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: PREVALENCE OF HPV GENOTYPES AND ASSESSMENT OF THEIR CLINICAL RELEVANCE IN LARYNGEAL SQUAMOUS CELL CARCINOMA IN A NORTHEASTERN STATE OF BRAZIL—A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall well-written.
Experimental design: How many HPV types were analyzed, and which ones?

The sample selected in the study is from a referral hospital. How representative is it of a community? Maybe only the advanced cancer are referred to the hospital, and hence the estimation reported in the study is biased given the association of HPV-associated laryngeal cancer with prognosis.

Overall, the sample size is small to run multivariable models and should be acknowledged as a limitation.

Given the small numbers instead of N, M, T stage-wise analysis, why didn't the authors analyze as distant metastasis yes/no, lymph involvement yes/no? Moreover, it's incorrect to choose no information as a reference category for the T stage analysis.

Was there any difference in histologic subtypes by HPV status?
Validity of the findings: The conclusion statement regarding the role of HPV as a risk factor in laryngeal carcinoma can not be derived from this study as there is no control group, and it's not a prospective study.
Additional comments: The discussion is too long and wordy.


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: PREVALENCE OF HPV GENOTYPES AND ASSESSMENT OF THEIR CLINICAL RELEVANCE IN LARYNGEAL SQUAMOUS CELL CARCINOMA IN A NORTHEASTERN STATE OF BRAZIL—A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Prevalence of HPV genotypes and assessment of their clinical relevance in laryngeal squamous cell carcinoma in a northeastern state of Brazil” by Santo de Aquina et. al, is reporting prevalence of HPV in laryngeal squamous cell carcinoma (LSCC) in a region specific manner. The prevalence of HPV in LSCC has been reported previously elsewhere as well. The only novelty in this work is that it is pertaining to a specific geographic location, northeastern state of Brazil, where there has not been any apparent reporting previously. In addition, the study could prove helpful in deciding the vaccination strategies against specific HPV genotypes prevalent in the region.   However, I have some concerns/questions about the manuscript as detailed below:

-In general, the data set presented here is small (n=82) especially when distributing across different stages of LSCC, the observations must be taken with caution.  There is too much of extrapolation based on the previously published data rather than the results presented in the manuscript.

-The results section of the manuscript  is too brief and seems giving more emphasis on the numbers which have already been mentioned in the Table 1 and are mostly not statistically significant, with p-values >0.05. The statistically significant parameters which are only 3 in the Table 1 should have been emphasized more or maybe shown as figure and the rest of Table 1 should have been either mentioned briefly and or added as supplementary.

-In the Discussion section, authors are applying the observations from other regions to discuss the results from northeastern state of Brazil where most of the parameters studied elsewhere could not be satisfied statistically.

-The important observation of relationship between T3/T4 disease stage and HPV+ incidence should have been explained further with validations from other similar studies.

-In the conclusion of the paper, line 266 to 268: “patients with HPV+ LSCC were generally found to be nonsmokers, to reside in the state capital, and to have a higher index of T3–T4 tumors”, the authors must specifically mention that there was no significant relationship observed in the first two cases (nonsmokers and residence) in their study. Also, make it clear in the abstract as well especially that there was no significant relationship observed between the economic, educational, occupational with the HPV LSCC in the presented data.

-The authors mentioned they could not detect HPV18 genotype (line 232 to 234), the second most common genotype in laryngeal cancers, in their patients. It is intriguing to know the frequency of HPV18 genotype reported in the country or in the adjacent regions?
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: PREVALENCE OF HPV GENOTYPES AND ASSESSMENT OF THEIR CLINICAL RELEVANCE IN LARYNGEAL SQUAMOUS CELL CARCINOMA IN A NORTHEASTERN STATE OF BRAZIL—A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 1

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


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

Input:
Title: COMPARATIVE FULL-LENGTH TRANSCRIPTOME ANALYSIS BY OXFORD NANOPORE TECHNOLOGIES REVEALS GENES INVOLVED IN ANTHOCYANIN ACCUMULATION IN STORAGE ROOTS OF SWEET POTATOES (IPOMOEA BATATAS L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript entitled “Comparative full-length transcriptome analysis by Oxford Nanopore Technologies reveals genes involved in anthocyanin accumulation in storage roots of sweet potatoes (Ipomoea batatas L.)” presented comparative RNA-seq analysis between two different sweet potatoes as well as pigment detection, based on which the author found out some candidate genes involved in flavonoid biosynthesis, aiming to provide basic information for future breeding. The overall research is relatively standardized, but lack of new ideas. Finally the author presented prosaic manuscript, in which the method and analysis results were regular without special points. What need to extensively modify were below.
Major point:
--Paragraph 2 in Introduction section, the author spent large content to introduce the RNA sequencing and focused on the 3rd generation sequencing technology, which was not necessary. The purpose of this research is to discover relevant genes involved in flavonoid biosynthesis between two different sweet potato cultivars simply named PFSP and WFSP rather than explore the development of RNA-seq technology, so the descriptions in introduction section should focus on the purpose of study rather than others. The same problem appeared in the discussion section. The author need to revised or removed. Similarly, in Figure 2, it was meaningless to introduce the ONT RNA-seq approach (Figure 2B) and the types of alternative splicing (Figure 2D), which had nothing to do with the aim of your research.
--Line 92-94, “In this research, the transcriptome profiles of storage roots of PFSP and WFSP at different developmental stages were identified using the ONT MinION platform in order to reveal the molecular mechanisms for anthocyanin accumulation in the two sweet potato cultivars”, molecular mechanism in this sentence is an overstatement because the study in only a preliminary exploration for anthocyanin accumulation in compared two cultivars. The author just provided some candidate genes involved in flavonoid biosynthesis, no relevant mechanism research were involved in this research. Therefore, the author need to revise the inappropriate wording statement throughout manuscript.
--In anthocyanin detection, it is not enough that the author only mentioned the method of UV-visible spectrophotometric measurements. The specific and detailed steps and parameters should be described in manuscript. How to extract anthocyanin from samples? Which wavelength was used in measurements?
--Figure 1, I am confused why the author separated the anthocyanin content of PFSP and WFSP into two figures. Putting Figure 1B and C together could more intuitively reflect the difference of anthocyanin content of PFSP and WFSP. Additionally, the anthocyanin content corresponded to the samples of seven periods and the content presented a dynamic change trend, why the author did not place seven corresponding phenotype of PFSP and WFSP? This would be more visible.
--Most of parts in discussion section were the simply repeated presentation of the results from the article, without the author’s own views or introducing new perspectives. The author should make extensive modifications in discussion section.
--The English language need to improve.
Minor point:
--Line 63, “The amount of ash...”, what is the meaning of ash here.
--Line 118, “firstly”, should be “Firstly”.
--Line 123, “ ...1D sequencing adapter...”, what is the meaning of 1D?
--Line 124, “ loadingto”, should be “loaded to”
--Line 128, “...minimum average read quality score of 7...”, what is the criteria for defining the read quality score?
--Line 218-219, “With respect to the group PL vs. WL 216 genes were up-regulated and 11 genes were down-regulated”, should be “With respect to the group PL vs. WL, 216 and 11 genes were up-/down-regulated, respectively”
--Line 231-233, the author identified transcripts based on the transcriptome data between PH and WH comparison, which meant the candidate gene screening were depended on the different expression of genes between different comparison. 3rd sequencing mainly provided specific sequence information while 2nd sequencing provided differential expression. So I want to know whether the author have perform 2nd transcriptome analysis.
--In conclusion section, the last sentence is the significance of your research rather than your study conclusion.


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: COMPARATIVE FULL-LENGTH TRANSCRIPTOME ANALYSIS BY OXFORD NANOPORE TECHNOLOGIES REVEALS GENES INVOLVED IN ANTHOCYANIN ACCUMULATION IN STORAGE ROOTS OF SWEET POTATOES (IPOMOEA BATATAS L.)
Review round: 1
Reviewer: 2

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


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: COMPARATIVE FULL-LENGTH TRANSCRIPTOME ANALYSIS BY OXFORD NANOPORE TECHNOLOGIES REVEALS GENES INVOLVED IN ANTHOCYANIN ACCUMULATION IN STORAGE ROOTS OF SWEET POTATOES (IPOMOEA BATATAS L.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The author have addressed my concerns carefully in the revised version that could be published.


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: COMPARATIVE FULL-LENGTH TRANSCRIPTOME ANALYSIS BY OXFORD NANOPORE TECHNOLOGIES REVEALS GENES INVOLVED IN ANTHOCYANIN ACCUMULATION IN STORAGE ROOTS OF SWEET POTATOES (IPOMOEA BATATAS L.)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I don’t think the authors have properly revised the manuscript. Not reflected in the main text. Also, there are still many mistakes.


