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

Input:
Title: COMBINATION OF CIRCULATING MIR-145-5P/MIR-191-5P AS BIOMARKER FOR BREAST CANCER DETECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Minor corrections:
Formula for expression stability
Verification of miRNA expression in tumor tissue and cell lines (for secretory hypothesis)
Not discussed about disadvantages of serum biomarkers, such as CA15.3
Was blood collected from patients under treatment?
What does a more representative sample (Line 279) entail?
How many freeze-thaw cycles of the stored plasma?
Line 19 need for new, accurate (comma missing)...
Line 20 The main goal of this study…
Line 22 was conducted using age-matched plasma specimens from 35 BC women patients and 30 healthy women samples (control group).
Line 24 The expression level of…
Line 26 as endogenous control…
Line 28 Moreover, ROC analysis…
Line 30 allows to accurately differentiate BC patients from healthy individuals.
Line 33 These findings need to be further substantiated using a more representative sample.
Line 37 Duplicated line 18. Rewrite please.
Line 37 Just like most cancers, early BC is asymptomatic.
Line 38 This has resulted in late detection of the disease, at which point no therapy is very effective.
Line 40 Mammographic screening of women, in the age range the most at risk to breast cancer, did make the tumor detection at early stages more common and therefore, caused significant reduction in mortality….
Line 43 Various molecular subtypes of BC make treatment of this disease more difficult and complicated.
Line 44 Kindly re-write the sentence for better clarity.
Line 49 miRNAs are a large class of…
Line 51 cellular development
Line 52 Many studies have highlighted differential expression of certain miRNAs in several cancer types, including BC.
Line 55 The property of miRNAs that they can be detected in both tumor cells and biological fluids (in a cell-free form) serves as a major advantage for using these molecules over other oncogenic biomarkers.
Line 56 Micro RNAs directly enter the bloodstream from primary or metastatic tumors by active secretion…
Line 60 High stability of miRNA in biological fluids also makes them a very suitable choice as cancer biomarkers.
Line 62 Several miRNAs have been revealed to contribute to the pathological mechanisms of BC progression and many of them have been recommended by previous research studies as diagnostic or prognostic markers.
Line 69 The aim of our study…
Line 70 To do so, we compared plasma levels of the six selected miRNAs between age-matched breast cancer patients (n= 35) and healthy women (n= 30) from Almaty and Almaty region in Kazakhstan. Please add reference as well.
Line 77 Venous blood of 30 patients…
Line 80 Clinicopathological…
Line 84 biomaterials…
Line 90 until the isolation of miRNA step…
Line 92 performed utilizing technique
Line 96 The pellet was discarded
Line 104 triplicates
Line 107 data was normalized
Line 110 Primary Processing of the results…
Line 119 Youden’s index method was used to calculate optimal cut-off points.
Line 123 analyzed used twice in the same sentence.
Line 127 inter-group
Line 133 According to GeNorm,…. are the best internal controls for…
Line 141 What is the significance of miR210-3p in this context?
Line 166 In the control group, the level of
Line 169 groups, categorized by other
Line 174 State the reason.
Line 180 were far behind with values close to each other
Line 232 ...reference although initially we selected it as target miRNA for the study in accordance with literature screening.
Line 268 We found associations of both factors
Line 275 30 +25 = 65
Line 279 need confirmation on a more representative cohort of samples.
Line 294 Duplicate of Line 279. Please edit.
Experimental design: Although the manuscript sheds light on previously unclear details regarding the miRNAs that are differentially expressed in the plasma of breast cancer patients compared to healthy control in Kazakhstan, it fails to address the following in its experimental design:

1) The authors do not reference the studies or the sources through which they identify the 6 miRNAs (noeither in methods nor in results) that they test as biomarkers for breast cancer in Kazhak women.

2) The study is claimed to be exploratory. However, the qPCR data needs to be validated via in vitro experiments -
a) Testing some of the tumors, derived from patients from whom plasma was collected for the study, in nude mice and conducting similar qPCR studies on mice plasma to verify differential expression of miRNAs.
b) Staining tumors excised from patients to determine whether the miRNAs identified in plasma as biomarkers are secreted by primary tumors or not.

3) Authors present no information whether mammography was used as a paired diagnostic test.

4) Raw data for RNA measurement should be presented, specifically A260/280 ratio, to determine any difference in the quality and purity of RNA extracted from patient and control samples.
Validity of the findings: No additional comments.
Additional comments: No additional comments


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

Input:
Title: COMBINATION OF CIRCULATING MIR-145-5P/MIR-191-5P AS BIOMARKER FOR BREAST CANCER DETECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: In line 98, reference the articles that supporting choosing the six miRNA.were they proved in circulation? Give at least one refernce for each miRNA.

Change "Mir" or "MiR" in line 325, 333, 356, 370 to "miR" and even screen figure legends and the whole text for that.

Add info about the healthy controls in the material and method where you specify that they undergone mammography and were older than 40 years old.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The article has drastically improved.


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

Input:
Title: COMBINATION OF CIRCULATING MIR-145-5P/MIR-191-5P AS BIOMARKER FOR BREAST CANCER DETECTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have incorporated the suggested changes regarding language.
Experimental design: na
Validity of the findings: na
Additional comments: Thank you for incorporating the recommended changes. The manuscript is good to go!


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: DESCRIPTION OF THE MICROBIOTA IN EPIDERMAL MUCUS AND SKIN OF SHARKS (GINGLYMOSTOMA CIRRATUM AND NEGAPRION BREVIROSTRIS) AND ONE STINGRAY (HYPANUS AMERICANUS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The focus of the manuscript is very interesting, the role and composition of microorganisms in the skin mucosa of fish is rarely studied. However, the analyses used and the results and discussion need to be improved. The main aims and objectives could be made clearer and the advancement made through this research could be highlighted further. I liked Figure 1 - would be nice as a graphical abstract. The figures/tables shown could be improved.

In general there are very few grammatical errors. But watch out for double spaces after full stops. Check that the line spacing is the same between each sentence.

Abstract

Line 2: Remove the word “as”.
Line 11: 16S rRNA gene.
Line 11-12: Next generation sequencing does not target the whole 16S rRNA gene. It is amplicon sequencing? What region of 16S was targeted using your specific bacterial primers? Was it a universal primer set? What sequencing technology? Pyrosequencing? Illumina Miseq?

The background sentences could be shortened. The abstract needs to be more conclusive and highlight the merit of the study.

Introduction

Line 44 – 47. The reference on line 44 can be removed as you can leave it at the end.
Line 50: Appears.
Line 55: Definition of polyandry?
Experimental design: Methods

Could be a bit clearer on how skin tissue samples were taken. Were the samples weighed? Was the same size/depth taken? These are all factors that will influence your DNA extraction and thus your sequencing results. How did you extract from the water samples? What method used?

Line 96: These primers are a universal primer set which amplify bacteria and archaea.

What error rate in the sequencing? How many sequences removed after quality control?

Line 101: Remove “The” and one “was”.
Line 104: Why were only 32 used? How did the DNA concentration determine what samples? Did some not amplify? Be more precise in your methods section.
Line 107: Quantified.

Why pairwise dissimilarity performed? Why Horn? Would Bray-Curtis dissimilarity matrix not have been more appropriate? Also consider stats that include Unifrac and Weighted Unifrac to consider phylogenetic distances. Was the data rarified or transformed in any way? How many total sequences were found?

N.B. You need to submit the raw sequences to Genbank and give the accession number.
Validity of the findings: Results

Line 139: Were they Archaea? Your primers target both.

Your first paragraph reads more like a methods section. One sentence should suffice, total number of sequences how many Phyla/OTU identified etc.

The results and discussion section need a lot of improvement. You need to consider species richness/evenness/ - alpha-diversity/ beta-diversity. Perhaps look to other microbial ecology manuscripts to give ideas to strengthen the results and discussion sections. Figure 2 should be in terms of the relative abundance in % terms so you can compare across the samples. Focusing on phyla level does not really give a lot of information. Table 1 could be supplementary. Is , meaning a decimal place? 3,182 reads from 32 samples is very very low. Consider investigating the sequence analysis further. How many total reads per sample before and after QC? Error rate of platform? More in depth analysis needed.
Additional comments: In general I think this is an interesting manuscript but the methods section needs a lot more detail. I think your sequencing analyses should be repeated and more rigorous analyses performed. The presentation of the data could be improved.


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

Input:
Title: DESCRIPTION OF THE MICROBIOTA IN EPIDERMAL MUCUS AND SKIN OF SHARKS (GINGLYMOSTOMA CIRRATUM AND NEGAPRION BREVIROSTRIS) AND ONE STINGRAY (HYPANUS AMERICANUS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: -While I did not factor this into my review, the paper needs a thorough editing. It is difficult to follow the author’s train of thought at times.

-The methods, in particular the bioinformatics, are very unclear and need revision for clarity.

-Not a single statistic is used in this manuscript. It is hard to draw conclusions without some significance testing.

-Overall, the figures need polishing they look straight out of R.

-Did the authors normalize their data for analyses? Given the very wide distribution of sequencing depth (fig 2), this should be done for beta diversity analyses.

