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: ASSESSING KINESTHETIC PROPRIOCEPTIVE FUNCTION OF THE UPPER LIMB: A NOVEL DYNAMIC MOVEMENT REPRODUCTION TASK USING A ROBOTIC ARM
Review round: 2
Reviewer: 1

Output:
Basic reporting: Is good and clear.
Experimental design: Is great.
Validity of the findings: Is acceptable.
Additional comments: I would like to thank the authors for addressing my initial comments. Following the revision to the article, the current version is a clearer, concise, and well-written manuscript.


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

Input:
Title: ASSOCIATION OF SLEEP DURATION AND SLEEP QUALITY WITH HYPERTENSION IN OIL WORKERS IN XINJIANG
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: The study investigates the relationship between sleep problems and hypertension.

In the Introduction, among the numerous factors associated with hypertension (L.41), the authors forget to include stress.

In the review by Bergmann et al. [The appraisal of chronic stress and the development of the metabolic syndrome: a systematic review of prospective cohort studieshttp://www.endocrineconnections.org DOI: 10.1530/EC-14-0031] many studies on the relationship between chronic stress and hypertension are reported.

Work stress is significantly associated with sleep problems.

Sleep problems, in fact, increase the sensibility to work stress [Magnavita N, Garbarino S. Sleep, Health and Wellness at Work: A Scoping Review. Int J Environ Res Public Health. 2017 Nov 6;14(11). pii: E1347. doi: 10.3390/ijerph14111347[, and stress increases sleep problems.

Sleep acts as a moderating factor in the relationship between stress and hypertension [Garbarino S, Magnavita N. Sleep problems are a strong predictor of stress-related metabolic changes in police officers. A prospective study. PLoS One. 2019 Oct 22;14(10):e0224259. doi: 10.1371/journal.pone.0224259].

This clarification is necessary to clarify the complexity of the factors influencing hypertension.

Unfortunately, the study did not take into account occupational stress, which can be important for hypertension and also for sleep problems.

This is a limitation of the study, which should be discussed by the authors.
Validity of the findings: The major limitation of the study is that it is a cross-sectional study. It is not legitimate to infer about causality in this type of study. In fact, there could be a reverse causation, the fact of suffering from hypertension could disturb sleep. Authors should discuss this point. They should avoid the causal interpretation of their findings, and change their claims non-purposely. It will be fair to say that impaired sleep is associated with hypertension, not that it causes hypertension.
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: ASSOCIATION OF SLEEP DURATION AND SLEEP QUALITY WITH HYPERTENSION IN OIL WORKERS IN XINJIANG
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author mentioned that the desert petroleum workers are a special group of people, the author should give more instruction of the population. Many similar studies has been determined previous and this study in some extent is lack of innovation.
Experimental design: Methodology section:
1) Many important variables should given more instruction, such as the definition of smoking, drinking and shift work
2) Lack of some important references, for example, PSQI questionnaire.
“The Pittsburgh Sleep Quality Index (PSQI) was used to assess participants' subjective sleep quality and sleep duration. The survey used the Chinese version of the Pittsburgh Sleep Quality Index (CPSQI), which was Cronbach's alpha of 0.89 and a KMO of 0.91. CPSQI consists of 7 factors and there are 19 items. Each item is scored from 0 to 3 according to the 4-level Likert scale. In this study, CPSQI ≤ 5 was considered ad poor sleep quality, whereas a score of >5 was regarded as poor sleep quality. Sleep duration was classified into “insufficient” (﹤7 h), “normal”(7- 8h), and “long”(>8 h) based on previous reports.”
Validity of the findings: Result section: Since this study is a cross-sectional study, it cannot be determined causal relationship. It not correct that poor sleep quality are risk factors for hypertension.

Discussion part: The content of the discussion is not clear and lack of logic.
1)The author has missed some discussion of the relationship between some variables and hypertension.
2) In addition the result of this study suggested the prevalence of hypertension was 15.3%, which was significant lower than the figure that 37% published in previous study (Jiang Lixin et al., 2017). The author should discuss the difference in the rate of hypertension.
Additional comments: The content of this study and the languages should be improved.


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

Input:
Title: ASSOCIATION OF SLEEP DURATION AND SLEEP QUALITY WITH HYPERTENSION IN OIL WORKERS IN XINJIANG
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is clear, but english could be improved.
Experimental design: 1)Many important variables should given more instruction, such as the definition of smoking, drinking and shift work
2) Lack of some important references, for example, PSQI questionnaire.
“The Pittsburgh Sleep Quality Index (PSQI) was used to assess participants' subjective sleep quality and sleep duration. The survey used the Chinese version of the Pittsburgh Sleep Quality Index (CPSQI), which was Cronbach's alpha of 0.89 and a KMO of 0.91. CPSQI consists of 7 factors and there are 19 items. Each item is scored from 0 to 3 according to the 4-level Likert scale. In this study, CPSQI ≤ 5 was considered ad poor sleep quality, whereas a score of >5 was regarded as poor sleep quality. Sleep duration was classified into “insufficient” (﹤7 h), “normal”(7- 8h), and “long”(>8 h) based on previous reports.”
Validity of the findings: Result section: Since this study is a cross-sectional study, it cannot be determined causal relationship. It not correct that poor sleep quality are risk factors for hypertension.

Discussion part: The content of the discussion is not clear and lack of logic.
1)The author has missed some discussion of the relationship between some variables and hypertension.
2) In addition the result of this study suggested the prevalence of hypertension was 15.3%, which was significant lower than the figure that 37% published in previous study (Jiang Lixin et al., 2017). The author should discuss the difference in the rate of hypertension (cite also: Magnavita N, Di Stasio E, Capitanelli I, Lops EA, Chirico F, Garbarino S. Sleep Problems and Workplace Violence: A Systematic Review and Meta-Analysis. Front Neurosci. 2019;13:997. Published 2019 Oct 1. doi:10.3389/fnins.2019.00997)
Additional comments: The paper is good, but you should improve some aspects of the paper.


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

Input:
Title: ASSOCIATION OF SLEEP DURATION AND SLEEP QUALITY WITH HYPERTENSION IN OIL WORKERS IN XINJIANG
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have modified the text, introducing new references. However, their work needs careful review, because some items are cited twice, and other references are inappropriate. For example, at line 261 the appropriate references are (Magnavita N, Garbarino S. Sleep, Health and Wellness at Work: A Scoping Review. Int J Environ Res Public Health. 2017;14(11). pii: E1347. doi: 10.3390/ijerph14111347), and (Garbarino S, Magnavita N. Work stress and metabolic syndrome in police officers. A prospective study. PLoSOne, 2015; 10 (12):e0144318. doi: 10.1371/journal.pone.0144318).

The sentence reported on line 263 "some studies have shown an ambiguous relationship among sleep, occupational stress, and hypertension" is inappropriate. The study cited [Garbarino S, Magnavita N. Sleep problems are a strong predictor of stress-related metabolic changes in police officers. A prospective study. PLoS One. 2019;14(10): e0224259. doi: 10.1371/journal.pone.0224259] indicates that sleep is an important moderator of the relationship between stress and hypertension or other components of the metabolic syndrome.
Experimental design: Research question well defined, relevant & meaningful.
Validity of the findings: Data are robust, statistically sound, & controlled.
Additional comments: The authors have modified the text, introducing new references. However, their work needs careful review, because some items are cited twice, and other references are inappropriate. For example, at line 261 the appropriate references are (Magnavita N, Garbarino S. Sleep, Health and Wellness at Work: A Scoping Review. Int J Environ Res Public Health. 2017;14(11). pii: E1347. doi: 10.3390/ijerph14111347), and (Garbarino S, Magnavita N. Work stress and metabolic syndrome in police officers. A prospective study. PLoSOne, 2015; 10 (12):e0144318. doi: 10.1371/journal.pone.0144318).

The sentence reported on line 263 "some studies have shown an ambiguous relationship among sleep, occupational stress, and hypertension" is inappropriate. The study cited [Garbarino S, Magnavita N. Sleep problems are a strong predictor of stress-related metabolic changes in police officers. A prospective study. PLoS One. 2019;14(10): e0224259. doi: 10.1371/journal.pone.0224259] indicates that sleep is an important moderator of the relationship between stress and hypertension or other components of the metabolic syndrome.


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: OUTCOMES OF CRITICALLY ILL END-STAGE KIDNEY DISEASE PATIENTS WHO UNDERWENT MAJOR SURGERY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some English language issues will likely be cleared up in the editing process.

A few of note:

1) line 16- 1%11% should be 1-11%

2) line 35- "We conducted a retrospective matched cohort" should be "We conducted a retrospective matched cohort study"

3) line 46- "criteria" should be "characteristics"
Experimental design: 1) mention in the abstract and in line 92 that "multivariable" logistic regression analaysis was performed.