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: COMPARATIVE FULL-LENGTH TRANSCRIPTOME ANALYSIS BY OXFORD NANOPORE TECHNOLOGIES REVEALS GENES INVOLVED IN ANTHOCYANIN ACCUMULATION IN STORAGE ROOTS OF SWEET POTATOES (IPOMOEA BATATAS L.)
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I think the authors have properly revised the manuscript based on the reviewer's 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: DOWNREGULATION OF THE ENHANCER OF ZESTE HOMOLOG 1 TRANSCRIPTIONAL FACTOR PREDICTS POOR PROGNOSIS OF TRIPLE-NEGATIVE BREAST CANCER PATIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this paper, Peng Wei et al. investigate the expression and transcriptional mechanisms of EZH1/EZH2 in triple negative breast cancer tissues. In addition, it has been analyzed the molecular signature as a prognostic value for risk stratification in patients affect by TNBC.

This work is well written and the english language is sufficient quality.
The figures and tables are enough clear. The citations cover the relevant literature adequately.
Experimental design: The methods using in this work are well described.

The statistical method seems valid and correct.
Validity of the findings: The conclusions are very little described  (only two lines). We could speculate the role of CCNB1 on immune system cells such as T and NK cells. It has been well described the role of these cells in tumor immunesuveillance. It would also be intersting to investigate the effect of pathenolide on Immune cells.
The impact on and the novelty of this potential therapeutic approach is not well described.
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: DOWNREGULATION OF THE ENHANCER OF ZESTE HOMOLOG 1 TRANSCRIPTIONAL FACTOR PREDICTS POOR PROGNOSIS OF TRIPLE-NEGATIVE BREAST CANCER PATIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors provide novel informaton of the potential role of EZH1 and EZH2 expression in breast cancer. The disruption of the expression of these molecules have been well documented in haematological diseases while less in solid tumors. Moreover,  the novelty of the paper is to suggest that while EZH1 upregulation wold seem to favor haematological diseases, in the present manuscript  is the downregulaiton of EZH1 that seems to correlate with a poor prognosis of TNBC.
It also of interest the  opposite result obtained  by analysis of EZH2 , suggesting a non reduntant  but rather  complementary  contribution of the two molecules to the disease .
Experimental design: The reviewer has a little expertise in in silico and clinical analyses , however data are produced by analyzing a high number of samples, and the diffferent type of statistical evaluations performed   seem to be coherent with the conclusions. However, it would have been important  to validate some of this data performing in vitro experiments . The effect of inhibitors of these moleculse could have been analyze on tumor BC cells( BC cell lines?) showing no perturbations in EZH1 and EZH2 expression and  then verify whether these cells would acquire a phenotype similar to that observed  in TBNC cells and whether the expression of genes  considered target of EZH1 or EZH2 activity resulted modified upon treatment wiht EZH1/2 inhibitors.
Validity of the findings: Again , data appear of great interest even if some in vitro analyses would have help to improve their validity.
Additional comments: NO further 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: DOWNREGULATION OF THE ENHANCER OF ZESTE HOMOLOG 1 TRANSCRIPTIONAL FACTOR PREDICTS POOR PROGNOSIS OF TRIPLE-NEGATIVE BREAST CANCER PATIENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Review
Downregulation of the enhancer of zeste homolog 1 transcriptional factor predicts poor prognosis of triple-negative breast cancer patients
The authors in this paper wanted to prove EZH-1 downregulation as a prognostic factor for risk stratification in patients with TNBC. They have used multiple molecular biology and bioinformatics tools, to prove the same. In addition to showing the downregulation of EZH1, they have also discovered downstream upregulated targets and pathways. Not only that, they have even done high throughput screening to identify the small molecule, Parthenolide, to sequester one of the up-regulated proteins, CCNB1, thus proving it as a potential therapeutic agent for TNBC patients. The research that went into this manuscript is commendable. However, I would like to draw the authors' attention to the following discrepancies.
Use colon (:) instead of a full stop at the end of subheadings.
Line 43: Please correct lack to lacks.
Line 58: Correct  66 TN to 66 TNBC.
Line 97: Correct “constructs” to “is”.
Line 111: Add  ‘to be” between proved and upregulated.
Line 135: Change were to was.
Line 138: “if they met any standard, as follows” - can be removed from the sentence.
Line 140: Please explain what is a special type of BC.
Line 154: The word “queried” might not be required, as it is implied already.
Lines 289 and 294: Suggest verifying if publication bias can be used in this context.
Line 418: Introduce “the” before response and hormone. Correct mutual to mutually.
Line 470: Correct researches to research.
Line 481: Correct transcriptional to transcriptionally.
Line 504: Correct stratifications to stratification.

General: Figure legends that represent combined results such as (A, B), (C–E), etc., should have proper labeling under the  images and/or should be separately notated under a general heading, with a separate mention. For Ex. Instead of the current legend, 1. (C–E) TNBC and non-TNBC tissues (area under the curve > 0.7). TNBC, triple-negative breast cancer, can be modified as EZH1 displayed moderate discriminatory ability in differentiating between D) TNBC and E) non-TNBC.
Experimental design: Introduction
Line 81 - 83: What is the stage of TNBC in non-Hispanic black women that is compared to Stage IV TNBC in Asian women?
Line 111: Correct miRNA193 to miRNA93.

Results and Figures:
EZH1 was downregulated in TNBC tissues:
    1. The text states that EZH1 is downregulated, while the legend says the opposite. Correct accordingly. Also, ad space between non-TNBC and TNBC labels on the y-axis.
2. Please mention  Fig 1C in the text. It will be helpful if the images are labeled below the graphs to specify the tissue samples (TNBC or non-TNBC),  from which the observations are made.
3. Correct line 81 (Fig 1D-1F) to (Fig 1C-1E).
4. Is it possible to put the figures 2 - 4 together under one figure for easy comparison and rearrange other important findings made in this manuscript?
Line 303: EZH1 may be a risk factor for poor prognosis of TNBC patients - EZH1 downregulation worsens prognosis, hence it is not a risk factor.
Figure 7D is encroaching on to Fig 7E. Can it be resized or reorganized?
Label below images 8B, C, and 8D, E as subtype 1 TNBC and subtype 2–3 TNBC, respectively.
Label 8 E, F, and G for their respective miR’s.
Parthenolide was identified as a potential therapeutic agent for treating TNBC patients: This is an important addition to the research, and I would recommend moving the Supplemental figure S8 to the main manuscript.
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: DOWNREGULATION OF THE ENHANCER OF ZESTE HOMOLOG 1 TRANSCRIPTIONAL FACTOR PREDICTS POOR PROGNOSIS OF TRIPLE-NEGATIVE BREAST CANCER PATIENTS
Review round: 1
Reviewer: 4

Output:
Basic reporting: This is a well-written article that described a down-regulation of the enhancer of zeste homolog 1 transcriptional factor, with the potential of predicting poor prognosis, in triple-negative breast cancer (TNBC) patients.

TNBC has been recognized as the most malignant subtype of breast cancer and lack effective biomarkers. In addition to reveal the prognostic molecular signatures, this study was designed to unravel the expression status and the prospective transcriptional mechanisms of EZH1/EZH2 in TNBC tissues.
Experimental design: The experiments were designed with target search from previously published and deposited datasets, tissue immunohistochemistry (IHC), applying a prognostic signature model to predict survival and risk probabilities in TNBC patients, determination of somatic mutations at EZH1/EZH2 loci, and prediction of down-regulation of EZH1 in activating cell cycle pathway.
Validity of the findings: This is a bioinformatic article. It was written based on reanalyses of existing data, with lacking of additional wet-lab studies. It is suggested that authors may conduct RT-qPCR to demonstrate the differential expression of EZH1/EZH2 and perform functional analysis to confirm the pathogenic impact of EZH1 on pathway(s).
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: DOWNREGULATION OF THE ENHANCER OF ZESTE HOMOLOG 1 TRANSCRIPTIONAL FACTOR PREDICTS POOR PROGNOSIS OF TRIPLE-NEGATIVE BREAST CANCER PATIENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Re-review
Downregulation of the enhancer of zeste homolog 1 transcriptional factor predicts poor prognosis of triple-negative breast cancer patients.

The authors in this paper wanted to prove EZH-1 downregulation as a prognostic factor for risk stratification in patients with TNBC. They have used multiple molecular biology and bioinformatics tools, to prove the same. In addition to showing the downregulation of EZH1, they have also discovered downstream upregulated targets and pathways. Not only that, they have even done high throughput screening to identify the small molecule, Parthenolide, to sequester one of the up-regulated proteins, CCNB1, thus proving it as a potential therapeutic agent for TNBC patients. The research that went into this manuscript is commendable.