-My main concern with this study is the data and the massive loss of data the authors report. The authors report 219k raw reads (line 136) and following filtering they use ~3.6k. What happened to the rest of the data? Was this just a bad run? What did the authors do to filter the data? More information is needed to evaluate this data.
Experimental design: This is fine overall. A better description of the methods is needed however
Validity of the findings: The results are reasonable. But it is hard
Additional comments: Line 80: please provide GPS coordinates for the sampled area. Also, a table would be nice summarizing the species and # samples.

Line 83: please provide GPS coordinates here too.

Line 87-88: how was the water collected and filtered.

Line 91: how long were the samples kept at 4C?

Line 95: I would suggest combine these two sentences into one

Line 97: 515/806 only amplifies the V4 region of 16S. Also I suggest moving ‘region’ to after ‘v3-v4’ to make the sentence flow better.

Line 97: capitalize the ‘s’ in 16S

Line 99: was this a single PCR reaction for each sample?

Line 101: this sentence needs to be rewritten. Also, outside of the water could the authors actually view skin metaG DNA on a gel?

Line 111: where was the sequencing performed?

Line 114: what parameters were used to filter the data?

Line 117: <20 sequences per sample doesn’t indicate poor quality, just poor sequencing depth. Do the authors mean a PHRED score <20?

Line 119: please elaborate on the methods used in QIIME

Line 120: OTU does not mean taxonomy. Do the authors mean the clustered the reads in OTUs and the assigned taxonomy? What methods did they use?

Line 131: did the authors deposit the sequences in a database (e.g. NCBI).

Line 120: what version of QIIME?

Line 122: ggplot is only a plotting package, not for analyzing diversity. Did the authors calculate diversity?

Line 124: I’m not sure what the authors are trying to say in this paragraph. They say MDS and then Horn similarity/PCoA. What analysis was used, make this clear. Also was the data normalized?

Line 129-130: this likely isn’t needed.

Line 140: should be ‘Bacterial’ not ‘Bacteria’.

Line 156: species-level assignments are hard with this dataset given the small fragment length (~250bp). I would suggest the authors avoid this type of analysis.

Line 161: did the authors try to add statistics to back this paragraph up?

It would be nice to talk about fish species difference in results/methods.

Discussion: the authors should, if they are going to talk about genera, provide some data showing the genera and not rely on the text to inform the reader.

Line 287: the authors do not report diversity estimates anywhere in this paper.

Figures and tables
Figure 1: In the caption, OTU’s should be OTUs. When the authors say OTUs at the phylum level, do them mean shared phyla? If so then they should revise the legend accordingly.

Figure 2: the color scheme on this is very taxing on the eyes, the colors are too similar to
discern between the phyla present. I would suggest using a different palette and simplifying it
to smaller number of taxa.

What does the y-axis mean? Number of reads?

The depth of sequencing is clearly different between samples so it makes comparing between
samples impossible. I would suggest the authors that the output from the QIIME script
“summarize_taxa.py” and use the relative abundances to make this plot.

Also, the phylum level is very coarse. It tells the reader very little about the system given the
diversity of functions bacteria are capable of in a single phylum. I would suggest the authors
do this at the order or family level to provide more information about the data.

What do the boxes around the bars mean?

Figure 3: is this a PCA or PCoA? The axes say PCoA, the legend says PCA and the methods say PCA. What method was used?


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: DESCRIPTION OF THE MICROBIOTA IN EPIDERMAL MUCUS AND SKIN OF SHARKS (GINGLYMOSTOMA CIRRATUM AND NEGAPRION BREVIROSTRIS) AND ONE STINGRAY (HYPANUS AMERICANUS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thanks to the authors for making the suggested changes. I appreciate the effort that went into the reanalysing of the sequencing data and the manuscript has improved considerably. I have revised my suggestion however to still consist of major corrections based on these changes to further improve the quality of the manuscript – but these should not be too difficult to address but would represent more than minor changes.

Your raw data needs to be submitted and given an accession number, it is possible to not make this public until the manuscript is published but it needs to be submitted before accepting for publication. Perhaps the editor can advise if you need to submit a letter if you want to avoid submission to a database for a specific reason?

1) Can you include OTU-level analyses?

"were assigned taxonomically to OTU against the SilvaV4 database"

OTUs are classified based on a cut off threshold -usually 97% similarity with each other. Is this what you did? The sequences of these OTUs are then aligned to the specific database you choose which is a separate step. Thus, OTUs are independent of the database and a true representation of your samples. Considering many OTUs could not be matched to the database including OTU-level analysis could provide a detailed picture of the microbiology of your samples.
-->The alpha and beta diversity measures in Table 2a and 2b don’t provide a lot of information. The authors discuss within the text but fail to address whether there are significant differences between the groups or samples? From Figure 4 it appears there is no significant difference? If so can add to Supplementary Information. Is there a difference between the water and the three fish types sampled? Also was the data rarefied to the lowest sequencing depth sample? These would make more sense performed at OTU-level ie. Not taxonomically assigned to the database.
-->Rarefaction curves need to be for each individual sample and ideally at OTU level rather than taxonomic assignment to order or family level.
-->Discuss diversity before discussing what is shared and any description of taxa found.

2) Can you describe your sequencing QC measures, each step and the sequences left after each output? Were chimeric sequences removed?

3) Figures

-->8 Figures is a lot to include. Are all these essential? Are all these necessary to back up your conclusions in the manuscript? -Table 1 is too large for inclusion within the manuscript. This could go in Supplementary Information along with the number of sequences that passed QC for each sample type. Can include a taxa plot (species above 1-2%) at family level for each individual sample instead of table?
-->For the shared Venns display and discuss one level only – or else OTU and then family (instead of order and family as a lot of your OTUs could not be assigned). Describing both family and order is confusing in these cases and adds no extra value. In methods you say OTUs but you show only show taxa.
-->Can PCA include all samples? PCA (Principle Component Analysis) and PCoA (Principal Coordinates Analysis) measures are different, which was used?
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Abstract:
Line 11: Remove sentence about collecting water samples.
Line 18: change to ‘and there was no significant difference’.
Line 21: Add the water collection sentence to here. Combine as one shorter sentence. E.g. sampling of the raw water revealed…..These were not observed in.
Line 21-23: What is the evidence for their function?

“We found potentially pathogenic bacteria in water samples such as Pasteurella spp., Haemophilus spp. and Halomonas spp. but these were not found in the tissue or mucus samples from any species.  We found some bacterial groups such as Flavobacterium, Pseudoalteromonas, Lactobacillus and Bacillus that could play a role protecting the animals from pathogenic infection”. -Can you back this up with further data? Perhaps a co-occurrence network analysis?

Results:
Why would you expect the water to have a lower diversity? Any evidence? Less DNA quantified?

Discussion:
Line 326: Remove sentence re. startling examples.
The discussion largely discusses genera identified but the results you display are not at genus level.

-->Some of the colours in your taxa plot colour scheme are too similar. The text for these colour codes needs to be increased in size.
-->Figure 6 and 8 say Order and Family? Which one?
-->Include the legend for the fish pictures in each figure where these are used.


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: DESCRIPTION OF THE MICROBIOTA IN EPIDERMAL MUCUS AND SKIN OF SHARKS (GINGLYMOSTOMA CIRRATUM AND NEGAPRION BREVIROSTRIS) AND ONE STINGRAY (HYPANUS AMERICANUS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The English used in the manuscript is correct, but sometimes there are long sentences that impede to understand everything at the first reading. I suggest to authors to make a review by a native speaker.
The references used in the article are very appropiated, and the structure of the article is correct for the publication.
Experimental design: The methodology used for perform the study is appropiated.
Validity of the findings: It is the first study assessing the microbiota of mucus and skin in 3 Elasmobranchs species, which is interesting to have a reference in future studies.
Additional comments: In the manuscript entitled “Description of the microbiota in epidermal mucus and skin of sharks and rays”, authors performed a description of the microbial communities from 3 Elasmobranch species. It is very interesting to have a reference for future studies, and thus this manuscript deserve to be published. However, it still needs some more work to clarify some aspects of the study.
One of the main concern that I have is about the different samples belonging to the nurse sharks. There are two blocks of samples: wild juveniles and captive adults. Both factors (habitat and age) may influence the microbiome, so I think that it is not correct to include those two groups as the same kind of samples just because they belong to the same species. Actually, if it is the environment which shape microbial communities, we could expect more similarities between the two shark species sharing the environment in the sea, than among wild juveniles and captive adults of the same species. Those factors should be taken in consideration in the analyses, because I’m sure that they are going to explain a part of the variance in the nurse sharks. However, it won’t be possible to distinguish among both factors since there are just those 2 categories.
I have major concerns with the statistical methods. Authors say that they performed Kruskal-Wallis tests, but they didn’t present results, in the general comparison nor in the group by group one. I guess that they decided if results were significant by looking at the box-plots, which is absolutely wrong. They speak about differences in diversity of different categories, but they don’t support this with statistics. In the Fig. 4 they say that there are no visual differences, but I don’t agree, since for example in E we could say that there is. But everything is speculation, if they don’t show the statistical results.
Another minor question is that when presenting the study, authors say that they worked on the taxonomical categories of Order and Family. However, in the Figure 1 for example, but also in the Discussion, they include the Phyllum and / or the Genus. Everything should be consistent. If you decide to work just on Order and Family, then remove results apart. And if you decide to include them, then say from the beginning.

