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: TRANSCRIPTOMICS ANALYSIS REVEALS THE EFFECT OF BROUSSONETIA PAPYRIFERA L. FERMENTED FEED ON MEAT QUALITY TRAITS IN FATTENING LAMB
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Transcriptomics analysis reveals the effect of Broussonetia papyrifera L. fermented feed on quality traits in fattening lamb” deals with an interesting topic of feeding strategy in the context of feed-food competition. The manuscript need
an English revision from native speaker. The introduction section lacks of previous paper on the same topic applied in ruminants; moreover, some information about the importance of transcriptomic analysis for understanding the molecular mechanisms activated by feeding strategy for meat production have to be included.
Experimental design: The methods have been well-conducted and conceived and give the opportunity of growing the knowledge about the molecular mechanisms involved in feeding strategy on physiology.
Validity of the findings: The results are in line with the objective of the paper; however, one missing point is represented by the best percentage of Broussonetia papyrifera L. fermented feed for fattening lambs considering both positive and negative aspects.
Additional comments: Point to point comments are listed below:

L33: Please change into “INTRODUCTION”.
L48: Please specify the country without “my”.
L60: Please define “HE”
L81: Please correct with “-80°C”
L89: For Zlatkis et al. reference, please add only the year between parentheses.
L97, 98: Please use “FAME”
L100: Add point after filtered.
L164: Please use the same character dimension for “(C4:0, C10:0, C6:0, C18:0, C12:0, C14:0; C20:0), and delete space after “:”. Pay attention to this suggestion along with the manuscript.
L174: Please use acronyms “MUFA” and “PUFA”
L178: The increasing it is not gradually for C17:1 and C20:1, because it increases only with 100% treatment. Please correct this sentence.
L203: Please change “B“ into “B. papyrifera” Pay attention to this suggestion with along the manuscript.
L205: Please substitute “.” with “,”. after “S4”. In my opinion, table S4 has to be included in the manuscript.
L243: Please substitute “B. fermenta” with  “B. papyrifera”.
L283: Please substitute “figure 2” with “Figure 7”. Moreover at L284, in the end, one more point has been added. Please delete it.
L304-305: In the discussion section it is strongly recommended do not add more results and P-value. Please correct or delete this sentence.
L313-316: I don’t agree with the increase of PUFA found in the LD muscle and the antimicrobial activity of flavonoids. The authors have to discuss better this point.
L348-350: Which is for the authors the best quantity of B. papyrifera supplementation in order to obtain the best meat quality?

For all tables, a column with P-value has to be added.
Table 2 caption: Please add an "analysis of fatty acids composition in LD muscle".
Figure 1. Poor quality, it is not possible to read the numbers that indicate muscle morphology.
Figure 4: Poor quality of the figure.


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: TRANSCRIPTOMICS ANALYSIS REVEALS THE EFFECT OF BROUSSONETIA PAPYRIFERA L. FERMENTED FEED ON MEAT QUALITY TRAITS IN FATTENING LAMB
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author has made changes on the grammar and language. Now the manuscript is clear and unambiguous.
Experimental design: This research meets the aims and scope of PeerJ journal.
Validity of the findings: All the data and results in this manuscript have been provided and robust, statistically sound and controlled.
Additional comments: The author has answered my questions and comments. I would like to support this manuscript to publish at PeerJ. Thanks.


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

Input:
Title: IDENTIFICATION OF KEY GENES AND BIOLOGICAL PROCESSES CONTRIBUTING TO COLITIS ASSOCIATED DYSPLASIA IN ULCERATIVE COLITIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Major comments:
1.	In the introduction, the authors should replace their study in the context of all the studies looking for early genetic changes contributing to cancer progression. For example, the characterization of premalignant lesions has been conducted in the context of breast cancer (See Ju et al in Cancers 2020) and it was shown that 70% of miRNA alterations occur during the initial progression from normal to a preneoplastic stage. This result is even more worth to mention as it support the results shown later by the authors.