The authors excellently addressed all the reviewers’ suggestions. In my opinion, the work that went into this paper is expansive. If all other reviewers and editors are satisfied with the additions, I suggest two publications instead of one. If there is a shortage of information for the second publication, they can easily add the supplementary figures as the main figures.
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: No additional comments


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

Input:
Title: CYCLOCYTIDINE HYDROCHLORIDE INHIBITS THE SYNTHESIS OF RELAXED CIRCULAR DNA OF HEPATITIS B VIRUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript addresses an interesting subject, suitable for publication in this journal. There are some concerns about the methodology and results in the presentation that needs to be addressed before providing a positive recommendation.
Experimental design: How was the Southern blot performed? Please add details about these experiments. What was used as a probe? Most importantly, how much DNA was loaded per lane? The figure legends stated that certain volumes were used per lane, but this is incorrect. The lanes should include the same amount of DNA, for comparison purposes.
Please include details about the densitometric analyses applied to southern blot images.
Importantly, there is no statistical analysis applied to results, and this is mandatory to draw conclusions. In the same line, a statistical analysis section should be included in the methodology.
Validity of the findings: Figures 3 and 4. I agree with the results presented as a ratio between both DNA forms, but I also suggest including the relative abundance of RC and NS and comparing them across the samples from cells treated with different concentrations of the compound. Results suggest that NS abundance is also affected by the drug.
Additional comments: Figure 1 should be deleted, adds no value to the manuscript content.


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: CYCLOCYTIDINE HYDROCHLORIDE INHIBITS THE SYNTHESIS OF RELAXED CIRCULAR DNA OF HEPATITIS B VIRUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear, concise, and well founded.
Experimental design: Simple and clear. Basic toxicity test, followed by measurements of drug inhibition. There could be additional work, but the work contained herein is excellent.
Validity of the findings: I had one major concern with figure 4C (see comments), but it should be easily remedied.
Additional comments: Small grammatical or spelling errors were noted. Otherwise the English and writing were excellent.


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: CYCLOCYTIDINE HYDROCHLORIDE INHIBITS THE SYNTHESIS OF RELAXED CIRCULAR DNA OF HEPATITIS B VIRUS
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this paper the authors tested the effect of cyclocytidine HCl on HBV DNA synthesis. They found that the cyclocytidine HCl decreased the synthesis of HBV rcDNA, and they hypothesize than this might be due to the inhibition of viral DNA polymerase activity.

General Comments

This is an interesting, straight forward work, with clear aims and an adequate experimental design. However, a number of observations should be answered by the authors before the manuscript is suited for publication.

A number of grammatical and language style mistakes were found throughout the manuscript. Thus, we suggest a deep review of English language.

The main deficiency of the present work is the lack of statistical analysis of data presented.

Introduction

The authors wrote that “Current anti-HBV agents primarily target the process of rcDNA synthesis, but cannot inhibit or destroy cccDNA formation”. It would be important to describe what is the actual effect of the mentioned anti-HBV agents, and why there is a need to look for new anti-viral agents.

Although cyclocytidine HCL is the agent evaluated in this work, the information provided about it in the Introduction section is very scarce, it is not clear why the authors selected this particular prodrug. Please write a clear justification to study cyclocytidine HCL as a potential anti-viral agent.
Experimental design: Materials and Methods

Brand names of relevant reagents are only cited in a few of them. Please indicate the brand name of the most important reagents included in the present work.

There is not a subsection of Statistical Analysis. To support your findings a basic statistical analysis should be performed, and described in the Materials and Methods section.
Validity of the findings: Results

Sub-section “Cyclocytidine HCl decreased rcDNA production”.  The authors declare that “20 µM cyclocytidine HCl appeared to moderately decrease rcDNA production”, “moderately decrease” is inconclusive, the authors must show a statistical analysis to support this observation.

In lines 145-146, the authors describe that “…the extended time (4 days) of treatment decreased the production of total core DNA that might result from cytotoxicity (Figure 3C, lane 3).”  It would be interesting to demonstrate that the effect on core DNA production is actually due to cytotoxicity, a simple assay incubating cells with 20 µM cyclocytidine HCl at different time points (including 2 and 4 days) evaluating cell viability with the MTT would provide a clear answer to this point.

Sub-section “Cyclocytidine HCl inhibited the synthesis of the positive strand of rcDNA”. Although the effect on rcDNA synthesis seems to be clear, a statistical analysis is needed to support the findings.

Figures

Figure 1. There is a number of inconsistencies in Figure 1 capture. For instance, the authors indicate “(left) Relaxed circular (rc) DNA.” , however there is no need for indicating “left” since the title “Relaxed circular DNA” is written. Same comment with the “right” section of the Figure. The authors do not declare what “POL” means. Please correct this Figure caption.

Figure 2.  Figure caption, the authors declare that “The Y axis represents the relative cell viability, and the X axis represents the dose of cyclocytidine HCl.”, again, there is no need for this phrase, since axis titles are clear enough.

Figure 3. The authors declare that “The data was presented as the mean±SD”, however the authors do not indicate whether the differences shown are significant. Statistical analysis of the results is needed.

Figure 4. The authors declare that “(B) The ratio of rcDNA to ssDNA was compared between the DMSO and the cyclocytidine HCL-treated groups. (C) The total amount of core DNA was compared between DMSO and cyclocytidine HCl treated group.”  In the actual figure it is not clear where DMSO treatment was used.

“The data was presented as the mean±SD.”  Again, the authors do not indicate whether the differences shown are significant. Statistical analysis of the results is needed.

Conclusions

Although the results showed that cyclocytidine HCl had a potentially favorable anti-viral effect the authors significantly limit the meaning of their findings by declaring that “A high test concentration (20 µM) may not be suitable for anti-HBV treatment, especially since highly efficacious anti-HBV drugs are already available…” then what is the relevance of the work?, what is the “light” that the results “shed on usage of an “old” drugs for a “new” application”, what are the perspectives of the work ?
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: CYCLOCYTIDINE HYDROCHLORIDE INHIBITS THE SYNTHESIS OF RELAXED CIRCULAR DNA OF HEPATITIS B VIRUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was improved, but still contains flaws that need to be solved.
Experimental design: My original question about the probe was not addressed. Please include the region coordinates where the probe is hybridizing.
The problem originally spotted with the DNA loading on gels still remains. The authors need to provide Southern blot images generated with the same DNA loaded per lane, otherwise, the comparison between lanes is meaningless.
Validity of the findings: Data are not valid, as southern blots were not generated with the same amount of DNA per lane.
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: CYCLOCYTIDINE HYDROCHLORIDE INHIBITS THE SYNTHESIS OF RELAXED CIRCULAR DNA OF HEPATITIS B VIRUS
Review round: 2
Reviewer: 2

Output:
Basic reporting: All comments have been properly attended
Experimental design: All comments have been properly attended
Validity of the findings: All comments have been properly attended
Additional comments: All comments have been properly attended


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: CYCLOCYTIDINE HYDROCHLORIDE INHIBITS THE SYNTHESIS OF RELAXED CIRCULAR DNA OF HEPATITIS B VIRUS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript was improved.
Experimental design: The manuscript was improved.
Validity of the findings: The manuscript was improved.
Additional comments: The manuscript was 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: ESTIMATING BITE FORCE IN EXTINCT DINOSAURS USING PHYLOGENETICALLY PREDICTED PHYSIOLOGICAL CROSS-SECTIONAL AREAS OF JAW ADDUCTOR MUSCLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is very detailed and clear. The text is comprehensive, provides a good background on the topic supported by relevant and adequate references and overall very professionally written.
Experimental design: The methodology is very interesting and offers and alternative and novel approach to estimated bite forces in extinct species and could also be applicable to modern species for which traditional bite force experiments are not possible.
The methods are described in detail and have been performed to a professional standard.
Validity of the findings: The results are clear and reported in detail. Their discussion is valid and supported by the results.
Additional comments: This is a very interesting study providing a new outlook to estimate bite forces in fossil species. I found the manuscript enjoyable to read and think this is a worthwhile contribution to the topic.

I have one more substantial point I would like to see included/discussed: How do the predicted bite force values obtained in this study compare to existing estimates? I appreciate that not all studied species have been the focus of previous work but there seem to be some differences in specific taxa. For example: https://www.nature.com/articles/s41598-022-06910-4 and https://peerj.com/articles/1093/
It seems the method under-/overestimates bite forces and a discussion where these differences could stem from would be useful.

Apart form that, I have a few minor points, questions, and suggestions but otherwise find the manuscript ready to be accepted.

Individual points:

- Line 40: Add here that bite force also depends on the skeletal morphology, not only on muscle anatomy and architecture alone.

- Line 114: Which part of the muscle body was sectioned? The region with the highest circumference?

