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: PERITUMORAL IMMUNE INFILTRATES IN PRIMARY TUMOURS ARE NOT ASSOCIATED WITH THE PRESENCE OF AXILLARY LYMPH NODE METASTASIS IN BREAST CANCER: A RETROSPECTIVE COHORT STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is quite comprehensive, including the well decribed groups of patients and the statistics. Sharing the anonymized patients data is appreciated (filename "Patients data_PeerJ.sav"). Having opened this file, patient number 124 is missing, so I can see 149 patients in sum, which would differ from the number of 150 provided in the abstract and the materials and methods part (line 34). The lost patient should be added in the file or the number n should be corrected to 149 and numbers n should be provided in the tables of the manuscript as there might be <nan> (or empty) values in some markers (".0" <zero> vs. " . " <nan>). However, I cannot totally exclude a formatting issue when opening the .sav file. The file is saved in the .sav format of SPSS, which can be opened and converted by open source software as "PSPP", too. Providing a text file as .csv would however allow a direct import in R, Python, Excel etc and would prevent formatting errors (e.g. from guys like me).
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The idea to dissect different lymphocytic phenotypes of the peritumoral area is much appreciated, which allows further correlations as well to define some kind of peritumoral immunological footprint (e.g. negative correlation between FoxP3 and ER etc.). This of course does not have to be considered but it could be and it would be already in your data.


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: PERITUMORAL IMMUNE INFILTRATES IN PRIMARY TUMOURS ARE NOT ASSOCIATED WITH THE PRESENCE OF AXILLARY LYMPH NODE METASTASIS IN BREAST CANCER: A RETROSPECTIVE COHORT STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed many points, however, they still neglect some of the latest findings - look at three studies in Nature 2020, by Cabrita et al., Petitprez et al. and Helmink et al..; or Sherene Loi KCO 2019.
sTILs at primary tumour site do influence outcome significantly - and there is a higher impact in patients with higher nodal disease.
The discussion needs to be structured, subheading would help to extract take-home messages.

Given the new addition " In this second multivariate model the immune populations of CD68+ macrophages were a factor associated with ALN metastasis (page 7, lines 181-188). " - should the authors now not revise their general statements and title:
“Peritumoral immune infiltrates in primary tumors are not associated with axillary lymph node metastasis in breast cancer: a retrospective cohort study"?
Experimental design: na
Validity of the findings: na
Additional comments: na


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: PERITUMORAL IMMUNE INFILTRATES IN PRIMARY TUMOURS ARE NOT ASSOCIATED WITH THE PRESENCE OF AXILLARY LYMPH NODE METASTASIS IN BREAST CANCER: A RETROSPECTIVE COHORT STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thank you for having addressed the issues mentioned. Please also correct the number of patients in the methods part of the abstract (still n = 150 vs. correct n = 149).
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: APPLIED PHYLOGEOGRAPHY OF CYCLOPIA INTERMEDIA (FABACEAE) HIGHLIGHTS THE NEED FOR ‘DUTY OF CARE’ WHEN CULTIVATING HONEYBUSH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present a well-written manuscript with a well-referenced introduction that suitably sets the scene for the study. The structure of the paper confirms to PeerJ standards.

Overall the figures are good and present the data well. However, there are a few errors in the captions (detailed below) and I found the colours used for haplotypes P and M are very similar and difficult to distinguish.

The raw data produced is supplied in Tables 2 and 4 and has also been deposited in GenBank. Additional information on the PCR protocol and the custom R script that were used is provided in the Supplemental Files.

Minor comments
Ln 74 – Change ‘I’ to ‘we’ (since there are two authors).

Ln 222- Change ‘all indels were reduced to single base pairs’ to ‘each indel event was reduced to a single base pair’

Ln 258 - Change ‘atpI-aptH intergenic spacer’ to ‘atpI-atpH intergenic spacer’

Ln 283 – should G’’’’’ts be Gst?

Ln 298 – Change (Potts, 2017a) to Potts (2017a)

Ln 351 - Change ‘I’ to ‘we’ (since there are two authors).

Ln 376 – Change to ‘Novel mutations and rare haplotypes become rapidly fixed in populations….’

Ln 433 – Acknowledgments – Change ‘me’ to ‘us’ (since there are two authors).

Ln 478 – The title of the Dray reference should start with ‘The’

Ln 498 – This reference is missing the journal.

Ln 518 – insert space between ‘GST’ and ‘and’

Ln 559 - Restio capensis should be in italics.

Ln 665 - Ambystoma tigrinum should be in italics and be followed by only one full stop.

Figure 1 caption – should ascensions be accessions?

Figure 2 caption – Change – ‘haplotypes connected by a single line differ by a one base pair’ to ‘haplotypes connected by a single line differ by one base pair’

Figure 4 caption – change demoted to denoted.

Table 3 caption – italicize C. intermedia

Table 4 – delete the word ‘various’
Experimental design: The research presented fits within the scope of PeerJ. The study has clear aims and examines these with appropriate sampling and analyses.  The methods are clearly explained, although I would like to see details of the outgroup samples added to the ‘Sample collection and DNA extraction’ section. The use of sequencing to confirm the HRM clusters and determine the level of false negative haplotype assignments is particularly commendable and it gives confidence in the quality of the data.
Validity of the findings: Overall, the conclusions are appropriate given the results and link back to the original research question in the introduction.

I have one question about the interpretation of the network. I found it interesting that three of the outgroup species (C. aurescens, C. longifolia and C. sessiflora) have haplotypes that, in the network, are nested within the genetic diversity detected in the study species. Are these species known to hybridise with the study species? Could chloroplast capture from these, or other closely-related species, be the source of some of the rare local haplotypes found in your target species? Chloroplast capture has been found in many overseas studies of plant phylogeography. I don’t think it would change the interpretation of the results or the conclusions but it is something that should be considered.
Additional comments: Overall a good solid study and a nicely written paper. I particularly like that clear conservation recommendations are provided.


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: APPLIED PHYLOGEOGRAPHY OF CYCLOPIA INTERMEDIA (FABACEAE) HIGHLIGHTS THE NEED FOR ‘DUTY OF CARE’ WHEN CULTIVATING HONEYBUSH
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think it is an important contribution because adds useful information about phylogeographic patterns of Cyclopia intermedia, used to produce the Honeybush tea. This species is endemic from the Cape Floristic Region (CFR), located along the southern Cape of Africa. Since the cultivation of this species is relatively recent, opportunities exist to guide future conservation and management based on the retrieved genetic patterns.
However, I consider there are some aspects of the manuscript that should be revised.
Authors should revise considerably  the English editing. I think some ideas are confused because of wording. In some places you used "I" instead of "We", please check.
In general terms I consider the introduction is sufficient and clear, the manuscript are appropriately referenced and is also well structured.
Authors do not mentioned if they have deposited or will deposit the sequences in the Genebank; they should do it.
Experimental design: I think the research question is well defined and the knowledge gap is well formulated. The hypothesis should be improved and Materials and Methods should include more detailed information about some issues.

Authors hypothesize that due to limited seed dispersal by ants, Cyclopia species will exhibit highly structured chloroplast genetic diversity as was observed in a previous study of another wild Cyclopia species. In other words, the hypothesis is raised in relation to an autoecological characteristic of the species. On the other hand, the topographic complexity and climatic heterogeneity of the study area are also mentioned in the introduction, but authors did not present an integrated hypothesis considering both factors (i.e. autecology and landscape and environmental characteristics). Thus, I suggest developing a hypothesis that includes both elements. Furthermore, I suggest including in Materials and Methods a description of the study area, since it is not mentioned how high these mountains are, which is the direction of the mountain ranges, and how is the distribution of Cyclopia intermedia within the mountain range. It is not clear for me if there could be altitudinal corridors for the species? Please, explicit how the topography could be a possible barrier for Cyclopia intermedia?
I consider that this will contribute to a better discussion of the results. For example, in the Discussion section the different basins and sub-basins are mentioned and it is difficult to follow it. I suggest including a DEM map (modifying Fig. 1) indicating the location of each basin, mountains, etc.  to better understand and discuss the retrieved genetic patterns.

