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: THREE-DIMENSIONAL FACIAL DEVELOPMENT OF CHILDREN WITH UNILATERAL CLEFT LIP AND PALATE DURING THE FIRST YEAR OF LIFE IN COMPARISON WITH NORMATIVE AVERAGE FACES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This was a prospective longitudinal 3D case-controlled morphometric study aimed at comparing 3D average faces of infants with unilateral cleft lip and palate with an age-matched control group at 3,6,9 and 12 months of age. The study is relevant and it enriches the body of published literature in this field. However, there are some areas in the manuscript that can be improved
Experimental design: Literature review
The present study was the first to compare average faces of infants with unilateral cleft lip and palate with age matched control group. However, there is another mixed longitudinal 3D case-controlled  study examining facial morphometric asymmetry of infants with UCLP and UCL with age matched controls using stereophotogrammetric camera and Procrustes analysis. Their 3D facial morphology of cleft infants was captured before and after surgical repair, and during the period of early growth. It would be appropriate to add it to the literature review and compare their findings with your results in the discussion, especially that there are differences in the timing of the applied surgical protocols and the technique used for soft and hard palate repair;
1-	Hood, C. A., Bock, M., Hosey, M. T., Bowman, A., & Ayoub, A. F. (2003). Facial asymmetry–3D assessment of infants with cleft lip & palate. International journal of paediatric dentistry, 13(6), 404-410.
2-	https://ethos.bl.uk/OrderDetails.do?did=1&uin=uk.bl.ethos.432864

Methodology
3D image acquisition: Elaborate on the image acquisition procedure; was the distance between the infants and the camera standardized? capturing the whole face from ear to ear to generate one point cloud. The number of included vertices in each image. How many megapixel were  in the texture map?
3D image processing
Procrustes transformation: elaborate on Procrustes transformation analysis
References
1-	Line 435: change the letter “o” in PLoS to a capital “O”
2-	In the reference  “Mancini, L., Gibson, T. L., Grayson, B. H., Flores, R. L., Staffenberg, D., & Shetye, P. R. (2019)  [line 436-439]; change the date of the publication from “Jan
1:1055665618771427” to “56(1), 31-38” to follow the used citation style
Validity of the findings: Construction of average faces provides an interesting perspective for diagnosis,
treatment planning, and comparison of facial shapes between controls and groups
with craniofacial deformities. This provides surgeons with valuable 3D templates in
planning the reconstruction of craniofacial anomalies. Orthodontists can use a soft
tissue reference for understanding facial soft tissue movement in relation to
dentoalveolar and skeletal changes occurring with treatment and growth. This might
develop to allow prediction of facial growth for sex and age group instead of relying
on 2D radiographic templates

Superimposing 3D faces provides a relevant tool in comparing and evaluating
surgical procedures outcome by superimposing pre and post-treatment facial images. Also, it can be employed in studying longitudinal soft tissue growth in cohort studies.
Additional comments: No additional comments


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

Input:
Title: THREE-DIMENSIONAL FACIAL DEVELOPMENT OF CHILDREN WITH UNILATERAL CLEFT LIP AND PALATE DURING THE FIRST YEAR OF LIFE IN COMPARISON WITH NORMATIVE AVERAGE FACES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors to be congratulated for this interesting piece of research which adds to our knowledge on the topic. The article is well written and the methodology is generally robust. The following are my concerns:
1. Methodology:
a. The lack of the errors of the method
the authors should provide an evidence of the reproducibility of the methodology, mainly the manual identification of the facial regions and the preauricular points used to identify the children's reference plane

b. It is not clear how the pose was standardised during capture?  was the labial seal readily achievable in all the cases and how open lips were accounted for in the 3D facial analysis

2.  Colour map

The authors used colour map to illustrate the morphological differences between UCLP cases and control, this lacks the directionality. The method dose not discriminate medial from lateral displacements not it separates upward from downward differences, these are essential to understated the pattern of dysmorphology between cleft and non-cleft cases.

3. Results:

a. I disagree that the asymmetry of the upper lip was completely restored, being 4 mm posterior to control.  According to Figure 3, residual asymmetry is clear and appeared on the right side, the non-cleft side according to the authors, and this should be more anterior than posterior according to the colour coding of the image.

b. The authors explained at 12 months there is a remnant asymmetry on both sides of the nasal ridge, Figure 3 doesn't not confirm this findings. It is not clear how the asymmetry was assessed in this study ? and if it affected both sides then I would expect, a display of  different colours, one blue and one red, of the nasal ridges which is not the case.

c. I am not sure of the authors  interpretation of the morphological differences that were found between the two groups from 3-6 months of age as an indication of significant greater growth of the full face, nose, cheeks of UCLP cases.  The nose is proven asymmetric according to figure 3, and cheeks are affected and displaced laterally on the cleft side, unfortunately  the used analysis doesn't disclose the directionality (medio-laterally) of the measured differences.  I also disagree with the statement of the significant more growth of the forehead in the UCLP cases. The axial rotation of the forehead along the axis of the facial reference used for detection of facial changes could lead to this findings.

d. Figure 3 doesn't support the claim that al all ages and mandible and chin were posterior to the control group. The captured images do not include the mandible! the ramus and body of the mandible were not included in the regions of interests! the blue colure at submental region is an area of prominence rather then deficiency according to the colour code.

4. Discussion
This section needs to be more focused and debate the two major findings of this study; the perceived greater facial growth at age 6 months of the UCLP case and the perceived mandibular deficiency at 12 months of the surgically manged cases. What is the biological bases of the overgrowth of the forehead in the UCLP cases?
Experimental design: see comments above
Validity of the findings: see comments above
Additional comments: see comment 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: THREE-DIMENSIONAL FACIAL DEVELOPMENT OF CHILDREN WITH UNILATERAL CLEFT LIP AND PALATE DURING THE FIRST YEAR OF LIFE IN COMPARISON WITH NORMATIVE AVERAGE FACES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors presented their research clearly, with a sufficient field background review and literature references. The tables, figures are fine and results were well presented.
Experimental design: 1. The authors aimed to investigate three-dimensional facial development of children with unilateral cleft lip and palate during the first year of life but the sample sizes were low as showed in the table 1 (13 in 3 months, 20 in 6 months, 16 in 9 months and 11 in 12 months) that the results could be biased due to the low sample sizes of the patient group. I suggest that the authors should increase the sample size of the patients.
2. The method of aligning 3D images based on Brons et al 2013 led to a biased data set towards the eye region, as indicated in line 214-215 "the least intersurface distance between UCLP and control facial morphology was noted in the region of the eyes".
3. Also because of the method of aligning 3D images, it was difficult to distinguish effects of facial growth and UCLP on the intersurface distance between UCLP and control facial morphology. I suggest the authors to clarify the ambiguity due to the aligning method applied.
Validity of the findings: The results of intersurface distance clearly showed the effects of surgical intervention of the cleft lip but the results failed to distinguish the effects of growth and UCLP. The differences between the UCLP and control could be originated from the intersurface difference in 3 months and the facial growth increased the difference up to 12 months; the same time the effects of UCLP on the difference were camouflage by the growth.
Additional comments: The major issue of the study is the low sample size of the patients. The other issue is the method of aligning 3D images based on Brons et al 2013 which led to a biased data set with least intersurface distance around the eye regions. Further the aligning method could not distinguish the effects on the facial growth and UCLP on the intersruface distance. The results showed clearly the effects of the surgical intervention on the upper lip but failed to provide effects of UCLP on the facial development. Minor issue is that the reference of Brons et al 2018 is not available yet.


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: THREE-DIMENSIONAL FACIAL DEVELOPMENT OF CHILDREN WITH UNILATERAL CLEFT LIP AND PALATE DURING THE FIRST YEAR OF LIFE IN COMPARISON WITH NORMATIVE AVERAGE FACES
Review round: 2
Reviewer: 1

Output:
Basic reporting: I would like to confirm that the authors had replied satisfactorily to the raised concerns. I would  like to congratulate the authors for their seminal work and recommend accepting the manuscript for publication
Experimental design: The added sentences had  met the reviewer expectations
Validity of the findings: The added sentences had  met the reviewer expectations
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: THREE-DIMENSIONAL FACIAL DEVELOPMENT OF CHILDREN WITH UNILATERAL CLEFT LIP AND PALATE DURING THE FIRST YEAR OF LIFE IN COMPARISON WITH NORMATIVE AVERAGE FACES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Satisfied
Experimental design: The authors agreed with my previous comments and I have no further comments.
Validity of the findings: The authors agreed with my previous comments and I have no further comments.
Additional comments: Satisfied with revised version.