- Line 116: Mention here what these calculations were based on.

- Line 126/128: "I" not "We"

- Line 132: Maybe explain the abbreviation PPM here again (I know it is in the introduction but the reader may have forgotten at this point).

- Line 281ff: Could you discuss briefly, how specific the model used here is. For example, is it only valid for dinosaurs or could it be transferred easily to modern and fossil mammals? Further, would it work of the sample size included a mix of species across a wide range of phylogenetically diverse species?

- LIne 314ff: Kind of following on from the previous comment. How adaptive is the model in general? Could other performance measures be analysed in a similar fashion? What would be required to do that?


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: ESTIMATING BITE FORCE IN EXTINCT DINOSAURS USING PHYLOGENETICALLY PREDICTED PHYSIOLOGICAL CROSS-SECTIONAL AREAS OF JAW ADDUCTOR MUSCLES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting is excellent in all categories. I  strongly suggest citing a few additional papers. Absolutely minimal suggestions for the writing.
Experimental design: The goal of the paper is so important that I suggest putting it up front in the abstract. The hourglass structure of scientific writing (broad contextualization to focus to broad implications) is fine, but we tend to bury the real lede. The reader might go from "bite force" to "Spinosaurus" and miss the paper's strongest value (see Validity below).

The commented manuscript calls attention some muscle vectors, which appear to extend farther anteriorly (if they were position vectors) than intuitively likely centroids of muscle pull (Cost et al. 2019, 2022).
Validity of the findings: The paper is tremendously useful in its findings that:
1. we can estimate bite forces with phylogenetic prediction based on an easy measurement (skull width);
2. sample size and performance estimates for some extinct taxa powerfully enhance estimates for other extinct taxa.

The predicted estimates in Table 1 are useful and interesting on their own, and the discussion of examples is appropriate and fascinating.

The dissections and measurements of muscle PSCA parameters are terrific as primary data and validation. I really like the groupings of muscle by in vivo function, rather than developmental classification.
Additional comments: The manuscript was interesting both operationally and philosophically. As suggested above, put the paper's rationale and goal in the first sentence of the abstract. I learned in engineering that if you bury the lede, people die. State the result or purpose immediately, and then (like good scientists) we can introduce the history of bridge building.

A peripheral and broad issue I'd like addressed is estimates of branch lengths from first and last occurrences. I can look at a familiar part of the tree in Figure 3 and see branch lengths that appear to radically contradict fossil evidence, and what we'd expect from finer-scaled phylogenies with more taxa.

I suggest commenting on this as a potential issue for the results, and cite it as a real strength of the method: later studies with refined branch lengths are likely to improve already excellent phylogenetic predictions. Or, tell me I'm wrong. Likely as not, but I've spent hours correcting occurrences from the Paleobiology Database.

If the author wishes, they might address the 5-10 fold discrepancy of bite force estimates for Deinonychus from the current method and bone indentation experiments. The issue is probably outside the scope of the paper.

The manuscript is an innovative, useful, and important methodological and biological contribution that I'd like to see published quickly.


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: ESTIMATING BITE FORCE IN EXTINCT DINOSAURS USING PHYLOGENETICALLY PREDICTED PHYSIOLOGICAL CROSS-SECTIONAL AREAS OF JAW ADDUCTOR MUSCLES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a well conceived and generally well written MS and I have no significant concerns with the design or findings. The author presents a method whereby researchers can generate predictions of jaw adducting muscle cross-sectional area based on simple linear measurements after correcting for phylogeny. These in turn can be used as bases for predicting bite forces. Results are largely in line with those based on actual muscle reconstructions.
This certainly provides a useful tool. However, it is also clear that in some instances the proposed method is less accurate than those based on reconstructions. This is acknowledged in the MS, but I do think that this should be clearly stated from the get go, i.e., in the Abstract. It may well be a minority of species to which this applies, but ultimately estimates of cross-sectional area based on reconstructions will be more reliable.
Couple of very minor points:
Line 90-91: "APhys for extant species were calculated from muscle architecture data collected predominantly from literature but also from dissections..." The actual number of extant species and range of taxa should be given here. As it is it's only available in the Suppl Data. "the" should be inserted before "literature".
Line 126: "We further applied muscle pennation...." This is a single author MS.
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: GABRP PROMOTES CD44S-MEDIATED GEMCITABINE RESISTANCE IN PANCREATIC CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Manuscript Number (#60030)
The manuscript is fairly well written and discusses how GABRP promotes CD44s-mediated gemcitabine resistance in pancreatic cancer. In this manuscript, the authors report the first link between GABRP and gemcitabine in pancreatic cancer.

Additional concern:
1.	Although this finding reports the first time GABRP as a downstream of the CD44 effector gene in gemcitabine-resistant pancreatic cancer cells, detailed information on how GABRP promotes CD44s mediated gemcitabine resistance is missing in the manuscript.
2.	The major issue is the use of a single cell line to validate the hypothesis tested. To validates the findings, at least two cell lines must be used to validate the hypothesis tested.
3.	Mechanism of action involved in CD44 directed GABRP expression affecting gemcitabine resistance should be included in the manuscript.
4.	Another major issue that needs to be addressed is the possibility of the non-specificity of shRNA. The knockdown data shown in the figures show the use of only include a single shRNA, and the Methods and Results do not mention how shRNA cells were made. Authors should use more than one shRNA for the study.
5.	Authors should also include a western blot of GABRP for figure 3B.
6.	Authors should show the GABRP protein expression in tumor tissues using immunohistochemistry or immunofluorescence or the pancreatic tissue microarray.
7.	Authors should proofread the manuscript for spelling mistakes.


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: GABRP PROMOTES CD44S-MEDIATED GEMCITABINE RESISTANCE IN PANCREATIC CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: Introduction is written very broadly, revise to fit to scope of the manuscript.

Manuscript writing is not meeting the publication standards.
Experimental design: Could you rewrite methods section in detail. FBS can not be used for culturing stem cells (CD44 high) and there is no mentioning of other media in this section.

Remove the word 'single clone' throughout the manuscript. Use standard nomenclature for the knockdown cell line.

Is it appropriate to use CD44 knockdown cells from CD44 High instead of using CD44 High and CD44 low cells, explain.
Validity of the findings: The manuscript has novel concept of establishing the role of GABRP in chemoresistance of pancreatic cancer stem cells.

However due to very less evidence/data shown in the manuscript, it is highly recommended to do more functional assays to support the hypothesis.
Additional comments: In fig1c, mention the p values.
In fig3c, present the data from one CD44 sh clone. It is always recommended to use two shRNAs rather than multiple clones of single shRNA.
In fig3d, confirm GABRP levels by western blot also.
In fig3, "CF-cl.21-shCD44-cl.1" "CD44 High single cloneshCD44 single clone cells" Replace with standard nomenclature.


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: GABRP PROMOTES CD44S-MEDIATED GEMCITABINE RESISTANCE IN PANCREATIC CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: No Comment
Experimental design: Experimental design is not that much strong enough to support the hypothesis. For example Functional role of CD44 shRNA in spheroid formation or any other functional assays like proliferation, migration and colony formation.

Authors were not mentioned how many replicates they did.
Validity of the findings: No Comment
Additional comments: "GABRP promotes CD44s-mediated gemcitabine resistance in pancreatic cancer" by  Chen chen group is quite interesting and very important in most of the cancers.
But the authors use most of the data from data sets (online), not from original experiments, whatever they use the data, that is good and enough to support the hypothesis, but I feel if it is more experimentally proven.
Here is the some comments to improve the manuscript.
1. Line 43 says some cancers, what are those cancers ?
2. Functional role of CD44 shRNA in spheroid formation or any other functional assays like proliferation, migration and colony formation.
3. Authors were not mentioned how many replicates they did.


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: GABRP PROMOTES CD44S-MEDIATED GEMCITABINE RESISTANCE IN PANCREATIC CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The authors have satisfactorily answered the raised concerns.


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

Input:
Title: GABRP PROMOTES CD44S-MEDIATED GEMCITABINE RESISTANCE IN PANCREATIC CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: By carefully analyzing GEO datasets authors identified GABRP overlaps between datasets, found that high expression of GABRP is associated with poor patient survival and finally provided strong evidence that CD44 and GABRP expression are significantly correlates positively. And in figure 3 authors proved this concept using western blot and RTPCT where over expression of CD44 increased GABRP expression and knockdown of CD44 reduced GABRP expression. So far it is very good. However, authors have not shown any experimental evidence that shows CD44 expression is directly responsible for GABRP expression in PDAC cells. Because there is an equal probability of CD44 being directly involved in GABRP expression or it could be indirect regulation. without having solid evidence authors can not claim "GABRP promotes CD44s-mediated gemcitabine resistance in pancreatic cancer"

Provide experimental evidence how GABRP is involved in Gemcitabine resistance.