Materials and Methods
Please introduce better the focal species. Is it an herb? a shrub?
Please specify the distance among sampled individuals and the minimum and maximum distance among sampled localities.
Author say that they amplified three non-coding chloroplast DNA loci. However, as you say in line 221, chloroplast DNA are maternally inherited and in tandem, thus the chloroplastidial DNA molecule is considered a single locus. In addition, in my opinion you amplified two non-coding regions (the atpI-aptH intergenic spacer and the ndhA intron), not three as is mentioned in the manuscript; the atpI-aptH intergenic spacer was amplified in two parts. Please modify.
Considering phylogeographic analyses, I think with this dataset authors could go further in unravelling the evolutionary history of the species, for example including demographic analyses. But, for the question posed, inferring haplotypes genealogy, their spatial distribution, and characterize the spatial genetic structure may be enough. The comment I have in this regard is in relation to the use of AMOVA. In this analysis, you define a priori groups of populations; so while partitioning can be significant, perhaps there is another barrier that further explains the genetic pattern obtained. Thus, I suggest to include some analysis such as BASP or SAMOVA where the population groups are identified a posteriori. Given that the patterns found were not expected, this type of analysis can help to infer some factor / barrier not previously considered.
Line 187-188 – How many replicates per sample?
Line 205-207- Please, inform how common was this situation
Line 226 - Did sequences retrieved many indels? Why indels were not coded? As suggested for example by Simmon and Ochoterena 2000, Gaps as Characters in Sequence-Based Phylogenetic Analyses
Line 237 - But you don’t have alleles. You mean, haplotypes? It is necessary to give more detail about the genetic distances and the richness estimator used.
Why did not use a genetic distance that consider nucleotide differentiation and haplotype frequency among populations? They are implemented in Arlequin or DNAsp and sure in R.
Line 239 - It takes indels as a fifth state?
Line 241 - Which analyses? Do you mean that significant association between these genetic and geographic distances were tested via Mantel Test? Please reword this sentence.
Line 244 - Did you correct this estimation based on sample sizes? This is important because of the difference in the sampling sizes among populations.
Line 247 -  Why did you consider only the longitude? And the latitude? Probably you can better explain how the study area and the area of distribution is to a better understanding
Validity of the findings: RESULTS
I consider results are not well stated and that there are some inconsistencies with the methodology.
In the Result section, Phylogeographic analysis, please include a description of haplotypes geographical distribution. I recommended to characterize the spatial distribution of haplotypes, while mentioning how genealogically close they are. This makes easier to build the underlying biogeographic scenario.
Haplotype network include out-groups as shown in Fig. 2, but it was not mentioned either in M&M or discussion. I suggest taking them out.
Could you please show the AMOVA results (FCT, FST, FIS, p values? In a table in the main manuscript or as supplementary material.
Line 258 – Please put “regions” instead of “loci”
Line 259 -  ndh or ndhA, please uniform throughout the manuscript
Line 259-260 Please move the line about haplotype spatial distribution to M&M
Line 287 – Among instead of within
Line 288 -  I suggest 9.0% structured among populations within mountain ranges.
Fig. 1. The study area it is not clearly indicated for me. What is it the white map? An amplification of the white are in the Africa map? What is obscure haplotypes? Please explain better. I also recommend to inset a picture showing the mountain ranges more clearly.
Fig. 2. Haplotype network include out-groups as shown in Fig. 2, but it was not mentioned either in M&M or discussion. I do not see any broken line as is mentioned in figure legend

DISCUSSION AND CONCLUSION
Authors conclude that all wild populations should be safeguarded from potential genetic pollution to some degree. Of course that this would be the best option, but I think author should propose an order of priority based on their study. For example considering haplotype diversity, private haplotypes, etc. Moreover, based on Fig. 1, it is evident that to the western side of the distribution range, there would be evidence of isolation among populations, but toward the eastern side there is some evidence of a greater connectivity among some populations. I think this pattern has management implications. I consider the conclusion could be more connected with the results obtained.

In the discussion section, basins and mountain range, are mentioned but they have not been well presented in the previous sections, so it is a little difficult to follow the reasoning of the discussion. As I mentioned above it is necessary to introduce better the geography and the biogeography of the study area. Including a figure where different mountain ranges, basins, etc be shown.

Line 294-297 I suggest moving these lines to M&M
Line 338-339 - Could you give a putative explanation for this pattern?
Line 370 – What process? Please, explain better what would be a physical barrier for C. intermedia…This idea should be also presented in the introduction
Line 379-380 -This could be a consequence of secondary contact area..
Line 405 - Probably this idea should be also proposed in the introduction, in the hypothesis
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: APPLIED PHYLOGEOGRAPHY OF CYCLOPIA INTERMEDIA (FABACEAE) HIGHLIGHTS THE NEED FOR ‘DUTY OF CARE’ WHEN CULTIVATING HONEYBUSH
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am satisfied that the authors have addressed my comments and the paper is suitable for publication.
Experimental design: n/a
Validity of the findings: n/a
Additional comments: n/a


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

Input:
Title: SIMULATING HIGHLY DISTURBED VEGETATION DISTRIBUTION: THE CASE OF CHINA’S JING-JIN-JI REGION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The reviewed manuscript is comprehended only with great difficulty. The text is on many occasions ambiguous and often lacking strong arguments. The text needs major improvements in English language, consistency and clarification. Both the introduction and the discussion are lacking a coherent structure.
Long difficult-to-understand sentences: line 48-52; line 213-217;  lines 321-328
Comma problems: line73 …
vague language: line 78-86; line 189, line 238-239
out of context: line 98-99,
rephrasing required: line 59 “smartly use vegetation”; line 69 “using information based on information..”; line 189 “through different model methods, we used different combinations…”; line 299 “a complex, multi-level, non-linear system, is one of the complex problems…;
repetition of methods: line 359-362
language problems: line 122; line 345

Manuscript uses broad and sufficiently varied literature, however it often relies on literature from a single book (Franklin 2010) without mentioning page numbers within this book (all instances when the book is cited) and often lacking reasoning (line 359).
Statement unsupported by reference: line 64; line 354
statement without reference: line 102-103

@Franklin, J. (2010). Mapping species distributions: spatial inference and prediction, Cambridge University Press.

Figures are generally in low resolution (and no hi-resolution/original resolution GeoTiff is provided). Color schemes used are difficult to distinguish and not color-blind friendly (example in Figure 4, where 39 different colors are used – impossible for human eye to distinguish).
Figure captions are cut-off (Figure 2,3,4).
Figure 1 is missing an overview-localization map (or marked large cities), for the readers who are not familiar with geography of China.

Raw data is partially shared, but point files are in uncommon format that is difficult to open using common computer programs (and not specified which program should be used). Additionally, the provided shapefile data for Vegetation Map (VMC) contains descriptions and encoding in non-latin and non-English characters, thus could not be explored further.
Experimental design: This research is within the scopes of PeerJ journal, however it seems very repetitive and little original with regard to the previous article from the authors Zhou et al. from the year 2016. There is no significant new findings or methodological advancements (default settings of an existing software) other than the produced vegetation map predictions (which should at least be provided in full resolution)
@Zhou, J., et al. (2016). "Comparison modeling for alpine vegetation distribution in an arid area." Environmental monitoring and assessment 188(7): 408.

The main flaw is in the fact that the authors are using relatively old vegetation survey data (that they claim is from year 1980 – line 121), which is then put through the modelling procedures using recent predictor variables (including vegetation indices from 2013). This could be causing a problematic temporal mismatch, especially since the study area is reported to be are heavily influenced by socio-economic development.

The software that was used to carry out the entire study is very little described (EnMAP-Box), and only mentioned at line 220. Describe the modelling platform early in this paragraph (line 202).  The default settings are used, but it would be useful to elaborate further on what are the settings. Why was it left on default? Other studies that only use the default settings?

Lacking description of how data between training and assessment points were split (Random subset?).

Not described how the vector vegetation map of China (VMC) was used as assessment. I must only assume that the vector map was first rasterized onto the same grid as the modelled data, and then tested with Kappa. Please explain further

Kappa was used as the only assessment method. It is known that Kappa is a threshold dependent metric, see Guisan 2017 page 242 for additional assessment methods

@Guisan, A., et al. (2017). Habitat suitability and distribution models: with applications in R, Cambridge University Press.
Validity of the findings: It is common in the Distribution Modelling (DM) research to provide all input data on a repository for possibility of future replication. It is highly advisable to do so in this study. See Araújo et al. (2019) for standards for DM
@Araújo, M. B., et al. (2019). "Standards for distribution models in biodiversity assessments." Science Advances 5(1): 1-11.

Conclusion paragraph both in the abstract and discussion is a word-by-word repetition of the results.
Line 329 – there is no explanation to support your statement.
Additional comments: I appreciate the last few sentences in discussion (line447-451) taking up the problem of human disturbance to the study area. This should be elaborated on in much greater extent.

The title should be reconsidered: “region that has been highly disturbed by social-economic development” it is not the core topic of this study, and the authors have avoided explaining/discussing or performing analysis on the human disturbances – rather they are modelling the potential vegetation

Shortcuts are not used consistently throughout the article (DT, RF, SVM and MLC sometimes written out, sometimes used with shortcut)

Use table to present the variable combinations (starting on line 187). Variable combination 11 and 12 are not sufficiently explained (give explicit examples – create a overview table).