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: INTEGRATING PHYLOGEOGRAPHIC AND ECOLOGICAL NICHE APPROACHES TO DELIMITATING CRYPTIC LINEAGES IN THE BLUE–GREEN DAMSELFISH (CHROMIS VIRIDIS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall the paper is well-written, the language and structure of the paper are easy to follow and the cited literature is sufficient and appropriate for the scope of this study.  I did find the figures a bit blurry and font sizes difficult to read; I encourage the authors to re-craft the figures with larger font and higher resolution lines, especially the map (Figure 1).
Experimental design: The examination of possible cryptic species in the marine environment using direct sequencing has become fairly common and the methods used are well-vetted. Again, the originality of this paper to past studies is the integration of ecological niche modeling to test phylogenetic patterns.  Nicely done.
Validity of the findings: The methods were provided with sufficient detail to replicate.  Greater sample size would have been ideal for several of the locations (Siapan only had 2 fish), but I can appreciate the difficulty of obtaining samples from such a broad geographic range.  The study’s overall sample size of several hundred provides enough power to give confidence to the drawn conclusions.
Additional comments: Thank you for this opportunity to review for PeerJ. I have read the manuscript ‘Integrating phylogeographic and ecological niche approaches to delimitating cryptic lineages in the blue-green damselfish (Chromis viridis)’.  This paper takes a complementary approach to examining the population-level/cryptic species-level relationship of a widely distributed coral-reef associated marine fish using a clever combination of molecular genetic data and ecological niche data.  I commend the authors for attempting this analysis. Although the ecological niche data did little to explain the observed genetic divergence among the C. viridis clades in this study, I believe it provides a replicable road map for others to integrate environmental and genetic data to explain marine biodiversity.  The paper already inspires re-thinking of how my lab will look at including ecological niche data in our pop gen work.

Overall the paper is well-written, the language and structure of the paper are easy to follow and the cited literature is sufficient and appropriate for the scope of this study.  I did find the figures a bit blurry and font sizes difficult to read; I encourage the authors to re-craft the figures with larger font and higher resolution lines, especially the map (Figure 1).

The examination of possible cryptic species in the marine environment using direct sequencing has become fairly common and the methods used are well-vetted. Again, the originality of this paper to past studies is the integration of ecological niche modeling to test phylogenetic patterns.  Nicely done. The methods were provided with sufficient detail to replicate.  Greater sample size would have been ideal for several of the locations (Siapan only had 2 fish), but I can appreciate the difficulty of obtaining samples from such a broad geographic range.  The study’s overall sample size of several hundred provides enough power to give confidence to the drawn conclusions.

I have several minor comments for the paper below.  I believe the paper should be approved for publication after considering the below comments, and those of the other reviewers.

Comments:
Line 73-76 – The authors cite possible cryptic lineages of C. viridis based on mtDNA data, and earlier mentioned how cryptic species are sometimes distinguished using morphology (Line 54). Are there examples where morphological data (or possibly other types of data) gives an indication of cryptic lineages in C. viridis?  That is data other then the mtDNA already mentioned?

Line 99 and Line 108: The authors amplify Cytb and Rag2 primers, yet referenced papers of Froukh & Kochzius used CR-A/XAN-DL-R primers and Messmer et al 2012 used CR-A/CR-E primers.  The Control Region (CR) arguably evolves faster than other parts of the mtDNA like Cytb (Avise et al. 1987). What is the authors justification for using Cytb and even Rag2 over CR which past authors have used and may provide a comparison with these other data sets?  Furthermore, the authors themselves highlight differences in substitution rates of the genetic markers used (Line 318-325). Again, why not include CR?

Line 242-244 – The authors bring up the role of water depth in helping to explain total variance in the PCA, however, I did not see that depth where fish were captured was included in the method.  Do I read this correctly that all C. viridis occur at 50m depth or less, and that all samples were obtained at these depths (seems likely given stated sampling method of SCUBA/snorkeling), and thus no further exploration was done of the data to tease apart role of depth in differentiating the clades?   The discussion on host switching leading to speciation (Line 294), although not testable due to the sampling scheme, further makes me wonder if environmental factors such as depth may lend themselves to explaining the divergence between the clades.

Figure 1 – Map is somewhat of low quality. Can this be improved upon? Further, I would suggest zooming in closer (cutting out the far north and south latitudes) to allow easier distinction of sites in Indonesia.


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: INTEGRATING PHYLOGEOGRAPHIC AND ECOLOGICAL NICHE APPROACHES TO DELIMITATING CRYPTIC LINEAGES IN THE BLUE–GREEN DAMSELFISH (CHROMIS VIRIDIS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is very well written and the integration of phylogeography and ecology would make it of interest to a broad audience. The literature references are comprehensive and relevant. The quality, format and arrangement of figures are all more than adequate to summarize their findings and support their conclusions.

I added some minor suggestions for improved clarity.
Experimental design: The experimental design is sound. Overall, this is a good study that address the role of niche divergence in the context of species divergence. Niche divergence is often stated as a mode for speciation in phylogeographic studies, but rarely ever tested in parallel.  I suggested as a slight addition to the BEAST results to explain the patterns seen with the Red Sea
Validity of the findings: The conclusions are well stated and the findings are valid and well supported. They linked very well to the
original research objective and placed in the context of previous research.
Additional comments: Lines: 128-129: How was this model determined? Please describe and add to the methods.

Line 227: Please list the 95% HPD ranges

Lines 312-313: What is the estimated age of the Red Sea population based on the BEAST analysis? It appears to be around 20,000 year which aligns well with the most recent closure of the Red Sea (Siddall et al., 2003)

Line 325: The 1.2 my age applies to Clade A not just to the Red Sea. The Red Sea appears to be much younger

Figure 2: change 0.9443 to 0.94 to keep consistent significant values

Figure 2, Haplotype networks: The network for cyt b is difficult to distinguish between the mutation tick marks and the circles that identify Red Sea haplotypes. There is room to blow up the size of the Rag2 network to make it easier to read

Figure S1: there is no caption. I am not sure what this figure represents

Suggested reference:
Siddall, M., Rohling, E.J., Almogi-Labin, A., Hemleben, C., Meischner, D., Schmelzer, I., Smeed, D.A., 2003. Sea-level fluctuations during the last glacial cycle. Nature 423, 853.


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: IDENTIFICATION OF SIGNIFICANT GENE AND PATHWAYS INVOLVED IN HBV-RELATED HEPATOCELLULAR CARCINOMA BY BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Xie et al. used gene expression data from diseased and healthy tissues to identify differentially expressed genes, aggregated using a rank-based method. They went on to perform enrichment analyses in terms of GO processes and KEGG pathways. They then created a PPI network from the DEGs and coexpressed genes and used a Cytoscape plugin to identify important genetic drivers (FOXM1, NEK2, RAD51AP1, ANLN and KIF18A) for the prognosis of HBV-related hepatocellular carcinoma, with survival analysis serving as validation for their findings. The findings are interesting and useful for further research into the diagnosis and treatment of the disease. The work sound but it has multiple problems which need to be addressed before publication.

The paper has poor English which make it hard to understand at times. For an example of spelling, on line 72 “tumor simples” should be “tumor samples”. In terms of grammar, lines 75 and 76 are not correct “difficult to obtain […] because of too many genes were identified”. There are cases such as line 323 where an abbreviation is used without prior definition, “CNV-driven”. I recommend that a native English speaker should read the manuscript and correct the mistakes to make it easier to understand. In the results section the cellular compartment “spindle” is quite meaningless if not mentioned the term fully (mitotic spindle astral microtubule end) or at least mitotic spindle.

The authors must take care to reference thoroughly, including every tool or software they use, and citing the paper and not just the web-link. For example, line 135 mentions STRING without citing the paper, as well as 124 with DAVID. In line 116 the authors mention using the limma R package and do not make any citations. Same with the used annotation databases (KEGG, GO). Also please cite R itself as well.

Figures and tables are generally good, however, I suggest to move some figures to supplementary and delete some altogether.

Move Figure 1 to supplementary.

Figure 2 is a useful figure, a minor point would be that volcano plots are conventionally plotted with logFC on the x-axis and -log10(adj.P.val) on the y-axis.

In Figure 3 the colour key must be corrected to show that 0 corresponds to white.

Figure 5A is not clear, I recommend that it be removed as it does not contribute to an increased understanding. Figure 5C has a mistake in the header “Sinple type”.
Experimental design: During the analysis, the authors made a mistake using gene names to map the probe sets. It is a dangerous practice because gene names are not unique. I strongly suggest redoing the analysis with mapping to any IDs which is in all the chips annotation files.

The methods lack the mention of how the authors handled when multiple probe sets are used for one gene. Please state that in the manuscript.

In line 130 they mention using P < 0.05 as a significance cut-off but do not specify whether this p-value is adjusted for multiple testing. Unclear whether this is a methodology issue or a typo.

The authors should describe why do they have chosen certain methods and cut-offs. For example (line 77) they use a rank aggregation algorithm but do not mention why they used this method and not others. A basic summary of other related methods and why the RRA method is the best choice, in this case, would greatly benefit the paper. This also applies to the choice of pathway database (KEGG and GO), why these and not e.g. Reactome or WikiPathway?