In fig 3 include CFPAC1 wild type cells as the reference in all the experiments.
Fig3 provide images  for invasion and colony formation assay.
Fig 3e provide westernblot for GABRP

Fig 3 Authors used only one cell line, reproduce using additional PDAC cell lines
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: TUCUXI-BLAST: ENABLING FAST AND ACCURATE RECORD LINKAGE OF LARGE-SCALE HEALTH-RELATED ADMINISTRATIVE DATABASES THROUGH A DNA-ENCODED APPROACH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and formatted. The figures are clear and explicit. Data and software have been made available when ethically allowed.
The only typo I could see was "macOS" instead of "MacOS" in line 115
Experimental design: The methodology is sound and addresses the research question well. The methods are generally well described. The main improvement I would suggest would be a better explanation of the "codon wheel" used for encoding, and how it relates to the alphanumeric encryption key. Based on figure 1, it looks like the encryption key only needs to be a number.
Validity of the findings: The manuscript describes an innovative method for comparing and linking collections of medical records by encoding specific fields as DNA sequences and comparing them using the well-known BLAST tool. BLAST is optimised for speed and memory efficiency and allows for imperfect comparisons, which makes it a good choice for this task. The conclusions are sound and supported by the data.
Additional comments: Data protection is presented as an advantage of the new method however it seems like the encryption method used would be reasonably easy to break. However none of the other methods that Tucuxi-BLAST replaces seem to have provide encryption so data protection does not seem to be a major issue in this use case. I would suggest that the authors clarify these points and explain whether data protection is necessary in this case or whether it is just an added benefit of the method.

The method also has been optimised for names and words present in the test datasets in Brazil/Portuguese. It would be useful to know how easily the method can be adapted to other languages/countries and to provide a protocol for doing so.


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: TUCUXI-BLAST: ENABLING FAST AND ACCURATE RECORD LINKAGE OF LARGE-SCALE HEALTH-RELATED ADMINISTRATIVE DATABASES THROUGH A DNA-ENCODED APPROACH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors made a tool for probabilistic RL that utilizes a DNA-encoded approach to  analyze and link massive administrative databases. This tool is valuable for epidemiological research in the medical field. The authors have presented a clearly written manuscript. The literature cited was sufficient. The structure, figures and tables were illustrative.
.
Experimental design: The manuscript is original since they utilize the Blastn algoritm to analyze and link massive administrative databases. The search is improved by allowing the generation of matches even with typing errors in the data bases.
The research question was well defined and relevant -yes
The rigorous investigation performed to a high technical and ethical standard- yes
Methods describes with sufficient detail and information to replicate- yes
The manuscript can be accepted for publication with revisions:
1.-Add a link for the git-hub site of Tucuxi-BLAST program availability in the introduction.
2.-When I try to run the program appear one error
./ tucuxi_blast: line1: version command not found
./tucuxi_blast: line 2: oid: command not found
This error have to be reviewed by the authors
They can be achieve this kind of problems through a forum or directly with a contact e-mail
3.- In the gitHub page you could have a more explicit help in order to know what are the input requirements and the output in a visual way
Validity of the findings: Meaningful replication encourages where rationale and benefit to literature is clearly stated- yes
The results were measure and compared with other tools availables on the web. the improvement in this tool is valuable since this tool can be used by a PC with 32 GB of RAM and not need dependenciies, which is convinient for people without eperience in linux.
Conclusions are well stated, linked to original research
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: TUCUXI-BLAST: ENABLING FAST AND ACCURATE RECORD LINKAGE OF LARGE-SCALE HEALTH-RELATED ADMINISTRATIVE DATABASES THROUGH A DNA-ENCODED APPROACH
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made changes that address my previous concerns. There are a few very minor typos in the changed paragraphs though that need to be changed for readability:

Line 319: codon wheel can be easily adapted to English language -> codon wheel can be easily adapted to the English language
Line 320: correspondent -> corresponding
Line 532: Codon wheel is used -> Codon wheel used
Experimental design: All good
Validity of the findings: All good
Additional comments: The changes address my concerns. The minor typos can be fixed by the editor and only impact readability


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: TUCUXI-BLAST: ENABLING FAST AND ACCURATE RECORD LINKAGE OF LARGE-SCALE HEALTH-RELATED ADMINISTRATIVE DATABASES THROUGH A DNA-ENCODED APPROACH
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comments
Experimental design: Methods described with sufficient detail & information: in this case it should be noted that the results folder should be easily identifiable, the Readme.md file should say where they are as well as in the body of the article.
Validity of the findings: I think it is an interesting program that can be used in various areas. I think that the algorithm used is very good and its application in other areas is very interesting.
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF AEROBIC EXERCISE ON EVENT-RELATED POTENTIALS RELATED TO COGNITIVE PERFORMANCE: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is generally written well and easy to read. The topic is interesting and important. Sufficient background was provided in the introduction. Tables and figures are clear. There are some places that should be improved.
1. It would be good to include discussion on how ERP paradigms, features of study populations and dosage and lengths would affect the results. The authors have briefly mentioned some of these either in main text or table, it might be worth the address a little bit in discussion and consider how these would inform future research.
2.  "Future studies should focus on comparing these aspects of aerobic exercise head-to-head". I am not sure what this means "head to head". Could you clarify.
3. As EEG is not very sensitive to structure change, it might be worth to link the current ERP findings with recent MRI findings in the discussion and see if there are consistent trend.
4. There are similar systematic review on this topic which mentioned in the introduction. Should the authors highlight what additional knowledge has bee added by this review.
Experimental design: See my comments above.
Validity of the findings: See above
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF AEROBIC EXERCISE ON EVENT-RELATED POTENTIALS RELATED TO COGNITIVE PERFORMANCE: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: The review presents a systematic review of exercise intervention studies on event-related potentials. The rationale for the review is well justified as there are not enough integrative views on this rapidly developing field. The manuscript is well written, and the review process seems to be adequate.

My overall perception of the work is positive, although some issues limit my enthusiasm for this manuscript in its present form. These are relatively minor comments, and my with presenting them is not to be critical but to help increase the impact of the review.

Background / context
1). The title, abstract, and to some extent, introduction suggest that the review concerns the effects of aerobic exercise on ERPs. However, the review focuses on ERPs in response to tasks that measure cognitive performance. So the title should be more specific.

In the method session, the authors clarify that they focus on ERPs associated with cognitive function, but I think that this is also too general. In my opinion, the presented review does not focus on cognitive function but on cognitive performance. For example, some studies rejected from the review concern the processing of emotional stimuli (e.g., Brush et al., Ligeza et al.). These studies used ERP / EMF as markers of attention towards emotional stimuli. Attention is also a cognitive function. I would suggest that authors consider framing this review around a more specific topic:  the effects of aerobic exercise on ERPs associated with cognitive performance, not with ERPs associated with cognitive functions, and certainly not an impact of exercise on ERPs in general.

2) I think that providing more rationale for concentrating on aerobic, but not other (e.g. resistance) forms of training could be useful.

3) The N2 component is often considered a marker of the inhibition process. I think it is worth adding this function while reviewing and discussing the results and the functional significance of the component.
Experimental design: 4) The authors divided interventions according to their exercise intensity based on % of HRmax However, not all the studies considered based intensity on % HRmax. How did the authors conclude about the intensity of exercise in these studies, when the intensity was based on a subjective (RPE) scale, ventilatory thresholds, or % HRR (heart rate reserve)?

5) I do not understand the reasons for excluding the following two Chang's studies (https://doi.org/10.1111/psyp.12784; https://doi.org/10.3389/fnhum.2015.001560. The authors provide "the wrong outcome measure" as a reason, but the measure of this study was the cognitive performance in typical tasks measuring cognitive functions with a combined behavioral and ERP measurement.
Validity of the findings: 6) I condemn that the authors distinguish between acute and chronic exercise effects. I think they may have discussed a little more about the possible mechanisms behind acute and chronic effects, as they seem to be different.

7) Relatively, I think it would be worth trying to make conclusions about the effects of different durations (e.g under 20 minutes vs over 20minutes) or modalities of exercise (e.g., running vs. cycling).
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF AEROBIC EXERCISE ON EVENT-RELATED POTENTIALS RELATED TO COGNITIVE PERFORMANCE: A SYSTEMATIC REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed all my previous remarks very carefully. It only remains for me to congratulate the authors on an exciting review!
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: FOOD TABOOS DURING PREGNANCY: META-ANALYSIS ON CROSS CULTURAL DIFFERENCES SUGGESTS SPECIFIC, DIET-RELATED PRESSURES ON CHILDBIRTH AMONG AGRICULTURALISTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper makes an important contribution to the literature by focusing specifically on food taboos in agricultural societies in a cross-cultural perspective. The manuscript needs significant revisions, particularly in the analyses and background literature (see comments below).