More specific comments:
L6: change to “from the water or from males’ mouth”
L6: “The role…” this sentence should be placed at the beginning, before talking about sharks, and as a general feature of fish. “…from colonizing the skin. However, the role…”
L9: “Tissues” is confounding. Authors has analysed just the skin, so I suggest to change tissue for skin all along the manuscript.
L14: “we analysed sequences… species” this sentences could be removed.
L16: “particularly” is in some group, not in all the fish samples. Maybe say the diversity was higher in fish than water. And remove “was high” if you don’t say in comparison to what? There are some reference values of diversity?
L18: comparatively to what?
L26: “changes in… conditions, including…”
L32: I suggest, for more clarity, to start the introduction as follows: “The first barrier of protection against microorganisms in fish is the mucosal immune system”
L71: remove “in the process”
L76: “…mucus, and the skin tissue, to assess differences among the fish microbiome and this of the surrounding water.”
L77: Change “Doing so…” to “This will help to understand the role…”
L78: “…we characterized the bacterial…”
L79: change from to in
L87: how can you know that they are healthy individuals? In those fish captured in the ocean, it is not possible to know. Maybe you could say "apparently healthy", or explain why you know they were healthy instead.
L95: More details on the sampling should be given. For instance, how did you cut the skin? With sterile scissors or could have had contamination among samples? How long lasted the manipulation? Fish were immediately released?
L100: continue breathing
L104: “and PCR amplification” in italics.
L105: “from all the entire samples collected” Remove in L106 “In all cases…”
L106: remove “for all samples”
L107: at which diameter of filtration? What does it means concentrated?
L108: include the sequences of the primers, and a reference.
L110: temperatures should be written as the first one: 94 ºC, 50 ºC.
L117: insert space 1.5 %
L133: (skin, mucus or water)
L140: comma after “family”
L146-152. It’s messy. You speak of normality, then Venn diagrams, and then Kruskal-Wallis. The normality analyses should be together with the Kruskal-Wallis since is part of the same.
The Venn diagram is not a statistical analyses, is just a representation of descriptive data, so I don’t think that it is necessary to say here.
Also, it is not clear if the K-W test was applied to all or only to the data that did not fit in a normal distribution.
Finally, it is not necessary to say that data were visualized in box-plots since this is part of the K-W test.
L179: It’s messy, the diversities are mixed. Say first the differences among fish and sample type, making the comparison of diversity among different samples, and then bacterial groups.
L193: which is the reason to expect a higher diversity in the fish than in the water? I think that is plausible to expect the opposite.
L210: change “appeared to be” to “are”
L221: The first speculative sentence should not be the beginning of the Discussion. It should be removed, or placed below, and start with “this is, to our knowledge…”
L239: I guess is the average of the alpha-diversity, if the variation intra category is not significantly lower than inter categories (statistics?)
L252: Cenarchaeales, which…
L258: change which to and
L264: analysis on microbiome
L267: were obtained
L279 and so on: In general, there is always a controversy in deciding if a bacterial taxon is pathogenic or not, because sometimes it depends not just in the species, but in the strain.  Here for example, Bacillus include numerous keratinolytic groups, which may cause wounds in fish skin.
L288: parenthesis
L371: “presents the first description of skin and mucus...”
L642-643: It’s OTUs, no OTU’s
Fig. 2. In the text, the figures are cited in minuscule (2a, 2b…), but in the graphs they are in majuscule. Be consistent along the manuscript.
In the name of the graphs, please include Fish species instead of species, because it could be confounded with bacterial species.
Fig. 4. Statistical results are needed.
Fig. 6. I don’t understand the legend and figure. It supposed to be the distance among different categories but I can’t see this in this graph, not differences among sample types. It’s like if they are mixed, is it the average?
Fig. 7. The skin of the stingray is absent?


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: DESCRIPTION OF THE MICROBIOTA IN EPIDERMAL MUCUS AND SKIN OF SHARKS (GINGLYMOSTOMA CIRRATUM AND NEGAPRION BREVIROSTRIS) AND ONE STINGRAY (HYPANUS AMERICANUS)
Review round: 3
Reviewer: 1

Output:
Basic reporting: Third Revision: Comments in attachment
Experimental design: No comment for this version
Validity of the findings: Line 499: Bioinformatics method – in your results you have the numbers of reads and how many remained after QC. Please add to this section. A lot of reads didn’t pass the QC. Mothur should tell you how many samples remain after each QC step and chimera removal please include if possible. Comment in the discussion about the high error rate and relate to the literature on the platform and other studies. I believe these primers (Caporaso) when used for salmon pick up a lot of host DNA and so have a high error rate so could you comment on the source of the read loss (ie chimeras, read length, host DNA contamination etc).
Recommended vevision: Sentences in discussion discussing error rate where you already discuss low % of assigned species. More detail on QC.

I don’t agree with having diversity measures to order level I think it is too high. In the previous version there was inconsistency between figures and text showing different levels so understandably choosing one level is easiest and is clearer, but for these analysis the standard is OTU-level or genus maybe family-level. For example in the results it is stated ‘Alpha-diversity was similar among species and among types of samples’ I’m highlighting this as I think that is likely you are just not looking deep enough and missing something interesting in your study. Unfortunately many of your reads did not assign at genus/family level. As you state only 16% of your reads were assigned taxanomically.
Recommended vevision: Test alpha-diversity at OTU level.

The rarefaction results I would like to see at read-level to see the variation per group but also per sample. Especially given how many reads were removed through QC. From reading the manuscipt there’s no way of knowing if this is even per sample. The rarefaction at this level is crucial here. In fact you mention ‘samples having 10 times the number of different orders found in other samples from the same type. Something similar was found for the samples from each species, meaning that there was high heterogeneity in samples used in this study (rarefaction curves for each species and sample type’ Supplementary Figure 2)
Recommended vevision: Rarefaction curve with reads after QC or OTU level per sample.

For the rest of the analysis it is fine to be at order level. The description of interesting genera in the discussion reads well.
Additional comments: Getting a paper to publication can be challenging at times and I’d like to thank the authors for making significant changes over a considerable time-frame. Overall these have improved the manuscipt greatly.

My main comments for this version which need addressing:

Line 499: Bioinformatics method – in your results you have the numbers of reads and how many remained after QC. Please add to this section. A lot of reads didn’t pass the QC. Mothur should tell you how many samples remain after each QC step and chimera removal please include if possible. Comment in the discussion about the high error rate and relate to the literature on the platform and other studies. I believe these primers (Caporaso) when used for salmon pick up a lot of host DNA and so have a high error rate so could you comment on the source of the read loss (ie chimeras, read length, host DNA contamination etc).
Recommended vevision: Sentences in discussion discussing error rate where you already discuss low % of assigned species. More detail on QC.

I don’t agree with having diversity measures to order level I think it is too high. In the previous version there was inconsistency between figures and text showing different levels so understandably choosing one level is easiest and is clearer, but for these analysis the standard is OTU-level or genus maybe family-level. For example in the results it is stated ‘Alpha-diversity was similar among species and among types of samples’ I’m highlighting this as I think that is likely you are just not looking deep enough and missing something interesting in your study. Unfortunately many of your reads did not assign at genus/family level. As you state only 16% of your reads were assigned taxanomically.
Recommended vevision: Test alpha-diversity at OTU level.

The rarefaction results I would like to see at read-level to see the variation per group but also per sample. Especially given how many reads were removed through QC. From reading the manuscipt there’s no way of knowing if this is even per sample. The rarefaction at this level is crucial here. In fact you mention ‘samples having 10 times the number of different orders found in other samples from the same type. Something similar was found for the samples from each species, meaning that there was high heterogeneity in samples used in this study (rarefaction curves for each species and sample type’ Supplementary Figure 2)
Recommended vevision: Rarefaction curve with reads after QC or OTU level per sample.

For the rest of the analysis it is fine to be at order level. The description of interesting genera in the discussion reads well.

Other comments:
Line 9: Remove ‘is considered’, remove sensentence from partlly (as you go on to say the same thing in the next sentence).
Line 334: change ‘place where each individual was captured’ to ‘sampling location of each individual’.
Line 349: Ion torrent method – were samples pooled to equimolar concentrations? Perhaps add more brief but relevant details from the stated protocols.
Line 519: What R version?
Line 572: Keep sentence with how many OTUs found (is this 22,803?) and then how many taxonomically asigned remove the first part and put into methods as described above.

Figures:

Figure 1: I think Figure 1 and Figure 2b can go in supplements as one figure.
Recommended new figure 1a and b: A)  Number of shared OTUs per sample type. I think this will be interesting as it tells us about the 84% of the community not tax assigned. Then Figure 2b) Number of shared orders per sample type.
Figure 5 should go to supplements as it is not needed. And the shark species cover parts of the plot the reader will want to see. See recommendation above for rarefaction curve.

Supplementary Figure 3: Labels on plot hard to read consider removing.

Image resolution quality may need to be improved for publication


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

Input:
Title: DESCRIPTION OF THE MICROBIOTA IN EPIDERMAL MUCUS AND SKIN OF SHARKS (GINGLYMOSTOMA CIRRATUM AND NEGAPRION BREVIROSTRIS) AND ONE STINGRAY (HYPANUS AMERICANUS)
Review round: 3
Reviewer: 2

Output:
Basic reporting: The wording and the English of the manuscript has substantially imporved. It is very clear.
Experimental design: Correct
Validity of the findings: It is the first study assessing the microbiota of mucus and skin in 3 Elasmobranchs species, which is interesting to have a reference in future studies.
Additional comments: Both in the Abstract and Introduction authors speak about the behaviour of sharks during copulation, and the risk of infections. However, there is no discussion at all about the implications of the results obtained in this situation.