How can predictor variables be used for assessment? Line 189

When listing out the three classification units, try to delimit the units with numbers (I. vegetation groups; II. vegetation types; and III. formations and subformations) to avoid double “and”


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: SIMULATING HIGHLY DISTURBED VEGETATION DISTRIBUTION: THE CASE OF CHINA’S JING-JIN-JI REGION
Review round: 1
Reviewer: 2

Output:
Basic reporting: I see many text errors thoughout the manuscript and some of the statements can definitely be written in a better way. I suggest that the co-authors whose first language is English carefully peruse the text. Most sentences are long and superfluous. Here are some of the few examples:
1. Line 19 - Vegetation distribution simulations could help to understand..may be better to write as: Vegetation distribution simulation is important to understand ….
2: lines 48 - 52: Very long sentence and difficult to understand. It can benefit from paraphrasing and breaking into two or three sentences.
3. Caption of figure 3 – “label e” is lacking … the Vegetation Map of the People's Republic of China in (e)
4. Similarly, on caption of figure 4, “label e” is lacking
5. Please supplement the 3763 observation points with corresponding vegetation groups, types, and formations and sub-formations.
Experimental design: The experimental design is good. But I have some concerns on resampling techniques and variable selections. Below I am also suggesting an alternative way of achieving the same objective.
Validity of the findings: The findings are valid but the objective is not clear. The paper will benefit by setting a clear objective - whther the primary goal of the paper is to improve the vegattion map of the study area or to explore and answer methodological questions. This should be clearly mentioned in the results and conlcusion sections too.
Additional comments: 1. The resampling techniques used: nearest-neighbor method is used to resample both the DEM and the climatic variables. This approach is commonly used for discrete variables such as land-cover. For continuous variables, bilinear or cubic techniques are commonly used. The authors should justify either how their approach may not affect their data and consequently their results or should use the right approach.
2. Variable selections: what is the criteria to select the four bioclimatic variables? Is there any objective way to determine the number of bioclimatic variables to be used as predictor variables? It is better to consider all the 19 bioclimatic variables, stack them together with the other predictor variables, extract their values at the 3763 observation point, compute pair wise correlation and consider only less correlated variables (R < /0.7/ is usually used as threshold; see Chala et al., 2017). This way, it is possible to be objective and avoid redundantly using correlated variables. It is also interesting to see the correlation among the topographic, spectral and climate variables.
3. Elevation is a proxy variable and does not have direct impact on vegetation.  Thus I don’t recommend using elevation as a predictor variable. It is also expected to be highly correlated with temperature related bioclimatic variables. Either the use of altitude as predictor variable should be justified or its correlation with the bioclimatic variables should be checked.
4. Probablity of using alternative approaches: cultural vegetation groups and the vegetation types included in this category are mainly shaped by anthropogenic impacts. Including this group in the model will definitely deteriorate model quality.  I am very curious what if you just use satellite images with higher spatial resolution such as sentinel and spot images and check how they perform in capturing the vegetation groups, types, formations and sub formations. That asks less energy and probably more reliable. That will also allow detecting anthropogenic driven changes and monitoring the land-use land-cover of the study area. Specially of the main objective of the paper is to explore the method that perfoms well, this vegetation types should be excluded from the model.
5. Checking the homogeneity of classes: It is also good to check the elevation ranges of each vegetation groups and types. If they cover wider elevation ranges, by dividing them into upper and lower elevation classes, it is possible to improve the performances of the algorithms (See Chala et al., 2017).
6. Figure 1 – it is great that the elevation range of the study area is provided. But it will be more informative if it is classified in to at least five reasonable elevation range classes. Can you please consider that?
7. The title can be kept short and precise – for example: Simulation of highly disturbed vegetation types: the case of Jing-Jin-Ji region, China
8. Last and finally: Is it possible to include the specific objective of the paper in the abstract – whether it is more interested in improving the vegetation map of the study area or exploring methodological issues. Just a sentence or two?
Reference: Chala, D., et al. (2017). "Migration corridors for alpine plants among the ‘sky islands’ of eastern Africa: do they, or did they exist?" Alpine Botany 127(2): 133-144.


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: SIMULATING HIGHLY DISTURBED VEGETATION DISTRIBUTION: THE CASE OF CHINA’S JING-JIN-JI REGION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article is much improved now except there are still some minor editorial mistakes.
Experimental design: This part is already improved
Validity of the findings: It is quite improved and good
Additional comments: The authors included (justified) most of the comments which were given on the earlier version. But stil there are few minor editorial mistakes. They are presented as follows:
18 - May be it can improved as: The primary goal of this study was to determine the best simulation method for a vegetation in an area that is heavily affected by human disturbance.
35 - "Variables Combination 7 produced the highest simulation accuracy". I think it is better to list the variables by name.
63 - "in conjunction": may be better to use in combination?
145 - "digital elevation model (DEM) (Zhao et al., 2018)" - may better to write it as - digital elevation model (DEM; Zhao et al., 2018).
148 - "1 km resolution" - may be better to put it as - at ~ 1 km resolution? I don't think the resolution is exactly 1 km. Please cross check this.


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

Input:
Title: PROGNOSTIC IMPLICATIONS OF METABOLISM-ASSOCIATED GENE SIGNATURES IN COLORECTAL CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper by Miao et al described the identification of prognosis-related metabolic genes for colorectal cancer using available TCGA and GEO datasets and evaluated their potential roles in disease pathogenesis. The manuscript is written well and covered all aspects of the area that need to be reviewed before publication. I have a minor comment which is described below and overall, I recommend this article for publication.

The resolution of figures 1, 2, 3, 5 and 6 are very poor. It is very hard for this reviewer to read the contents of figures. Please expand all the figure legends and appropriately describe X and Y axis in the figures.
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: PROGNOSTIC IMPLICATIONS OF METABOLISM-ASSOCIATED GENE SIGNATURES IN COLORECTAL CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: - There still has some grammatical errors and typos. The authors should re-check and revise carefully.
- Quality of figures needs to be improved.
- Background and literature review are weak, need to add more information.
- The authors should provide source codes for reproducing the results. It is very important.
Experimental design: - The flowchart (Fig. 1) looks unclear. For example, did the clinicopathology data come directly to DEG analysis? As I known, it is maybe only gene data.
- ROC curve or AUC has been used in previously biomedical works such as PMID: 31277574 and PMID: 31362508. Therefore, it is suggested to add more references in this description.
Validity of the findings: - It is easy to see that the performance results are not quite significant (i.e., very low AUC). Therefore, it's hard to convince that this gene set is significant.
- Also, ROC curves showed that the model is not so good.
-
Additional comments: No comment.


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

Input:
Title: PROGNOSTIC IMPLICATIONS OF METABOLISM-ASSOCIATED GENE SIGNATURES IN COLORECTAL CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: In this manuscript “Prognostic implications of metabolism-associated gene signatures in colorectal cancer” Miao et al. have identified a prognostic model in  colorectal cancer patients based on metabolic genes. They showed that risk score that authors have identified based on TCGA is robust across GEO datasets and correlated with clinic pathological characteristics. Authors have performed multiple bioinformatics analysis to characterize the risk model but completely lack rationale and key methodological details which makes manuscript hard to follow in its current form. Following are the points to be addressed:

Major comment:

1. Abstract of the manuscript is verbose and lacks specificity. Abstract should be written in a clear and consise way, explaining the research question and results/conclusions.

2. Authors have not mentioned how prognosis associated metabolism related genes were identified? Was comparison pf CRC patients was done with normal? Line 86 -> Define what is data extraction and integration here? What pathways are enriched in Differentially expressed PRMGs? Does these metabolism associated genes are also differentially expressed in different subtypes of CRC?

3. What is the significance of using Gene Set Cancer Analysis
when mutation patterns in genes can be easily mapped using TCGA mutation information of CRCs? A simple bargraph representing the mutation frequency can also show the importance of picked genes. Cite appropriate references to discuss the importance of selected genes and there known role in CRC if any.

4. Line 65-> which genes? Explain why there is a need of a new prognostic model? Define PRMGs in introduction section of the manuscript. What is rationale of using metabolism related genes?

5. What is advantage of using risk score over patients clinical characteristics when five year survival statistics is similar with either (Figure 3)?

Minor comments:

6. What level of TCGA was used for the analysis? Does authors perform expression normalization?

7. Table1: Mainly pathway -> Pathway

8 Figure quality is very poor and text is unreadable.

9. Figure1: Figure quality is very poor. 1E what does X and Y axis indicates?

10. How patients were divided into low-high risk groups? Are these six prognostic genes are known prognostic genes for other tumor types?

11. Why flowchart of study design is Figure 7 of the paper? It should be the first figure explaining the schematic of study design.


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: PROGNOSTIC IMPLICATIONS OF METABOLISM-ASSOCIATED GENE SIGNATURES IN COLORECTAL CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: My previous comments have been addressed satisfactorily.