1.	The English used in the manuscript should be improved to enhance readability of the text. There are several instances of minor grammatical errors. Below are a few examples, but this is not an exhaustive list.
•	Ln 29: should be data “were analyzed” not “was analyzed”
•	Ln 46: “mean” should be “means”
•	Ln 56: comma should be inside the quotations
•	Ln 147: “human” should be “human’s”
•	Ln 168: “we used reliance on” should be “we relied on”

2.	In the opening paragraphs about evolutionary theory and pregnancy diet, the authors should discuss and cite the classic works of Margie Profet, Sherman, Flaxman, and Fessler, since they were the “trailblazers” (so to speak) on this topic, and formulated a model referred to as the “maternal fetal protection” hypothesis. See references below.

Flaxman, S. M., & Sherman, P. W. (2000). Morning sickness: a mechanism for protecting mother and embryo. The Quarterly review of biology, 75(2), 113-148.

Fessler, D. T. (2002). Reproductive immunosuppression and diet: an evolutionary perspective on pregnancy sickness and meat consumption. Current anthropology, 43(1), 19-61.

Profet, M. (1992). Pregnancy Sickness as Adaptation: A Deterrent to Maternal Ingestion. The adapted mind: Evolutionary psychology and the generation of culture, 327.

3.	The reference for “eating down” is dated (ln 60-62). Please provide a more up-to-date reference if possible, because some sources suggest that it is no longer as common as once though (especially in India). Below are two references that discuss eating down and might have some relevant references cited within. One of them is a recent paper on pregnancy fasting, which provides more nuance to the practice of eating down.

Chanchani, D. (2019). Maternal and child nutrition in rural Chhattisgarh: the role of health beliefs and practices. Anthropology & Medicine, 26(2), 142-158.

Placek, C. D., Jaykrishna, P., Srinivas, V., & Madhivanan, P. (2021). Pregnancy Fasting in Ramadan: Toward a Biocultural Framework. Ecology of Food and Nutrition, 60(6), 785-809.

4.	The post hoc analysis can be presented later, after the other results are presented. There is no need to put that information on ln 119.
5.	I don’t think the information about this being a dissertation project is necessary for publication purposes (123-128).
6.	Taboo reasons: why did the authors only choose those categories? The first one (big baby/difficult delivery) is very specific while the other two categories are not. Did abortion come up? Or what about specific physical abnormalities?
8.	After reading the entire paper, I think the authors should cite the references I mentioned above about eating down and fasting, as they are highly relevant to the findings of the study.
Experimental design: N/A
Validity of the findings: I am a little confused about how the authors analyzed the data. Was it on the studies, or the taboos themselves (which equaled 263). If it is the latter, then I would like to see more sophisticated statistical analyses that control for geographical region.
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: FOOD TABOOS DURING PREGNANCY: META-ANALYSIS ON CROSS CULTURAL DIFFERENCES SUGGESTS SPECIFIC, DIET-RELATED PRESSURES ON CHILDBIRTH AMONG AGRICULTURALISTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is well written. The literature review is generally sufficient, though I would like to see more included on food aversions. The article is well structured and presented.
Experimental design: This is adequate, though again, aversions could be interesting (if not necessary. The results are well-presented, especially considering that they are not statistically complex (a breath of fresh air).
Validity of the findings: The findings appear valid, though they could be presented perhaps more holistically. This is a nice article.
Additional comments: Major comments
1.	I agree that this is an under-studied topic and yet certain references are missing, e.g., to Profet and Flaxman and Sherman, that speak to the importance of individual food aversions, as opposed to cultural taboos. The likely overlap in functions of aversions and taboos could be discussed with greater clarity for novice readers. The focus on subsistence is novel and interesting; it seems almost silly in hindsight that it has not been pursued previously.
a.	Could search terms include items related to food aversion, in addition to taboos?
2.	It might be interesting to see more of the qualitative results and/or lower N associations. There are good reasons to avoid meat and other animal products during pregnancy, none of which are described here, presumably because they are lumped under ambiguous physiological outcomes. The reasons for these taboos are thus obscured by the binning/aggregation of data. I’d like to see some of these, even if they are not central to the paper.
3.	Given that the focus of this paper is on cultural taboos, it might be nice to see some consideration of non-health-related reasons to engage in taboos. Are there any relationships between taboos and family composition? Patri/matriliny? Food availability/insecurity? Signaling?
4.	Relatedly, while the contribution of (over)nutritional risk is important, the article should probably also consider risks associated with epidemiological environments and energetic balance. This would certainly present a more thorough, life-history-oriented framing.

Minor comments
5.	Line 46: Are all taboos concerned with dangers? Are they sometimes about control (e.g., menstrual huts/associated taboos)?
6.	123-8: I found this somewhat jarring – do we need to know about the history of supervision on this project?
7.	140 ff: Do any of the papers refer to non-industrialized versus industrialized agricultural contexts? Would market-based subsistence add a useful test of ideas presented about carbohydrates and baby size?
8.	201 ff: Should “no” and “unspecified” physiological complications be separated?
9.	257: The proportion of animal versus plant taboos is roughly identical in agricultural populations. Why should this be, if animal products are likely safer and/or concerns are related to the size of the baby?
10.	360-3: Surely, maternal energetic output matters here, too? Is it really only carbohydrate intake that determines the size of the baby?
11.	364 ff imply to me a sort of ideological catch-up to mismatch between nutritional surplus and fetal nourishment. In other words, I would more generally expect mothers to be encouraged to eat more, not less, during pregnancy, given the extra energetic burden.


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: FOOD TABOOS DURING PREGNANCY: META-ANALYSIS ON CROSS CULTURAL DIFFERENCES SUGGESTS SPECIFIC, DIET-RELATED PRESSURES ON CHILDBIRTH AMONG AGRICULTURALISTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Improved, but could alter the text in the manuscript to situate more solidly within existing literature, rather than just adding citations.
Experimental design: Adequate, but subject to limitations, which are generally under-described, both in the first iteration and in this revision.
Validity of the findings: See above. Limitations require more thorough discussion.
Additional comments: Notes – Peer J Revision 68928

Abstract “useful for detecting biological pressures” is less cautious than it should be given the limitations acknowledged in the response to review. The data are suggestive of perceptions of problems, but not necessarily biologically meaningful ones, particularly given the tenuous links between nutrition and actual outcomes.

l.45-6: Pregnancy is more than just a period that ensures successful delivery; it sets the stage for later development, etc.

l.60-1: It would still be useful to characterize the prevalence of “eating down” versus “eating up” for audiences that aren’t familiar with these specific arguments. There are a lot of cultures where women have specific avoidances but are not encouraged to eat less overall.

l.67-9: I agree – these are not mutually exclusive interpretations of patterns, but it is probably more balanced to acknowledge the possibility that the cultural taboos do not necessarily have an obvious biological function – at least not a direct one.

l.80-1 – Again, agreed – but this has implications beyond just successful delivery.

l.87 ff – The new text is helpful here, but I think it is better to consider the possibility that aversions/fasting may serve different purposes in different times and places and the same purposes in any given context.

l.175-8 – I understand aggregating categories due to small N (as per the response letter), but it would be good to address associated limitations both early and late in the paper. “non-agriculture” is a highly heterogeneous bin consisting of potentially very different hazards for pregnant women and infants. This should be acknowledged and discussed.

Methods, general comment: It’s always a little disappointing to me when authors do not investigate alternative analyses or methods, particularly when reviewers converge on similar issues. The comments regarding the narrowness of the search from both reviewers suggest to me that a wider base for meta-analysis is appropriate and would resolve certain issues associated with sample size as well as how this work is situated within relevant literatures. There is very limited engagement with reviewer comments in the work presented, despite responses in the review.

Results: The “control analysis” if I understand it correctly investigates associations across three different gross geographic areas. What motivates these bins? And why is this geographic aggregation the only thing controlled for? Why not just present controlled analyses from the get-go?

Discussion: This does not do enough to incorporate the authors’ responses to review. Changes are very minimal compared to justifications in the response letter.

Summary: I like this paper and think it makes a valuable contribution, but I still think it could be stronger. Even if the authors do not want to adjust methods and analysis, they should be more cautious in their interpretations of the data, guiding future researchers to areas of nuance that remain under-investigated here.