L21 and so on: If you decided to keep only the results on Orders, do not include the Genera.
L23-24: There is an error in the redaction of the sentences: “Bacillales,  However…” There is no sense.
L24-28: Please rewrite those results since it is not clear what you mean.
L107: 90 % and 4 ºC, and be consistent all along the manuscript.
L196: Remove “and” after 28.
L238: due to a…
L231: Add: “or fish in general”
L410: Remove “and”
Fig. 8: were due to a…


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: DESCRIPTION OF THE MICROBIOTA IN EPIDERMAL MUCUS AND SKIN OF SHARKS (GINGLYMOSTOMA CIRRATUM AND NEGAPRION BREVIROSTRIS) AND ONE STINGRAY (HYPANUS AMERICANUS)
Review round: 4
Reviewer: 1

Output:
Basic reporting: Covered in previous revisions.
Experimental design: Covered in previous revisions.
Validity of the findings: Covered in previous revisions.
Additional comments: Thanks to the authors for being very patient throughout this review process and for sticking with it - and gaining additional training in bioinformatics. I found the paper really interesting and a valuable contribution to the field so well done - only some minor comments below.

Note: These line numbers are from the tracked word document version:

Line 17: Define OTUs when you first mention in the text.
Line 24 + 25: Give the P value
Line 155: Is that the right reference for Qiime?
Line 177: Reference J Oksanen - 2019 for Vegan package – Vegan without all caps.
Line 212: change to occurrence
Line 340: change , to full stop
Line 350: Within text change “Alpha-diversity for OTUs” to Alpha-diversity at OTU-level” – same for others such as order level.
Line 453: Remove startling, perhaps start the sentence from the word “More”
Line 472: Do you show that in this study? Or you mean the reference you are citing?
Line 410:I would perhaps move around this paragraphs – you talk about pathogens 410-456, then have a paragraph on co-occurrence then pathogens again 472-506. So perhaps move the co-occurrence to fit in above when discussing the make up of the communities. It could be shortened to add further up.
Line 844: Is it meant to be (N#). In the figure legend?
Line 819: for OTUs is mentioned twice
Supplementary figure 3 – it’s unclear what the samples are as the reader won’t know your sample IDs. You will need to add a description to the legend. Or you can overlay a text box in powerpoint and re-save the image.


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: DESCRIPTION OF THE MICROBIOTA IN EPIDERMAL MUCUS AND SKIN OF SHARKS (GINGLYMOSTOMA CIRRATUM AND NEGAPRION BREVIROSTRIS) AND ONE STINGRAY (HYPANUS AMERICANUS)
Review round: 4
Reviewer: 2

Output:
Basic reporting: Very well written, everything is correct in this new version.
I have just a suggestion. L280: Change the comma to a full stop.
Experimental design: Everything is correct.
Validity of the findings: No additional comments
Additional comments: No additional comments


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

Input:
Title: DESCRIPTION OF THE MICROBIOTA IN EPIDERMAL MUCUS AND SKIN OF SHARKS (GINGLYMOSTOMA CIRRATUM AND NEGAPRION BREVIROSTRIS) AND ONE STINGRAY (HYPANUS AMERICANUS)
Review round: 5
Reviewer: 1

Output:
Basic reporting: Addressed in previous revisions.
Experimental design: Addressed in previous revisions.
Validity of the findings: Addressed in previous revisions.
Additional comments: Thanks to the authors for making all these suggested changes through previous drafts. The manuscript is much improved through these revisions and represents a valuable piece of work for the research community.


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: FUNGAL SURVIVAL UNDER TEMPERATURE STRESS: A PROTEOMIC PERSPECTIVE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The review collects fungal proteomics work produced during the decade 2008-2018 with a special focus on proteome rearrangements as triggered by temperature stress. The manuscript is written in a clear and unambiguous English and is of interest, however there are some recommendations below to strengthen and improve its quality.

A number of reviews on fungal proteomics are available documenting stress response, host-pathogenic interactions, biotechnological applications or describing the advancement of proteomics techniques applied to the investigation of the fungal proteome. As compared to those, this manuscript offers an insight into the very specific topic of temperature stress, which had not been the main subject of a review on fungal proteomics yet. The introduction adequately explains the subject of the review, describing the crucial role of fungi in soil microbial communities as well as the importance of protein biomarkers of fungal stress for the monitoring of environmental changes.
Experimental design: The article content is within the aims and scope of the journal. The review collects the results of not more than 10 studies produced between 2008 and 2018, which reflects the limited number of proteomics studies specifically including temperature stress hitherto available. Two considerations on this regard: 1_ I understand the aim to focus on a specific time span, however I fear that excluding the last two years of research will have an impact on the completeness of the review, along with making it not thoroughly up-to-date. Two works, among others, that focus on heat stress, would be worth a mention i.e. Zou et al., 2018 DOI=10.3389/fmicb.2018.02368  and of Deng et al., 2020 https://doi.org/10.1002/mbo3.1012. 2_One of the aims of the manuscript is to provide an overview of the implications of temperature stress for fungal responses within the ecosystem to current climate change challenges. However, the authors do not expand on this aspect other than in the introduction, whose content sets expectations about a manuscript primarily dealing with soil fungi and their potential as biomarkers of global warming.
Validity of the findings: No comment.
Additional comments: Line 41: “global ice loss will”. Here the verb is missing.
Line 61: Please consider changing “play” with “plays”
Line 94: “have analysed on fungal temperature”. Please consider removing “on”.
Line 115-118: the authors state that the objective of the review is “to provide an overview on the effects of temperature stress on filamentous fungal proteomes”. Nonetheless, some of the species listed in the review, e.g. Cryomyces antarcticus (microcolonial RIF) and Exophiala dermatitidis (black yeast), are not exactly filamentous fungi. I would suggest clarifying this in the text.

Lines 145-159: This paragraph is part of a subchapter entitled “High temperature stress” and references to psychrophiles are made using M. psychrophile and F. endolithicus as examples. To be thorough and give the reader a better idea of the stress, I’d suggest adding the optimal temperatures of growth of both the yeast and the fungus to the text.

In the case of F. endolithicus the authors should provide more details. “The proteome abundances of another psychrophilic fungus, F. endolithicus, assessed using a classical 2D gel electrophoresis (non-comparative) approach, showed a reduction from 284 to 224 protein spots”. In relation to the exposure to what temperature did this occur?

Line 165: Please clarify in the text what RanA is, in order to make the statement “Increased levels of
RanA expression suggest that there was an additional signalling mechanism involved during phase transition of P. marneffei”, understandable.

Line 166-167: “Gauthier (2017) reviewed the molecular strategies used by thermally dimorphic fungi (Gauthier 2017).” The authors do not provide any details about the findings of the cited research. Please consider expanding on it.

Line 169: Please consider changing “a species whose growth is optimum at 37°C but maximum mycotoxin is produced at 28°C.” with: A species whose growth optimum is.., but highest levels of mycotoxin are produced at

Line 201-203: I’d suggest rephrasing this sentence and improving the wording
Lines 226-231: I suggest splitting this very long sentence in two parts.
Lines 247-248: “Protein structures are modified with subtle changes in the amino acid composition, thus remaining functional extremely cold conditions.” Here, the preposition (e.g. “under” or “upon”) is missing.
Line 248: Please use the past here.

Line 254-260: Please pay attention to when to use the extended or shorten species name. Generally, the full name should be used the first time that a species is mentioned in a text. In the second and subsequent uses of the name, the name can be abbreviated. Please check this throughout the text.

Line 271: Please consider changing “Decreased levels of heat shock proteins” with Decreased levels of proteins involved in the response to heat stress”.

Lines 273-275: The authors cite the work of Tesei et al (2015) stating the following: “Exophiala dermatitidis lacks a typical stress response under exposure to non-optimal temperatures, possibly because the cost of utilising alternative metabolic pathways may outweigh the benefits of much slower growth rates”. This sentence is not entirely clear. According to Tesei et al. (2015) a much slower growth rate is a consequence of the down-regulation of the metabolism that black fungi undergo upon low temperature stress.

Lines 280-281: The authors state that “Mrakia psychrophila has been shown to respond to low temperature stress in a very similar fashion to other cold-adapted fungi.” However, chaperones and energy metabolism pathways are found to be upregulated in the yeast upon exposure to low temperature, which is in contrast with what observed in E. dermatitidis, the study cited just before in the text. What do the authors mean by cold-adapted fungi? Psychrophilic fungi? Please consider specifying this in the text.

Line 289: Please consider writing the extended name of F. velutipes, since it is the first time that it is mentioned in the text.