The authors mention various cut-offs, such as in line 138 when selecting significant modules, but do not justify why they chose these cut-offs. Were other parameters tried, with the final selection being the optimal choice? It is not clear in this case.

The authors used STRING database for PPIs but they not mentioned whether they used any filters regarding STRING. STRING collects the data by text-mining which made it inherent for false positive predictions. Have the authors considered this?
Validity of the findings: The findings, especially with the validation from an outside database, are extremely good and are an important addition to determining HCC outcome. However, these findings are depended on the used cut-offs, methods and gene mapping. The authors should redo the analysis or justify their cut-offs.
Additional comments: It is valuable work but needs some adjustments.

The main concerns raised are:
1.	Incorrect mapping from probesets to genes - it can make the findings different
2.	Methods not being described or justified sufficiently
3.	Poor use of English language and grammar - makes the paper difficult to read
4.	Insufficient or incorrect referencing
5.	Some figures being unclear or not needed

Please address these before 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: IDENTIFICATION OF SIGNIFICANT GENE AND PATHWAYS INVOLVED IN HBV-RELATED HEPATOCELLULAR CARCINOMA BY BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Article figures (especially Figure 5 and 6) are low resolution (at least the versions I had access to). Large part of the network in Supplementary Figure is missing.
Experimental design: The authors perform meta-analysis of the 4 different gene expression studies using Robust RankAggreg (RRA) method. I found several problems with data preprocessing and also some missing details of methods.

The used gene expression studies are referred as microarray experiments, however one of them (GSE94660) is RNAseq (which has no mayor consequence in the meta-analysis, but should be corrected). Also the authors write (line 113) that "gene expression were subjected to log2 transformation". Based on Fig. 1 it looks like that same used data (Fig.1 C,E and G?) were already log transformed, so this should be checked (as a second log transformation can influence the filtering criteria for differentially expressed genes (DEG)).

Identification of DEGs can be influenced by different non-biological effects (like batch effects), so some control for this in the individual datasets would be important to see (like a PCA plot of different samples coloured by tumor / control etc.).

In Figure 8 the authors performed survival analysis on cBioPortal. However the information about the used cancer dataset (TCGA?) and genomic profile (mutation, CNV alteration of gene expression alteration) is missing from the manuscript. If they used gene expression alteration, it would be important to see whether the decreased survival is associated with over or under-expression of the selected genes.
Validity of the findings: In Figure 9 the authors show association of ANLN and KIF18A with different tumor phenotypes (as Grade and Vascular Invasion). However I was not able to find the significance of this difference in the text or in the Figure. It would be also interesting to see this analysis with the other selected genes.

As the authors mention, there are other studies investigating HBV-related HCC gene expression (like reference Zhou et al. 2017, or Zhou et al. 2014 Int J Clin Exp Med ) Some more detailed comparison with these other studies (overlap of identified DEGs of functions) would be important information.

The authors performed KEGG pathway analysis with the DEGs. Comparing the enrichment of KEGG pathways with over and under expressed genes (like for GO terms) would be also an important information.
Additional comments: The manuscripts describes a meta-analysis of 4 different HBV-related hepatocellular carcinoma (HCC) gene expression studies and performs functional analysis of the identified differentially expressed genes (DSGs). However as they compare only HBV-related HCC samples against control (also from HBV infected patients) they analysis does not contain specific information about the HBV-related HCC (compared to other HCCs like HCV related or toxic) but HCC generally. This should be more emphasised 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: IDENTIFICATION OF SIGNIFICANT GENE AND PATHWAYS INVOLVED IN HBV-RELATED HEPATOCELLULAR CARCINOMA BY BIOINFORMATICS ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: my concerns have been answered
Experimental design: 1) Original question: In Figure 8 the authors performed survival analysis on cBioPortal. However the information about the used cancer dataset (TCGA?) and genomic profile (mutation, CNV alteration of gene expression alteration) is missing from the manuscript. If they used gene expression alteration, it would be important to see whether the decreased survival is associated with over or under-expression of the selected genes.

Response:Our research is to identify differentially expressed genes. The oncomine database was used to performed survival analysis of these genes. If we want to study the mutation and CNV alteration of gene expression alteration of gene, more work needs to be done.

Question: The authors say in their answer that they used “The oncomine database” for survival analysis. However based on Methods, they refer cBioPortal for survival analysis (line 146), and Oncomine for “interactional correlation of the expression level of hub genes and tumor grades, satellites, and 148  vascular invasion” (line 148). Which one is correct? If they used cBioProtal, I still did not get answer which dataset they used it. In cBioProtal there are 7 different HCC datasets (from MSK, INSERM, MSK, AMC, RIKEN, and 2 from TCGA), so it is an important information for the reader (and for the reproducibility of the analysis) which dataset they used. Also they say in results that “HBV-related liver cancer with ANLN and KIF18A alteration showed worse disease-free survival“. As far as I know non of these dataset is specific to HBV related cancer (but generally HCC), so the term HBV-related liver cancer should be used with caution here.
Validity of the findings: 2) Original question: In Figure 9 the authors show association of ANLN and KIF18A with different tumor phenotypes (as Grade and Vascular Invasion). However I was not able to find the significance of this difference in the text or in the Figure. It would be also interesting to see this analysis with the other selected genes.

Response: Figure 9 (now is Figure 8) is from Oncomine. Although there are no results of statistical analysis, we can still find that the expression of these two genes is correlated with the phenotype of tumor. In survival analysis, only these two genes (ANLN and KIF18A) alteration showed worse disease-free survival and worse overall survival. Secondly, these two genes are the focus of this analysis. So, we want to study the relationship between their expression and phenotype of tumor. When we searched for the correlation between the expression of other genes and the phenotype of tumors in the same dataset (Wurmbach Liver dataset), some of them lacked statistical data, while others did not show a significant correlation with the phenotype of tumors (the results were not shown).

Question: I still think that if the authors write “In the Wurmbach Liver dataset, higher expression of ANLN and KIF18A had significantly correlated with tumor grade, satellites, and vascular invasion”  (line 220), than they should use some statistical analysis to support their conclusions.

3) Original question: The authors performed KEGG pathway analysis with the DEGs. Comparing the enrichment of KEGG pathways with over and under expressed genes (like for GO terms) would be also an important information.

Response: Previous studies have confirmed that comparing the enrichment of KEGG pathways with over and under expressed genes (like for GO terms) will lead to a reduction in the number of meaningful signaling pathways.

Question: Could the authors cite some relevant literature for this claim?
Additional comments: The authors have answered some of my questions, but at other points the clear answers are still missing.


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: IDENTIFICATION OF SIGNIFICANT GENE AND PATHWAYS INVOLVED IN HBV-RELATED HEPATOCELLULAR CARCINOMA BY BIOINFORMATICS ANALYSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have answered my questions.
Experimental design: The authors have answered my questions.
Validity of the findings: The authors have answered my questions.
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: ALTITUDINAL GRADIENTS IN MAGELLANIC SUB-ANTARCTIC LAGOONS: THE EFFECT OF ELEVATION ON FRESHWATER MACROINVERTEBRATE DIVERSITY AND DISTRIBUTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article is written in a easy and clear form, and have the structure of a cientific investigation. It uses the appropiate literatura, and demostrate that the cientific group dominates the topic. The figures that they used are very good.
Please write always et al in italics.
The figure 2 indicates temperatura between february 2015 to march 2016, it must be congruent with the methogology described (there, the autor indicates from March 2015-abril 2016).
All the proposed target are well met.
Experimental design: The experimental design is clear and concise, and it leads to obtaining and answering the questions listed
In future, it would be appreciate if the studies include also other chemical parameters, such as MODC (material organic disolved colored), ortophosphate, phosphorous total, Nitrogen total, NH4, NO3, NO2. We know that with all antropic activities, the values ot this parameters will increase in time, so it is necessary to have a baseline.
Validity of the findings: The statistics test are well used and are appropiate to answer the questions.
Additional comments: I congratulate the authors because of the effort shown in this study, and they have shown that good results can be achieved with basic tools.


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: ALTITUDINAL GRADIENTS IN MAGELLANIC SUB-ANTARCTIC LAGOONS: THE EFFECT OF ELEVATION ON FRESHWATER MACROINVERTEBRATE DIVERSITY AND DISTRIBUTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting and very nice study in which authors assessed freshwater macroinvertebrates communities in lentic environments along an altitudinal gradient in southern South America. The manuscript is well written, clear and concise, figures are high quality and methods are technically sound. I strongly recommend it for publication.
Experimental design: It’s a descriptive study. Methods are adequate.
Validity of the findings: Authors provide a description of richness and composition of macroinvertebrates communities along the altitudinal gradient examined. They found that elevation has substantial effects on community structure and function. This information is very welcome for the study area.
Additional comments: Some minor comments:

Figure 1: Although not incorrect, I suggest converting geographic units from UTM to degrees (Decimal Degrees or Degrees, Minutes, Seconds) for easy referencing, since the authors express the location of the Magellanic Sub-Antartic region using degrees (introduction: lines 69 to 72). This would apply also to coordinates provided in Table 1.