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: PROGNOSTIC IMPLICATIONS OF METABOLISM-ASSOCIATED GENE SIGNATURES IN COLORECTAL CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: Authors have addressed all my concerns


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

Input:
Title: MAYFLY EMERGENCE PRODUCTION AND BODY LENGTH RESPONSE TO HYDROLOGY IN A TROPICAL LOWLAND STREAM
Review round: 1
Reviewer: 1

Output:
Basic reporting: See "General comments for the author"
Experimental design: See "General comments for the author"
Validity of the findings: See "General comments for the author"
Additional comments: Dear Editor,

I have read and reviewed the paper entitled “Hydrology influences body length, but not benthic and emergent biomass of Ephemeroptera in a tropical lowland stream” by Gutiérrez-Fonseca & Ramírez, which has been submitted to PeerJ for your consideration (manuscript number 47058).

The authors study the traits declared in the title for a specific mayfly species (Ephemeroptera: Leptophlebiidae) called Farrodes caribbianus. The interannual variability in precipitation that characterizes the tropical stream they study (Carapa stream) is presented as a stressor, yet precipitation does not seem to influence asymetry which is known to respond to environmental stress. In contrast, body length (but not biomass) is affected by hydrology. The authors use monthly data for years 2002 and 2003 to make their case.

The Introduction section is well written and easy to follow. I only made few specific comments, most of them really minor. Nevertheless, predictions are described as hypotheses, although this could easily be fixed with some rephrasing. Also, I think adding a couple of references (particularly recent references, if possible) at specific places (see comments below) would be beneficial. Methods are also well written and very well explained. Only some minor suggestions apply here as well. Results indicate absence of effect of environmental variables on biomass or asymmetry, but a significant effect on body length.

One drawback of this study is that it might be problematic to have a short time series (two years) to prove any influence of interannual variability on biomass and asymetry, in my opinion. On the other hand, it is true that even with a short time series the authors were able to find a clear (inverse) relation between body length and precipitation. But the interpretation of results (i.e. the Discussion section) is still not convincing, in my view.

For example, with respect to biomass, it is stated that high floods may be responsible for microdistributional changes which in turn may result in changes in biomass. However, this was never proven with the precipitation data that the authors have at their disposal. With respect to asymmetry, no environmental variable was significant in their model. The explanation given in this case relies on a possible trade-off between body size and asymmetry, in the sense that the priority would be to preserve symmetry and thus function, at the expenses of body size variation. It can be reasonable to argue that body size changes are less critical to fitness than deviations from symmetry might be. However, the connection between the two in more mechanistic terms is unclear to me and might not be so straightforward. A more detailed explanation is needed, in my opinion. Also, I wonder if it could be possible that other environmental variables that weren’t measured are actually responsible for the observed variability in body size. Also in relation to body length, it isn’t very clear why body length (and not biomass) is responding to precipitation.

Overall, I don’t see this manuscript ready for publication in its present form, but maybe the authors are able to provide a more convincing case after major revision. This should include other forms of measuring precipitation at appropriate scales of analysis. The Discussion section needs to improve substantially so as to address the comments mentioned above (see also the specific comments provided below).

Sincerely,
the reviewer.

Specific comments:

Abstract:

Line 38: “Our multi-year study”. However, this study only covers two years: 2002 and 2003.

Lines 39-40: “unrelated to hydrological fluctuations, but potentially related to trophic interactions (e.g. fish predation)”. However, the absence of relation to hydrogical factors could change if precipitation were estimated otherwise (i.e. at other scales of analysis). On the other hand, the relation to “trophic interactions (e.g. fish predation)” has not been proven with the data available. Overall, the absence of relation to hydrological fluctuations could result from considering precipitation at the wrong scale of analysis (e.g. single events may have more influence than average rainfall) and the potential influence of fish predations is weakly supported by results. Both issues undermine the conclusions drawn.

Introduction:

Lines 49-51: “In lotic environments, much attention is given to understand the impact of environmental variables on aquatic organisms at multiple levels, from individuals to the community”. However, this reads like if this were a particular feature of studies focusing on lotic environments. I would rephrase this to highlight that lotic environments are not an exception in this regard.

Line 53: I think adding one more recent reference here would be a good idea.

Lines 76-78: “The first two cases of asymmetry are related to genetic changes, while fluctuating asymmetry (FA) is caused by environmental stressors”. I think there should be a reference here.

Line 77: “fluctuating asymmetry (FA)”. This abbreviation “FA” is seldom used in the text, tables and figures. Therefore, I would eliminate “(FA)” from here.

Lines 93-95: “we used data collected during two years (2002-2003) to determine temporal patterns of emerged Ephemeroptera and the benthic standing-stock biomass”. I think the temporal span (2002-2003) might be too short to draw conclusions about the effect of interannual environmental variability on biomass and asymetry.

Line 98: “(Leptophlebiidae, Domínguez, 1999) adults”. I would eliminate “Leptophlebiidae” from here because it reads strange together with a reference. And, also “adults” could be placed somewhere else to facilitate reading. Perhaps the entire sentence could be rephrased as follows: “we examined departures from perfect symetry in adults of the Leptophlebiidae Farrodes carribamus (Traver) comb. nov. (Domínguez, 1999), ... ”.

Lines 102-104: “We hypothesized a peak in benthic and emergent biomass during the low rainfall season, due to a decrease in the risk of mortality from drag during floods”. I think the “hypothesis” here is the “decrease in the risk of mortality from drag during floods” while the “peak in benthic and emergent biomass during the low rainfall season” is the “prediction” that results from this hypothesis. I would rephrase this so as to be more accurate in this regard.

Lines 104-106: “We also hypothesized that potential changes in adult symmetry and body length would reflect nymph development instability due to exposure to environmental stressors during their lifespan, and their ability to buffer environmental disturbances”. I think here the “hypothesis” is that “exposure to environmental stressors during their lifespan relate to nymph development instability” while the “prediction” resulting from this hypothesis is a “change in the adult symmetry and body size”. I would rephrase this a little bit as well.

Methods:

Line 113: “1563ha”. Space needed.

Line 115-116: “Long-term average annual precipitation (1963-2000) is 4314 mm, ranging from 2809 mm in 1995 to 6164 mm in 1970”. Can this be updated to present dates (e.g. from 1963 to 2019)?

Line 120: “100m. Space needed.

Line 136: “Three core samples (0.006 m2 each)”.

Line 151: “AFDM”. Please develop the meaning of this for non-specialized readers.

Line 171: “abbreviated to BL”. However, this abbreviation is never used in the text, nor in Tables or Figures. Therefore, I would keep using “body length” all the time and eliminate this explanation here.

Lines 176-177: “all measurements were taken three times from the same image at a random order”. I didn’t get this. So, the same image is used to perform three measurements at a random order. Does this mean that many different images were displayed at a random order so that in the end all images were shown three times? I think this needs some more detailed explanation.

Lines 183-186: “biomass better reflects potential changes in mass production”. This is circular. I would eliminate this. In fact, I would eliminate the entire paragraph (Lines 183-186) because it doesn’t add anything relevant for the purpose of this study.

Line 206:  I would replace “FA” by “fluctuating asymmetry”. The definition of “FA” was made in the Introduction section (Line 77) but seldom used elsewhere in the text. I didn’t remember what it meant when I found it here again for the first time.

Line 208: I would replace “FA” by “fluctuating asymmetry” also here.

Results:

Line 222: “the 2002 and 2003”. I would eliminate “the”.

Lines 229-230: “The AIC analysis used to determine the relative importance of environmental variables on the biomass of emerging adults showed no support for any model”. Maybe other environmental variables not registered in this study are responsible for biomass variability?

Lines 230-231: “This trend was consistent for mayflies collected during 2002 and 2003”. I don’t think this is necessary because this study is about mayflies, and about the years 2002 and 2003. This was made clear in the Introduction and there is no need to repeat it here. In fact, in may be confusing as it reads like if there were other invertebrates or other years available.

Line 241: I would replace “FA” by “fluctuating asymmetry”.

Lines 244 and 246: “Appendix 1”. Where is this Appendix? I don’t find it in this pdf which is the only file associted to this submission that I can find online. See also comments for Table 2.

Line 247: I would replace “FA” by “fluctuating asymmetry”.

Line 251: I would replace “FA” by “fluctuating asymmetry”.

Discussion:

Line 256: “Our 2-y study”. I would write it as “Our two-year study” as it reads better.

Lines 260-261: “our hypothesis that mayfly biomass would peak in the dry season of La Selva was not supported by our findings”. This is the “prediction” that was not observed. The hypothesis not supported is the “decrease in the risk of mortality from drag during floods”

Lines 264-266: “we found a strong negative relationship between body length and precipitation variability at La Selva. This relationship was consistently significant for males, females, and both sexes combined”. How do the authors interpret this finding? Is it related to e.g. resource availability which would decrease with precipitation? Or maybe related to the necessity of synchrony in emergence, which perhaps obligates nymphs to stay longer (or shorter) periods under water and therefore grow more (or less)? Do they have an explanation for this? I think the explanation given in Lines 310-317 suits well right here.

Lines 274-275: “these studies measured productivity (g/m2/y), while we reported standing-stock biomass (g/m2)”. Can the authors develop this a little bit more? Are these measurements comparable? How?