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: WIDE SPACE SOWING ACHIEVED HIGH PRODUCTIVITY AND EFFECTIVE NITROGEN USE OF IRRIGATED WHEAT IN SOUTH SHANXI, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English used throughout the manuscript. Lieterature is OK.Figures and results are also fine.
Experimental design: This portion is fine.
Validity of the findings: OK
Additional comments: I have gone through the exhaustive article on Wide space sowing achieved high productivity and effective nitrogen use of irrigated wheat in south Shanxi, China.
1.	The “Title” of the article is appropriate and there is no need of any change.
2.	All the selected relevant aspects have been well placed under the respective sub-title of the article.
3.	Abstract is not well written; it is only a mere conscript of the study. Better would be to give some introduction followed by the gap in knowledge, hypothesis, general results and then conclusion. The abstract is the only part of the paper that the vast majority of readers see. Therefore, it is critically important for authors to ensure that their enthusiasm or bias does not mislead the reader.
4.	The introduction resembles that of a review article and not that of a research article. What’s the gap of knowledge? Which is the scope of the manuscript? What hypothesis have been made? The introduction should be revised accordingly.
5.	Experimental section:. A more succinic yet complete writing should be done. Moreover the author state that a statistical analysis has been made. I believe that the authors should give more details about the analysis performed,
6.The scientific background of the topic is poor. In "Introduction" and "Discussion", the authors should cite recent references between 2016-2020 from JCR journals.
Fahad, S., Sönmez, O., Saud, S., Wang, D., Wu, C., Adnan, M., Arif, M., Amanullah. (Eds.), (2021e.) Engineering Tolerance in Crop Plants Against Abiotic Stress, First edition. ed, Footprints of climate variability on plant diversity. CRC Press, Boca Raton.
Fahad, S., Sonmez, O., Saud, S., Wang, D., Wu, C., Adnan, M., Turan, V. (Eds.), 2021b. Climate change and plants: biodiversity, growth and interactions, First edition. ed, Footprints of climate variability on plant diversity. CRC Press, Boca Raton.
Fahad, S., Sonmez, O., Saud, S., Wang, D., Wu, C., Adnan, M., Turan, V. (Eds.), 2021c. Developing climate resilient crops: improving global food security and safety, First edition. ed, Footprints of climate variability on plant diversity. CRC Press, Boca Raton.
Fahad, S., Sönmez, O., Saud, S., Wang, D., Wu, C., Adnan, M., Turan, V. (Eds.), 2021a. Plant growth regulators for climate-smart agriculture, First edition. ed, Footprints of climate variability on plant diversity. CRC Press, Boca Raton, FL.
Fahad, S., Sönmez, O., Turan, V., Adnan, M., Saud, S., Wu, C., Wang, D. (Eds.), 2021d. Sustainable soil and land management and climate change, First edition. ed, Footprints of climate variability on plant diversity. CRC Press, Boca Raton.
Fahad S, Saud S, Yajun C, Chao W, Depeng W (Eds.),  (2021f) Abiotic stress in plants. IntechOpen United Kingdom 2021. http://dx.doi.org/10.5772/intechopen.91549
Fahad S, Hasanuzzaman M, Alam M, Ullah H, Saeed M, Ali Khan I, Adnan M. (Eds.) (2020) Environment, Climate, Plant and Vegetation Growth. Springer Nature Switzerland AG 2020. DOI: https://doi.org/10.1007/978-3-030-49732-3
Fahad S, Abdul B, Adnan M. (Eds.), (2018b) Global wheat production. IntechOpen United Kingdom 2018. http://dx.doi.org/10.5772/intechopen.72559


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: WIDE SPACE SOWING ACHIEVED HIGH PRODUCTIVITY AND EFFECTIVE NITROGEN USE OF IRRIGATED WHEAT IN SOUTH SHANXI, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1.	In the References section, the format of the References is wrong, please correct the references format.
Journal reference format: List of authors (with initials). Publication year. Full article title. Full title of the Journal, volume: page extents. DOI (if available).
Example journal reference:
Smith JL, Jones P, Wang X. 2004. Investigating ecological destruction in the Amazon. Journal of the Amazon Rainforest, 112:368-374. DOI: 10.1234/amazon.15886.
2.	In the Discussion section, please mention how your results compare to “Effects of Reduced Application of Nitrogen Fertilizer on Yield Formation and Nitrogen Use Efficiency of Winter Wheat With Wide-Range Sowing” which was published very recently.
3.	What do ** and ns mean need to be added in Table 4, 5, 6 and 7.


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: WIDE SPACE SOWING ACHIEVED HIGH PRODUCTIVITY AND EFFECTIVE NITROGEN USE OF IRRIGATED WHEAT IN SOUTH SHANXI, CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Wang and associates provides a thorough comparison of N-related parameters between two wheat sowing methods under various N applications.

The writing is globally clear, but a careful proofreading by a fluent English speaker is necessary, as many sentences are not correct.
Especially (but nor limited to), few sentences are not understandable: lines 273-274 (“Wheat crop … both growing season”) ; lines 296-298 (“With the increase in N rate … respectively”) ; lines 338-339 (“Crop yield was determined … ways to increase”).

The manuscript is well structured, with sufficient context and appropriate references. Clear figures, data and raw data are provided.
Mentions to supplementary file should be added in the Result part.
Experimental design: Research question is well defined and investigation is sound.
Methods are globally well described. Calculation methods and definitions of Harvest Index and Nitrogen Harvest Index should be added.
Validity of the findings: Results are carefully analyzed and conclusions are supported by the results.
Additional comments: Additional minor comments:
-	Line 93: “of fertilizer N absorbed” should be replaced by “of supplied N fertilizer”
-	Table 1 (soil analysis): how many replicates were done? Have all the sub-plots been tested?
-	Figure 1 suggest that plant density was different between the sowing methods, whereas text state that it is similar. Please clarify this, maybe by correcting the figure.
-	Lodging in DS with N300 should be clearly stated in Results part (probably at several places). Indeed, it affects most of the measured parameters, as well as the comparisons between the sowing methods (it affects the statistical analysis, as sowing methods are compared globally instead of per N-condition); this effect should clearly appear (especially lines 219-220, 248-249, 250-252 and 259-260).
-	Lines 227-228, “The stimulating effects … those under DS”: I don’t understand what this sentence is describing, and don’t identify these effects on Table 4.
-	Lines 232, 239: mistakes in Fig numbers.
-	Table 6: I am surprised by the result of statistical analysis for GNC% between N180, N240 and N300 for WS in 2017-2018. Is the table correct?
-	“N uptake at maturity” (and related phrasings) is misleading, as the uptake is defined as N quantity taken up during a unit of time. It should be replaced by “total N taken up during life cycle” or (better) “total N quantity at maturity”. “Pre-anthesis uptake” and “post-anthesis uptake” are fine, as they refer to a period of time, not to a timepoint. Note: N uptake at maturity stage is actually null, as the plant is dead and cannot take N up anymore.


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: WIDE SPACE SOWING ACHIEVED HIGH PRODUCTIVITY AND EFFECTIVE NITROGEN USE OF IRRIGATED WHEAT IN SOUTH SHANXI, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
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: WIDE SPACE SOWING ACHIEVED HIGH PRODUCTIVITY AND EFFECTIVE NITROGEN USE OF IRRIGATED WHEAT IN SOUTH SHANXI, CHINA
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: WIDE SPACE SOWING ACHIEVED HIGH PRODUCTIVITY AND EFFECTIVE NITROGEN USE OF IRRIGATED WHEAT IN SOUTH SHANXI, CHINA
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I would like to thank the authors for their answers to my comments.
However, I am not fully satisfied by some of the answers:

Reviewer #3, comment #1: despite the improvement, the manuscript would still gain from a careful proofreading by a fluent English Speaker.

Reviewer #3, comment #5: please modify Figure 1, to make clear there is the same plant density between sowing methods. Indeed, some readers will not fully read the Materials and Methods section, and this figure would thus be misleading.

Reviewer #3, comment #6: many measured parameters were affected in the 2 points where lodging occurred (DS with 300 kgN/ha in 2017-2018 and 2018-2019). The authors pinpointed this without mentioning the lodging : grain yield (lines 213-214), total dry weight at maturity (lines 243-244), harvest index (lines 246-248), pre-anthesis and post-anthesis dry matters (lines 256-257), grain N content (lines 268-269). As the lodging might be the cause of these observations, it should be mentioned in most of these paragraphs.