Table 2: Please check the superscript in cm2.

Tables 4 & 5: I suggest including the meaning of column headings within the abbreviations in the legends.


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: ALTITUDINAL GRADIENTS IN MAGELLANIC SUB-ANTARCTIC LAGOONS: THE EFFECT OF ELEVATION ON FRESHWATER MACROINVERTEBRATE DIVERSITY AND DISTRIBUTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The text is unambiguously written in technically correct English. Background, relevant literature and article structure is appropriate. Figures and tables conform to an acceptable format.

The results are only partly relevant to fulfill the aims because of reduced sampling coverage (please see experimental design).
Experimental design: The research is original and falls within the aims and scope of the journal. Its goal is to describe and compare aquatic macroinvertebrate communities along altitudinal gradients on Magellanic sub-Antarctic lagoons. Four lakes/ponds along an altitudinal gradient of 680 m (lower site 20m, highest 700m) were sampled in one occasion. The relevance of the goal relates to the harsh and poorly known environment sampled, and constitutes a great effort to fill the gap in basic knowledge of the area. Nevertheless the number of gradients (transects) and sites (and probably sampling dates too, depending on hydrologic regime) should be increased to attain the objectives. Random noise caused by absence of replication and reduced sample size (number of sites, dates and gradients) could cause spurious or inconsistent results that do not conform to the regional diversity pattern.
Validity of the findings: Methods are described with sufficient detail except the following points:
1) Biological sampling consisted of a "standard sampling" (1 min of net sweeping in four predefined habitats) and a "complementary collection" to "get a comparable and representative sample of each habitat". All samples were analyzed together. The standard sampling is comparable between sites, since they were standardized by time (minutes of net sweeping) but are not readily comparable with other studies that mainly use densities (standardized by area). The complementary collection may vary between sites so even inside the reduced sampling universe of this study, they are not comparable. The authors should compare sites only if similar sampling effort were carried out.
2) The method followed to assign each taxon to its corresponding functional feeding group (FFG) is not mentioned. For example, in the results, figure and table it can be read that the mayfly found in this study (Meridialaris chiloeensis) was assigned to the shredder category, but published works (e.g., Diaz Villanueva & Modenutti 2004, DOI: 10.1002/iroh.200310694) have determined Meridialaris chiloeensis to be a grazer. The assignation of remaining taxa should be also checked. Two ways of facing the assignation of taxa are commonly used: 1) assignation by bibliography (e.g, Rodriguez et al 2011 Rev. Biol. Trop 59: 1537-1552 ), and 2) gut content analyses (e.g., Tomanova et al. 2006, Hydrobiologia, 556, 251-264). If the first is the case it is recommendable to use regional literature since geographic variations in diets are commonly reported. The second approximation is undoubtedly more accurate, since any particularity of the regional fauna or differences between instars of a given taxon can be readily acknowledged. The manuscript should be completed with this information. An introductory  paragraph about the relevance of FFG to attain the proposed objectives would be welcomed as additional background.
Conclusions would gain strength and wider validity if more gradients (and sites) are added to the study.
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: NEUTROPHIL–LYMPHOCYTE RATIO IS PROGNOSTIC IN EARLY STAGE RESECTED SMALL-CELL LUNG CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This manuscript needs substantial revision.
Figure shown in manuscript is contradictory to the interpretation of the result.
This manuscript is very badly written which makes it very difficult to understand.
Figure used in this manuscript lacks figure legend.
Data doesn't support the finding of this manuscript.


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

Input:
Title: NEUTROPHIL–LYMPHOCYTE RATIO IS PROGNOSTIC IN EARLY STAGE RESECTED SMALL-CELL LUNG CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Neutrophil-Lymphocyte ratio is prognostic in early-stage resected small-cell lung cancer” is a poorly-written manuscript. Overuse of abbreviations made this article difficult for reading. I strongly recommend authors to take help of professional English Language editors. This manuscript is a retrospective study aimed to investigate the prognostic value of neutrophil to lymphocytes ratio (NLR) and platelet to lymphocyte ratio (PLR) in Caucasian patients with resected small cell lung cancer (SCLC). Several studies already reported the prognostic value of NLR & PLR in SCLC. This study has a very narrow scope and may be interesting for oncologist who are mainly interested in SCLC.
Experimental design: No comment
Validity of the findings: Need further supportive study to confirm these results. But this study is well designed and statistically validated.
Additional comments: Major
1)	Inclusion of the flow diagram of the study will be useful.
2)	Research question was not clearly established. Background also need to be better written
3)	Some part of the introduction is irrelevant especially Lines 63-67.
4)	Poorly-written manuscript. Overuse of abbreviations made this article difficult for reading. I strongly recommend authors to take help of professional English Language editors.
5)	Introduction and discussion look similar with same references which looks unnecessary.


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: NEUTROPHIL–LYMPHOCYTE RATIO IS PROGNOSTIC IN EARLY STAGE RESECTED SMALL-CELL LUNG CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: English grammar /synatx used across the manuscript is acceptable. Minor grammatical errors have to be fixed in introduction section.

A lot of abbreviations do not have full forms in the text : for example
-Mention LNLR full form in abstract/background :
-What is ES and LS ?? early and late stage?
-CHT RT full forms should be stated in text

Authors have provided sufficient background in the introduction. However the second half of the introduction requires major revisions in terms of flow. The literature about the discrpencies in the levels of NLR and PLR and patient prognosis has to be explained better. Right now it is messy and reader can get lost! Authors should also clearly discuss the possible reasons that could result in these opposite conclusions for all the studies. For example different race or study design or treatment regimen etc..

Authors have to clearly state how similar or different their analysis is from existing data. Authors do specify that this cohort is caucasian compared to Asian cohorts used in other studies cited. Why do they think that this analysis will be useful/conclusive?

Ethics statement is provided and raw data is shared.

Figures require better annotation. X axis unit is not mentioned. P value should be stated on charts.

In Table 1
-Only gender has total but no other category?
-What is pT, pN in table1?
-“Column N%” should be relabeled for more clarity!

Other minor grammatical errors :
"Consecutive patients" with histologically confirmed and surgically resected SCLC evaluated between 2000 and 2013 at three centers were retrospectively analyzed. NLR and PLR at diagnosis was used to categorize patients into "high" (H) and "low" (L) groups based on ROC analysis
In a large cohort of 2,476 "SCLC patients underwent" surgical resection and chemotherapy had improved five-year overall survival (OS) compared to those who received nonsurgical therapy alone
Experimental design: Research question is defined but how this research will fill a knowledge gap is not clearly stated. This can be elaborated further.

The individual cohort sizes were not very big in the study so the authors combined three cohorts to get about 155 patients for subsequent analysis. While this number is not very big it is still a reasonable size for this kind of study.

Authors should comment on any issues faced when using data from multiple institutes. For example data from all three cohorts were measured/graded the same or comparable way across different centers (For example NLR and PLR calculations, histological grade etc) ?

Statistical analysis described in Methods section seem appropriate for the study. Why was p value less than 0.008333 used for significance?Authors should state what was the p value used for Spearman correlation analysis.

Authors have to provide more details for each statical analysis in the results section. Details are very messy and not clearly explained.

Authors should clearly state how NLR and PLR were calculated? Were they log transformed for symmetric distribution?

Authors should clearly state in results section (before Table 1!) how NLR and PLR were classified as Low or High. Authors mention in Methods section that they use the ROC curve (instead of using a cutoff). This analysis has to be further explained clearly in the results with the appropriate figures. this section is fundamental for the entire paper and is not present in the Results section.

Where is the data/figure for lines 187-190 of results?

In describing table 1 there is no mention of “vascular involvement, tumor necrosis, peritumoral inflammation, or tumor grade” in the results text..All results stated in figures/tables/ should be discussed and vice versa.

In this paper all chemotherapy treated patients across different institutes were grouped together for all analysis. If the data is available, drugs administered to patients should be specified. If patients were grouped by specific drugs administered would there be differences between groups?

The results section under Prognostic factors for OS and DFS is extremely unclear. All the data mentioned in the text is not supported by the figure. For example authors state the following :
 "Univariate survival analysis identified significantly longer OS in patients with lobectomy and segmentectomy (vs. pneumonectomy, median OS, 56.2 vs 31.7 months, p=0.0015), pN 0-1 (vs 195 pN 2, median OS, 64.9 vs 30.3 months, p=0.001), and LNLR (vs HLNR, median OS, 74.8 vs 196 44.5 months, p=0.033, Fig 1A). " However Fig 1A only has survival curve for LNLR vs HLNR survival.

Authors should clearly state the individual variables used for the multivariate analysis.

Authors state that "Thus higher NLR indicates a higher grade in tumor stage. Also, higher PLR correlates with a higher NLR value" While the p values are significant the r values are seem very low to make these statements? (r=0.23 or r=0.035?)