2) The authors state they performed multivariate analysis with only a few variables (age, SOFA-renal score, ESKD) to avoid "overparameterization". However, it is standard practice in these types of studies to perform univariate logistic regression with all relevant variables, then identify those that are significant or approach significance (usually with P value <0.1) and include them in multivariate analysis. It would be preferable if the authors could do this. If not, I accept their explanation of "overparameterization", but this should also be mentioned in the methods section when discussing the multivariate logistic regression analysis.

3) I would classify the ICU comorbidities as post-surgical outcomes and include them as secondary outcomes, rather that baseline characteristics. As such, I think they would benefit from being in a separate table instead of included in Table 1.
Validity of the findings: 1) in the abstract, the sentences "Patients baseline characteristics between the ESKD and non-ESKD were similar as in priori matching criteria, which were all p>0.05). Pre-existing diabetes mellitus and hypertension were found more in ESKD significantly (p=0.03 and 0.04, respectively)" contradict each other. It should be mention that baseline characteristics were similar "except for" diabetes and hypertension.

2) It would be worthwhile for the authors to analyse and discuss the causes of mortality in the ESKD and non-ESKD cohorts and if there was a difference herein. I can see that the cohorts did not differ in terms of incidence of comorbidities during their ICU stay. Therefore, which comorbidities actually led to the increased mortality of ESKD patients?
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: OUTCOMES OF CRITICALLY ILL END-STAGE KIDNEY DISEASE PATIENTS WHO UNDERWENT MAJOR SURGERY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Discussion section needs to be revised. Also, the overall paper can benefit from English editing. See my comments for the details.
Experimental design: There are several minor concerns regarding the choice of matching criteria, as well as outcome variables. See my comments.
Validity of the findings: Findings can be further validated using similar works from the literature. I mentioned a few areas for improvement in my comments.
Additional comments: See the 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: OUTCOMES OF CRITICALLY ILL END-STAGE KIDNEY DISEASE PATIENTS WHO UNDERWENT MAJOR SURGERY
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a retrospective study conducted with ESRD patients underwent major operation in single institute. The manuscript is well written, but still lack of something need to elucidate. 1. As the title mentioned, the study groups were ESRD and no-ESRD, however in the ESRD group in post-operative ICU care, what did the patients receive dialysis therapy, still HD or CVVH?
2.In the comparison group, ie, no-ESRD patients, was there any AKI, acute kidney injury, and need further transient treatment to stablize their hemodynamic, fluid status, intake/output...?
3. What were the cause of death in this study? And it is better to present in tables to offer more details.
Experimental design: well
Validity of the findings: This is a retrospective study conducted with ESRD patients underwent major operation in single institute. The manuscript is well written, but still lack of something need to elucidate. 1. As the title mentioned, the study groups were ESRD and no-ESRD, however in the ESRD group in post-operative ICU care, what did the patients receive dialysis therapy, still HD or CVVH?
2.In the comparison group, ie, no-ESRD patients, was there any AKI, acute kidney injury, and need further transient treatment to stablize their hemodynamic, fluid status, intake/output...?
3. What were the cause of death in this study? And it is better to present in tables to offer more details.
Additional comments: This is a retrospective study conducted with ESRD patients underwent major operation in single institute. The manuscript is well written, but still lack of something need to elucidate. 1. As the title mentioned, the study groups were ESRD and no-ESRD, however in the ESRD group in post-operative ICU care, what did the patients receive dialysis therapy, still HD or CVVH?
2.In the comparison group, ie, no-ESRD patients, was there any AKI, acute kidney injury, and need further transient treatment to stablize their hemodynamic, fluid status, intake/output...?
3. What were the cause of death in this study? And it is better to present in tables to offer more details.


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: OUTCOMES OF CRITICALLY ILL END-STAGE KIDNEY DISEASE PATIENTS WHO UNDERWENT MAJOR SURGERY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further issues
Experimental design: No further issues
Validity of the findings: No further issues
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: OUTCOMES OF CRITICALLY ILL END-STAGE KIDNEY DISEASE PATIENTS WHO UNDERWENT MAJOR SURGERY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Insufficient context and not enough motivations based on existing gaps. This concern was raised initially and unfortunately not being addressed sufficiently.
Experimental design: Choices of variables not being supported from the literature. Also, experiment details are lacking as being mentioned in the first round.
Validity of the findings: Since majority of key variables (based on the literature and other studies) are missing, I would be concerned about generalizing the outcomes and 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: OUTCOMES OF CRITICALLY ILL END-STAGE KIDNEY DISEASE PATIENTS WHO UNDERWENT MAJOR SURGERY
Review round: 3
Reviewer: 1

Output:
Basic reporting: All my previous comments have been satisfactorily addressed.
Experimental design: All my previous comments have been satisfactorily addressed.
Validity of the findings: All my previous comments have been satisfactorily addressed.
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: RE-SEARCHER: GUI-BASED BIOINFORMATICS TOOL FOR SIMPLIFIED GENOMICS DATA MINING OF VCF FILES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript was very well structured and written.
Experimental design: The primary aim of the project was clearly defined and meaningful. The project provides a new GUI tool for working with VCF files. The methods description was clearly and the tool designed is easily accessible at github.
Validity of the findings: Authors designed and wrote this GUI-base bioinformatics tool for mining VCF files. The tool designed is easily accessible at github and the tests work.
Additional comments: In this manuscript the authors described a GUI-base bioinformatics tool for simplified genomics data mining of VCF files. This tool would be a great help to researchers lacking command line computation skills. Under Materials & Methods, the authors described how to convert genotype format (line 86). It seems the authors did not discuss multiallelic variants (https://gatk.broadinstitute.org/hc/en-us/articles/360035890771-Biallelic-vs-Multiallelic-sites#:~:text=A%20multiallelic%20site%20is%20a,two%20or%20more%20variant%20alleles.), which are common in cancer sample.  Additionally on the tool's github website, the authors seemed to label multiple nucleotide variants as multiallelic variants (under Features section, item 5, bottom graph), which could confuse and mislead potential users.


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: RE-SEARCHER: GUI-BASED BIOINFORMATICS TOOL FOR SIMPLIFIED GENOMICS DATA MINING OF VCF FILES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a paper about a tool to visualize vcf files. Authors explained the tool in a clear manner in the manuscript. Existing tools were mentioned and compared. Figures and table are clear.
Here are some suggestions for the authors:

1. The reviewer looked at the github for the tool and first couldn't access as the link provided in line 122 is incorrect. The link provided in the abstract is correct.
2. The reviewer did not find an .exe file in the github although it was mentioned in the manuscript that the tool works with .exe file. The code is available, but for the users who do not have any background in programming or bioinformatics this could be very problematic. The authors need to make sure their github page is user friendly for all users with diverse backgrounds as they mentioned that this tool would be useful for a "broader biomedical community".
3. Please include clear instructions for installation. No instructions for installation was found in the github page.
Experimental design: The authors mentioned that they are working on a web-based tool. The reviewer suggests to include the web-based tool in this version if possible. It would be very useful for the users with no programming background.
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: RE-SEARCHER: GUI-BASED BIOINFORMATICS TOOL FOR SIMPLIFIED GENOMICS DATA MINING OF VCF FILES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Authors used professional english and also cited the literature references wherever needed.
Experimental design: This is not an original research article. There are already various other software available for this purpose.
Validity of the findings: I don't think this is a novel research idea and will not make any further impact and neither will be more useful for researchers than previously published software.
Additional comments: In this manuscript, authors developed a software called "re-Searcher" to process and visualize VCF files, which is not giving any additional advantage over previously published software. Only unique functionality of "re-Searcher" is to convert numerical genotypes to alphabetical genotypes, which is also not sufficient to claim a manuscript.


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

Input:
Title: RE-SEARCHER: GUI-BASED BIOINFORMATICS TOOL FOR SIMPLIFIED GENOMICS DATA MINING OF VCF FILES
Review round: 2
Reviewer: 1

Output:
Basic reporting: This manuscript was very well structured and written.
Experimental design: The primary aim of the project was clearly defined and meaningful. The project provides a new GUI tool for working with VCF files. The methods description was clearly and the tool designed is easily accessible at github.
Validity of the findings: Authors designed and wrote this GUI-base bioinformatics tool for mining VCF files. The tool designed is easily accessible at github and the test exampless work.
Additional comments: In this manuscript the authors described a GUI-base bioinformatics tool for simplified genomics data mining of VCF files. This tool would be a great help to researchers lacking command line computation skills. The revision addressed my concerns from the previous version.


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

Input:
Title: RE-SEARCHER: GUI-BASED BIOINFORMATICS TOOL FOR SIMPLIFIED GENOMICS DATA MINING OF VCF FILES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors responded to all comments well.
Experimental design: The webpage with the user interface looks good. The github page has been improved.
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: CENTROMERE PROTEIN N MAY BE A NOVEL MALIGNANT PROGNOSTIC BIOMARKER FOR HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: none
Experimental design: none
Validity of the findings: none
Additional comments: The 2 GEO Datasets used in this study is already analyzed and published in other reputed journals. Hence this is a reanalyzed  study and no new investigations are carried out. The author mentioned  automated GEO2R tool to extract Up and Down regulated  DEGs but limma R package to extract DEG's. The Key Experimental DEG Tables for each dataset  is omitted in this manuscript, hence this work might be not a novel study. Heatmaps for each datasets are also omitted. Valcano plots are not clear and incomlete. GO and Pathway Enrichment analysis Plots are not clear and incomplete and it should be in Tabular form with gene, gene counts and statistical parameters. The PPI n/w construction is not clear and incomplete. Module analysis is also omitted. The Target Gene-mirna and Target Gene - TF n/w construction is omitted. ROC  analysis validation of selected  hub genes are omitted.