Lines 282-285: “the lack of abrupt peaks in mayfly emergence found in our study suggests that mayfly emergence is aseasonal. A possible factor explaining the stability in adult emergence is fish predation on newly emerged adults”. However, the authors also mention that this isn’t happening in other studies on tropical streams. Can they provide data about fish abundance or fish predation rate comparing their study to previous studies? If not, this explanation seems to have weak or no support.

Lines 285-286: “Wesner (2016) showed that fish predation decreases the number of insects reaching riparian ecosystems by 40% on average”. Would this 40% be enough to produce aseasonality? What this number would be in this study? Do the authors have an estimate?

Lines 287-289: “Our focal stream is inhabited by the insectivorous poeciliid, Priapicthys annectens, which is abundant (4-14 individuals/m2, Small et al. 2013) and could have negatively affected mayfly emergence”. What about the other studies? Are predation rates or fish abundance lower in those streams having seasonality in emergence?

Lines 292-294: “However, observed peaks in benthic biomass occurred in both dry and rainy seasons in La Selva. This may be due to high floods during the rainy season eliciting microdistributional changes in macroinvertebrates”. However, biomass wasn’t related to any of the environmental variables considered (Lines 229-230), which included “montly precipitation” (Line 165). Thus, I think the results don’t support this interpretation. Maybe monthly precipitation is not accurate enough and other measurements of precipitation may support better this view?

Lines 310-317: This paragraph would be better placed right after Lines 264-266 where an explanation was missing.

Line 300: I would say “In contrast” (or something similar) instead of the more neutral term “Meanwhile”.

Conclusions:

Lines 332-333: “Body length, and not bilateral asymmetry, proved to be a better indicator of environmental stress”. However, what type of stress is normally associated to asymmetry in other studies? Is it anomalous precipitation or is it pollution? In fact, the second hypothesis (see comments for Lines 104-106 for hypothesis definition) was not very well discussed, in the sense that it isn’t that clear whether or not this hypothesis is rejected (as it is done with the first hypothesis, see comments for Lines 260-261). It is true that a trade-off between body length and asymetries is mentioned, rather than truly discussed in the context of the environmental variables in this study (Lines 315-317). In any case, maybe it would be helpful to develop a little bit in the Discussion section which types of environmental stressors are more likely to actually induce asymmetry.

References:

I wouldn’t use DOI numbers as many reference don’t have and so it feels weird (i.e. non-standard) that some have (the more recent) and some don’t (the older). I would use DOI only in case the manuscript has been accepted for publication (or it is already published as EarlyView) but doesn’t have volume and pages assigned yet. However, this is just a personal view and in fact I didn’t even check the journal instructions with regard to this specific detail. But in my opinion DOI numbers make the reference list less neat and do not contribute to clarity in this regard.

Tables:

Table 2: I would specify also here that “Side” is the fixed component and “Individual” the random component of this ANOVA analysis. In this table the authors provide p-values (for the variable “individual” and the categoy “male”) in the form of “p>0.001”. I assume that the correct Table 1 is the one uploaded as “Supplementary Materials”. In fact, this is the only information I was able to find in the “Supplementary Materials”. I would therefore replace Table 1 in the main text by Table 1 in the Supp Info. Also, I was expecting to find “Appendix 1” as part of the Supplementary Materials, but I didn’t find it there nor elsewhere.

Supplementary Materials:

See comments for Table 2.

Sincerely,
the reviewer.


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: MAYFLY EMERGENCE PRODUCTION AND BODY LENGTH RESPONSE TO HYDROLOGY IN A TROPICAL LOWLAND STREAM
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see below
Experimental design: Please see below
Validity of the findings: Please see below
Additional comments: General

This manuscript reports the results of a study of mayfly nymphs and adults in a small first-order stream on the Caribbean slope of Costa Rica. Biomass of nymphs was assessed with a core sampler and biomass of adults with a 2m2 emergence trap monthly over 2 years (nymphs) and weekly (adults). At the same time, 8 environmental variables were recorded monthly (nitrogen as nitrate and ammonium, soluble reactive phosphorous, temperature, pH, conductivity, discharge, precipitation).  The hypothesis addressed was that there would be a peak in benthic and emergent biomass during the low rainfall season due to reduced mortality from drag during floods.  To test this, precipitation was measured rather than flows within the stream because previous research has “demonstrated their influence on LSBS streams”.  Secondly, the authors hypothesized that hydrological stresses suffered by nymphs of the most abundant mayfly, Farrodes caribbianus, would produce bilateral asymmetry in adults and changes in body length. Benthic biomass was variable but was not reflected in emergence biomass which was relatively constant. No evidence of bilateral asymmetry was observed but adult body length was negatively correlated with average precipitation in the preceding 159 days (an estimate of the duration of the nymph stage).  Overall the questions addressed by this study are interesting, the study design is suitable for addressing the questions and the results appear to be appropriately analyzed and interpreted.  Data are clearly presented in tables and figures and the writing is clear.  I recommend that this paper be published after consideration of the questions and suggestions below.

Details
- nymph biomass was estimated from monthly triplicate core samples in leaf-covered runs in a 100m reach of the small (1m wide, 25 cm deep) Carapa Stream.  Can you say anything about spatial variance within this reach so the reader may understand how representative each monthly sampling was and the likelihood that the data in Figure 1 represent temporal variance, as suggested, rather than spatial variance or some combination of spatial and temporal variance?
- when a mayfly nymph emerges from the substrate and leaves the water, where would I find that individual relative to where it had lived in the substrate as a nymph?  In a fast flowing stream I would expect it to leave the water some distance downstream from where the nymph had been in the substrate because the stream would carry it before it had a chance to reach the surface and fly free. I don’t have a feel for this in your stream because I don’t know how fast the flow is or how long the nymph would be in the water column before it is able to break the surface and fly free.  The relevance of this is that, if you want to relate emergent adults to nymphs, you would want to place your emergence trap some distance downstream of the patch of stream in which you sampled nymphs wouldn’t you?  Can you tell us whether you took this into consideration in your sampling design?
- It would be helpful to describe the relationship between rainfall and flow discharge or velocity in the stream to bolster the argument that precipitation relates to increased forces being exerted on nymphs in the substrate.  How strong is the correlation between precipitation and current speed and what temporal lag is there between a rain event and increased flow in your creek?
L70-71 – The logic of this sentence is not clear. Do you mean that bilateral symmetry is known to be best in some way (what way? Reproductive success?) and so any deviation from bilateral symmetry puts the animal at some disadvantage? Given that environmental stresses can produce deviations from bilateral symmetry, measurements of bilateral symmetry are accepted as an index of environmentally relevant perturbation? You go on to make that argument for body length but not explicit for asymmetry.
L76-78 – include references to support this statement
L79 – add “s” to alter
L85 – no need for hyphen between long and periods.  Add “days” after 165
L86 – add “d” to compare
L97 – add “98” after nov. (?)
L104 - define drag – just force of water movement?
L125 – pH of 3.62 is very low – why did the El Nino event of 1997-98 produce this effect?
L151 – define AFDM (presumably Ash Free Dry Mass?)
L170 – change were to was to agree with a suite
L176 – did the same person make all measures?  How close were the measures? Can you provide a measure of variance?
L201 – data were (rather than was)
L204 – add s to model (?)
L233 – tell us why you used a Spearman’s rather than Pearson’s correlation
Figure 2 – I presume the grey band is a 95% Confidence Interval? Explain in figure caption
Table 2 – define all acronyms in Table caption so table can stand alone (i.e., be understood without reference to text)
	- should all > symbols actually be <?
	- Provide degrees of freedom for tests


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: MAYFLY EMERGENCE PRODUCTION AND BODY LENGTH RESPONSE TO HYDROLOGY IN A TROPICAL LOWLAND STREAM
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study presents a substantial amount of data that can potentially provide interesting results if the study would have been framed in a stronger conceptual framework. Unfortunately, the presented manuscript fails to convince and suffers from the lack of a strong theoretical background. Some statements by the authors are not supported by any literature. For example, in lines 71 to 78, genetic causes are presumed to lead to directional symmetry and antisymmetry while fluctuating asymmetry is presumed to be caused by environmental stressors. Would that refer to phenotypic plasticity? Stronger arguments are needed.
Experimental design: My main concern is how hydrology was handled and considered as a stressor for the benthic communities. The variable chosen by the authors as a proxy for hydrology was average precipitation, and later on, they expand in the discussion with direct links to flood as the main cause for change in F. caribbianus total body length. While the relationship between precipitation and body length was significant, it is very important to remind the reader that this decrease in body size was established only on 117 individuals. A very low number of individuals to be analyzed considering this taxon to be the most abundant mayflies in the study site. Also, precipitation can be highly correlated to changes in air temperatures. Did the authors examine such correlation? How can the authors be certain that precipitation (then flow), is behind the change of total body length? wouldn’t it be the temperature?
Validity of the findings: The findings are partially speculative and fail to establish a clear causality relationship between hydrology and body length.
Additional comments: Here are few more minor mistakes that need to be addressed, even if at that point I did not thoroughly examine the manuscript for grammatical and typographical mistakes.