In Table 3 was analysis done for 189 patients (as stated in table title)? Authors state NLR/PLR data only available for 155 patients in methods section?
Validity of the findings: Please see section above for details to be verified to ensure the data and analysis is robust.

Discussion and Conclusion is reasonable but requires better flow and organization (in discussion section)
Additional comments: This study has the potential of being useful to the scientific community because of the valuable patient information for caucasians with SCLC. However in its current state the manuscript is very difficult to read and comprehend. While the aim of the study is clear and the introduction is reasonably explained, the results sections require major revision! The results have to be stated more clearly and in an organized manner. Appropriate results have to be clearly tabulated, figures have to be shown for all data reported in the text, all data in the tables have to be mentioned in the results. While the statistical approaches  described in the Method section seem appropriate for the study, the details are not clearly mentioned in the Results section for each analysis.


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

Input:
Title: NEUTROPHIL–LYMPHOCYTE RATIO IS PROGNOSTIC IN EARLY STAGE RESECTED SMALL-CELL LUNG CANCER
Review round: 1
Reviewer: 4

Output:
Basic reporting: This study attempts to demonstrate prognostic significance of preoperative NLR and PLR in patients with resected SCLC. The finding of this study is novel, but there are several issues in this study as follows. In summary, they need to revise this manuscript correctly.
Experimental design: 1)	In the Introduction section, rationale of the study is unclear, particularly regarding prognostic significance of preoperative NLR in patients with resected NSCLC. Preoperative high NLR was reported to be significantly associated with poorer survival specifically in NSCLC (Please refer to “Takahashi Y et al. Ann Surg Oncol 2015;22 Suppl 3:S1324-31.” and “Takahashi Y et al. World J Surg 2016: ;40(2):365-72.”). Please do not ignore important literature for this topic.
2)	The authors should describe the inclusion and exclusion criteria for patient selection.
3)	The study cohort should be homogeneous especially in terms of treatment protocol. However, the study cohort seems to be very heterogeneous in terms of adjuvant chemotherapy and radiotherapy. At least we would know whether NLR is a prognostic factor in each patient group (adjuvant therapy + or -).
4)	Also, patients with stage III SCLC should be excluded in the survival analysis because they have a very poor prognosis. Or, they may show survival curves of each stage.
5)	Are there any differences in NLR and PLR between the institutions. The analytical method can be different in each institution.
6)	In Figure 1, what does the X-axis represent? They should put unit in the X-axis.
Validity of the findings: 7)	In the Discussion, biological background of correlation between NLR and inflammation as well as immuno-microenvironment seems to be insufficient. The careful discussion should improve quality of their novel finding.
8)	The Cut-off value may be arbitrary unless it is referred from previous study. Thus, they may add this description as one of the limitation of their study.
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: NEUTROPHIL–LYMPHOCYTE RATIO IS PROGNOSTIC IN EARLY STAGE RESECTED SMALL-CELL LUNG CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This manuscript provides an interesting data to consider neutrophil to lymphocyte ration as a prognostic marker for multimodal treatment for SCLC.
The authors have  clarified the issues raised in previous review. However, discussion part is very lenghty which needs to be  polished and crisped.
Line 246-255 is not required for the context of current study.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND CLASSIFICATION OF THE HSF AND SHSP GENE FAMILIES IN PRUNUS MUME, AND TRANSCRIPTIONAL ANALYSIS UNDER HEAT STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript requires some further editing to correct grammar throughout.
Experimental design: The introduction provides a general review of plant Hsfs and Hsps, but it is not clear how this material sets up the rationale for this study, or the gaps in knowledge that this study fills.  The data provided in this study are clearly unique, but I think the authors could do a better job of explaining how their work connects to and expands on what is known about plant Hsfs and Hsps.  The authors could also be clearer on why this study focuses on the small Hsps specifically.  How is it beneficial to add information on these genes for P. mume?

Phylogenetic analysis: I suggest describing the settings used in Mega to produce the phylogenies.  If the default settings were used that should be stated.

qRT-PCR design: There are a number of details that I think are important to include in the methods section.  It would be helpful if the authors stated whether primers were designed to produce products spanning introns to reduce the chance that genomic DNA was amplified (for genes that contain introns).  Were products from each primer pair sequenced to determine if the correct gene was amplified?  Was melt curve analysis used to ensure a single product was generated?  Were negative RT and water negative controls used?  Has EF1a been validated as a good reference gene for P. mume or at least other plant qRT-PCR experiments?  If not, why was this gene chosen?  What range in Cq value was accepted within a single technical triplicate (0.5 or other value)?  The use of two reference genes would improve the strength of these data, and could be considered for any future experiments.

I would suggest some other revisions to the description of the qRT-PCR design as recommended by the MIQE guidelines.  These suggest referring to control genes as “reference genes” and the use of Cq instead of Ct. Were efficiencies calculated for each primer pair?
Validity of the findings: The data in the paper are well presented and described.  Figures are effective.  Conclusions resulting from the figures are valid.  The descriptions of upregulation and downregulation in the qRT-PCR data seem valid as the changes in expression are so large for many of the genes.  It is a bit confusing that each y-axis differs depending on the amount of gene expression change, which visually makes it difficult to compare the extent of expression change from gene to gene.  Perhaps the authors can clearly note that y-axis scales differ to allow presentation of all data in one figure.  It might also be helpful to clearly state the fold difference that was considered significant.  Is it appropriate to use an ANOVA to show statistical significance for these changes?

Most of the first paragraph of the discussion reiterates information from the introduction.  I would recommend using this first paragraph to stress the contributions of this current study.  Line 251 beginning paragraph 2 could more clearly state that it is similarities between P. mume and Arabidopsis that was conserved.

Paragraph 3 starting on line 258 seems to restate some of the findings, but could better explain the importance of these findings.

Paragraph 4 restates a result.  Is this loss of the beta 6 sheet found in any other plants or eukaryotes?  Or is this a unique finding in P. mume?

Paragraph 5 starting on line 270 restates information from the introduction on cellular localization.  Since no cellular localization data are provided for expression of the P. mume genes I am not sure if this information is relevant in the discussion.

The discussion on timing of gene expression during heat stress also restates the results.  How do these results compare to what is known about timing of heat shock response in other eukaryotes? What do these data add to our understanding of heat shock response in plants?  Why might these different temporal patterns exist?
Additional comments: The authors present a novel data set that will be of interest to researchers working on plant heat stress response.  The data appear valid and well presented.  Most of my comments, detailed in the other sections of this review, relate to the written presentation of the paper and the design and analysis of the qRT-PCR experiment.

Here are a few other minor comments:

Abstract – “Heat” in first sentence should probably be lower case.  Authors could clarify what they mean by “outstanding features” in the second sentence.  While heat shock factors (HSPs) is defined in the abstract, sHSP is not.  I would define that “s” stands for “small”.  When authors state that certain Hsfs became “prominent”, under what conditions were they prominent?  I assume this means after heat shock, but that could be clarified.  “PmHsp” should not be italicized.

Line 33: Change to “relieved”

Line 128: Change to RNase and DNase

Line 214: PmHsf and PmsHsp does not refer to specific genes in this sentence, so I do not think they should be italicized.


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: GENOME-WIDE IDENTIFICATION AND CLASSIFICATION OF THE HSF AND SHSP GENE FAMILIES IN PRUNUS MUME, AND TRANSCRIPTIONAL ANALYSIS UNDER HEAT STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language should be improved to ensure that an international audience can clearly understand your text. Some examples where the language could be improved include lines line 37, 148-155, 156, 159-162, 163-165, 259-260 and 282-284. – the current phrasing makes comprehension difficult. I would suggest writing short sentences and asking a native English-speaking reader to read the manuscript before submission.

Line 176 – 177 - The length of the introns varies among the Hsf genes – for example, PmHsfX and PmHsf4 have smallest (?bp) and longest (3196bp) intron respectively.

Line 282: exist should be exhibit

Specifying the acronym small HSP (sHSP) in the abstract (instead of line 51) to help the readers to understand it early.

Fig 1 A: Because the chromosome start and end is clear from the scale, it is not required to mention with each chromosome. For each chromosome please mention the position of the centromere.
Experimental design: The Prunus mume genome database (http://prunusmumegenome.bjfu.edu.cn/) is not accessible. That creates difficulty to run some analysis by myself.

Line 166 – It is not clear why there is a difference between phylogenetic analysis and annotation? What are the (de)merits of this information?