Line 389: typo. “HSP60 has alos been”

Table 2:  In this table, for each of the cited studies, both the temperature of exposure and the temperature of optimal growth of the fungus are listed. In the case of E. dermatitidis, both 37 and 45°C are indicated as T optimum, however the correct temperature optimum is solely 37 °C. In Tesei et al. (2015), following growth at 37°C, E. dermatitidis was exposed to 1 or 45°C for 1 hour (short-term exposure) or 1 week (long-term exposure) in order to monitor the yeast response to both low- and high-temperature stress. Protein profiles from the different temperatures of exposure were compared in order to assess protein modulation.
Please consider editing the table as well as the text, accordingly. The portion of Table 2 describing proteins and pathways affected by the exposure of E. dermatitidis (with optimum at 45°C) to 1°C shall be edited to point out that 45°C is not the fungus temperature optimum, but one of the experimental conditions.
Please also cross-check the part of Table 2 displaying up and down regulated proteins at 1°C as compared with the optimal temperature of 37°C, as e.g. acetate metabolism is listed among up-regulated pathways but instead it exhibits lower levels at 1°C.


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

Input:
Title: FUNGAL SURVIVAL UNDER TEMPERATURE STRESS: A PROTEOMIC PERSPECTIVE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall well written and informative. Some minor grammatical errors (e.g., fungi proteomes in abstract should be fungal proteomes, various should be various). Good overview of relevance and importance of mycological studies and fungal organisms within communities. Language is passive at times (e.g., can be due to, been made based on the), such statements should be minimized to strengthen the impact of the manuscript. Some proof-reading of acronyms and shortening of scientific names before proper names are introduced need to be addressed.
Experimental design: The authors state previous work and reviews available and highlight the uniqueness of the current manuscript. The survey methodology is unbiased in coverage of the subject and is mostly comprehensive and refers to other reviews for research not covered in this manuscript. Sources are adequately cited.
Heat shock proteins are discussed in the introduction and their relevance and importance is presented but then they are not highlighted again until the last section. Perhaps move the section on the impact of temperature stress on HSP to after the introduction if they are important to the studies presented in the Review.
Validity of the findings: The studies are well reported and results are interpreted well with some suggestions for future work provided. Tables 1 and 2 are well-organized and helpful for the reader. Notes in the General Comments section address concern over arguments and focus established in the abstract and introduction that are not clear throughout the manuscript.

The concluding remarks section would be strengthened by highlighting specific information gained by conducting the current Review
Additional comments: The title refers to temperature stress in general, but the abstract emphasizes agricultural applications and the main body of the text focuses on agricultural and medical fungal species. Providing some clarity on the emphasis in the abstract would help the reader follow the direction of the manuscript. Why emphasizing agricultural relevance? It’s a good topic and important but a little more justification would help the reader.
Several of the highlighted studies seem to use qualitative analyses to determine in proteins are increased or decreased in abundance by reporting presence/absence. It would be valuable for the authors to discuss limitations of such studies where absence may be technical variation/error if quantification values are not available, or increased abundance could be attributed to differences in staining between two gels if no normalization is provided. Such statements could be provided in the last paragraph of the ‘high temperature stress’ section as this is paragraph reads as a discussion of the studies reported to date.

Specific comments:
Abstract:
-	What is the difference between a traditional vs. systematic review process?
-	How can PTM in stress pathways identify potential biomarkers? The connection is not evident, although later in the introduction the importance of profiling proteomes for biomarkers is clear but the connection to PTMs remain elusive.
Line 41: ice loss will ? (missing word)
Line 71: is there a reference for anti-freeze proteins (other points in this sentence had references)
Line 92: metabolic differences relevant to heat stress?
Line 94: ‘In the last 20 years…’ proteomics has been used to study fungal temperature stress and all references are within the past 10 years. Seems more accurate to state within the past 10 years. As, the methodology focuses on a 10 year period of study.
Line 97: In general, the field of proteomics was established in the late 90s, but the sentence states ‘over the past 4 decades’, I would argue that over the past two decades is more accurate.
Line 102: why highlight the use of isotopic tags? Many other approaches exist. Are these approaches generally used for heat stress proteomics? Why is pooling samples an advantage? This paragraph is informative but seems to jump around in focus.
Line 111: suggest defining what the ‘traditional approach’ is compared to other approaches. Why was a traditional approach selected?
Line 258: check usage of fungal names (e.g., Penicillium chrysogenum is spelled out by abbreviated in a couple lines above) same as in line 273, 354
Line 266: DIGE and nLC-ESI-MS/MS need to be spelled out
Line 337: What advances in proteomic technologies have been made? Reference to specific aspects would be helpful.
Line 369 HSP used previously, not need to be redefined here
Line 394: cAMP-PKA mentioned in above sections but only shortened in this paragraph. Same with MAPK.
Line 424: PTM discussed but not mentioned in the Review. Why is this relevant now? References to support?


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: FUNGAL SURVIVAL UNDER TEMPERATURE STRESS: A PROTEOMIC PERSPECTIVE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Review article entitled ‘Fungal survival under temperature stress: A proteomic perspective’ uses a limited range of studies sketching the effect of temperature range on different fungi. The followings are the comments to improve the article.

Authors have considered temperature as a stress factor to compile the review; A temperature range is favorable for growth, and below/above of that rage is /are considered as stress. When the optimum/temperature range for growth has been shifted to a 'temperature' referred to as 'temperature stress', the author should discuss the criteria and to strengthen the manuscript discuss it with specific examples?
Experimental design: Table-1 indicated that 7 studies have been included for the basis of this review? A column needs to be put in for “favorable temperature range of growth”

Line 401-407 Hsp90 has also been implicated in drug resistance? Add this point?
Lamoth F, Juvvadi PR, Steinbach WJ. Heat shock protein 90 (Hsp90): A novel antifungal target against Aspergillus fumigatus. Crit Rev Microbiol. 2016;42(2):310-321. doi:10.3109/1040841X.2014.947239
Line 69-75 A recent review on temperature/cold stress/heat shock proteins has summarized well on fungal biology is missing in the introduction section or elsewhere?
Tiwari S, Thakur R, Shankar J. Role of Heat-Shock Proteins in Cellular Function and in the Biology of Fungi. Biotechnol Res Int. 2015;2015:132635. doi:10.1155/2015/132635

In dimorphic fungi ‘temperature’ is the key factor for transition/dimorphic switch of fungi from one to form to another form.  Why the author did not consider those fungi e.g., Paracoccidioides, etc, where temperature stress/thermoregulated fungi will be addition/ relevant to the reader.

Concluding remarks; It misses the punch line or the outcome of the review should be brought in here to strengthen the review article.
Validity of the findings: No comments
Additional comments: As above


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

Input:
Title: THE PERFORMANCE OF A MODIFIED EWMA CONTROL CHART FOR MONITORING AUTOCORRELATED PM2.5 AND CARBON MONOXIDE AIR POLLUTION DATA
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. For example, in line 82, the sentence “Xt is tth observation” is not correct in English grammar.
Experimental design: The question research well defined. Research methodology is correct and there are some isuues which are not clear.
Validity of the findings: 1.	In the introduction part, there are no the literature reviews about the using CUSUM and EWMA charts with PM2.5 and carbon monoxide air pollution data. The literature reviews in this matter should be added in the introduction.
2.	Why the author select a modified EWMA chart with the PM2.5 and carbon monoxide air pollution data? Could you give the reason?
3.	In line 82, the range of parameter   is not correct.
4.	In the upper and lower control limits for the EWMA chart, the formula of H value or the method for finding the H value should be explained.
5.	In the upper and lower control limits for the modified EWMA chart, the formula of L value or the method for finding the L value should be explained.
6.	In the upper and lower control limits for the EWMA and modified EWMA charts, what is the criteria for setting the value of the smoothing parameter  ?
7.	In Eq.6, how do you set the value of k? Explain more in details.
8.	In line 112, “UCL = b” Is it correct? What is the value of b?
9.	In line 131, the details of M are not appear. (It is showed in line 137)
10.	In line Eq.19, the typing of the formula is mistake.
11.	In application part, the ACF and PACF for fitting the MA process should be shown and analyzed in the paper.
12.	The estimation method for estimating the parameters of MA(3) process should be mentioned.
Additional comments: This paper proposed new knowledge and apply to a real data set. It is suitable for publication in your journal. There are some isuues which is not clear.


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

Input:
Title: THE PERFORMANCE OF A MODIFIED EWMA CONTROL CHART FOR MONITORING AUTOCORRELATED PM2.5 AND CARBON MONOXIDE AIR POLLUTION DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript proposes a modification of the EWMA control chart for monitoring air quality (PM2.5 and CO). The application is based on data collected in Thailand during 2020. The topic is relevant, interesting and it could potentially be a good application or a case study. The authors have submitted some codes and the dataset.

However, I found that the article is poorly written, the sections are not well connected and it fails to put in context this new approach. This manuscript would need a lot of work, heavy rewriting and copyediting to make it publishable.
From a practical point of view, some of these results would be very hard to reproduce. The authors should keep practitioners in mind.

I am listing here some of the main issues but not all of them.

The introduction should highlight the importance of air quality and in particular monitoring the concentrations of PM2.5, but there is not a proper literature review.
For example, in L27-39 the authors introduce what is PM2.5 and the main sources of contamination. This needs proper referencing to the relevant literature.
In L51-55 the same. The authors make some claims no properly justified with evidence/citation e.g. : “the population has been exposed to dangerously high PM2.5 pollution levels”

L64 This sentence is not grammatically correct:
“Thus, if the Shewhart control chart is applied to serially correlated
observations, the performance in detection of the process change will be poor due to the sequentiality of the numerical data values recorded for a variable over a specified period of time with data points recorded on a regular basis.”