Lines 187 to 190: More information on the model selection needs to be provided in the manuscript. Referring to published literature is not sufficient.

Line 221 to 223: Emergence represents 24.4 to 14.9% of the benthic biomass? please clarify.

Line 85: Add “days”, after range from 26 to 165 …
Line 123: Silt and clay are fine sediments not only sediments.

Table 2: When using codes in the table please introduce them in the caption, and no need for a separate table for that.
Figure 2: Need to specify in the caption that this data represents only Farrodes caribbianus.


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: MAYFLY EMERGENCE PRODUCTION AND BODY LENGTH RESPONSE TO HYDROLOGY IN A TROPICAL LOWLAND STREAM
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors' revision of their manuscript addressed all of my concerns (and from my reading most concerns raised my the other two reviewers) adequately with the exception of three:
1. Nymph biomass was estimated from monthly triplicate core samples in leaf-covered runs in a 100m reach of the small (1m wide, 25 cm deep) Carapa Stream. Can you say anything about spatial variance within this reach so the reader may understand how representative each monthly sampling was and the likelihood that the data in Figure 1 represent temporal variance, as suggested, rather than spatial variance or some combination of spatial and temporal variance?
2. did the same person make all measures? How close were the measures? Can you provide a measure of variance
3. data were (rather than was)
      I consider these sufficiently minor that I am happy to recommend acceptance of the manuscript at this point.
Experimental design: as above
Validity of the findings: as above
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: MOLECULAR CHARACTERISTIC OF ACTIVIN RECEPTOR IIB AND ITS FUNCTIONS IN GROWTH AND NUTRIENT REGULATION IN ERIOCHEIR SINENSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the manuscript entitled "Molecular characteristic of activin receptor IIB and its functions in growth and nutrient regulation in Eriocheir sinensis", the authors cloned the full-length cDNA of EsActRIIB, and further evaluated the possible role of EsActRIIB in the growth, molting and nutrition regulation with RNAi. Overall, this study is interesting and meaningful. However, there are some drawbacks regarding the materials, results and English writing that seems to be inappropriate to potentially publish the manuscript.

My main concerns are about the introduction, materials & methods, analytical methods:

1. The authors spend a lengthy text describing the role of ActRIIB in vertebrates, but the study on invertebrates is rarely mentioned in the manuscript. The authors indicate that the TGF-β superfamily consists of a large number of structurally and functionally related cytokines subfamilies, however, the authors fail to indicate whether the same family identification relates to the invertebrate ActRIIB receptors. Is the ActRIIB receptor studied in this paper closely related to the members of the mammalian family of receptors?

2. The relationship between the background and study purpose described by the author is very vague, which caused this research seems not to make much sense. Line 116-126: " Unfortunately, in E. sinensis aquaculture, the weight gain rates of crab individuals after molting display significant difference even in the same culture environment, which results in low production of this highly demanded crab…The present research was performed…" this does not seem to be sufficient support for conducting this study. I recommend re-working on the introduction and discussion so that the potential importance of these results might become more obvious.

3. Line 134: "…sampled from four healthy individuals of crab at the four mentioned molting stages", does the authors mean that there is only one individual per molting stages?

4. Fig 4: The authors first examined the distribution of the EsActRIIB mRNA expression levels in 11 tissues (four molting stages), and then analyzed the differences in Mw, Mc and Mp tissues at different molting stages. However, the tissue distribution results show that the EsActRIIB mRNA expression levels in G, I, Tg and Ep are significantly higher. So why didn't the authors choose to analyze these tissues? In addition, I seriously question about the last four results (Fig 4 Hp, Mw, Mc and Mp). Their analysis methods are wrong and unscientific.

5. Table 1: In the control group, the molting interval time of juvenile crabs (4.54 ± 0.85 g) has reached more than 50 days (50.14 ± 7.44). The authors need to explain and clarify what causes the molting cycle to take so long? Are these results scientific? Are these crabs healthy? Are the culture conditions suitable? In addition, the authors need to add the following details in the "Materials Method" section: month, culture conditions, monoculture or mixed culture, etc. These are very important and essential information.

6. In this study, the single molting cycle of juvenile crabs even exceeded 50 days, so I am skeptical of the results and conclusions obtained from this study, as well as their objectivity if the authors cannot explain the reason logically.

Other concerns：

7. The entire manuscript requires extensive editing for format errors and English grammar, and in order for the language to meet with the published standards, it was recommended that some native English speaker should be involved in revising the manuscript. For examples, the abbreviations used for the first time need to coexist with the full name, such as line 42, 53-56. Non-first-time Latin names need to be abbreviated, such as line 59. These are examples taken only from the Abstract. Also, Latin names should be in italics, such as line 411, 435, 439… The authors need to check the entire manuscript.

8. There are no "keywords" found in the manuscript. The authors need to confirm it.

9. The authors describe on line 95 that "blocking ActRIIB signaling reduces fat contents", while " inhibition of ActRIIB signaling enhances obesity and obesity-linked metabolic diseases" on line 99. I wondered about the relationship between ActRIIB signaling and lipid metabolism, and what the authors really  wants to express.

10. Line 130: If the authors would like to make a statement about the health of the crabs, they must also indicate how the health of the crabs was assessed.

11. Line 130: The authors need to supplement the crab's weight (mean and standard error) in the manuscript, only "juveniles" is ambiguous and inaccurate.

12. Line 133: Much of the information provided by the authors about sample collection is ambiguous. For examples: Are the "gill" collected by different crabs the same type? As far as I know, the front and back gills seem to play different roles. The same comments can be made for the "walking leg muscle".

13. Line 131-133: The authors divide the molting cycle into premolt (PrM), molt (M), postmolt (PoM) and intermolt (InM) stages, what are the criteria and methods for classification? And (Chen et al., 2017) is not an initial citation, the authors need to verify and provide accurate information.

14. Line 144: What does "he" mean?

15. Was the possibility of genomic DNA contamination taken into account in the total RNA samples?

16. Did the authors sequence the product(s) of their qPCR reactions or products obtained otherwise by using the primers they used for qPCR? What is the length (size) of their qPCR products? The authors detect what they believe to be the EsActRIIB mRNA in all organs/tissues examined but have not determined if the products of their PCR reactions are indeed the EsActRIIB mRNA. The same comments can be made for the reference genes.

17. Line 192: In the "RNA interference conduction" section, why did you choose this injection concentration and volume?

18. Line 223: Is it 48h after the first injection? The authors need to clarify it.

19. Line 313-316: Since the author's data analysis is unscientific (Fig 4), the results obtained are also unreliable. Therefore, this part of the discussion is not credible.

20. Line 344: "…the lipid metabolism…be blocked", does the authors mean that lipid metabolism is not working?


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: MOLECULAR CHARACTERISTIC OF ACTIVIN RECEPTOR IIB AND ITS FUNCTIONS IN GROWTH AND NUTRIENT REGULATION IN ERIOCHEIR SINENSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are small errors (quite a lot) in the writing, some sentences are ambiguous (some of them have been high lightened in the annotated PDF)
Should add some references closely related with this study. Such as:
田志环，焦传珍，成永旭*，吴旭干. 中华绒螯蟹Akt (EsAkt)的cDNA克隆、序列分析及表达特征研究，水产学报，2018，42（4）：485-494.
田志环，焦传珍*，成永旭.中华绒螯蟹Smad3 (EsSmad3)的cDNA克隆、序列分析及表达特征研究，中国水产科学，2018，25（2）：316-324.
Identification of a transforming growth factor‑β type I receptor transcript in Eriocheir sinensis and its molting‑related expression in muscle tissues, Molecular Biology Reports (2020) 47:77–86.
Zhihuan Tian, Guangchun Lin, Chuanzhen Jiao*.Identification of an S6 kinase transcript in the Chinese mitten crab Eriocheir sinensis, and its molting-related expression in muscle tissues. Fisheries Science, 2019, 85:737–746.

Should add discussion on the nutrient changes in the RNAi groups, especially on the amino acid. what’s the molecular story under this result?
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This study is very interesting. On whether knocking down Myostatin signal in crustaceans will promote or inhibit the animal’s growth, there were conflict results reported before. This study supports the promotion view, it is inspiring on the potential application in Aquaculture.