The figure legend of fig 8 and 9 is telling there are 3 biological replicates, however, line 138 (method section) suggesting there are three technical replicates. Moreover, line 124 is suggesting there are three biological replicates. Authors need to clarify at one place how many biological replicates and technical replicates are used in the qPCR experiment.
Validity of the findings: The discussion section is not written in a very thoughtfully way. There are lots of redundancy between the discussion and the introduction section. It is more focused on other plant’s Hsf and sHsp (i.e. what we already know) rather than what new things we are learning for P. mume. This section should include, what is the significance of the current study to improve/ help in conservation of P. mume.
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: GENOME-WIDE IDENTIFICATION AND CLASSIFICATION OF THE HSF AND SHSP GENE FAMILIES IN PRUNUS MUME, AND TRANSCRIPTIONAL ANALYSIS UNDER HEAT STRESS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript “Genome-wide identification and classification of Hsf and sHsp gene families in Prunus mume, and their transcriptional analysis under heat stress” attempts to provide structural as well as functional characterization of Hsfs and sHsps in P. mume. Overall, the manuscript conforms to the structure recommended by the journal viz. Introduction, Materials and Methods, Results, Discussion, Conclusions. I found the manuscript very well written. The overall English comprehension and grammar of the article are good with very few typos and ambiguous sentences. Figures are of high quality. The methods and results section is explained in satisfactory detail. However, there are few suggestions that would be useful for the readers and are important to address to make the manuscript scientifically thorough. More specific suggestions include:

1.	In line 3 of the abstract, please be specific about the “outstanding features of Prunus mume”.
2.	I suggest to combine paragraph 1 and 2 of the introduction as a single paragraph as both talks about Hsfs and demands continuity.
3.	An ambiguous sentence in line 32. Please rephrase.
4.	Incorrect grammar in the sentence at line 61.
5.	In line 90, the sentence is confusing such that if the author has done genome sequencing of Prunus mume or was it done already? Please rephrase.
6.	In line 113, please correct neighbor-joining (NJ) methods to “neighbor-joining (NJ) method” as it is one method.
7.	Remove extra comma in line 186 in “(Fig. S1,)”
8.	P. mume is not italicized in Table 1 legend.
9.	In the legend of Table 2, “MEME tools” should be singular.
Experimental design: 1.	In the introduction, I suggest the author to rephrase the sentences from line 81 -  87 to be in active voice. For e.g.” In this study, we performed genome-wide identification of Hsf and sHsp ……” etc.
2.	In the “Methods” section, please indicate the version of all the software used for the analysis, wherever applicable. For e.g. MEME, MEGA etc.
3.	In MEME analysis, authors have reported motifs with different length. Please include all the parameters of MEME analysis in the method section.
4.	A detailed characterization of Hsf and sHsp in P. mume is missing. In particular, the author can provide a figure with distribution of Hsf and sHsp in  P. mume among different classes based on DNA binding domains and sub-cellular localization.
5.	Author has provided a comparative analysis of  P. mume with Arabidopsis. In addition, a bar chart indicating the number of Hsf and sHsp in different species (if possible, for different category) would be very informative in the context of the present study. It would help readers to understand the discussion section that also includes below lines - “The number of Hsf genes in pear and apple was nearly twice as much like that in peach, strawberry, and Mei flower, which may be the result of the whole genome duplication (WGD) event in the Maloideae. Similar situation was observed in Populus trichocarpa, Zea mays (Lin et al., 2011; Zhang et al., 2015)”.
6.	Is there any class of Hsf and sHsp that is more prominent or specific to P. mume or similar lineage? Please comment.
7.	The result section is written using passive voice. I would suggest the authors read the result section and rephrase the sentences with the active voice to present the findings of the study in an exciting and interesting manner.
Validity of the findings: The study is experimentally sound and well presented. For further comments refer section Experimental design.
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: GENOME-WIDE IDENTIFICATION AND CLASSIFICATION OF THE HSF AND SHSP GENE FAMILIES IN PRUNUS MUME, AND TRANSCRIPTIONAL ANALYSIS UNDER HEAT STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made edits to address my comments and the grammatical issues throughout appear to have been addressed.  The introduction does a better job of setting up the rationale and importance of the study.

My comments about reporting in the discussion have also been addressed.
Experimental design: The authors have responded to my comments and added details about their phylogenetic and qRT-PCR analysis.

The authors addressed my questions about their qPCR design in their rebuttal letter, but I think some of this information should also go in the Methods section.  These additions would help address PeerJ’s guidelines that qPCR analyses follow the MIQE guidelines.

Specifically, I recommend stating that each qPCR primer pair was tested by end-point PCR to show that they produced a specific product of predicted size.  But I would also add that these products were not sequenced to confirm their identity.  While I don’t necessarily suggest that this work be repeated to sequence each product, I would strongly recommend that this be done in future studies.  I also recommend that the authors state that primer efficiencies were not calculated, and suggest that this be done in future studies, and that melt curve analysis be completed after each reaction.

I also recommend stating what controls were done.  It appears that water non-template controls were used, but not negative RT controls.  This should be made clear in the Methods section.  It would also be helpful to add the citation supporting the use of the reference gene and to state what Cq variation was acceptable between technical triplicates.
Validity of the findings: The authors have added a statistical analysis of gene expression differences.
Additional comments: I appreciate the time taken by the authors to revise their manuscript and address my suggestions.  The manuscript is much improved.  I believe that there are still some needed changes to the description of the qPCR analysis.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND CLASSIFICATION OF THE HSF AND SHSP GENE FAMILIES IN PRUNUS MUME, AND TRANSCRIPTIONAL ANALYSIS UNDER HEAT STRESS
Review round: 2
Reviewer: 2

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


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND CLASSIFICATION OF THE HSF AND SHSP GENE FAMILIES IN PRUNUS MUME, AND TRANSCRIPTIONAL ANALYSIS UNDER HEAT STRESS
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed my final suggestions.


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: ASSOCIATION NETWORK ANALYSIS IDENTIFIES ENZYMATIC COMPONENTS OF GUT MICROBIOTA THAT SIGNIFICANTLY DIFFER BETWEEN COLORECTAL CANCER PATIENTS AND HEALTHY CONTROLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have done a tremendous job in re-writing the manuscript. However, there are still some major concerns that have not been adequately addressed.

First, there are still problems with grammar throughout the manuscript. For example, line 65, “…importantly, however, disruption of gut microbiota has been linked to a number of gastrointestinal.”

Second, on line 62 the authors state, “…gut microbiota…the most capable of influencing human physiology, metabolism, nutrition, and immune function…” This is an assumption. Is there a citation for this statement? As a counterpoint, an infection starts off as having very low abundance but can have an outsourced influence on health and the immune system. Likewise, communities in Cystic Fibrosis are 3-4 orders of magnitude less than the gut but can have devastating immune-mediated effects.

Third, in the abstract on line 54-55, “…changes in microbiota composition may increase the risk of colorectal cancer.” Without formal testing of an odds ratio or similar measure it is hard to accept this claim at face value (i.e. does a higher or lower network centrality lead to lower odds of having an adenoma or carcinoma). This sentence needs to be revised.

Fourth, there are at least 3 papers that have looked at metagenomics and CRC. There has also been a recent meta-analysis in this area. One of these papers (Hannigan et al.) shows that 16S does better than metagenomics in classifying those with and without CRC. The authors need to do a better job of providing a clear rationale for their current study over simply looking for enzymes/compounds.

Fifth, I do not understand how probiotics fit into the introduction or into CRC treatment. Especially since probiotics do not persist in the gut community. Again, the authors are keeping the introduction very broad and use, "disease" rather liberally. This has the consequence of making it hard to understand the main message that they are trying to make. I strongly suggest the authors think carefully over whether they want this presented as a general methods paper or a CRC specific paper.

Sixth, the authors state in a response, “Data standardization is the normalization of data…” I understand that this is what normalization is supposed to do. However, there are many ways to get to this. What approach was used needs to be clearly stated. For example, a Z-score normalization may not improve anything if there is a lot of 0 inflated values.

Seventh, figure 1 is still not easily understandable. This graph is still not clear with respect to what the authors are trying to highlight. I understand what it is they are getting at but this is not clearly shown and raises more questions about the robustness of the analysis if a difference can be found when so many connections are still present.

Eighth, with respect to table 1 this is really what I want a range for. It should be possible if I understand the manuscript to be able to generate either a confidence interval or SD for these measures.
Experimental design: The authors responded to one of my statements with, “The purpose of this project is to detect the differences of enzymes between healthy people and cancer patients, and to deduce the differences of microorganisms from the biochemical reactions, metabolic functions and enzymes involved in biochemical reactions of intestinal microorganisms.” This specific goal that is stated by the authors is not accomplishable with the present data. Having strictly 16S data cannot elucidate the biochemical reactions, metabolic functions, or enzymes involved in the biochemical process. It can infer via known genomes what is likely to be happening. However, this database dependent approach does not necessarily accurately represent the authors’ goals. Additionally, they have not provided enough reasons as to why imputation of metabolite gene abundance is better than 16S given they trace back organisms via the imputed genes to species/genera that would have been identified via 16S.

The authors need to discuss how their method improves upon what was done previously with the data by Zeller et al or reference how it compares to standard practice for processing and analyzing metagenomics
Validity of the findings: Most of the references given by the authors for their algorithm concern dynamic or time-dependent processes. Naturally, the authors need to address how this algorithm performs in identifying novel associations in cross-sectional data (i.e. how does it compare to the original analysis).