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: CENTROMERE PROTEIN N MAY BE A NOVEL MALIGNANT PROGNOSTIC BIOMARKER FOR HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: I have carefully reviewed the manuscript entitled: " Centromere protein N may be a novel malignant prognostic biomarker for hepatocellular carcinoma" by Wang et al. In this work, Wang et al. have identified CENPN as an important differentially expressed gene in hepatocellular carcinoma and the upregulation of CENPN was shown to be related with the low survival rate and advanced T classification of hepatocellular carcinoma patients. Moreover, in vitro study of cell proliferation and the damaging effect of X-rays on CENPN knocked down cells further justifies CENPN as a potential novel malignant prognostic biomarker for hepatocellular carcinoma in vitro. This paper is clearly written and well organized. The introduction and background are reasonable. Figures and tables are comprehensive and helpful. I have few minor concerns that could be addressed to improve the quality of this study.

1. Is there any specific reason behind choosing  HepG2 and Huh7 cell lines among all the available  hepatocellular carcinoma cell lines to study the oncogenic role of CENPN?

2. Was the effect of CENPN on major EMT markers and transcription factors checked in hepatocellular carcinoma cells? It would be worth checking the effect of CENPN knockdown on the levels of EMT markers in HCC cells to understand the oncogenic potential of CENPN.
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: CENTROMERE PROTEIN N MAY BE A NOVEL MALIGNANT PROGNOSTIC BIOMARKER FOR HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this study, Wang et al. proposed a bioinformatics analysis to understand the role of centromere protein N in hepatocellular carcinoma (HCC). They also validated the results by in vitro experiments and confirmed that this protein might be a novel prognostic biomarker of HCC. In my opinion, the study is well-prepared and holds the potential for publication. I raise some major comments to improve the study as follows:

English language should be improved. Currently, there are still some grammatical errors and typos such as:
- There is an urgent to discover ...
- ... liver cancer continues to be one of the leading 10 causes for cancer-related fatalities ...
- The level of expression of the hub genes in clinical samples were compared with those in - ...

An important concern of this study is that the authors missed discussing a lot of literature review related to bioinformatics analysis on HCC.
Experimental design: It needs to provide more information for methodology. Source codes should be provided for replicating the methods and results.

The authors selected different microarray data from GEO as well as TCGA. Did they concern about the batch effect removal among different data?

There must have space before the reference number.

GO database or analysis has been used in previous studies such as PMID: 31277574 and PMID: 31921391. Therefore, it is suggested to provide more references in this description.
Validity of the findings: The findings are good. The authors also validated the predictive performance using in vitro experiments.
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: CENTROMERE PROTEIN N MAY BE A NOVEL MALIGNANT PROGNOSTIC BIOMARKER FOR HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this study, Wang et al. have done bioinformatics analysis and in vitro experiments to study centromere protein N's role in hepatocellular carcinoma (HCC). The revised manuscript is well written, and the authors have added more updated references. Moreover, the authors have well addressed the reviewers’ concerns.
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: CENTROMERE PROTEIN N MAY BE A NOVEL MALIGNANT PROGNOSTIC BIOMARKER FOR HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 2

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


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

Input:
Title: SPHENOFONTIS VELSERAE GEN. ET SP. NOV., A NEW RHYNCHOCEPHALIAN FROM THE LATE JURASSIC OF BRUNN (SOLNHOFEN ARCHIPELAGO, SOUTHERN GERMANY)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The structure of the manuscript is well organized and presented in clear and technically correct English. Figures, illustrations and captions have a good quality and are easy to understand. Bibliography is extensive and sufficient to provide a good background for the introduction and discussion of the work.
Experimental design: As mentioned above, the manuscript is well organized; however, it was frustrating that the authors did not include a phylogenetic analysis considering that their specimen is very complete and allows observing many taxonomic features worth to be included in a data matrix. If they were describing a specimen that only consist in fragments of dentary or maxilla, I could understand their decision to not to perform phylogenetic analysis, but this is not the case. I suggest including a parsimony analysis in order to improve their work.
Validity of the findings: After reviewing the manuscript, I found the results and conclusions of this work convincing and interesting enough to be considered for publication in PeerJ. I think the authors provide a well written and very detailed description of a fascinating specimen that provides remarkable information about some skeletal elements poorly studied in fossil rhynchocephalians as is the case of the pectoral and pelvic girdle. As mentioned before, the description of the new taxon is very detailed; but I felt that the diagnosis presented is somewhat plain. Therefore, in order to improve their work I suggest changing the current diagnosis for a differential diagnosis.

For example:
Sphenofontis velserae is moderated sized taxon that differs from other sphenodontians by the following unique combination of features. Proximally-constricted and strongly distally-expanded transverse processes of the first sacral vertebra (not present in any other fossil rhynchocephalian or in the extant Sphenodon); anterolaterally-oriented transverse processes of the first caudal vertebra (a similar transverse process is present in Sphenodon but ………).

Regarding to the taxonomic validity of the new taxon, I would like to make it clear that a) I have no doubt that the specimen represents a new genus and species, b) that this new taxon confirms the high taxonomic diversity of the Late Jurassic sphenodontians, and c) that Sphenofontis can be accurately referred to Neosphenodontia, and it likely can be placed within the Sphenodontinae. However, I was surprised with the fact that the authors did not include a phylogenetic analysis to provide more support to their conclusions on the taxonomic placement of Sphenofontis. As far as I am concerned, the authors present a work of an overall great quality, however, I suggest that before the final acceptance of their work, to include at least a basic parsimony analysis to test the phylogenetic position of the new taxon. If a phylogenic analysis is presented, I think is not necessary to do a further discussion on rhynchocephalian relationships, it is fine if they only point out if the results confirm the placement of Sphenofontis within the Neosphenodontia and the Sphenodontinae. An additional figure with a phylogenetic tree can be incorporated in the main manuscript, but it is also fine if the authors include it as part of the supplementary material.

Other comments and suggestions to the authors are as follow:

The authors cite several times the work of Romo-de-Vivar-Martinez et al., in press; nevertheless, that work was already published last year. Please, correct that in the text and in the references.

•	Romo-de-Vivar-Martínez, P. R., Martinelli, A. G., Paes Neto, V. D., Scartezini, C. A., Lacerda, M. B., Rodrigues, C. N., & Soares, M. B. (2019). New rhynchocephalian specimen in the Late Triassic of southern Brazil and comments on the palatine bone of Brazilian rhynchocephalians. Historical Biology, 1-9.

Simões and colleagues recently published a review of sphenodontian phylogeny which also discusses a little bit about mosaic evolution in Sphenodon. I suggest the authors take a look of that work.

•	Simões, T. R., Caldwell, M. W., & Pierce, S. E. (2020). Sphenodontian phylogeny and the impact of model choice in Bayesian morphological clock estimates of divergence times and evolutionary rates. BMC biology, 18(1), 1-30.
Additional comments: This is a very interesting study of a specimen that I considered fascinating when some pictures were published in a previous work. I am glad to see this specimen finally described, but I recommend taking into account my suggestions in order to improve your work and give more support to your findings.


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: SPHENOFONTIS VELSERAE GEN. ET SP. NOV., A NEW RHYNCHOCEPHALIAN FROM THE LATE JURASSIC OF BRUNN (SOLNHOFEN ARCHIPELAGO, SOUTHERN GERMANY)
Review round: 1
Reviewer: 2

Output:
Basic reporting: 'no comment
Experimental design: 'no comment
Validity of the findings: 'no comment
Additional comments: The manuscript is well written, however, take into consideration that English is not my native language. The description is good, and I think  is necessary continue producing this type of papers. Although when using the terms "Slender", "Robust", "longer", "Short" without a reference either in relation to the proportions of the bone element in question, or between said element in relation to other bone elements, it becomes quite The meaning of these terms is subjective and I think we should stop with this. Throughout the text the authors make mention of some proportions, a table could be attached that contains them and they are used as a reference in the descriptions of the bone elements.

Although, a phylogenetic analysis is not necessary to describe a new species, and even less with a detailed description and good comparison like the one presented in this work (and finally the authors mention that it will be done in the future). It seems to me that for the first part of the discussion it would help a lot, and in this case it would be necessary, to be able to effectively test the hypothesis about the phylogenetic location, mainly into the Sphenodontidae and to locate how the new taxon described relates to the other taxa. It would also help to map some characteristics that have been highlighted throughout the text and this would contribute to the understanding of the relationships and evolution of the group. I recommend checking out the new article:  Simões, T. R., Caldwell, M. W., & Pierce, S. E. (2020). Sphenodontian phylogeny and the impact of model choice in Bayesian morphological clock estimates of divergence times and evolutionary rates. BMC biology, 18(1), 1-30.