What you are trying to say here is that Shewhart control charts assume randomness in the data.

L73 What studies are the authors referring to?
Experimental design: The presentation of the statistical methods is inadequate. For example, in Line 78 the authors attempt to describe the EWMA control chart, which is basic in statistical quality control technique. The description is ambiguous and not statistically sound e.g. “This chart design parameters are the multiple of width of control limit and smoothing parameter”. You could say that the statistic is obtained using a smoothing parameter lambda and it depends on previous realizations.

Some of the derivations and proofs could go into the appendix. For example from L113 to 161 the authors could leave what is relevant for the reader.

With so many issues in this section, is difficult to assess if there are methodological flaws too.

In L102: Here again, this is inadequate e.g. “In time series analysis, the moving average process (MA) is one of the common approach for modeling univariate time series.”
L115: What do you mean by “Change the variable, we obtain”? Please clarify
Validity of the findings: The application or case study is vaguely presented.
Why the PM2.5 and CO were measured at different time points? January the PM2.5 and May the CO. Aren't these variables correlated?
What is the justification for selecting only 31 data points?  The study would be more robust if the time series was larger.
L237 Do you mean > 50?
Additional comments: No additional comments


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

Input:
Title: THE PERFORMANCE OF A MODIFIED EWMA CONTROL CHART FOR MONITORING AUTOCORRELATED PM2.5 AND CARBON MONOXIDE AIR POLLUTION DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: A modified exponentially weighted moving average control chart is proposed to detect small changes when the observations are autocorrelated with exponential white noise.  The performance of a modified exponentially weighted moving average (modified EWMA) control chart is illustrated through the average run length evaluated by explicit formulas. The solution accuracy is verified with a numerical integral equation method. The efficacy of the modified EWMA control chart to monitor PM2.5 and carbon monoxide air pollution data is compared with the standard EWMA control chart.

Many descriptions of the manuscript are not clear. The comments are the following.

Comments:
1.            Why do the modified EWMA consider only Xt-1 not Xt-1, Xt-2,….?
The reason should be given to convince readers, although its performance looks better.
In fact, the review does not feel it is a good approach, and it is not new.
2.     What is the distribution of X?  The authors did not mention it. However, it is important.
3.     Eq. 12 should be expressed clearly.
4.     Why are the control limits symmetric? The distribution of X is not mentioned.
If it is normal distribution, then the control chart is not new.
5.     In the numerical example, no distribution of the data is described or tested.
What is the AR(q) model? Does the author make the model adequate checking?
6.     What are the in-control and out-of-control data for pm 2.5 and CO gas data?
7.     So far, many modified EWMA control charts have been discussed in the literature.
The modified EWMA control chart should compare with the out-of-control detection speed with the existing EWMA control charts.


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

Input:
Title: THE PERFORMANCE OF A MODIFIED EWMA CONTROL CHART FOR MONITORING AUTOCORRELATED PM2.5 AND CARBON MONOXIDE AIR POLLUTION DATA
Review round: 3
Reviewer: 1

Output:
Basic reporting: The english grammar in this paper has been improved already.
Experimental design: Some mistakes have been corrected.
Validity of the findings: no comment
Additional comments: This paper is suitable for publishing in the journal.


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

Input:
Title: THE PERFORMANCE OF A MODIFIED EWMA CONTROL CHART FOR MONITORING AUTOCORRELATED PM2.5 AND CARBON MONOXIDE AIR POLLUTION DATA
Review round: 3
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: A modified exponentially weighted moving average control chart is proposed to detect small changes when the observations are autocorrelated with exponential white noise.  The performance of a modified exponentially weighted moving average (modified EWMA) control chart is illustrated through the average run length evaluated by explicit formulas. The solution accuracy is verified with a numerical integral equation method. The efficacy of the modified EWMA control chart to monitor PM2.5 and carbon monoxide air pollution data is compared with the standard EWMA control chart.
      This is an application of using the EWMA chart with a time series model in air pollution data. It looks that the detection performance of the time series model and modified EWMA control chart is quite good compared to the traditional EWMA control chart. It shows a good application in air pollution data analysis.
However, the modified EWMA control chart is not new.  There are many related papers about the modified EWMA control chart, some may be more effective in abnormal detection. The authors should cite them in the manuscript before the manuscript can be published. Furthermore, the distribution of error in the time series model is not normal but exponential distribution. It should be explained in a revised manuscript. Under the exponential error term, the EWMA control chart should have asymmetric control limits, not symmetric control limits.
This revised manuscript is not completely responsive to the referees’ comments. For easy reading by referees, usually,  a revised manuscript should reply to referees’ comments point by point.
The reviewer suggests accepting the revised paper before the above suggestions can be considered in 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: POLYESTER OR EPOXY: ASSESSING EMBEDDING PRODUCT EFFICACY IN PALEOHISTOLOGICAL METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: I had several questions, all which are minor or easy to address:

1) I know it seems like a silly question, but was the chicken a roast chicken or raw? Could argue cooking affects the bone.

2) Note: Acetone can also damage the fossils, so be careful that your wash didn’t affect the mineral integrity of the fossils.

3) Curious, but would there be a difference between “fresh” extant specimens and museum-based extant specimens?

4) For the difference in penetration between the two embedding mediums, was there a correlation with which one was usually used with a vacuum? I know a number of paleohistologist who swear by vacuum impregnation.

5) Can you state what you’re desired thin section thickness range is? That way you could say “you can’t get beyond X thickness with this, but that’s OK since we only need to get to Y thickness for histological analysis”.
Validity of the findings: no comment
Additional comments: Thank you for writing a techniques papers. I feel these are much needed in paleontology.


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: POLYESTER OR EPOXY: ASSESSING EMBEDDING PRODUCT EFFICACY IN PALEOHISTOLOGICAL METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the manuscript by Heck and colleagues conforms to journal standards.  The literature review is sufficient to give enough background to a non-specialist, and the writing is excellent.  I do think that an additional figure is needed to show how polyester resin and epoxy perform for modern bone sections.  Given the amount of space allotted to the preparation of modern bone, the lack of images from modern bone histology is surprising.
Experimental design: The design could use some refinement.  My main suggestion relates to the variation in grinding pressure and quality of the section.  In the revision, please address how grinding pressure was standardized or justify why it’s not an important factor.

I’m also curious about the use of cyanoacrylate adhesive.  It’s not an ideal adhesive for applications involving high shear stress (e.g., grinding sections).  Two-ton epoxy has much better shear strength.  However, the “confetti” artifact is a concern.  My immediate thought was contamination from the starch powder used in gloves.  But that would appear as round granules with the Maltese cross artifact under crossed polarizers.  My second thought was that the confetti represents the slow crystallization of epoxy.  If correct, you should be able to remelt them by heating the part A component of epoxy (resin) to 50 degrees C for a few hours.

Can you discuss why specimens are allowed to air dry before embedding?  Trapped air in your sample causes transmitted light to scatter away from the objective lens, in effect cloaking that part of the sample.  Sections with lots of trapped air need to be ground thinner to transmit enough light for imaging.  And the thinner one needs to grind the section, the more likely it will peel.
Validity of the findings: The study seeks to determine the cost-effectiveness of polyester resin when used to embed fossils for undemineralized histological sectioning.  The results suggest that modern and fossil bone sections can be ground thin whether they are embedded in polyester resin or epoxy.  Therefore, polyester resin, as the less expensive of the two, is more cost-effective, especially when embedding large bones.  I generally agree with the conclusion and can attest to the cost-effectiveness of the polyester resin as an embedding medium (I’ve used it for nearly 30 years).
Additional comments: See annotated PDF


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: POLYESTER OR EPOXY: ASSESSING EMBEDDING PRODUCT EFFICACY IN PALEOHISTOLOGICAL METHODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for adding clarification to your methods.


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

Input:
Title: CHANGES IN SPRINT PERFORMANCE AND SAGITTAL PLANE KINEMATICS AFTER HEAVY RESISTED SPRINT TRAINING IN PROFESSIONAL SOCCER PLAYERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Changes in sprint performance and sagittal plane kinematics after heavy resisted sprint training in professional soccer players.

The aim of this study was investigated whether heavy resisted training affected sprint performance, kinetics, sagittal plane kinematics, and spatiotemporal parameters in professional soccer players.

The authors performed nine-week protocol of resisted sprint training in professional soccer players with a resistance that induced a 60% velocity decrement from maximal velocity for one group and a second group that used a 50% velocity decrement, and a control group that continuing training as normal with no systematic non-specific acceleration training.

General comments

The positive characteristic of this design is that the intensity was determined taking as reference the decrement of velocity when the subjects trained with a resisted sprint. However, unfortunately, there are many aspects that, in our opinion, invalidate the design.

One of the design problems is that only two levels of independent variable are analysed (60 and 50% velocity decrement). This implies that we cannot deduce which load produces a better positive effect. The lighter load used tends to give a better result, as it seems that this load is the only one that presents some significant differences with respect to the control group. This means that probably lower loads could be better for improving sprint, but this is not analysed in the study, so the design does not provide enough relevant information on resisted sprint training.