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: MOLECULAR CHARACTERISTIC OF ACTIVIN RECEPTOR IIB AND ITS FUNCTIONS IN GROWTH AND NUTRIENT REGULATION IN ERIOCHEIR SINENSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript looks very messy, which makes it difficult for me to find and
review the author's changes in the text. The author needs to highlight the revised
content in yellow and indicate the line number, rather than the current form.
Experimental design: See comments for the author
Validity of the findings: See comments for the author
Additional comments: Although the authors have responded to the reviewer's comments, there are still some
comments that need to be corrected before publication:

Line 183: "...we collected the relative tissues of four crabs..." This sentence is still
ambiguous.
Regarding previous question "The authors first examined the distribution of the
EsActRIIB mRNA expression levels in 11 tissues (four molting stages), and then
analyzed the differences in Mw, Mc and Mp tissues at different molting stages.
However, the tissue distribution results show that the EsActRIIB mRNA expression
levels in G, I, Tg and Ep are significantly higher. So why didn't the authors choose to
analyze these tissues?", the author's explanation is far-fetched. According to the
author's response, I would like to know what was the purpose of the author's initial
examination of the EsActRIIB mRNA expression levels in 11 tissues?
In addition, the analysis method seems to be incorrect and unscientific despite the
revision made by the authors (Fig 3 Hp, Mw, Mc and Mp), and it is suggested that the
authors discuss the analysis method carefully. If possible, the authors should upload
the original data.


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: MOLECULAR CHARACTERISTIC OF ACTIVIN RECEPTOR IIB AND ITS FUNCTIONS IN GROWTH AND NUTRIENT REGULATION IN ERIOCHEIR SINENSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: non
Experimental design: non
Validity of the findings: non
Additional comments: As I had mentioned before, the method the authors used to normalize the real-time quantitative RT-PCR data is a single internal control gene method (known from the reference the authors cited). However, the real-time quantitative RT-PCR data should be accurately normalized by geometric averaging of multiple internal control genes due to the multiple internal control genes (β-Actin, S27, and UBE) were used in the present manuscript. The authors should revise the calculating method and calculate the relative gene expression by Vandesompele, et al. (2002).

In addition, the authors described in the text that the relative expression level of target genes were estimated by the 2-ΔΔ△△Ct method according to the Livak & Schmittgen (2001). However, when I reviewed the original data, I found that the authors did not use this method, therefore, the authors need to check and correction.

(Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biology, 2002, 3(7): 1-12.)


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: GENOMIC DIVERSITY AND EVOLUTION, DIAGNOSIS, PREVENTION, AND THERAPEUTICS OF THE PANDEMIC COVID-19 DISEASE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript presents an attempt of comprehensive representation of the literature concerning SARS-CoV-2, with three-pronged approach: 1) structure and evolution, with a review of mutational landscape; 2) treatments; 3) development of the vaccines. While this manuscript is having certain educational value in its broad coverage, at this point its scope is too broad for a journal paper. I would recommend selecting just one of these "topical areas" and go further in depths. If I may suggest, mutation/evolution aspect of this paper is developed best, may be authors should concentrate on that, while making forays into adjacent areas (i.e. connection between mutations in particular epitopes and vaccine properties of these epitopes). Also, diagnostics approaches may be included, as they are directly related to analysis of viral sequences.

As it is written now, the treatments portion is very sketchy and does not go beyond naming candidate treatments, and the vaccine portions somehow not discussing natural anticoronavirus immunity, and recent studies about antibody levels and strengths of immunity post-infection. These parts are underdeveloped, and, therefore, should be reserved to future. separate, manuscripts.
Experimental design: the Review is well organized, but is too broad. In its broadness, it is missing important aspects (i.e. immunity, mechanisms of antiviral action for named compounds). I propose that authors should narrow the scope while increasing the depths and interpretation
Validity of the findings: as such, this study does not argue any particular point of view except stating that coronavirus research is actively developing area. Because of that, conclusions and perspectives part seem trivial. Also, Fig.1 is completely unneccessary.
Additional comments: this is a good effort, which needs a bit of focusing before publication. One cannot cover entire Universe, one will be spread too thin!


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: GENOMIC DIVERSITY AND EVOLUTION, DIAGNOSIS, PREVENTION, AND THERAPEUTICS OF THE PANDEMIC COVID-19 DISEASE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The review (article Id 48746) submitted by Houque et al. reported on i)  Genome analysis and evolution of SARS-Cov-2, ii) diagnostic tools and iii) updating the therapeutics and vaccine development against COVID19. The review is well organized and well written and I recommend it publication in PeerJ. I have two main comments:
1-	The authors mentioned that the S-Protein of SARS-Cov-2 possesses furin cleavage site at S1 and S2 boundary (Walls et al., 2020). This finding was also published by Coutard et al (2020). This furin-like cleavage is absent in betacoronaviruses of lineage b including SARS-CoV sequences. I suggest if the author can discuss this findings deeper to shed lights on the potential origin (Can this a natural evolution for Coronaviruses?), the consequences, and the impacts on treatment. Based on this finding, is SARS-CoV-2 a manipulated laboratory construct?
I do not believe also that any type of laboratory-based scenario is plausible, based on the available data,  but I think it will be great if the authors discussed this point in more details.
These links might be helpful (of course only publications can be cited)
Andersen, K.G., Rambaut, A., Lipkin, W.I. et al. (2020): The proximal origin of SARS-CoV-2. Nat Med 26, 450–452.
Coutard et al. (2020): The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Research 176: 104742
Follis, K. E., York, J. & Nunberg, J. H. Virology 350, 358–369 (2006)
https://nerdhaspower.weebly.com/blog/scientific-evidence-and-logic-behind-the-claim-that-the-wuhan-coronavirus-is-man-made
https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748

2-	Figure (1), I suggest to delete it. The review is very rich and there is no need for this figure.

Other comments:
-	Line 83: please add the reference
-	Line 181: Please delete the word spike (use the abbreviation “S”), the full name was mentioned in line 103
-	Line 130: Please delete the word “and” before antifungal treatment
-	Line 143: Again S protein Not spike protein
-	Line 173: Please use the abbreviation ICTV, the full name was mentioned in line 63
-	Lines 187-188: Please use abbreviations of S, N, E, and M; also please delete fig 1.
-	Line 191: Please delete  “open reading frame” and use abbreviation, it is mentioned in line 85
-	Line 196: 29,844 (coma)
-	Line 197: 29,752 and 30, 119 (coma)
-	Line 197 (add aa after 7,078 to be 7,078 aa)
-	Line 201-202: use abbreviations E, M, and N
-	Line 202: please use a uniform writing:  beta coronavirus, betacoronavirus or beta-coronavirus?
-	Line 237: S
-	Line 241: coma after the word respectively
-	Line 253: should be: National Center for Biotechnology Information (NCBI)
-	Lines 258-260: Please explain your findings in more details
-	Line 272: coma after respectively.
-
-


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: GENOMIC DIVERSITY AND EVOLUTION, DIAGNOSIS, PREVENTION, AND THERAPEUTICS OF THE PANDEMIC COVID-19 DISEASE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Please refer to general comments to authors. Issues noted.
Experimental design: Please refer to general comments to authors. Issues noted.
Validity of the findings: Please refer to general comments to authors. Issues noted.
Additional comments: In this manuscript, the authors’ goal is to write a “comprehensive review on genomic diversity and evolution, diagnosis, prevention, and therapeutics of the SARS-CoV-2.” The overall constructive criticism is that it is an overambitious review with frequent superfluous sentences. There are many major points for authors’ consideration.

•	The review contains many superfluous sentences that do not add critical analysis or provide scientific insights. Example in Lines 605- , “We expect researchers and/or companies who are working round the clock will bring a new SARS-CoV-2-based vaccine from isolated virus particle or gene sequences to clinical testing within a short period of time.”
•	COVID-19 is the disease, and SARS-CoV-2 is the virus. The terms are not interchangeable as authors suggested.
•	Lines 184-185: “The positively-sensed single-stranded RNA SARS-CoV-2 virus (Ahmed et al., 2020) has a genome size of 26,000 and 32,000 bases (Abdelmageed et al., 2020). It shares more than 80% sequence identity to the previously reported human coronaviruses.” The genome size that the authors provide is the range for the coronavirus genus. Authors should look carefully to precise data on the SARS-CoV-2 genome length.
•	The authors stated: “Genomic analyses have indicated that the virus, popularly named as corona, is probably not a purposefully manipulated laboratory construct.” “Corona” may be is considered a popular name for the virus these days, as authors claimed. However, this information should not be considered in a scientific review. In addition, the authors lost the opportunity to discuss the scientific reasons why SARS-CoV-2 is unlikely a laboratory construct.
•	There are many paragraphs that lack focus. As an example, lines 113-119.  Do the authors want to write on entry, virulence, or epidemiology?
•	Figure 2 needs some revision. ORF is not present in all human pathogenic CoVs. Also, in MERS, ORF4a is shown twice; please correct the second for 4b.
•	Figure 4 shows “The dynamic curve showing daily increase in complete genome sequences of SARS-CoV-2 strain (s) from different patients across the globe, and being submitted to the reference databases. The data were collected from China National Center for Bioinformation 2019.” However, in Lines 462-464, authors mentioned Fig 4 as “The new cases of active acute infections are being added to the open COVID-19 database every day.
•	The manuscript must be edited throughout. There are multiple grammatical errors, including mistakes in the use of “are” vs. “is” are very frequent. Example: Lines 115-116: “However, the virulence mechanisms of the SARS-CoV-2 is not yet clear.”