I think it is important to improve some figures or to append some made with the help of a stereoscopic microscope since some structures mentioned in the text are not visible or are difficult to find. Or even in the interpretive drawings to point out some structures such as foramina, the autotomy plane, notch, ridges, etc. since sometimes it is difficult to see them or I do not know they see them in the photographs.

Finally, they could append one more figure to accompany the Intruduction sections, and Geological and Palentological Context, this figure should be a stratigraphic column (or several columns if they are the case and the relationship between them) where the mentioned fossils are located stratigraphically in the text, and wich  they have been found in what is known as the Solnhofen Archipelago.

Finally, in the manuscript, attach some 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: SPHENOFONTIS VELSERAE GEN. ET SP. NOV., A NEW RHYNCHOCEPHALIAN FROM THE LATE JURASSIC OF BRUNN (SOLNHOFEN ARCHIPELAGO, SOUTHERN GERMANY)
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript represents a detailed description of a new taxon from the Solnhofen Archipelago. The authors are to be congratulated on a very thorough job and an excellent comparison with the known record of Early Mesozoic rhynchocephalians. It will be a valuable contribution to our understanding of  the increasingly complex radiation of the sister group of lizards.

There are very few things that need to be considered in the revision, although I would like to see at least some discussion of its potential habit – such a beautifully preserved specimen does at least require comments on the limb and body proportions when compared with other members of the group.
Experimental design: The approach is excellent and the figures clear and well-composed.

I am assuming that it is not possible to CT scan the specimen? You might like to comment on this as it would have potentially offer  a solution for better understanding of the dorsal cranial morphology.
Validity of the findings: For the diagnosis of the new taxon, I completely agree that the “waisting” of the sacral rib on the first sacral is probably quite unique. However, I wonder if the small medially placed tooth on the maxilla might be aberrant? After all we are only dealing with one individual.

I have a handful of questions about your identification and comments on different elements:

L. 164. Difficult to interpret but I guess I have to take your word that you have identified the frontal correctly. It just looks a bit like the anterior ramus of the pterygoid and its position would make sense in this context.

L. 299. Is this not the medial view of the right quadrate so that what is labelled as the qj is the internal (columnar) part of the quadrate. The elements of the right side, although somewhat disarticulated are surely preserved in medial view as you indicate in L. 278?

L. 571. Additional teeth on the two maxillae seem to be different on either side. On the left it is the third successional tooth that is reduced and positioned more medial, whereas on the right side it is indeed the fourth successional tooth that is small and medial in position.

L.905. The femora seem relatively short to me. I guess it depends upon your point of reference - but when compared to the forelimbs. It would be worth comparing the proportions of the forelimbs and hindlimbs to other rhynchocephalians. Likewise the pedal digits seem short. I could be incorrect, but comparison with the forelimb:hindlimb ratios in other taxa/ specimens would be instructive.

L. 1036. I tend to agree that the dentition is unique and indicative of a new taxon, but should perhaps add the caveat that this is based on just one (not completely mature) individual, and that some variation in dentition might be expected.

L.1164. This is the only real comment that hints at further studies on functional morphology. As suggested above, it would be helpful if the authors could at least make some comments on limb proportions in the new taxon in this respect.
Additional comments: For the most part the text reads fairly well, but I have made a few suggested changes to phrasing in the attached PDF where I have felt there is either some ambiguity or the expression is particularly clumsy.


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: ECOSYSTEM SERVICES SHOW VARIABLE RESPONSES TO FUTURE CLIMATE CONDITIONS IN THE COLOMBIAN PÁRAMOS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a very well written manuscript and covers an important topic in an innovative fashion. Citations are fine, as is the organization and presentation of the findings.

Title is a bit succinct, as there are many high mountains in the Andes that do not have paramo and in fact are quite different. For that matter, there is paramo in Costa Rica, so not necessarily an Andean phenomenon.

More could be said about the effects of the assumptions described on page 6 of the ms.

Page 14 of ms talks of many other GCMs giving different findings, and illustrated in Figure 4. This seems like a lot of work done and very important to climate scientists, so could use some more text and explanation here.

I think the conclusions section is redundant and could be shortened.
Experimental design: I think the research design is fine, although as always, choosing just 100 or so species from a very rich flora seems arbitrary. I know this is always a criticism of those working in the tropics, and so it may be necessary and the authors seem aware of the seeming arbitrariness.

Why is the study area described as from 1000 m in elevation and above? Seems like data is from the paramo flora, at much higher elevations. Also, in general topography seems overlooked as a factor, given that higher up should be smaller areas and indeed eventually the topography will end and some species will go extinct due to mountaintop extinction. I was surprised topography was not part of the experimental design or the discussion.
Validity of the findings: Generally the more leaf area, the more evapotranspiration, and hence more water loss from the soil. So, the ecosystem service proposed for the Puya seems unlikely, and just an impression by people who have not quantified water and soil relations.

I think habitat is an important ecosystem service, as it provide refuge and food to many animals. This aspect does not seem to be captured in the ecosystem serviced and/or their discussion.
Additional comments: Well written and well done analysis. I think the title misleads a bit however, making it seem like it covers all the Andes of South America.


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: ECOSYSTEM SERVICES SHOW VARIABLE RESPONSES TO FUTURE CLIMATE CONDITIONS IN THE COLOMBIAN PÁRAMOS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The hypotheses are clearly stated in the Introduction but only in the Conclusions do we clearly see what happened. I would suggest putting the main results of these hypotheses in the first paragraph of the Discussion so the reader can clearly understand what was expected and what happened.

Minor comments
1. Introduction – I think that the Intro could be tightened up. For instance, paragraphs 1 and 2 could be consolidated into one.
2. Line 82 – add a comma. “As a result, it is projected that at the species level, between 10 and 47% “.
3. It could be useful to number the hypotheses (lines 97, 101) and relate to them in the Discussion more clearly. They are only clearly mentioned in the Conclusions.
4. Line 107 – I suggest using the word “could” instead of “are going to”. These models and projections are still hypotheses of what “could” happen in the future.
5. Line 189 – low and high páramo… based on whose definition?
6. Line 190 – add dot in “m.a.s.l.”
7. Line 225 – Should Fig S1 be cited here? It was a little confusing. I think it is best to cite it where the Mahalanobis distance model is being described (along Fig. 3, line 255)
8. Line 306-307 – rephrase to “minimum, mean and maximum temperatures as well as …”.
9. Line 309 – I would, again, suggest using “could” instead of “will” since there are many other factors (e.g., biotic interactions, land use change) that could influence these trajectories.
10. Lines 313-322 – It’d be good to report percentages. What % of species show a decrease/increase/no change?
11. Line 323 – this is interesting but it should not be in the Results.
12. Line 325 – “most, if not all ecosystem services, is a decline…” (add commas).
13. Figure 6 and 7 legend – please say what the colors (orange, blue, green, yellow) mean.
14. Line 418 – rewrite to “some species from the upper Andean forest could potentially be colonizing”.
15. Recommendations i) and iii) could be consolidated since they are both about monitoring. If the authors want them separate, they should go in sequence.
Experimental design: 1. The authors reviewed literature in order to derive the ES provision by páramo plants. However, they do not show how they did the search (keywords for international/indexed literature) or how many papers/documents they revised. I suggest explaining this more clearly and adding a supplementary material with the references (corresponding to what is described in lines 147 to 151).
2. Related to the previous comment, how were species placed in Cook’s categories? Only based on what is reported in the literature or did the authors include their own knowledge?

Minor comments
1. Line 178 – “several databases”. I think this has to be explained in more detailed. If not in the main document, the process should be described in Supplementary material.
2. Line 220 – seasonality of temperature or precipitation, or both? From Figure S2, since the axis is described as drought, I would guess seasonality of precipitation? But you refer to seasonality as a whole… Please clarify this and make it consistent (also in line 345).
3. I think it would be good to explain why the authors chose E. boyancensis in Figures 3 and 4. Given that it is a species that can be found in a greater altitudinal range (based on line 194-195), why did they choose that species as an example?
4. Line 437 – literature review should have a table in the Suppl. Material and a method describing how it was done.
5. Lines 450-460 – I think it is positive that the authors acknowledge the lack of data since this is a major issue in studying and understanding the potential effects of climate change.
Validity of the findings: 1. Although the authors state in their first assumption (lines 158-160) that based on plant uses, ecosystem services can be inferred, I have some (conceptual) trouble with extrapolating from a few plant species to ES such as water regulation, erosion, nutrient cycling (n=3) and soil formation (n=5). Especially since the species number is low and I do not understand how can that be representative of an ES at a large scale (you are predicting at the regional level). I don’t disagree that individual species can have impacts on these ES but, are these numbers enough to talk about ES at the scale proposed? Should the authors maybe focus on ES with higher “n” and related to food, medicine, materials…?
2. Related to the previous comment, I’m also worried about drawing conclusions about Gene sources and Agroforestry when n=1 (also valid for cultural ES). For instance, Gene sources and Agroforestry are mentioned in the Abstract and Results (lines 332, 339, 341) as examples of ES with substantial negative responses… but with n=1 is it really valid to draw this conclusion?
3. Is there a criterion for a minimum number of species to reliably represent an ES?
4. I think it is important to remember that climate is just being defined by temperature and precipitation (and their variables). Climate, is much more comprehensive than just these two variables, especially for páramo plants where topography, soils, aspect, winds, etc. also play a crucial role. Therefore, in the paper (maybe in the assumptions/ discussion/conclusions) it should be acknowledged that projecting future climate change scenarios with just temperature and precipitation is still limited and that páramo responses may be much more complex due to all these factors.