2.	In the differential expression analysis, the authors wrote that they only compared CAD to control but in figure 2 show UC compared to control. That brings me to a comment that apply to the manuscript overall, the authors should make 3 clear comparisons that reflect the steps of cancer progression:
-	control vs UC,
-	control vs CAD,
-	UC vs CAD.
I think they have the dataset and the opportunity to detail the genetic changes happening early in the carcinogenesis if they show these comparisons. That will create also more continuity in their manuscript as they used the 3 groups again in their hub genes analyses. Furthermore, it will be a great addition to quantify how similar are UC and CAD samples overall. How many genes show an increase in expression only in UC or CAD? Venn diagram are a useful tool to present that type of data.

3.	Even if the dataset used does not contain a CRC group. It will be easy to look for the genes they identified in another CRC dataset. Rather than a review of the literature, I think that will serve better their purpose of demonstrating that the identified genes play a role in carcinogenesis.

4.	Their explanation of the correlation between the modules and the progression of UC to dysplasia is a little confusing, they should rephrase and detailed more. What do they define as “progress” in Figure 3C for example?

Minor comments

1.	Could the authors pool the two datasets to increase statistical significance as they showed that they give similar results? Notably, that will increase their chances to identify differences between UC, CAD and controls.

2.	It is not clear how the gene significance threshold and the module membership cut-off were determined. If there is a reference, please cite it.

3.	The English language should be improved to ensure that an international audience can clearly understand the manuscript. Some examples where the language could be improved are the abstract and lines 54, 58, 67, 194, 201 – the current phrasing makes comprehension difficult.

4.	Figures 1B and C and figures 5B, D: Authors should keep the same color for each group across the figures.

5.	Figure 2B: It is impossible to read the Genes names, please provide a figure with a better definition.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In this manuscript, the authors proposed an interesting study based on data mining to look for the genes and biological processes involved in the transition from inflamed to premalignant lesions and ultimately to colorectal cancer. They have realized a substantial work of biostatistical analyses and the genes they identified could stimulate further experimental studies. However, I believe that there is a lot of room from improvement of the current manuscript. I detail the major issues I found below, as well as some other minor concerns, and suggest some ways to improve the manuscript.


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

Input:
Title: IDENTIFICATION OF KEY GENES AND BIOLOGICAL PROCESSES CONTRIBUTING TO COLITIS ASSOCIATED DYSPLASIA IN ULCERATIVE COLITIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: The sample size is small to jump into a conclusion and warrant further explanation and to also recommend for further study with larger sample size . Details of UC patients such as age, smoking status, intensity of colonic inflammation (based on endoscopic score), disease duration, concomitant medications taken (any potential chemoprophylaxis such as mesalamine), co-existence of primary sclerosing cholangitis,family history of CRC or UC are important factors towards UC-CAD and neoplasia. These parameters need to be mentioned in the manuscript.
Validity of the findings: Mitochondrial dysfunction that potentially lead to UC-CRC warrant further explanation in this study in the context of different disease activity and disease duration. I recommend also to compare with CRC arises from the polyp (sporadic CRC) in terms of mitochondrial dysfunction issue.
Additional comments: Overall is good but the sample size is very small and the validation is not determined. Need to take into account that sporadic CRC occurs slightly younger age group in Asian population and UC is low prevalent too in Asian countries. Hence, the possible pick rate of UC-CRC in the low prevalent area is very low and it might not be cost-effective and useful in daily clinical practice.