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

Input:
Title: GENOMIC DIVERSITY AND EVOLUTION, DIAGNOSIS, PREVENTION, AND THERAPEUTICS OF THE PANDEMIC COVID-19 DISEASE
Review round: 2
Reviewer: 1

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


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

Input:
Title: SERUM PROTEINS DIFFERENTIALLY EXPRESSED IN EARLY- AND LATE-ONSET PREECLAMPSIA ASSESSED USING ITRAQ PROTEOMICS AND BIOINFORMATICS ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: The use of small sample (4 per sub-group) size should be justified.
Validity of the findings: 1. Were the top three differentially expressed proteins equal in all the samples of the sub-groups?

2. LRP1, APOC1, and APOL3 were linked to which gestational sub-group?

3. Whether the differentially expressed proteins are placenta-specific needs be clarified. If not, how do you justify them as a biomarker?

4. Power analysis should be carried out to determine the acceptability of the findings.
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: SERUM PROTEINS DIFFERENTIALLY EXPRESSED IN EARLY- AND LATE-ONSET PREECLAMPSIA ASSESSED USING ITRAQ PROTEOMICS AND BIOINFORMATICS ANALYSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the authors analyze a set of serum samples obtained either from control individuals or patients diagnosed with early and late preeclampsia. Differential quantitative proteomics experiments have been carried out using iTRAQ 8-plex isobaric labeling as an experimental approach, followed by LC-MS analysis of the samples. The main objective of this work is to describe proteins differentially expressed between the control and problem (preeclampsia) samples in order to find new clues related to this pathology. This is an objective of great biomedical interest, since preeclampsia has a great impact on the human population, causing high economic and human costs, and for which clear and unambiguous biomarkers useful for diagnosis and prognosis have not yet been found.  Both the introduction and, above all, the discussion of the results adequately pose the problem and the experimental context. In addition, the manuscript is well written and easy to understand. For everything mentioned above, it is a manuscript of potential interest for publication. However, significant improvements must be done, particularly in the data presentation format, as described below.
Experimental design: The experimental design proposed by the authors is not novel, but it is adequate to try to obtain the answers sought, although it could be objected that the number of samples used and representative of each condition is somewhat low. Likewise, it would be advisable to include some experiments to validate at least some of the so-called differentially expressed proteins identified here, either using MRM directed proteomic techniques or other approaches based on the use of antibodies, such as Western Blot analysis. The validation should ideally be performed on a cohort of samples not related to that used in the first phase.
Validity of the findings: MAJOR POINTS:
The presentation of the experimental data requires further improvement. For example, in the results file corresponding to LOPE, 8 data columns (supposedly corresponding to 4 control samples + 4 LOPE samples) are displayed. However, in the EOPE file, 16 data columns are shown, and apparently it includes all (8) the control samples and both 4 LOPE samples + 4 EOPE samples. Please modify this as it only serves to create confusion. In addition, the authors should indicate in the table which samples correspond either to the control or preeclampsia groups.

Lines 135-140: Data processing and protein identification and quantification analysis methodology is confusing and contains some spelling mistakes, for example, writing Unipro instead of UniProt. It should be explained why using ProteinPilot to analyze the results instead of using other alternatives recommended by Thermo (please note that data were obtained in a Thermo Orbitrap Fusion Lumos mass spectrometer) such as ProteomeDiscoverer. Other common alternatives, and by far more used by the proteomics community, include freely available software like MaxQuant.

In addition, there is no coincidence when the proteins found in the result files and the protein numbers mentioned in the text are compared. Thus, 65 and 132 proteins, respectively, are shown in LOPE and EOPE files, values that do not match the total number of proteins identified in both data sets (413 for EOPE, 470 for LOPE) or the proteins described as differentially regulated (70 and 24 for EOPE and LOPE, respectively). I do not understand why quantification values corresponding to all the proteins, either differentially expressed or not, identified in both data groups (EOPE and LOPE) are not shown. I suggest that the authors present the data so that we can:
1) Have the quantification values of all the proteins identified in each data set.
2) highlight the values corresponding to differentially regulated proteins in each data set independently.

Regarding the quantification values presented in the table, it is well known that the number of decimal numbers should reflect the accuracy of the technique used. It is not a proper practice to present the data with such a high number of decimal numbers, since differential quantitative proteomic analysis using iTRAQ 8plex is far from reaching such high accuracy. I suggest the authors to use 2 or 3 decimal numbers in the tables, because it will help to better understand the data.

MINOR POINTS:

Line 118: dithiothreitol final concentration
Line 118: iodoacetamide final concentration
Line 120: UA? Please detail
Iine 125: 75 micrograms of sample are obtained but the protocol starts with 60 micrograms (line 117): please clarify this point

Discussion:
I was surprised that the authors did not include in the discussion any mention to the protein “cDNA FLJ75401, highly similar to Homo sapiens endoglin”, described as differentially expressed protein in the EOPE dataset, since this protein has been previously described as a putative preeclampsia marker (Venkatesha S, Toporsian M, Lam C, Hanai J, Mammoto T, Kim YM, Bdolah Y, Lim KH, Yuan HT, Libermann TA, Stillman IE, Roberts D, D'Amore PA, Epstein FH, Sellke FW, Romero R, Sukhatme VP, Letarte M, Karumanchi SA. Soluble endoglin contributes to the pathogenesis of preeclampsia Nat. Med. 2006 Jun; 12 (6): 642-9.). Particularly interesting would be to discuss the presence of this protein in the EOPE but not in the LOPE dataset.
Additional comments: no comment


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

Input:
Title: SERUM PROTEINS DIFFERENTIALLY EXPRESSED IN EARLY- AND LATE-ONSET PREECLAMPSIA ASSESSED USING ITRAQ PROTEOMICS AND BIOINFORMATICS ANALYSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript entitled “Serum proteins differentially expressed in early- and late-onset preeclampsia assessed using iTRAQ proteomics and bioinformatics analyses” by Tu and his co-authors reported the differently expressed serum proteins in early- and late-onset preeclampsia. The manuscript reported some new findings that are associated with preeclampsia.

The English language used is a professional standard.
Literature and references are sufficient.
Article structure, figures, and tables are sufficient and standard.
Experimental design: The only limitation is the small number of samples.
Validity of the findings: The data provided are robust and statistically sound. The authors explained their findings in a proper way.
Additional comments: The manuscript entitled “Serum proteins differentially expressed in early- and late-onset preeclampsia assessed using iTRAQ proteomics and bioinformatics analyses” by Tu and his co-authors reported the differently expressed serum proteins in early- and late-onset preeclampsia. The manuscript reported some new findings that are associated with preeclampsia.


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: SERUM PROTEINS DIFFERENTIALLY EXPRESSED IN EARLY- AND LATE-ONSET PREECLAMPSIA ASSESSED USING ITRAQ PROTEOMICS AND BIOINFORMATICS ANALYSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: See attachment
Experimental design: See attachment
Validity of the findings: See attachment
Additional comments: See attachment


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: SERUM PROTEINS DIFFERENTIALLY EXPRESSED IN EARLY- AND LATE-ONSET PREECLAMPSIA ASSESSED USING ITRAQ PROTEOMICS AND BIOINFORMATICS ANALYSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Please see my first report
Experimental design: Please see my first report
Validity of the findings: Please see my first report
Additional comments: The authors have made an effort to introduce the changes and modifications suggested in the previous version. As a consequence, I understand that the overall quality of the manuscript has improved substantially and that now it can be considered acceptable for publication. I would just like to suggest a few minor changes to the text that are detailed below.
Line 33: What is exactly the meaning of the expression “an absolute log10 fold change (i.e., >1.2 difference)”? It seems confusing to me. Could it be expressed more clearly?
Line 48: Use Downregulation instead of “The downregulated”
Line 83: What does it mean R 2007?
Line 127: The expression “All the remaining samples were used” is unclear
Line 128: Use  "After labeling with iTRAQ" instead of "After labeling". In addition it should be specified the iTRAQ version (4plex? 8plex?) across the manuscript.
Line 134-135: methyl cyanide is correct but very few people use it. It is much popular to use acetonitrile.
Line 155: please specify the reference of the kit used
Line 156: use "manufacturer instructions"  instead of "kit instructions"
Line 197: It is more appropriate to use "interact" instead of writing "coordinate with one another"
Line 231: the expression "we used ELISA to identify again" is grammatically wrong, but it is also meaningless. Please correct.
Line 255: Use  "previous evidence"  instead of  "much evidence"
Line 278: Factor VII instead of factor VI?
Line 284:" consumes" is inappropriate in this context, please use a more adequate expression.
Line 291: Use "support" instead of "add to"
Line 339: please use "previous studies have shown that" instead of "a study shows that"
Line 340: please use "which is involved"  instead of "which involved"
line 342: please use "were similar"  instead of "was similar"