Minor comments
1. Lines 378-383 – I think that while climate is important, climate here is only defined by temperature and precipitation. It is important to acknowledge that many other climatic variables are important (e.g., wind, topography, soils, etc.; WorldClim 2 has data for solar radiation, wind, water vapor pressure). I also consider that in some place in the discussion it should be acknowledged that these analyses are based on the current land use (which could also change, among others, with climate change).
2. Conclusions – how much do people actually use these species? There’s some emphasis on people suffering losses from reductions in SE (which is not unreasonable), but, are there are any studies/numbers supporting this claim?
Additional comments: This is an important study proving valuable information on ecosystem services (ES) of the páramos. It has a great amount of work on species modelling, ES, and the possible impacts of climate change (RCPs) on those services. I have not yet seen as study like this for the páramos, and I think this study is of relevance in our understanding of this key ecosystem. I also liked the Recommendations sections.
However, although the authors acknowledge their assumptions, as well as several of their analyses’ shortcomings, I have some areas of concern. I believe that addressing them could improve readability as well as understanding of this manuscript. I would like to apologize in advance, if I misunderstood certain areas of the manuscript.


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

Input:
Title: ECOSYSTEM SERVICES SHOW VARIABLE RESPONSES TO FUTURE CLIMATE CONDITIONS IN THE COLOMBIAN PÁRAMOS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Well written. The rewrite and the answers to previous reviewer comments were excellently and completely done.

All findings are well demonstrated; figures and tables all necessary.

In fact, there are still very worthwhile findings put into supplementary information. I agree with those designations, but always regret having an article split up into the main article and supplementary materials produced at great effort, but not read by typical users of the article.
Experimental design: Fine as done. This part was fine in previous submission and is now even better explained and options taken defended.
Validity of the findings: All is fine in the rewrite in regards these criteria.
Additional comments: Fine as is.


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: DOWNREGULATION OF BOTH MISMATCH REPAIR AND NON-HOMOLOGOUS END-JOINING PATHWAYS IN HYPOXIC BRAIN TUMOUR CELL LINES
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript by Crowman et al., the regulation of repair genes after hypoxia was examined. The results are not completely new, as similar studies have already been carried out on other tumor cell lines (e.g. Meng et al., 2005).

Critique
1) Four cell lines were used for the investigations. Why these lines? Do they have a different DNA repair or p53 status?
2) Although some repair genes are downregulated under hypoxic conditions, as Fig. 2 shows, there are different genes found to be downregulated in the cell lines. Thus ligase 4 is strongly downregulated in U87, but not in the other lines. Is there one or more genes that are downregulated in all 4 lines? How generalisable are the findings? How robust are the data (line 129) in terms of generalization. Is the downregulation of a specific gene related to the p53, IDH1 or MGMT status of the cells?
3) No data are provided on tumor material. Can the results be transferred to tumors?
4) Please insert a table in which the genes are clearly and transparently listed that are downregulated in the 4 lines. These cannot be seen in Figs. 1, 2 and 3.
5) MMR genes are cell cycle and proliferation dependently regulated. Thus, MSH2, which stabilizes MSH6, is positively regulated by E2F1. This transcription factor is down in non-proliferating cells. Therefore, it is very important to measure the proliferation level of the glioma cells after cultivation under hypoxic conditions. Are the effects observed a by-product of slowing down proliferation? Is the same observed if cells are cultivated under low serum condition? Please add data on cell's doubling time and cell cycle distribution under normal and hypoxic conditions.
6) line 216. The downregulation of MMR and DSB repair is discussed here and elsewhere in the mancript. Therapeutically, these repair pathways exert opposite effects. For example, a weakening of the TMZ effect is to be expected through downregulation of MMR, while an increase in the effect is to be expected after downregulation of HRR (as HRR is a major TMZ-protection mechanism). Possibly both effects neutralize each other. This should be discussed.
7) line 231: For cisplation and MMR quite old data are cited. Work published later by Jiricny and others showed that MMR is not clearly involced in the repair of cisplatin induced toxic adducts such as inter- and intrastrand crosslinks (for extensive discussion see Koeberle et al., Cisplatin resistance: preclinical findings and clinical implications, BBA, 1806, 172-182, 2010). This paragraph needs update.
8) line 238: MMR is essential for TMZ-induced apoptosis. Again, a quite old paper was cited. Please describe briefly why MMR is essential (there are many reviews on this, for a recent one see Kaina and Christmann, DNA repair in personalized brain cancer therapy with temozolomide and nitrosoureas, DNA Repair, 78. 128-141, 2019).
9) line 282: E2F1 regulates MSH2. Is downregulation following hypoxia mediated by upregulation of HIF and in turn downregulation of E2F1? I recommend to show in a western blot the expression level of E2F1, using the same cell extracts.
10) line 247: NHEJ, a highly error prone repair process,... This is not true, at least the general statement. Only the backup B-NHEJ pathway (also designated as Alt-NHEJ) was shown to be error-prone (XRCC1, PARP-1 and ligase III dependent), the major NHEJ pathway is largely error free.

Minor comment
11) line 10:  Use the generally accepted terminology for repair genes, including MGMT. The full name is O6-methylguanine-DNA methyltransferase (MGMT).
line 111-113: This is a very superficial description of the function of MMR, NHEJ and HRR in temozolomide and radiation responses in GBM. At no point in the manuscript could I find a mechanistic description of MMR in TMZ-induced effects. Please describe in more detail. It is important to note that MMR does not remove TMZ-induced damage, but leads to futile repair cycles that activation death pathways.
Experimental design: see above
Validity of the findings: see above
Additional comments: see above


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

Input:
Title: DOWNREGULATION OF BOTH MISMATCH REPAIR AND NON-HOMOLOGOUS END-JOINING PATHWAYS IN HYPOXIC BRAIN TUMOUR CELL LINES
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this manuscript, authors studied the effect of chronic hypoxia on DNA damage repair gene expression in different glioblastoma (GBM) cell lines. They used the NanoString system to characterize the gene expression profile in cells treated with normoxia, moderate or severe hypoxia for up to 5 days. Several gene groups (apoptosis, NHEJ, HRR and MMR) were mostly found to be downregulated in four different GBM cell lines examined. Further, downregulation of PMS2 in MMR, and LIG4 in NHEJ were confirmed by RT-PCR and Western blotting. Finally, HIF inhibitor Acriflavin recovered the downregulation of LIG4, indicating that this process is mediated by the HIF-dependent mechanism.
The manuscript describes an important point on how cells respond to chronic hypoxia, and the molecular mechanism of gene downregulation under such condition. However, it is not clear how significant this machinery is upon GBM treatment. Following points should be addressed and modified before acceptance.

Points to be addressed
1. What would be the effect of Temozolomide on GBM cells cultured under chronic hypoxic condition compared with normoxic condition?
2. What would happen to the expression level of PMS2 and LIG4 upon reoxygenation?
3. Authors should show an entire expression profile of individual genes included in the Nanostring analysis.
4. In Figure 5, what is the expression level of LIG4 in normoxia? Statistical analysis should be done.


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: DOWNREGULATION OF BOTH MISMATCH REPAIR AND NON-HOMOLOGOUS END-JOINING PATHWAYS IN HYPOXIC BRAIN TUMOUR CELL LINES
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a revised version. A detailed criticism and comments were made in the primary review.
Experimental design: See above.
Validity of the findings: Significant findings were reported. The data are worth to be published.
Additional comments: The authors answered all of my questions and made satisfactory corrections in the manuscript.


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

Input:
Title: DOWNREGULATION OF BOTH MISMATCH REPAIR AND NON-HOMOLOGOUS END-JOINING PATHWAYS IN HYPOXIC BRAIN TUMOUR CELL LINES
Review round: 2
Reviewer: 2

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: The reviewer wonders if the chronic hypoxic treatment renders U-87MG cells sensitive to TMZ treatment due to changes in gene groups involved in DNA repair pathways.
It is not clear how the authors performed the experiment in point #1, but if it is without hypoxia, then they should examine the effect of chronic hypoxia.


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: CONTINUOUS BUBBLE STREAMS FOR CONTROLLING MARINE BIOFOULING ON STATIC ARTIFICIAL STRUCTURES
Review round: 1
Reviewer: 1

Output:
Basic reporting: -- Introduction does a good job of providing background, context and highlighting key literature in the field.