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

Input:
Title: IDENTIFICATION OF KEY GENES AND BIOLOGICAL PROCESSES CONTRIBUTING TO COLITIS ASSOCIATED DYSPLASIA IN ULCERATIVE COLITIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors made major revisions to their article and I am satisfied with their answers. I do not have more 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: AMBIENT OZONE POLLUTION AT A COAL CHEMICAL INDUSTRY CITY IN THE BORDER OF LOESS PLATEAU AND MU US DESERT: CHARACTERISTICS, SENSITIVITY ANALYSIS AND CONTROL STRATEGIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Most of the results presented in the manuscript are lack of evidence. In addition, some description in the manuscript is unclear and confused. The following presented some examples:
1.	Lines 187: The value of wind speed has been illustrated before.
2.	Lines 172-173: Why the temporal variations of O3 were not discussed for HGEP site (the urban background site)?
3.	Lines 187-188: does it mean that the meteorological conditions in April and September 2019 were not significantly different from those in 2017 and 2018?
4.	Lines 1910197: Much more evidence was needed to demonstrate that the sustained increase in O3 from 3-8 September 2019 was mostly related to local emissions.
5.	Lines 236-237: As mentioned above HGEP is an urban background site not a traffic-intensive area.
6.	Line 253: What are the units for the valley values?
7.	Line 254: What are the valley values in past?
8.	Line 253-259: To highlight the difference between regional transport and local emissions, it is suggested to compare backward trajectories between EP3, EP4, EP6 and EP1, EP2, EP5.
9.	It is suggested to provide a figure to present the time series of air pollutants from 2017 to 2019.
10.	It is suggested to provide a figure to provide the statistics of VOC species, NOx, CO and NOx in different years.
11.	It is suggested to provide a detailed description for the confirmation of PMF solution.
12.	It is suggested to highlight the difference between local emissions and regional transport instead of just simply providing the classification of backward trajectories.
13.	The results in the manuscript are lack of logic.
Experimental design: no comment.
Validity of the findings: As the results of this study were not presented properly, the findings of this study could not provide much useful information for the O3 research community and air quality management departments. Furthermore, the science values of this study needs to be highlighted.
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: AMBIENT OZONE POLLUTION AT A COAL CHEMICAL INDUSTRY CITY IN THE BORDER OF LOESS PLATEAU AND MU US DESERT: CHARACTERISTICS, SENSITIVITY ANALYSIS AND CONTROL STRATEGIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Professional articl strucle, figures and tables.
Sufficient field background.
Experimental design: Research question well defined.
Rigorous investigation performed to high technical and ethical standard.
Validity of the findings: All underlying data have been provided.
conclusions are well stated.
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: AMBIENT OZONE POLLUTION AT A COAL CHEMICAL INDUSTRY CITY IN THE BORDER OF LOESS PLATEAU AND MU US DESERT: CHARACTERISTICS, SENSITIVITY ANALYSIS AND CONTROL STRATEGIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript investigated ambient O3 pollution characteristics and sensitivity analysis over Yulin, a typical coal-chemical industry city in China. The manuscript highlight O3 concentration reductions are achievable through precursor control, but more effort is needed to reach the 30% and 40% reduction control targets. Overall, the methodology and results seem well presented. I recommend the manuscript be considered for publication only after my following comments being addressed.
Abstract:
“OBM, EKMA, RIR” may make the readers confused. Pls give the full names.
Introduction:
Line 63-68: As the author just mentioned the O3 pollution over BTH and YRD, what about the O3 pollution over the Guanzhong Plain? Can the author make a review about the O3 pollution across China, not just BTH and YRD?
Line 79-87: For this paragraph, the author should make a review about the current atmospheric pollution studies over Yulin, for example, PM pollution?
Results:
Line 222: What is the objective of this spatial distribution of O3 pollution? More discussion is needed.
Line 357-358: Pls re-write this sentence.
In addition, I don’t quite understand the sensitivity analysis? What do you mean by sensitivity analysis of O3 pollution?
Table:
Add SD.