Many kinetic and kinematic variables are analysed in the study, but the real effect of interest is the change of velocity in the sprint. Therefore, all other indicators are secondary if the training effect does not translate into more velocity in the sprint.
It seems that the objective of measuring the kinetic and kinematic variables is to "prove" that the loads used do not cause adverse changes in the sprint technique. But if speed is improved, what does it matter whether or not there were adverse changes in technique, especially in team sports players? In addition, the authors state that they found no relationship between changes in some of these variables and improvements in sprint performance (lines 508-09). This commitment to analysing numerous variables in addition to velocity leads the authors to present a disproportionate length of four pages of test results without including tables and figures, while only three pages of discussion.
Very often the authors talk about the differences or not of the kinetic and kinematic variables, when the reliability tests of these measures had very poor results in most cases. In fact, in some cases the value of the lower limit of the 95% CI of the ICC is zero or even negative. We believe that it is not appropriate to make deductions about the effect of variables that are so unreliable.
In relation to the reliability problem, the procedure for calculating the ICC and the SEM is not indicated in the manuscript. This is especially important because the results can be quite different depending on the procedure used. In addition, the ICC values considered as "good" are extremely low, which is related to the values indicated above of zero or negative in the lower limit of the confidence interval.
It is not correct to implement two statistical tests to know the differences between and within groups. You cannot apply an ANCOVA to detect differences between groups and then a "t" test to contrast differences within groups. You should have applied a 2 (measurement) x 3 (group) repeated measurement ANOVA to check differences between and within groups with a single test.
Nor is it appropriate to consider the two experimental groups as a single group for comparison with the control group. It is clear that with only two groups, one experimental with a larger number of subjects, and one control group, it is more likely to find statistically significant differences, but what the authors intend to explain with this analysis. This type of decision does not seem reasonable.
How should we interpret some results as "within-group spatiotemporal analysis showed that HS50% increased maximal velocity step rate (p <0.05, d = 1.50), however, the improvement remained under the minimal detectable change" (Lines 48-50).
The authors decide to apply a sled sprint distance of 0-15 m for the HS60% group and 0-20 m for the HS50% group in order to use a standardized time under tension. This apparent control of an independent variable, which presents equalization problems, introduces the sprint distance as a strange variable, when this variable is easier and more precise to control. Therefore, this does not seem to be an adequate decision if we want to improve the knowledge about the effect of resisted sprint training.

Other comments

Lines 175-77.  What was the reason for carrying out all training and testing sessions inside on artificial turf, and made the post testing outside? At best this increases the probability of introducing error into the measures

Lines 192-94 “There was no ordering of the control group, however, the sprint performance was predicted to be similar due to earlier consultation work with the team”

We think this sentence is strange and should have been clarified. What is meant by "team consultation work"?

Lines 225-29 “Load-velocity tests were completed under one unloaded and 3 loaded conditions with one sprint per load (50%, 75%,100% of BM) for both HS groups, outlined in previous literature27. The load-velocity data was then fit with a least-square linear regression to generate an individualized load-velocity profile for each athlete. Thereafter, the individual load corresponding to a 60% and 50%-velocity decrement of maximal velocity was calculated”.

The fact that the tests had been done previously in this way does not guarantee that the procedure is appropriate. If we do not use light loads, the adjustment will always tend to be linear, while including light loads the trend may be different. Therefore, the load corresponding to each decrement of velocity could be different from the one selected in the study

Lines 353-54 “At baseline population variance was not significantly different for any variables”

This statement speaks of pre-training data, but several people were eliminated at the end of the study, so this information should be about the final sample.

Lines 501-03 “The results showed no changes in the kinematics or any other variables in early acceleration, which is in contrast to previous light load literature showing slight increases in trunk lean”

What is the importance of leaning the trunk a little more or not?

Lines 515-24. In our opinion, according to the results of the study and the deficiencies observed in the design, the last paragraph of the discussion seems to us to be highly speculative on the strength or speed profile and the recommendation to train with one load or another

Lines 527-28 and 530-36. The fact that the limitations are exposed is appreciated, but these limitations were already known before the training and are of maximum importance for the design, then the study should not have been done without eliminating most of the limitations.


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

Input:
Title: CHANGES IN SPRINT PERFORMANCE AND SAGITTAL PLANE KINEMATICS AFTER HEAVY RESISTED SPRINT TRAINING IN PROFESSIONAL SOCCER PLAYERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	Basic Reporting
The writing is clear and unambiguous, with professional English used throughout. The article includes sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge. Relevant prior literature is appropriately referenced. The structure of the article conforms to an acceptable format of ‘standard sections’. Figures are relevant to the content of the article, of sufficient resolution, and appropriately described and labeled. All appropriate raw data (and more) have been made available.

Further basic reporting comments in approximate order of importance:

1.1.	Please proof-read for minor typos. In a number of places letters are missing (for example, showing --> showed in line 45, seem --> seems in line 87, a --> an in line 90, subjects --> subject’s in line 112 and 115…)

1.2.	Line 247: There is an inconsistency between for example ‘1.5-m’ here and ’22.5 m’ on line 249. I prefer the space but consistency is the main goal here.
Experimental design: 2.	Experimental design

This is original primary research within the aims and scope of the journal. The research question is well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap. The methods are described with sufficient information to be reproducible by another investigator. All data are available as supplementary materials, including all additional reliability analyses and additional information such as training schedules. This facilitates greater inspection of the data and findings by the interested reader and should be commended. The research was conducted in conformity with the prevailing ethical standards in the field.

There are some limitations in the study design (e.g. control group from a different team so not fully matched, small sample size leading to uncertain conclusions based on lack of effect, poor reliability of some measures used, etc.) but these are mostly discussed sensibly and are mostly a consequence of conducting longitudinal training interventions within a professional soccer club.

Further comments in approximate order of importance:

2.1.	The statistical power of the study needs to be considered in more detail. A small sample size may be justified on the basis of the population used, but inferences should be made accordingly.  This is especially true given that conclusions are drawn on the basis of no significant differences in kinematics. An under-powered study due to small sample size is less likely to identify any true differences and this makes this conclusion more likely. I fully appreciate that sample size is a trade-off when dealing with longitudinal interventions in professional sporting settings, but this should be discussed more explicitly and should be taken into account when drawing conclusions. For example, you could state in the methods what you will consider to be evidence of no difference (evidence of absence rather than absence of evidence). As a guide, please see this recent editorial in Journal of Sports Sciences: https://doi.org/10.1080/02640414.2020.1776002

2.2.	There are a lot of tests performed. How did you control for multiple statistical comparisons within the study?

2.3.	I really like the use of the minimal detectable change to inform inferences. However, I think the manner in which this is used can be made more objective and be outlined in the methods. For example, are differences only to be considered meaningful if they are greater than the MDC? Does the mean difference or the entire confidence interval need to be greater than the MDC? Clear and consistent standards would improve these inferences. It sometimes seems like the MDC comment is added on to the end of a result statement as an afterthought. As a guide, please see this recent editorial in Sports Biomechanics: https://doi.org/10.1080/14763141.2020.1782555

2.4.	Lines 206: Is there any possible benefit to investigating split times such as 10-20 m or 20-30 m to investigate different phases rather than only 0 – x m?

2.5.	Line 218: Is a 100% horizontal and 0% vertical GRF direction the optimal?

2.6.	Post-testing was performed outside, while pre-testing was performed inside – is there any data to support what difference this would make? Any reliability data for example? If this effect is systematic then this could either blunt the observed pre-post changes or make them seem larger – it is therefore of interest to be quantified.
Validity of the findings: 3.	Validity of the findings

The data on which the conclusions are based is provided. I was able to quickly recreate some of the figures in the manuscript from the supplied data. As discussed above, the validity (or the reader’s confidence in the validity) could be improved by greater objective detail around the interpretation of: a) magnitudes of difference in relation to the MDC; and b) no significant effect (is this evidence of no effect, evidence of no effect above a certain size (e.g. no meaningful effect), or just an uncertain finding?

Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: 4.	General comments

The aim of this manuscript was to investigate changes in sprint performance and the potential underlying mechanical changes after integrating two different heavy resisted sprint training loading variables within a professional soccer setting. This is a very interesting, novel study with clear practical applications. There are some limitations in the study design (e.g. control group from a different team so not fully matched, small sample size leading to uncertain conclusions based on lack of effect, poor reliability of some measures used, etc.) but these are mostly discussed sensibly and are mostly a consequence of conducting longitudinal training interventions within a professional soccer club. I feel that once any possible adjustments are made the remaining limitations in study design will be an acceptable trade-off for this type of applied sample and intervention. It is extremely unlikely that such an applied question could be answered in a professional sporting setting whilst fully controlling everything – the teams / players would be unlikely to provide access and engage.

Further comments in approximate order of importance:

4.1.	 Can exact (3 decimal places) p-values be given wherever possible? There are many instances where ‘p < 0.05’ or ‘p > 0.05’ are stated but the exact values would allow better inferences to be made.

4.2.	Line 260: What was the reliability of the digitisation process?

4.3.	Line 285: Throughout the manuscript, 1-2 sessions per week would be more accurate than 2 per week, especially as the average was around 10-11 in 9 weeks.

4.4.	Line 301: I expect this is sprint time rather than ‘time under tension’?

4.5.	Line 435: Confidence intervals around the d effect sizes would be useful to indicate the level of precision.

4.6.	Line 361: It seems odd to group multiple tests together into one set of confidence intervals – this results in a very wide range being reported.