-- Methods and Results
*  Figure captions lack sufficient detail. A reader should be able to follow the manuscript narrative via figure captions, and not need to refer to the main text for key information. Figures 1–7 in particular are lacking in key background and contextual information. E.g., it needs to be made clear that Figures 4 and 5 relate to scouring and settlement disruption, respectively.

*  Statistically significant differences should also be indicated in the figures.

-- Minor formatting and style corrections are needed throughout, e.g.,
*  commas after e.g. and i.e.
*  spaces between values and unit symbols.

-- Structure conforms to PeerJ standards.

-- Raw data are supplied.
Experimental design: -- The research question is well-defined, and the authors highlight key knowledge gaps the study attempts to address.

-- Methods
*  Authors have been forthcoming in identifying limitations and logistical issues around experiments, and have explained the rationale behind work-arounds.

*  Some content requires greater clarity or further information:
Lines 141–147: the order of operations is hard to follow, and could be explained more clearly.

Lines 182–184: some details are lacking on how taxa were assessed on experimental panels.
Validity of the findings: -- The data are robust and statistically sound.

-- Conclusions are well stated, and the authors do a good job of not over-interpreting the results.
Additional comments: The manuscript "Continuous bubble streams for controlling marine biofouling on static artificial structures" sets out to address some key questions around the use of aeration to prevent marine biofouling on submerged surfaces. Over the past ~ 10 years, aeration / bubble curtains has been touted as an environmentally-friendly (i.e., non-biocidal) tool that could be used to manage marine macrofouling on stationary structures and vessels, but this approach has received relatively little attention. The authors highlight the key papers in this field, and point out that there are some substantial knowledge gaps regarding the development and use of bubble curtains:
•	Mechanism of action -- does turbulence disrupt organism settlement, or do shear forces scour larval recruits off?
•	What air flow rates are effective at preventing macrofouling accumulation, and how is this influenced by organism and substrate type?
•	How frequently should bubble streams be applied, and for how long?
•	What's the potential for bubble stream systems to contribute to underwater noise pollution?

Through various experiments, the authors provide some insights towards these questions. The findings provide operational guidance to a first approximation, and will be of interest to municipalities, ports and marinas seeking to trial or test this biofouling management tool. This is also a solid contribution to the field of biofouling prevention technologies, and it will be of interest to researchers as it provides insights towards the mechanisms by which bubble curtains affect biofouling (e.g., via disruption and scouring).

The Introduction and the Discussion read well, the but figure captions lack sufficient detail, making it difficult for the reader to interpret and understand the Methods and Results.


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

Input:
Title: CONTINUOUS BUBBLE STREAMS FOR CONTROLLING MARINE BIOFOULING ON STATIC ARTIFICIAL STRUCTURES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In a set of lab and field experiments, Hopkins et al. present an investigation of the effects of bubble streams on the scouring and settling disruption of marine organism settlement and growth on fouling panels. Panels composed different materials, compared with and without bubbling, and with and without anti-fouling coatings are tested. Sonic analyses were conducted in the field to determine the frequency bandwith and sound pressure emitted from the bubbling mechanism as a way of determining whether underwater sound might disrupt marine mammals and fishes. The authors put their results in the context of previous literature that investigates biofouling and air bubble/curtains effects, indicating that previous work has focused on scouring vs. settlement disruption. They also discuss the possibility and limitations of real-world bubbling systems scaled to the size of marinas.

Overall, this paper addresses the issue of how to prevent/minimize biofouling on static structures in the absence of shear forces that are produced when ships are propelled through the water, which are critical to modern anti-fouling coatings. With the global phasing out of tributyl tin, an element that is toxic to wide range of marine biofoulers, many of the new coating formulae rely on physical forces for efficacy. The investigators thus address an important limitation of modern coatings on stationary vessels and structures, focusing on the possible mechanistic effects of continuous bubbling on scouring and larval settlement disruption.

The manuscript seems to draw on appropriate literature to set the stage and context for their experiments and they discuss previous work that has examined the effects of bubbling on biofouling effectively.

The experimental approaches are fairly well described and appear sound. However, there are a number of aspects of the presentation of the data and analytical findings that need improvement.
Experimental design: The experimental and methodological designs were sound, but replication was limited in some instances (e.g., n=2 for treatments described in Fig 7). Simultaneous replication treatments for some experiments was limited by apparatus and the investigators repeated the experiment through time. There was mention of the importance of the temporal variable in relation to results, but the model results and statistics for all results were relegated to the Supplementary Materials. Below are some specific comments on issues that

Figures
The captions on some of the figures should to be revised to better describe what is being presented and highlight what a reader should take away from each. Figs 1-3 describe experimental apparatus and setup, and are reasonable. Although on Figure 3, it might be helpful if the labelling for FR-IS1000 and FR-IS1100 could be differentiated more clearly in another way (e.g., FR #1, FR #2). This was also problematic in Fig 6 and the associated Results section, where the single digit in the formal name was lost to this reader until the second or third pass. More obvious differentiation would make message more accessible and obvious. The authors might consider making this differentiation at lines 168-19 and/or 201-202.

Although opinions vary, my opinion is that figures that describe measurements/results should be able to stand alone from the main text and be fully interpretable. I found the data figures (Figs 4-7) to be lacking sufficient information. For example, Fig 4 (lab settlement results) present box plots but don’t provide any information about statistically relationships or differentiation. Text from the Results section (lines 270-275) indicate this differentiation, but then reference Table S3. Include and state statistical differentiation among box plot data distributions in Fig 4 – it would make for a far more compelling figure. Likewise, Figs 5-7 are summaries of the data, but could be improved if some notation (reflected in the figure captions or graph legends) specified how apparent differences/similarities. It is not clear to me that the information included in Fig 5 warrants a figure, a table (including statistical comparisons) may be preferable. Likewise, Figs 9 and 10 are only introduced in the Discussion section, so Supplementary Materials may be a more appropriate place for these.

Methods
The distance below the experimental fouling panels varied among lab and field experiments. In the lab the distance was 400mm, but 50mm in the vertical panel field experiments and 300mm in the horizontal and angled panel field experiment. There was no apparent rationale or justification for use of different distances from diffuser heads to the test panels, but it is logical to assume that concentration and vigor of bubbles is lessened with greater distance from diffuser. Some comment on why these depths were chosen, or justifying why they are comparable would be useful. Likewise, I had expected some discussion of the behavior or air bubbles underwater, specifically the effects of pressure and dissolution of gas into surrounding water has on bubble volume. Likely at the shallow depths considered in these experiments, the effects may be negligible, but if one considers scaling up bubbling systems to marina/harbor sizes, depth may become a factor.
Validity of the findings: The investigators draw conclusions that do not overstep their experimental findings, so thus seem valid. I provide some detailed suggestions in Experimental Design and General Comments to Authors that are meant to clarify and bolster their reporting. It strikes me that the manuscript could be more hard-hitting if some changes to the figure captions and main text were edited, as suggested.
Additional comments: Below are some detailed suggestions for edit that I think would strengthen the manuscripts and make it more accessible to readers.

Model organisms
Although the authors refer readers to details of Ciona and Crassostrea spawing and larval rearing in the Supplementary Materials, the question of larval competence is critical when considering settlement ability and success/failure. Some of this is addressed in Supplementary Materials, but a little more detail describing the importance of timing and competence on settlement success might be helpful in the main text.

Results
Although data analyses are described in Methods section on lines 242-262, including descriptions of multiple GLM models, there are no such results reported in the manuscript proper! The only numerical results that are reported in the paper are summary statistics: central tendencies and measures of variance. All modeling and statistical details are buried in Supplementary Materials, where many readers will never venture. The authors need to revise and expand their Results section to include more quantitative descriptions of their findings.

Vertical surfaces
Results are summarized in the text but it would be would be helpful and have more impact to reference a figure or consolidate the results in a table that includes the qualitative scoring as well as the % cover.

In the Methods and Results section, the authors may be able to better differentiate the field trials of horizontal and angled plates of various treatments if they described them as A) three trials (n=1 per treatment) replicated the experiment through time (i.e., the angled and flat panels summarized in Fig 6) and B) a separate experiment compared POLY with and without FR-IS1000 panels in the horizontal position (n=2 for each of four treatments). As is, it is confusing since a “final” trial is not similar to the previous experiment.

Figure 6 is the summary of a huge amount of work across a year, but the authors don’t seem to wring out as much information that the figure possesses. Again, some indications of statistical comparisons among treatment make the analysis more robust. Figure 7 reflects the results from two replicates, but there is no indication that the values reported are an average, since there is no measure of variance reported for the two replicates of each treatment. Experiment figures should all report sample sizes.

The apparently important temporal aspect of the horizontal/angled plate is mentioned, but glossed over and readers are referred to the Supplementary Materials for details. Given the generally well known importance of seasonality on marine invertebrate reproduction and settlement, any interaction between treatment and time should be further presented and discussed.