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: AMBIENT OZONE POLLUTION AT A COAL CHEMICAL INDUSTRY CITY IN THE BORDER OF LOESS PLATEAU AND MU US DESERT: CHARACTERISTICS, SENSITIVITY ANALYSIS AND CONTROL STRATEGIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: This manuscript has clear and unambiguous, professional English used throught.
Experimental design: The Experimental desigh has sufficient detail and information to replicate.
Validity of the findings: The conclusions are well stated, linked to original research question.
Additional comments: No additional comments


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

Input:
Title: GENOME-WIDE IDENTIFICATION, CLASSIFICATION, AND EXPRESSION ANALYSIS OF THE HSF GENE FAMILY IN PINEAPPLE (ANANAS COMOSUS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article includes sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge.
Experimental design: Research question well defined, relevant and meaningful. The study also opens several important questions that probably the authors will address in the future.
Validity of the findings: Conclusions are well stated and linked to original research question.
Additional comments: The manuscript entitled “Genome-Wide Identification, Classification, and Expression Analysis of the HSF Gene Family in pineapple (Ananas comosus)” reports about identification of 22 AcHSF genes in pineapple, analysis of the gene and protein structures, as well as HSF functions in various developmental processes or regulatory pathways. The manuscript was easy to read and in general is well described. However, I have several comments about the manuscript:

L48-49. ….act as repressors of the expression of heat-inducible… It seems like the word was lost (after heat-inducible).
L90. This sentence should be corrected (to obtained…). At the same time, it remains unclear which plant DBD domain was used for screening.
L96. What does it mean the Chromosome NO.? Probably it means location on the chromosome. This phrase should be modified.
L104. The same comment.
L117. Authors described the gene. The AcHSFs should be written in italics.
L124. Conserved domains and motifs analysis were performed for proteins. It means that AcHSFs should not be written in italics in this case.
L136-137. The transcriptomic data of pineapple in different organs and developmental stages……… This phrase should be corrected.
L161. Please check the name of this section.
L282. The resulted… (probably, the results).
L308. …..in respond to the induction of heat shock. Probably, ….in response to the induction of heat shock.
L350. … to other HSFAs. Please present names in a universal form such as HSF-A in all text.
L361. The HSF should be written in italics. The tomato and apple should not be written in italics.


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

Input:
Title: GENOME-WIDE IDENTIFICATION, CLASSIFICATION, AND EXPRESSION ANALYSIS OF THE HSF GENE FAMILY IN PINEAPPLE (ANANAS COMOSUS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the submitted manuscript the authors give a comprehensive description of the HSF transcription factor family in Ananas and their expression characteristics.

The provided background information are sufficient. Most of the tables and figures are in a self-explanatory form, but some of them would need some improvement. In Fig. 5 p-values are given, but there is no explanation for what they have been assigned to. In Table 3 the acronym nd has not been explained. For Table 4 I must admit that it does not make sense to me. It seems that in this table the different motifs found in HSFs have been compared with each other. The sense of this comparison is not apparent and does not contribute any further understanding of the protein family. Therefore, I would suggest to delete it.

The language used in this manuscript needs thorough correction. In some cases words are missing (like in line 45, 182) or they are wrong (like line 48, 225, Table 1 "genome" instead of "genomics"). But there are a lot of passages which are misunderstandable (e.g. lines 190-200; see also section 3). Please give the text to a native english speaker for thorough correction.
Experimental design: The experimental design of the presented study meets the criteria defined by the journal. Therefore I have no comments, except for the description of the promotor sequences which seems to be rather unclear. According to the information given in lines 190-200 there were no upstream sequence information available for 14 HSFs. But for three of them (A4, C1b and B4c, lines 211-213) a lack of elements have been described for which no sequence information available. Please check this part.
Validity of the findings: No comment.
Additional comments: As stated above I would strongly recommend thorough text editing.


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

Input:
Title: ASSOCIATION BETWEEN GONADAL HORMONES AND OSTEOPOROSIS IN SCHIZOPHRENIA PATIENTS UNDERGOING RISPERIDONE MONOTHERAPY: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study explored the relationship between serum levels of gonadal hormone and bone mineral density or osteoporosis in patients with schizophrenia treated with risperidone, which is one of the significant clinical issues. This study is well designed with relatively large sample. However, some methodological deficiencies remained. Comments to this manuscript have been listed as follows for quality improvement.
Experimental design: The design is well addressed.
Validity of the findings: The findings are interesting.
Additional comments: Abstract:
-Result: (Bonferroni corrected P's < 0.05) should be modified.