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

Input:
Title: IDENTIFICATION AND SUBCELLULAR LOCALIZATION ANALYSIS OF MEMBRANE PROTEIN YCF 1 IN THE MICROSPORIDIAN NOSEMA BOMBYCIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors cloned and expressed a transmembrane protein Ycf1 from Nosema bombycis, and prepared monoclonal antibody, investigated its localization using the antibody, and found that knocking-down Ycf1 could inhibits proliferation of N.bombycis, suggest that Ycf1 is an essential gene in  N.bombycis, and could be a target for development of anti-N.bombycis drugs. They studied the location and function of Ycf1, it is important to understand N.bombycis infection in silkworm. The article was well-written, and figures and tables were well prepared. But the English needs to be improved by a proficient English speaker or a professional editing service. And the format of references is not correct and uniform, some author names are not correct.
Experimental design: The study is within Aims and Scope of the journal, their hypothesis is clear, and confirmed the hypothesis using proper research methods such as monoclonal antibody, indirect fluorescence assay, and RNAi, the methods were described with sufficient detail.
Validity of the findings: they provided enough data/result to conclude that Ycf1 is an important membrane protein in proliferation of N.bombycis, confirmed their hypothesis.
Additional comments: some references are not correct, such as  in line 306-308, 316-319, 345, 432-441, 448-450
in line 80, the concentration of the primer may be not correct
in line 99, the antibiotics should be written in full name, and the concentration is not clear
in 124-127, the sentences should be rewritten. The antibody could be labeled with a dye but not another antibody.
in 168-170, the sentences should be rewritten


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

Input:
Title: IDENTIFICATION AND SUBCELLULAR LOCALIZATION ANALYSIS OF MEMBRANE PROTEIN YCF 1 IN THE MICROSPORIDIAN NOSEMA BOMBYCIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this work, the authors screened a putative membrane protein Ycf 1 from the N. bombycis. However, this manuscript should be further improved before ready for publication.

Major revisions

1) The Introduction chapter is composed of one big paragraph, which logic and contexts are not clear enough, and should be revised to make it more clear.

2) Figure 1 is the exactly the same as the prediction of transmembrane helices by the TMHMM Serverv.2.0, which screenshot is not appropriate to be directly used as a result.

3) Sections 3.3 and 3.4 could be integrated, because both sections describe the same thing and show quite similar results.

4) lines 281-283: Zheng et al. did not report that the NbTMP1 plays an role in spore germination, but just speculated that it may be involved in proliferation in their discussion.

Minor revisions

1) The “N.bombycis” should be “N. bombycis”.

2) The “ml” should be “mL”.

3) Line81: “ddH2O” is not correct.

4) Please used 24h, 72h, 96h and 24hpi, 72hpi, 96hpi properly. The two expressions have completely different meanings.

5) Species names in references should be in italic.
Experimental design: 1) Lines 155-157, the BmGAPDH is not an appropriate reference to determine the function of Ycf1 in N. bombycis proliferation.

2) Figure 4, the subcellular localization of Ycf 1 was just detected by using the Ycf 1 antibody and DAPI, which are not sufficient to indicate that Ycf 1 locates on plasma membrane. This could be improved by using cell membrane dye.
Validity of the findings: 1) Section 3.6, why was the expression of N. bombycis β-tubulin highest at 24 hpi, but lower at 48, 96 and 120 hpi? How to confirm that the silkworm midguts were infected?
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: IDENTIFICATION AND SUBCELLULAR LOCALIZATION ANALYSIS OF MEMBRANE PROTEIN YCF 1 IN THE MICROSPORIDIAN NOSEMA BOMBYCIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Chen et al. identified one putative membrane protein Ycf1 in Nosema bombycis and IFA and RNAi results suggested that this protein may involve in the proliferation of N. bombycis in silkworm cells. Here are my comments/suggestions.
What is the full name of Ycf 1? It seems the sequence of Ycf 1 is very similar to CQ1 (2 nt and 1 aa difference). Did the function of CQ1 have been studied in N.bombycis or other related microsporidia? If yes, what is the function of CQ1? It is better to provide this information in the introduction.
In the discussion, the authors should mention how Ycf 1 can be used to prevent the pebrine disease in sericulture.
The manuscript is very difficult to read and there are many language and grammar errors.
Some figures are not necessary or could be combined. Fig.2 could be deleted. Fig.7 and Fig.8 should be combined.
Experimental design: In the RNAi experiment, information about how to synthesize siRNAs and the concentration of siRNAs used for injection needs to be provided.
Validity of the findings: It would be better if the authors could provide the count number of N.bombycis spores in midguts of silkworm after knockdown of Ycf 1 gene.
Additional comments: n/s


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

Input:
Title: IDENTIFICATION AND SUBCELLULAR LOCALIZATION ANALYSIS OF MEMBRANE PROTEIN YCF 1 IN THE MICROSPORIDIAN NOSEMA BOMBYCIS
Review round: 1
Reviewer: 4

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: The "merge" in row B of Figure 4 does not seem to result from objective superposition. Moreover, besides the two ends of the meront marked by the authors, there are many other places where you can see green fluorescent signals. All of these may significantly reduce the credibility of the data. A more convincing result is recommended (transmission electron microscopy observations can be one of the alternatives).

The authors are invited to explain and discuss why RNAi had a significant effect at 96 h, while the spore proliferation was not inhibited (Figure 7), and why there was no significant effect of RNAi at 120 h but spore proliferation was significantly inhibited (Figure 8).
Additional comments: The manuscript "Identification and subcellular localization analysis of membrane protein Ycf1 in the microsporidian Nosema bombycis" by Chen et al., identified a membrane protein Ycf 1 from microsporidia N. bombycis, found Ycf1 was distributed on the plasma membrane of spores throughout the life cycle of N. bombycis. The authors further found that the proliferation of N. bombycis was significantly inhibited after silencing the expression of Ycf1 by RNAi technology, and they concluded that Ycf1 was involved in the proliferation of N. bombycis. However, the reliability of several key experimental results obtained in this study remains to be determined, which challenges the scientific validity and reproducibility of the study. In terms of writing, the manuscript generally resembles a first draft and needs to be rewritten for the introduction and discussion sections. Therefore, I do not recommend publication at this stage. See below for more specific comments.

1.The authors are invited to reorganize the abstract to reduce background statements and increase the presentation of important results, highlighting the innovation and the significance of this study.
2.Refine the introduction to clarify the rationale for conducting this study and its purpose and significance.
3.Line 115. NP40 lysate is usually tricky to extract the complete total protein of microsporidia. The authors should rephrase "this is the total protein of N. bombycis".
4.138 lines of "IFA" first appeared in the manuscript should provide the full name.
5.141 lines. The authors should provide the dose for 3 μL of siRNA used in the study.
6.The predicted molecular size of Ycf 1 is 34.43 kDa. However, the Western blotting in Figures 2 and 3 showed that the recombinant protein expressed by E. coli and the protein from N. bombycis was 52 kDa. I am puzzled whether the E. coli expression system undergoes phosphorylation or glycosylation modifications of proteins as the authors claim? Considering this, I would challenge the authors' statement that they are studying Ycf 1, casting doubt on the validity of the subcellular localization results of the Ycf 1 protein. Please provide compelling evidence, a plausible explanation, and an appropriate discussion for the significant difference in molecular weight. Extracting the total protein (including spore protein) with strongly denatured lysates and then analyzing by Western blotting and using mass spectrometry to identify hybridization-positive bands is recommended.
7.The "merge" in row B of Figure 4 does not seem to result from objective superposition. Moreover, besides the two ends of the meront marked by the authors, there are many other places where you can see green fluorescent signals. All of these may significantly reduce the credibility of the data. A more convincing result is recommended (transmission electron microscopy observations can be one of the alternatives).
8.In Figure 5 and Figure 6, are the anti-Nbactin and anti-SWP30 the monoclonal antibody prepared by the authors, and please describe its source in Materials & Methods. A co-localization investigation of Ycf1 and Nbactin protein in mature spores is recommended.
9.Figure 7 should provide data for a nonsense fragment negative control. More importantly, the author should analyze statistically not only between treatments but also along the time axis. Please provide a supplementary (or additional) figure with the control data and the RNAi data shown separately for the readers to evaluate the effect and reliability of the RNAi treatment during the period of the experiment. In addition, please explain why Ycf1 expression was reduced at 48 h compared to 24 h and why RNAi interference had no significant effect.
10.Figure 8. Likewise, the authors should show as a supplementary figure with the control data and the RNAi data shown separately for the readers to evaluate the effect and reliability of the RNAi treatment during the period of the experiment and whether the proliferation of spores within the host cells follows a defined pattern reported in the previous study.
11.The authors are invited to explain and discuss why RNAi had a significant effect at 96 h, while the spore proliferation was not inhibited (Figure 7), and why there was no significant effect of RNAi at 120 h but spore proliferation was significantly inhibited (Figure 8).
12.From the discussion section, the manuscript seems to be still like a first draft, without sound, in-depth and more meaningful analysis and discussion. In addition to what the reviewers suggested in the previous points of comments, the authors were invited to provide a full discussion based on the purpose and significance of this study, the biological function of Ycf1, and the other valuable results and the limitations and shortcomings of the study.
13.Manuscript has serious grammar problems. Please check it throughout.