In reference to the results presented by figure 3, It is hard to make the case that these enzymes are creating metabolites that are exacerbating CRC. There seems to be a very good association with simply a loss of a specific species (e.g. Bifidobacterium longum). Is it possible for the authors to address or mention that the results could simply be a good marker of the loss of a bacterium rather than the pathway that creates a metabolite of the importance of worsening disease?
Additional comments: Overall, I think the authors have done a tremendous job in improving the manuscript. However, I do not think they have appropriately addressed some of the major concerns that I originally had. I think it would be beneficial for Dongmei and colleagues to think about whether this is supposed to be more of a method or approach-based study or a disease-centered study.


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

Input:
Title: ASSOCIATION NETWORK ANALYSIS IDENTIFIES ENZYMATIC COMPONENTS OF GUT MICROBIOTA THAT SIGNIFICANTLY DIFFER BETWEEN COLORECTAL CANCER PATIENTS AND HEALTHY CONTROLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: line 65, truncated, should add 'diseases' may be to 'gastrointestinal'
line 106, 'constraint-based method'
Experimental design: the investigation is not rigorous

section 'Topological attributes of metabolic association networks' lines 188-234: it is not clear to me what the authors did here in relation to what is curated from literature, looks more relevant to the discussion section more than results

line 238: 'the standard deviation and mean of 13 enzymes' how were these 13 enzymes extracted ?

starting line 256: it is not clear if the authors did any statistical analysis to compare the abundance of K02003-producing microbes for instance, stacked bar charts could be used for visualization but an appropriate statistical test still needs to be done.
Validity of the findings: lines 184-187 'nodes of which showed enrichment in the colorectal cancer environment, e.g., KI01251
(adenosylhomocysteinase) (Figure 1C), were absent in the metabolic association network of
healthy samples, but present in the association network of the adenoma environment' is there any other kind of high throughput data that can support this finding ? for instance, how about looking for RNAseq data from colorectal patients and healthy subjects, or metabolomics data for colorectal patients ?
Additional comments: lines 107-109 the description of the limitations of constraint-based methods is not very accurate. 'The constraint-based method requires establishing a series of ecological constraint conditions, but this involves unknown parameters and requires performing repeated calculations'. constraint-based methods are based on imposing biologically relevant constraints rather than parameters.

Figure 1: which networks is for healthy subjects vs colon cancer vs adenoma patients ? labels should be indicated in the figure legend as well. Also a more detailed description needs to be added to the legend (e.g. mention details about 1-2 key nodes enriched in healthy vs colorectal patients)

Figure 2: Y-axis scale shows that the abundance is almost scarce, also the relative abundance looks different indeed, are these raw or normalized values ?

I would strongly recommend that the authors try to validate the relative abundance of the enzymes they are focusing on in other datasets. The paper as such presents more of preliminary findings that need to be asserted at other levels.


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: ASSOCIATION NETWORK ANALYSIS IDENTIFIES ENZYMATIC COMPONENTS OF GUT MICROBIOTA THAT SIGNIFICANTLY DIFFER BETWEEN COLORECTAL CANCER PATIENTS AND HEALTHY CONTROLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made some modest improvements to the overall manuscript. I have some concerns that I think are not adequately addressed by the this or previous revisions. Perhaps this iteration of comments will be beneficial in helping the authors consider the pros and cons of the current approach.

Need to correct the title. It is misleading since the authors did not actually perform metabolomics. They imputed gene/enzyme abundances.

There are still minor grammatical errors throughout (e.g. the phrase "may predict for human disorders" in the abstract).

Line 63: microbial communities "causing" colorectal cancer is controversial statement. It has not been shown that the microbiota causes the initial host genetic mutations in key genes like APC or micro-satellite instability. In fact, many of the "microbiota causative" studies require either a chemical or an initial KO of genes for the cancer phenotype to occur.  Additionally, the following sentence is one theory as to how the microbiota might contribute to cancer.

Weiss et al. 2015 citation: This paper does not state that eLSA has good performance for the task the authors are using it for. First, this was done on compositional data which Weiss et al. state the algorithm has problems with. The current paper uses a compositional based approach. Second, Weiss et al state that none of the methods are great at detecting interactions between species at that there is room for improvement. It is not clear based on this paper whether eLSA would do better than the previous approach taken by Zeller et al. when using compositional enzyme abundances.
Experimental design: The previous work would have also analyzed the metagenome and presumably also included enzymes that the author's say they focused on. They need to compare their work to what was previously found. Is theirs more believable or was the approach the previous authors took sufficient? It is not clear why this is a better approach than what was already published.

HUMAN2 can also pick out human KEGG annotations. The authors should be reporting how often the KEGG annotations overlapped with human pathways/enzymes.
Validity of the findings: The authors state, “The network topology of metabolic association network exhibited significant difference between the healthy and CRC environments.” It is not clear how the authors can make this call when there is no SD or confidence interval for the three groups. Essentially, the authors are describing an n of 1 experiment if this is based on their network analysis. As I understand the methods, based on the author's responses each sample did not have a network created but they combined them all into a single network.

Using imputed metabolites to show that you have identified bacteria common in metagenomic analysis seems to me to be a little circular. The data set is metagenomics and differences in abundances are known to cause differences in metagenomic analysis.  Hence finding key bacteria that the initial data set identified after imputation is almost to be expected and more of a control rather than a reportable novel finding. Using an actual metabolomic data set and finding this would be more convincing that the tools used here would be useful over standard approaches.
Additional comments: Line 57 "undoubtedly" is conjecture.


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: ASSOCIATION NETWORK ANALYSIS IDENTIFIES ENZYMATIC COMPONENTS OF GUT MICROBIOTA THAT SIGNIFICANTLY DIFFER BETWEEN COLORECTAL CANCER PATIENTS AND HEALTHY CONTROLS
Review round: 3
Reviewer: 1

Output:
Basic reporting: title: using the word 'enzymatic' twice is redundant. May be just 'network analysis ...etc'
Experimental design: It is still not clear to me how the authors ended up with those particular 13 enzymes. What is the initial number of enzymes tested ? how much were differentially abundant and how much were not ? all these details need to be clearly outlines.

I still have the same concern about the  relative abundance values, are these expected to be that low ? what is the distribution of the abundance and relative abundance of all tested enzymes ?
Validity of the findings: the findings presented as such are meant to motivate further investigation but not robust on their own.
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: ASSOCIATION NETWORK ANALYSIS IDENTIFIES ENZYMATIC COMPONENTS OF GUT MICROBIOTA THAT SIGNIFICANTLY DIFFER BETWEEN COLORECTAL CANCER PATIENTS AND HEALTHY CONTROLS
Review round: 4
Reviewer: 1

Output:
Basic reporting: I have no further comments
Experimental design: I have no further comments
Validity of the findings: I have no further comments
Additional comments: the authors have addressed most of the comments I had, thank you


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

Input:
Title: MANIFESTATIONS OF MUSCLE FATIGUE IN BASEBALL PITCHERS: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: Reporting can be improved to make sure this review meets the standard expected of systematic reviews, based on the PRISMA guidelines, i.e. introduction should be more concise, methods and results should include risk of bias assessment, justify weighted average approach to data synthesis, ensure flow chart numbers are correct, provide a table with study characteristics for all included studies, avoid duplication of information in tables and text, ensure information in the discussion is consistent with the results. Please see General Comments section for specific details and strategies to address these issues.
Experimental design: The major concerns with the design are the lack of risk of bias assessment and inclusion of non-pitcher based studies (i.e. swimmers, volleyball, tennis players). These other sports were included in the search methodology but then they are not mentioned in the results and discussion, so it is unclear how they have been integrated. I strongly suggest removing these studies from the eligibility criteria and results. Please see General Comments section for specific details and strategies to address these issues.
Validity of the findings: The authors offer interesting insights based on the findings of their review, but greater care needs to be taken to clearly identify when there is and is not strong evidence to support their claims. It is important to know when evidence is weak or non-existent because it provides direction for future research. Please see General Comments section for specific details and strategies to address these issues.
Additional comments: The authors have chosen an interesting topic to review and I congratulate them on the effort involved in screening 31,860 abstracts. I think this manuscript needs to be improved for publication and I have provided the following specific feedback for the authors that I hope they will find useful.