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

Input:
Title: CONTINUOUS BUBBLE STREAMS FOR CONTROLLING MARINE BIOFOULING ON STATIC ARTIFICIAL STRUCTURES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors present a multi-stage study (several lab and several subsequent field trials) to fill knowledge gaps surrounding the efficacy and practical uptake of bubble streams to prevent the accumulation of biofouling on static artificial structures. They are successful at achieving these goals and at providing meaningful progress toward additional work that can build off of these results. There are, however, a few areas that need to be addressed to provide more clarity on the methods used and the overall readability of the manuscript.

Clarity:
- The Materials and Methods section appears to have been written out of sequence, with acronyms and abbreviations being used paragraphs or pages ahead of where they are formally introduced. Please review this entire section sequentially and adjust as appropriate. There are other issues with the description of the laboratory trials, but I will address those in the comments on Experimental Design.

- Lines 352-353: Sentence structure makes this statement unclear. Please clarify.

- Line 422: Should there be a "were" inserted between "forces" and "created?"

- Line 443: For clarity, please refer to the Naval Base experiment as "continuous bubble treatment." This makes it easier for a reader to understand to what you are referring, rather than making them refer back to the methods.

- Lines 458-459: For clarity, please refer to the Naval Base experiment as "continuous bubble treatment." This makes it easier for a reader to understand to what you are referring, rather than making them refer back to the methods.

Figures:
- The description for Figure 1 on line 111 states that two diffusers were placed on the bottom of the tank, but only one diffuser is visible in the figure. Presumably there is another diffuser in parallel with the displayed diffuser, but the figure should reference the paired diffusers or present a side view of the tank to display both diffusers. I think I understand the layout, but it is not clear and I should not have to guess when trying to understand the design.

- The caption for Figure 1 should describe the acronyms and abbreviations used in the figure, including FR-IS1100 and ACR (See Figure 4 for correct descriptions). Also in Figure 1, the diagram demonstrating the design for the scouring experiment does not show the five FR-IS1100 panels with Ciona. I understand from reading the rest of the manuscript that the Ciona failed to settle and remain on these panels and were, therefore, excluded from the analysis, but that fact comes much later than the introduction of the figure and description of the design. This leads to a lack of clarity when trying to review the figure while reading the description of the design. They do not add up and a reader doesn't find out why until much later. This can be addressed by including the panels in the figure and indicating that they were excluded from the analysis.

- The caption for Figure 2 should describe the acronyms and abbreviations used in the figure, including CONC and FR-1100SR (See Figure 4 for correct descriptions).

- The caption for Figure 3 should describe the acronyms and abbreviations used in the figure, including CONC, POLY, and FR-1100SR (See Figure 4 for correct descriptions).

- The caption for Figure 6 should describe the acronyms and abbreviations used in the figure, including CONC, POLY, and FR-1100SR (See Figure 4 for correct descriptions).

- The caption for Figure 7 should describe the acronyms and abbreviations used in the figure, including POLY and FR-1100SR (See Figure 4 for correct descriptions).

- The caption for Figure 8 should describe the acronyms and abbreviations used in the figure, including POLY and FR-1100SR (See Figure 4 for correct descriptions).
Experimental design: As indicated above, the materials and methods section appears to have been written out of sequence, making it difficult to follow what was being done. Please proofread this entire section to ensure that it flows sequentially.

Examples:
- In the description for the scouring experiment on lines 147-149, you describe the addition of an algal culture as a food source for the Crassostrea larvae, but do not mention anything about feeding of the Ciona larvae.  In the next section describing the settlement disruption experiment, line 159 indicates that Ciona larvae are lecithotrophic and, therefore, did not require feeding. This should have come earlier.

- The abbreviation CONC is used on lines 170 and 174 without reference to what it means. It is not until the description of the field trials on line 200 that you define CONC as an abbreviation for concrete. This should have come earlier.

- The acronym for the foul release coating FR-1100SR was introduced on line 125 and is then used several times before being reintroduced on line 169. This is not necessary and suggests that these subsections were written separately and then combined without proofreading.

The subsection describing the scouring experiment is not clear and appears to contradict itself in several places. My understanding is that the panels were soaked with larvae for periods of 3 hours and 120 hours and then were subjected to 10-minute bubble treatments (or control) before being evaluated for retention. I am not clear on what is meant by the sentence on lines 143-145 and the references to 120-hour "treatment" on lines 147 and 150. I understood the 120 hours to be the settlement time before treatment and not the duration of the treatment, but the language on lines 147 and 150 is unclear and confusing. Please read this entire subsection and revise as necessary to ensure clarity.

Additional questions and comments about the methods:
- Please explain why the flow rates described on lines 118-120 were selected. Was this arbitrary or was this based on prior knowledge of previous work?

- Lines 135-136: Please provide the density of Ciona and Crassostrea larvae that were used in the settling tank.

- Lines 137-138: Please explain why the 3 and 120 hour time periods were chosen for settlement. Was this arbitrary or was this based on prior knowledge of previous work?

- Line 143: Please explain why 10 minutes was selected as the duration of the bubble treatment. Was this arbitrary or was this based on prior knowledge of previous work?

- Lines 149-150: Please provide more details on how the panels were inspected. Were the entire plates assessed for retained larvae?

- Lines 158-159: Please clarify if 20 L of Isochrysis galbana was added to both tanks (meaning it was split between the two tanks) or if 20 L was added to each of the tanks.

- Lines 176-177: Please describe how many divers were used and whether they independently assessed for Level of Fouling. How was the final category determined? By consensus?
Validity of the findings: The conclusions are well-stated. I would have liked to see a bit more here about the potential for future studies related to the formation and retention of large bubbles on the underside of flat structures and the influence of surface design. The idea is briefly mentioned on lines 371-374, but it would be useful to highlight here again with a bit more direction for future studies.
Additional comments: Overall, I think this work achieves the goals it set out to achieve and will contribute to the progress of this type of approach to preventing the accumulation of biofouling on these structures. The carryover benefits of minimally fouled infrastructure are important in reducing the spread of nonindigenous species as this can reduce the propagule supply and subsequent recruitment on vessels within these marinas.

I have a couple of additional comments that did not fit the other comment categories:

- Lines 442-450 contemplate scaling up the equipment and energy costs associated with treating an entire marina, but there is no reference to what that could look like at full scale. I suggest including the scaled up equipment and electricity costs for the entire marina that you worked in to give the reader a sense of how practical it would be and to contemplate what could be done to make those costs manageable.

- Are the authors aware of any local or regional sound restrictions within marinas? It would be helpful to put the underwater noise results into the context of what would be allowed to better interpret the results.


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

Input:
Title: CONTINUOUS BUBBLE STREAMS FOR CONTROLLING MARINE BIOFOULING ON STATIC ARTIFICIAL STRUCTURES
Review round: 2
Reviewer: 1

Output:
Basic reporting: -- Introduction does a good job of providing background, context and highlighting key literature in the field.

Methods and Results
-- Figure captions have been revised and now provide sufficient detail that reflects the narrative of the manuscript.
-- Statistically significant differences are indicated accordingly.
-- Minor formatting and style issues have been corrected.
-- Structure conforms to PeerJ standards.
-- Raw data are supplied.
Experimental design: -- The research question is well-defined, and the authors highlight key knowledge gaps the study attempts to address.

Methods
-- The authors have been forthcoming in identifying limitations and logistical issues around experiments, and have explained the rationale behind work-arounds.
-- The revisions provide better clarity around the sequence of experimental steps and the characterisation of biofouling on experimental panels.
Validity of the findings: -- The data are robust and statistically sound.
-- Conclusions are well stated, and the authors do a good job of not over-interpreting the results.
Additional comments: The revisions have substantially improved the clarity of the manuscript.

This is a solid contribution to the field of biofouling prevention technologies, and it will be of interest to researchers as it provides insights towards the mechanisms by which bubble curtains affect biofouling (e.g., via disruption and scouring).


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: CONTINUOUS BUBBLE STREAMS FOR CONTROLLING MARINE BIOFOULING ON STATIC ARTIFICIAL STRUCTURES
Review round: 2
Reviewer: 2

Output:
Basic reporting: I am satisfied with the adjustments and additions that the authors made to address my comments. I have no further comments.
Experimental design: I am satisfied with the adjustments and additions that the authors made to address my comments. I have no further comments.
Validity of the findings: I am satisfied with the adjustments and additions that the authors made to address my comments. I have no further comments.
Additional comments: No additional comments


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

Input:
Title: THE HYPOFERREMIC RESPONSE TO ACUTE INFLAMMATION IS MAINTAINED IN THALASSEMIA MICE EVEN UNDER PARENTERAL IRON LOADING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The work aims at verifying the effect of LPS and iron treatments, separately and together, on various indices of iron status in a mouse model of b-thalassemia and wild type controls. The results show that many indices respond similarly  in the two types of mice, with the exception of Hepcidin, ERFE and DMT1 mRNAs. The work seems rather unfocused and the significance of the findings are unclear. This is evident even in the title with the obvious statement that "LPS alters iron homeostasis in mice....".
Experimental design: It is unclear why they they induced  iron supplementation via intramuscular injection, that is an unusual way, since most studies use intravenous injections. Blood transfusions would have been a better model.  The authors should indicate a reference for the treatment.

