Nagumo Neurons Model Using Multistep Generalized Differential Transform Method, Abstract and Applied Analysis 2014, 276279 (2014)",
Experimental design: Please see the comments on basic reporting
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: CONFIDENCE IN THE DYNAMIC SPREAD OF EPIDEMICS UNDER BIASED SAMPLING CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors show that they have worked on the text, but additional work is needed to make it more accessible to the reader. Several sections are short and not connected, there should be a flow on the paper that leads to the main story. Additionally, I find lines 80-82 hard to understand.
There are interesting references. The authours should check the reference in line 48, I guess the author of that is World Health Organization. Also it would be good to include references in the first paragraph of the introduction, and cite the seroprevalence study performed in Spain that illustrated the fraction of asymptomatic infected by COVID-19.
There are a couple of spelling typos: L94 deponds->depends, L 144 possibly -> possible.
There are many figures. I recommend to select the most relevant and keep the rest for appendix/supplementary materials. Figure 4 is not cited between figs. 3 and 5.
Experimental design: The research question is very interesting and sound in the field.
The methods need some work. For example, I am missing a mathematical definition of \lambda in lines 91 and 92, it should be specified that equations (1,2,3) correspond to the unbiased case. Additionally, the matching between the mathematical model of epidemic spreading and the variables I_1, I_2 and H is done in the appendix, while I think that it is critical to include this in the main text. Additionally, when introducing SIR and SAIR, the compartments and the rates of transitions between them should be defined.
Validity of the findings: I am missing some discussion on the limitations of the work (for example, the absence of the exposed state in the dynamics).
Additional comments: I have additional suggestions to improve the manuscript:
Some limit cases and values of B (when it is introduced in the methods) would be really illustrative to provide the reader with references about this value.
Figure 1: if 5% of 300 million is infected and you want to find 90%, please mark with a horizontal line the limit case 0.05*0.9*300,000,000.
Lines 143-149: please specify that in this case B is constant.
Figure 3: I am missing a label in the color bar.


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: CONFIDENCE IN THE DYNAMIC SPREAD OF EPIDEMICS UNDER BIASED SAMPLING CONDITIONS
Review round: 1
Reviewer: 3

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


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

Input:
Title: CONFIDENCE IN THE DYNAMIC SPREAD OF EPIDEMICS UNDER BIASED SAMPLING CONDITIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thanks to the authors for addressing the reviewer's comments. From my side, there are just two minor remarks:
-Line 89: this should be rephrased to be grammatically correct: "We
therefore introduce non-dimensional a bias parameter"
-Figure 3: please change the size of the number font such that they do not overlap.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Thanks for improving the manuscript.


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

Input:
Title: CONFIDENCE IN THE DYNAMIC SPREAD OF EPIDEMICS UNDER BIASED SAMPLING CONDITIONS
Review round: 2
Reviewer: 2

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


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

Input:
Title: RESUMPTION OF DONOR-ORIGIN SPERMATOGENESIS IN SENESCENT GOLDFISH CARASSIUS AURATUS (LINNAEUS, 1758) FOLLOWING SPERMATOGONIAL CELL THERAPY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English language should be improved to ensure that an international audience can clearly understand your text. Meanwhile, the format of the manuscript should be optimized.
The flowing is the minor issues in the manuscript.
Line 49: the year should be 2004.
Line 49-51: Please revise this sentence, because the meaning is similar to the sentence from Line 47-48.
Line 80: “500L” should be “500 L”
Line 123: “produce spermatozoa from the transplanted cells;” should be “produced spermatozoa from the transplanted cells;”
Line 134: “Forty fish” should be ‘Forty fishes”
Line 149: “8-µm” should be “8 µm”
Experimental design: No conmment
Validity of the findings: No comment
Additional comments: This manuscript reported that the spermatogonia stem cell therapy could extend reproductive lifespan of fish. These findings in fish are useful and meaningful. In the following, there are some major issues. The author should complete all these issues before acceptance.
Major issue:
1. The author should use “*” to indicate the significance.
2. Please optimize the written English in the help of native speaker.
3. The author should make a table to present all the tests of 12 years old fish and the fish after transplantation in figure 2 if they have the images.
4. The image A looks different from image C at the size in figure 3, whether they are at the same magnification.
5. Why the resolution of figure 4 was lower compared with others?
6. The author should draw a schematic diagram for the genotyping in figure 8.