ABSTRACT
“is a primary reason for injury in baseball pitchers” – too strong a conclusion based on the evidence you have collated
“Findings of interest were not limited to baseball pitchers” – this does not make sense, given the aim and the conclusion, the review should be limited to pitchers.
INTRODUCTION
Line #64: “Fatigue can lead to performance decrements and the corresponding cumulative loading…” – I don’t think the “performance decrements” component fits in this sentence, I suggest just focussing on cumulative loading or better explain how the performance decrements are associated with the cumulative loading.
Line #68-74: restructure this information so that it flows better from the preceding sentences. This paragraph is long, so it will be easy to lose the reader if you jump around too much. These final 2 sentences need to be clearer and more concise to improve readability.
I don’t believe Table 1 is needed. Tables are not expected in the introduction of a journal article.
Line #79: “factors involving the brain and central nervous system” – this is too vague. Please be more specific or just say “due to central and peripheral neural mechanisms”.
Line #78-109: Too much detail here about each individual study. Please make this paragraph more concise so that the important information is better highlighted to the reader e.g. studies showing similar findings can be described together. Introduction sections of literature reviews do not need to be long.
Line #92-93: Good point.
METHODS
You should provide an operational definition of fatigue in the methods section. In the intro you define fatigue as “as a decrease in force generating capacity” but throughout the review you indirectly infer fatigue from high pitch counts, lack of rest, etc.
Line #122: There is no risk of bias assessment, so the review is not consistent with PRISMA. Please justify why this was not done and revise the statement about PRISMA guidelines accordingly.
Line #139-140: “not limited to baseball (swimmers, volleyball, and tennis players)” – I strongly disagree with the use of other sports. Your review objective and search terms are baseball specific, so it does not make sense for your included studies to not be baseball specific. Reviews need to be specific otherwise it is too difficult to draw strong conclusions. Additionally, there is no mention of non-baseball specific findings in the results so it is not clear how they have been incorporated. You should remove the non-baseball studies from the review.
Line #147: separate the data extraction and study selection paragraphs as per the PRISMA checklist. Please also make it clear that data extraction was done by one or multiple reviewers. It is unclear from the current wording.
RESULTS
Line #165-166: 74 minus 7 does not equal 65. Please clarify the numbers here.
Figure #1: several values do not add up. i.e. In the ‘Initial Screening Process” section, 38+10+11+6+6 = 71 not 72; in the ‘Secondary Screening…” section it is not clear how you go from “abstracts screened for eligibility (n=29)” to “full-text articles screened for eligibility (n=9)” to “Articles included (n=26)”. It would be clearer if the side arrows branched off of the descending arrows so that it is clearer which step they came before and after.
In general, these errors suggest that you should undertake a thorough cross-checking of you values to make sure there are no other errors/discrepancies in the final publication.
Line #169: not sure what is meant by “full-text was unavailable for use”. Please make this clearer.
Participants and Study Designs: this information should be in a table summarising the general characteristics of the included studies. The table should have (at a minimum) sample size, participant info, study design and duration (if relevant), outcome measures, key findings. This information is needed to be consistent with PRISMA.
Line #187-189: “The study designs varied within the selected literature, among which included four epidemiological studies, three retrospective analyses studies, two included crossover designs, and one longitudinal study.” – this begs the question, what was the study design of the other 16 included studies?
Line #192: “tiredness” – vague term, please clarify/define further.
Line #208-233: Much of this information is straight repetition of findings and values from Table 2. This paragraph should be condensed to avoid duplication of information from the table. i.e. synthesise the findings and make statements relating to the overall trends i.e. “There were significant changes in kinematics at the shoulder in X studies, elbow in X studies, hip in X studies … The variables that were most commonly affected were X, Y, and Z.”
Table 2: Include a description of the fatigue protocol used in each study. Switch the table to landscape orientation to facilitate this. Describe the age/level of pitchers for each study. Report key null findings (if relevant) as well as significant findings, otherwise it’s a biased representation.
Line #238-263: same feedback from Line #208-233/Table 2 applies here with regards to Table 3.
Table 3: you present weight averages here (i.e. a form of meta-analysis) but this is not described in the methods or highlight in the results texts.
Line #270-274: This is too descriptive to be in the results section. Include in the introduction/discussion if you must.
Line #274-298: Summarise these studies in a table and then succinctly highlight the key findings in-text to improve readability. You should present the findings based on the hierarchy of evidence i.e. RCT first (high quality evidence), survey last (low quality evidence). Talk about the results of studies that used similar study designs together i.e. currently you jump back and forth between longitudinal studies and survey studies and RCTs.
DISCUSSION
#304-306/Figure 2: you don’t have strong enough evidence to claim this. You have studies showing fatigue effects biomechanics and fatigue effects performance but nothing demonstrating that biomechanical changes caused by fatigue are causing performance changes or injury risk (if this information exists it does not appear to be included in your review). You can still propose your theoretical framework but need to highlight the parts that are based on evidence and the parts that are not. This is not necessarily a bad thing because it highlights areas of future research (i.e. the parts of your framework that lack evidence). Please revise the discussion section accordingly.
Line #310: This explanation does not fit with your results which suggest “results showed not only a significant reduction in ball velocity with fatigue,” (Line #254).
Line #311: Not sure what you mean by “command” here. Please make this clearer?
Line #313: these subsequent sentences don’t fit with the start of the paragraph. It’s not clear why you are even bringing up EMG at this point because you haven’t mention it once in your review up until this point. I suggest removing (or condensing and moving to later in the discussion) and elaborating more on the opening concept of this paragraph, which was relationship between fatigue, biomechanics and injury.
Line #337-340: as above, why are you now bringing up EMG.
Line #350-352: need to be careful with matching references to statements like this. The study you have referenced did not investigate injuries so doesn’t support your statement.
Line #356-363: again, this does not support your statement in line #310 that biomechanics change to maintain performance, i.e. here biomechanics have led to reduced force.
Line #363-370: This section is meant to be about kinetics (i.e. based on the subheading) but here you are talking about muscle activity. You’ve only referred to one study about kinetics.
Line #373-374: again, this does not support your interpretation at line #310 about biomechanical changes helping to maintain performance.
Table 4 – this appears to be results copied from 1-study that has been published previously. It would be more appropriate to summarise the findings of this study in-text and include a reference. This table should be deleted. It does not add anything to the interpretation.
Line #401 – this is probably the key paragraph (i.e. injury seems to be a big focus of your overarching review objectives) so you need to make it clearer what the current interpretation of the literature is, i.e. do we have any studies that actually link fatigue with increase injury risk? They must be highlighted first at the beginning of the paragraph. The weaker, survey-based studies can be mentioned later because they provide a low level of evidence that is not convincing in this case.
Line #426: “This can lead to an increased risk of injury in baseball pitchers.” – you have not provided strong enough evidence to make this claim, i.e. please soften your wording e.g. indirect evidence suggests there is a relationship, but there have been few longitudinal studies that have directly investigated it, and fatigue is often inferred from pitch counts, etc.


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

Input:
Title: MANIFESTATIONS OF MUSCLE FATIGUE IN BASEBALL PITCHERS: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is well written and provides significant background and context on the relationship between fatigue, injury and performance in baseball players. I have provided specific comments on the basic reporting provided within the manuscript with suggestions to the authors for improvement.

Specific comments:
•	Minor typographical error: Line 68: Major League Baseball should be spelled out in its entirety prior to using the MLB abbreviation.

•	Referencing error: Line 434: The authors cite Passan (2016) as support for a claim that $1.5 Billion is spent annually in Major League Baseball on pitching talent. This reference is not included in the reference list. Additionally, I believe the authors are refereeing to “The Arm: Inside Billion-Dollar Mystery of the Most Valuable Commodity in Sports” By Jeff Passan (2016). This $1.5 billionfigure from the reference actually appears in the first sentence of the inside cover of the book, I suggest that the authors track that figure from the book back to its original source for the reference.

•	Typographical error Lines 288-293: The authors describe a “randomized control trial” by Freeston et al. (2016) where 13 junior baseball players were tested on two occasions, seven days apart. The referenced study actually describes a “randomized crossover trail” as indicated in the title of the referenced article “Indicators of throwing arm fatigue in elite adolescent male baseball players: A Randomized crossover trial”.

•	Article structure: Results section: Thank you for the authors for including the PRISMA checklist in their supplementary file. However, although the authors have included synthesis tables for kinematic (Table 2) and performance changes (Table 3) with fatigue in pitchers a more detailed synthesis table is need for a systematic review (item 20 in PRISMA checklist). I suggest combining all reviewed articles into a large table with different sections for Kinematic changes, performance changes and injuries associated with fatigue in baseball pitchers. This table should also include a risk of bias score for each reviewed article (see comment in experimental design).
Experimental design: The manuscript in its current form is more of a narrative review that used a systematic search protocol. For this to be a true systematic review the following changes should occur.

•	According to the PRISMA statement by Moher et al 2009, “A systematic review is a review of a clearly formulated question that uses systematic and explicit methods to identify, select and critically appraise relevant research, and to collect and analyse data from the studies that are included in that review” (pg. 264).  Although the authors have indicated on their PRISMA checklist that they have adhered to item 12 (risk of bias in individual studies). A more robust appraisal should be employed incorporating a tool to assess the quality of the studies. The Downs and Black checklist or a modified version of, is commonly used for this (Downs SH, Black N. The feasibility of creating a checklist for the assessment of the methodological quality both of randomised and non-randomised studies of health care interventions.

•	A synthesis table that includes all reviewed literature and the risk of bias score (see previous comment in basic reporting sections)
Validity of the findings: Conclusions are well stated and link back to the research questions
Additional comments: No additional comments