4.7.	Table 2: It may be beneficial to include the MDC within these tables for comparison.

4.8.	Line 122: Try to be more specific than ‘unwanted’ – do you mean an increase or decrease in what parameters?

4.9.	Line 32: Should ‘heavy resisted training’ include the word ‘sprint’ if it is abbreviated to HS?


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

Input:
Title: CHANGES IN SPRINT PERFORMANCE AND SAGITTAL PLANE KINEMATICS AFTER HEAVY RESISTED SPRINT TRAINING IN PROFESSIONAL SOCCER PLAYERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: All comments are listed below in "General Comments for the authors"
Experimental design: General comment: there are some inherent challenges with conducting a “perfect” experiment when completing a training study (scheduling & logistics, illness & injury, etc.), especially in a professional sports environment. However, it appears the investigators have handled the potential confounding variables in a rigorous manner, and the Methods are comprehensive and provide a good description of the data collection/analysis and intervention protocols.
Validity of the findings: No major issues here. All other comments listed below in "General Comments for the authors"
Additional comments: Overview and Summary Comments:
•	This study aimed to evaluate the effects of heavy resisted sprint training in male professional soccer players. This investigation is of high interest because it is rare to have a training intervention with professional athletes as the subject population. Moreover, the improvements in sprint acceleration elicited by the training intervention(s) are notable, both with respect to the improvement in sprint performance with no detrimental effects on kinematics/technique, which has always been a key question regarding heavy resisted sprint training. The authors should be commended for conducting such a comprehensive investigation with intriguing and impactful findings for the fields of sports performance and applied biomechanics.

•	Some minor comments and suggested revisions are listed below with the aim of strengthening the manuscript.

Abstract
•	Line 33: after “spatial-temporal parameters” it would make sense to add the phrase “in male professional soccer players” to indicate the sex of the subject population, as I don’t think sex of the subjects is mentioned elsewhere in the Abstract.
•	Lines 38: the phrase “with no systematic non-specific acceleration training” does not read smoothly to me. Perhaps delete the term ‘non-specific’ (?).

Introduction
•	Line 87: minor typo, change “seem” to “seems” or “is”
•	Line 106: The second paragraph of the Introduction is somewhat lengthy, perhaps divide into two separate paragraphs to make it easier to follow. I would suggest starting a new paragraph at Line 106: “Interventions with heavy loads…”
•	Lines 106-116: perhaps include reference to recent publication by Cahill et al. on resisted sled-pull training in male HS athletes
o	Cahill, Micheál et al. (August 2020). Influence of Resisted Sled-Pull Training on the Sprint Force-Velocity Profile of Male High-School Athletes, JSCR. doi: 10.1519/JSC.0000000000003770

Methods
•	General comment: there are some inherent challenges with conducting a “perfect” experiment when completing a training study (scheduling & logistics, illness & injury, etc.), especially in a professional sports environment. However, it appears the investigators have handled the potential confounding variables in a rigorous manner, and the Methods are comprehensive and provide a good description of the data collection/analysis and intervention protocols.
•	Line 151: To summarize the experimental design at the beginning of this section, I would suggest starting this paragraph with a sentence similar to the following: “A pre-test, post-test experimental design utilizing three training groups was used to examine the effects of heavy resisted sprint training in professional male soccer players.”
•	Lines 278-309: If it is possible to provide a little more description on the training activities of the CON group, that may be useful.

Results
•	Lines 361-363: is there any explanation or reason suggested for the poor reliability found for the sprint F-V profile slope?
•	I would double-check the post-test maximum velocity contact time value for Subject HS60_6 (“Raw Data” excel file, “Sprint Kinematics” worksheet, cell A7.). If I’m interpreting the data correctly, it lists a post-test max velocity contact time of 0.163s for a subject running ~9 m/s, which seems unlikely.
•	In Table 3, I would suggest listing the contact time values to three decimal places  provide the reader with enhanced resolution/clarity.
•	Minor suggestion for Results and Discussion: I would suggest replacing the word “Immediate” with “Acute”

Discussion
•	Lines 473-476: This section “However, as Pmax was…” does not read clearly in its current form. Table 2 presents that neither HS60 nor HS50 had statistically significant increases in F0 values compared to CON, but it is also presented that combined HS had increases in F0 that were statistically significantly greater vs. CON (Table 2 and Figure 3A). Based on the latter statistic, it seems that on the whole with both resisted groups combined, HS led to increases in F0 when compared to CON, correct? From this reader’s perspective, the sentences in Lines 473-474 vs. 475-476 appear to contradict each other as presently written. Modified/additional text may help here.
•	As listed in Table 3, it’s fascinating that both HS60% and HS50% and significant and large ES improvements in F0 but that HS50 had a slightly better improvement in V0 (albeit not quite significant). In line with this, both HS groups significantly improved Pmax but HS50 to a larger magnitude (ES) than HS60 (although not significantly different HS50 vs. HS60).
o	To me, the fact that HS50% had equally large improvements in F0 while still increasing V0 to a larger degree than HS60% lends supporting data to the previous suggestion that HS50% is the optimal load for maximizing acute power but also, evidence that HS50% is the optimal load for longitudinally enhancing power (i.e., per prior work from Cross et al., reference 27). Although it is written  that anything in the range of 45-65% velocity decrease could be beneficial (Lines 490-492), the fact that HS50% may indeed be optimal is perhaps something that could be discussed more.


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

Input:
Title: CHANGES IN SPRINT PERFORMANCE AND SAGITTAL PLANE KINEMATICS AFTER HEAVY RESISTED SPRINT TRAINING IN PROFESSIONAL SOCCER PLAYERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: In relation to a comment and response for Reviewer 1: Reporting the F and df values for the post hoc tests would be a good addition.

Line 254: The brackets around m/body length should be removed.

The plural of subject is subjects. In multiple places subject’s is used incorrectly.

Line 342: The – can be removed between good and excellent as it already says ‘to’

Lines 378-392: Could these p values be reported to 3 decimal places? Especially those that are < 0.1
Experimental design: No further comments on experimental design.
Validity of the findings: Reading through the changes and responses to other reviewers, I believe that the statements (including within the conclusion) around adaptations potentially being maximised with a 50% velocity drop resistance should be downplayed. Your results support statements in some places that 50% velocity drop is more effective than 60% velocity drop but you cannot say that 50% is optimal or likely optimal. The results support that the ‘optimal’ is < 60% but we do not know any more than that from the current study design. The comparison to typical maximal power regions of the force-velocity curve support your claims but it should be made clear where these claims are speculative.

I would still like to see further clarity around the pre-post kinematic changes and the influence of sample size. The lack of kinematic changes is a major finding within the study and so we must ensure that the results support this conclusion. Reporting the actual p values here would be beneficial. Unless I have missed it somewhere (I apologise if this is the case!) it still says simply p > 0.05: ‘No main effects or interaction effects were found for pre and post sprint kinematic variables for both early acceleration and upright sprinting (p > 0.05)’. The reader is therefore unclear whether the p values are 1.00 or 0.051. If the results are not close to significance then this perhaps gives greater confidence that the inferences would not change with a greater sample size. This is important because the lack of significant main effects prevents post hoc tests from being performed. It appears from the confidence intervals (i.e. those that do not cross zero) that had post hoc tests been necessary, some parameters may be significant (or at least very close to significance after controlling for multiple comparison). For example, for 60%: ACC CM angle at touchdown; ACC contralateral hip angle at toe-off and touchdown; MAX CM distance to toe at toe-off and touchdown; MAX CM angle at toe-off and touchdown. I therefore just want to be clear that the results are being interpreted with the necessary caution. I believe greater reporting of exact p values / effect sizes will help with this, along with discussion of the level of (un)certainty around these findings. Whether there is or is not a change in kinematics, this should not affect the ability to publish. It is simply important to make sure the findings are clear. To be clear, I am not suggesting that you perform post-hoc tests if the main effects do not support these - I just wish to see a bit more information to help evaluate the likelihood of a type II error and would like to see this discussed when results are interpreted and conclusions formed. It is likely that the effect sizes in kinematics may differ to those in kinetics or split times. These likely effect sizes (from previous studies) could also support the sample size and / or the interpretation of non-significant results.

I still believe that the conclusion should reflect the 1-2 sessions per week performed in the study. I don’t believe you can say ‘preferably twice per week’ when your study does not show this to be preferable compared to once per week. 1-2 or ‘at least once’ reflects the study more accurately in my opinion. It is important that the conclusions and practical recommendations are supported by the study – a lot of the conclusion paragraph is detailing what was done in the study, rather than parameters that were systematically perturbed and investigated. You can therefore say that this type of intervention is successful in improving certain parameters. However, I think you need to be clear where your recommendations are based on a single effective method and we do not know if they are the most effective method (e.g. number of sessions per week, session duration, number of sprints, taper period, etc.).
Additional comments: Thank you for your detailed response and the many changes made to the manuscript. I believe the manuscript has improved and I hope that my few remaining comments can be relatively easily addressed.

Your statements / general principle of a more horizontal GRF being better is valid as long as a greater % horizontal is always beneficial within the range of values reported. From the values in the table this is likely the case. I suggest including a statement such as ‘A more horizontally oriented ground reaction force was considered beneficial within the range of values reported in this study’.