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: RESUMPTION OF DONOR-ORIGIN SPERMATOGENESIS IN SENESCENT GOLDFISH CARASSIUS AURATUS (LINNAEUS, 1758) FOLLOWING SPERMATOGONIAL CELL THERAPY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The study by Majhi SK et al reported the spermatogenesis in senescent goldfish by spermatogonia cell therapy. Overall, it is interesting and adds knowledge into the field. Prior to acceptance for publication, the following comments should be addressed:

Major concerns:
1.	In ‘cell therapy procedure’ subsection, the authors did not have a proper control group. It should be processed as did a cell suspension for therapy.
2.	It is well known that trypan blue is of reproductive toxicity. So the authors are strongly suggested to redo the experiments without use of trypan blue in cell therapy.
3.	The authors used 5X10^4cells in cell injection. Why? Did the authors try other doses? If yes, what are the results?
4.	Line 123, which the layer contained spermatogonial cells? The authors should clearly point out.
5.	Figure 5 is wrongly presented. In the figure, the relative mRNA levels of vasa are shown, so the value of one group (for example sexually mature group) is required to set as 1±SE.
6.	How many samples were used for gene expression analysis? (line 99)
7.	The authors need to provide the evidence that the primers (actin and vasa) are suitable for qPCR analysis.
8.	Line 154, there were 25 therapy-treated males and 5 control males. Why only 5 control males were used? The discrepancy of sample size is too big and the results are skeptical.

Minor concerns:
9.	Too many figures. It is recommended to combine some related figures into one. Figure 1 and 8 are better to be deleted or added as supplementary figures.
10.	Tables 1 and 2 should be reformed by three-line tables.
11.	There are some English language errors. The authors need to double-check the MS.


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

Input:
Title: RESUMPTION OF DONOR-ORIGIN SPERMATOGENESIS IN SENESCENT GOLDFISH CARASSIUS AURATUS (LINNAEUS, 1758) FOLLOWING SPERMATOGONIAL CELL THERAPY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Dear editor and authors
I reviewed a paper Resumption of donor-origin spermatogenesis in senescent goldfish Carassius auratus (Linnaeus, 1758) following spermatogonia cell therapy.
The authors confirmed that senescent fish are capable to restore spermatogenesis from transplanted spermatogonia. The topic and results are quite interesting as fundamental research and I can recommend the paper for publication after some revisions.
The main massage of the paper shouldn’t be misleading. It can be far applicable for aquaculture (line 73), since the germ cells are derived from sexually potent males and the spermatogenesis of germ cells from senescent fish itself was not restored.
Fig 6 F: Authors showed spermatozoa labelled by PKH26. Firstly, I don’t believe that the fluorescent dye can remain from spermatogonia after proliferation and differentiation until spermatozoa and secondly according to the scale bar, the spermatozoa should have about 400 µm in length, which is not possible. Goldfish spermatozoa has about ten times less.
Authors didn’t mention how many males were used for genotyping (blood and sperm from senescent fish after transplantation). Please provide the number.
Authors evaluate the age of senescent fish from scales, could they provide a picture to support this evaluation?
Experimental design: no commnet
Validity of the findings: no commnet
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: RESUMPTION OF DONOR-ORIGIN SPERMATOGENESIS IN SENESCENT GOLDFISH CARASSIUS AURATUS (LINNAEUS, 1758) FOLLOWING SPERMATOGONIAL CELL THERAPY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The manuscript and figure were greatly improved according to the reviewer's comments. However，the author should draw the primers designing diagram to indicate how the primers distinguish the donor and recipient DNA. If the author could accomplish it, I suggest the manuscript to be accepted.


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

Input:
Title: RESUMPTION OF DONOR-ORIGIN SPERMATOGENESIS IN SENESCENT GOLDFISH CARASSIUS AURATUS (LINNAEUS, 1758) FOLLOWING SPERMATOGONIAL CELL THERAPY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors have improved the manuscript according to the comments and therefore I can suggest it for publication.