A sensitive index of inflammation other than IL6 is important to verify the success and extent of LPS treatment. And also to verify whether the intramuscular iron treatment induces inflammatory response. The liver CRP mRNA is one of the possible indices.
Validity of the findings: no comment
Additional comments: The discussion is far too long and unfocused. Rather than a summary of all previous studies on effect of iron and LPS in cells and mice, the discussion should deal with the comparison between WT and BKO mice and possibly provide an interpretation on the novel findings, such as the blunted hepcidin response and the higher levels of Fam132b and of Slc11A2 in BKO mice.


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

Input:
Title: THE HYPOFERREMIC RESPONSE TO ACUTE INFLAMMATION IS MAINTAINED IN THALASSEMIA MICE EVEN UNDER PARENTERAL IRON LOADING
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: In the manuscript the authors report the effect of iron loading and acute inflammation on iron homeostasis in wild-type and  Hbbth3/+ (thalassemic) mice. They show that inflammation, independently of parenteral iron loading, modulated the expression of hepcidin and iron transport molecules in both WT and thalassemic mice.  The hypoferremic response to acute inflammation was maintained in iron-loaded Hbbth3/+ mice and was, at least  partially, independent on hepcidin regulation.

Minor comments:

1.	Methods part in the abstract should be better described, while respecting the requirements of the journal – more information about treatments (iron loading, acute inflammation) instead of statistics.

2.	Some abbreviations, while explained in the text, should also be defined in the abstract (e.g. FPN1, ERFE).

3.	The authors are advised to use the designation Hbbth3/+ instead of BKO, both in the text, tables figures and legends.


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

Input:
Title: THE HYPOFERREMIC RESPONSE TO ACUTE INFLAMMATION IS MAINTAINED IN THALASSEMIA MICE EVEN UNDER PARENTERAL IRON LOADING
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The answers to the points I raised are satisfactory and the manuscript improved.
As a title, I think that the suggested one below gives a better description of the work than the present one.
“The hypoferremic response to acute inflammation is maintained in thalassemia mice even under parenteral iron loading”,


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF THE TREHALOSE-6-PHOSPHATE SYNTHASE (TPS) GENE FAMILY IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript shows the identification, characterization, and expression analysis of Trehalose-6-phosphate (TPS) genes of cucumber under different stress conditions. The manuscript structure is acceptable, however, there are points that should be check.
Experimental design: The authors mention that they used three technical replications (line 161 "Each reaction was repeated three times") for qRT-PCR-, but they do not comment how many biological replicates and how many plants they used for the analysis. This information is essential to support the statistical analysis and conclusions. The authors must add this information to the manuscript.

Line 87-95 Identification of TPS candidate sequences is not clear enough, it is suggested to define clearly the characteristics of TPS candidates, what were the characteristic domains or sequences that were searched in the HMMsearch analysis.
Line 91-92 How do you define an uncertain sequence? What was the goal of re-examining the sequences in Pfam and CDD?

NJ method for reconstruct the evolutionary relationship is weak method. ML o Bayesian methods would be preferred.

It is not clear why using root as calibration sample to calculate the relative gene expression (Fold change) in the different tissues. If the authors determine the relative gene expression only using the reference gene (CsActin) would give a better picture of how these genes are expressed in the tissues.
Validity of the findings: Line 209 It is not clear why the sequences were divided into six subgroups.

Lines 268-269 It is not clear why the authors indicate that CsTPS genes are involved in cucumber growth and development, they evaluated the expression at only a developmental stage.

The expression analysis of TPS genes in response to different types of stress shows interesting information, however, the discussion of these results is weak. It is suggested to perform a hierarchical clustering of the results of gene expression to identify common expression patterns, likewise, the authors must deepen the discussion of these results.

Lines 343-345 Is there previous evidence that plants have a low resistance to oxidative stress? If so, the authors should include references to complement this part.
Additional comments: For a better understanding of data presented, the authors should improve figure 3. It is suggested to add information that indicated which motifs represent each characteristic domains of the TPS proteins.

Line 279 This sentence is no 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: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF THE TREHALOSE-6-PHOSPHATE SYNTHASE (TPS) GENE FAMILY IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The sentence of the whole article is smooth, but there are still a few sentences that need to be further revised. It is suggested to ask foreign teachers to help modify it.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1. It is too simple to describe the expression patterns of genes under different stresses. The expression patterns of all genes under a single stress condition should be analyzed one by one. Then, in addition to the induction of genes which are more sensitive to stress. The author should express the content of the chart in the expression analysis more in words.
2. The discussion section needs to focus on one or two topics rather than on every study result. for example, focus on the expression pattern of one gene in different crops.
3. The study of transcription factors should be from the aspects of identification, chromosome location, phylogeny, structure, expression patterns, cis-elements in the promoter, and so on.
4. The full description of the cis-acting element can be placed in the test method and does not need to be described in the study result.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF THE TREHALOSE-6-PHOSPHATE SYNTHASE (TPS) GENE FAMILY IN CUCUMBER (CUCUMIS SATIVUS L.)
Review round: 1
Reviewer: 3

Output:
Basic reporting: Improvements in English are required. Most significantly the paper does not consider the full literature regarding role of the trehalose pathway. See my fuller comments further down.
Experimental design: Seedlings only are used for expression analysis, yet the trehalose pathway may be particularly important in reproductive development, so the full picture does not emerge.
Validity of the findings: See above regarding comments on expression analysis. Critical function is overclaimed based on expression analysis in seedlings.
Additional comments: Genome-wide identification and expression analysis of the trehalose-6-phosphate synthase (TPS) gene family in cucumber (Cucumis sativus L.). Dan et al.
The paper documents information based on available bioinformatics data and expression analysis to assemble an analysis of the trehalose phosphate synthase (TPS) gene family in cucumber.
The writing is largely quite good and clear throughout with only a few language errors (see comments at end for examples of where improvements are necessary). However, consideration of the trehalose field in the paper is very limited and does not represent the current status of knowledge and sense of momentum that exists in the research area. In the Introduction and throughout the paper trehalose 6-phosphate (T6P) as critical component of the trehalose pathway is barely considered except for one reference to TPS1-T6P signaling pathway in tomato in the Introduction (Chen et al. 2017). There is a published example in cucumber that is not cited (Zhang et al. 2015 T6P and SNF-1 related protein kinase are involved in the first fruit inhibition of cucumber Journal of Plant Physiology 177, 110-120) which provides valuable context. The authors should refer to this and include it in interpreting their own results. T6P has been implicated in the regulation of sucrose homeostasis and in integrating sucrose supply with growth and development and coordination of source-sink relations in plants and crops. The trehalose pathway has been shown to be involved in improving stress resilience, but this may be through T6P rather than trehalose i.e. in adjusting sugar levels e.g. Nuccio et al. 2015 doi.org/10.1038/nbt.3277; Griffiths et al. 2016 doi.org/10.1038/nature20591. For an overview see T6P signalling and impact on crop yield doi.org.10.1042/BST20200286. As the trehalose pathway is so strongly linked to sugar metabolism, it is a pity that sucrose was not used in the studies that determined gene expression responses to different to different factors. TPSs of Arabidopsis can be sugar repressed or sugar induced. A major function of the trehalose pathway is in reproductive development and it is a shame again that expression levels of the TPSs were not determined during reproductive development and in fruit as this where the trehalose pathway may have a particularly strong role in yield formation in cucumber and in crops generally. However, 200 µmol m-2 s-1 irradiance may be too low for fruit formation. Cucumber requires sunny conditions, particularly for productive fruit formation. Do measurements of gene expression in cucumber seedlings grown at 200 µmol m-2 s-1 irradiance give a true picture for the crop?
The role of trehalose as stress protection compound is possible, but trehalose doesn’t accumulate to high enough levels to perform a major role of stress protectant and mode of action of trehalose is less clear than that of T6P including in cucumber (Zhang et al. 2015). The paper considers stress in terms of a more extremophile response whereas ongoing mild stress interspersed with moderate and severe stress plus periods of recovery is more of a reality for much of agriculture. Fruit production in cucumber will be the most stress-sensitive developmental phase from flowering to harvest, yet this aspect is not considered because seedlings only are used for gene expression analysis.
Line 354 and in the conclusion. The paper states that critical roles for TPSs have been established. Yet there is no direct evidence in this paper that TPSs perform critical roles in cucumber. This can only be assessed based on studies in other species. Expression studies are not enough to confirm critical function.
Line 368 speculation that TPS2 and TPS4 have catalytic activity. This is based I guess on the assumption that class I TPSs have catalytic activity? No direct evidence is supplied in the paper, though. How could this conclusion be better supported?
 “Plant stress resist ability” line 360/ 361, replace with “plant stress resistance”.
In the Introduction are osmosis and superoxide adverse environments?