Main text:
There are some small spelling errors and grammars in this article, please pay attention to them. Some of them have been presented as follows.

Introduction:
-Paragraph 5: Please introduce the study hypothesis in this paragraph.

Materials and Methods:
Participants:
- DSM-IV should be added with reference.

-Please polish the following sentence, “They were excluded if they: (1) had other diagnosed psychiatric disorder besides schizophrenia or a lifetime substance abuse/dependence disorder; (2) severe cardiovascular, hepatic, or renal diseases that affect bone metabolism, such as diabetes and hyperthyroidism; (3) were pregnant or breastfeeding; (4) history of bone fracture within one year prior to the enrollment”.

Statistical analysis:
-The author should specify dependent variables and independent variables in stepwise multiple logistic regression analysis .

Discussion:
-Paragraph 3
“The above results in accordance with the phenomenon of high incidence rate of osteoporosis in postmenopausal women and support the role of estrogen involved in bone metabolism”
-This sentence should be polished and some references should be added.


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

Input:
Title: ASSOCIATION BETWEEN GONADAL HORMONES AND OSTEOPOROSIS IN SCHIZOPHRENIA PATIENTS UNDERGOING RISPERIDONE MONOTHERAPY: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is well written.
Experimental design: The experimental design is reasonable and clear.
Validity of the findings: The findings are novel.
Additional comments: This is a well organized study that examined the relationship between serum levels of gonadal hormone and bone mineral density or osteoporosis in schizophrenia patients treated with risperidone. The sample size is large enough. Finally, the authors found a high prevalence of osteoporosis and significant reduced BMD in schizophrenia patients with risperidone monotherapy. This study has great clinical implications. Here are some concerns that may help improve the quality of this manuscript.

1) The main text should start from a separate page from the Abstract.

2) Introduction: the sentence "In the clinical practice, risperidone is a widely used second generation antipsychotic (SGA), and also a prolactin-elevating SGA compound." The cited study was published in 2012. Please update the reference.

3) Introduction: please introduce the study hypothesis, if any.

4) Method: "(1) had been diagnosed with schizophrenia according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV)" Please double check the diagnostic criteria. In China, the ICD-10 was used in clinical practice.

5) Statistics: please specify dependent variables and independent variables in multiple logistic regression analysis.

6) Discussion: "In addition, we only recruited inpatients with the same diet structure and similar physical intensity, thus, to minimize the disturbance of those factors on our conclusions." This sentence addressed a limitation, rather than strength.

7) There are some minor spelling errors in the text. For example, "SGAare" and "detailedinformation". Please insert space in betwee.


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

Input:
Title: ASSOCIATION BETWEEN GONADAL HORMONES AND OSTEOPOROSIS IN SCHIZOPHRENIA PATIENTS UNDERGOING RISPERIDONE MONOTHERAPY: A CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: none
Experimental design: none
Validity of the findings: none
Additional comments: The authors have addressed my concerns.


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

Input:
Title: IDENTIFICATION OF POTENTIAL GENES IN UPPER TRACT UROTHELIAL CARCINOMA USING NEXT-GENERATION SEQUENCING WITH BIOINFORMATICS AND IN VITRO ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Literature references are sufficient and updated.
Number of tables should be reduced.
Experimental design: The authors address an interesting issue on one of the less studied urological cancers,  UTUC. Literature need more reports on this issue. However, the manuscript must be improve before its publication.
Validity of the findings: Conclusions are not supported by the results.
Additional comments: In this manuscript the authors aim to investigate potential biomarkers and molecular mechanisms to determine suitable diagnostic and therapeutic strategies. The authors address an interesting issue on UTUC. However there are a few questions to be answered.

Abstract: The abstract does not reflex the study design, it should be rewritten. It has to be clear what is performed and with what samples. In results section the authors mention bladder cancer, but what samples? Sample size does not appear. It is mentioned diagnostic but in the conclusions prognostic is referred.

Methodology: Initially two tumour samples are analyzed (NGS). Why a T1 and T3 tumours are selected? These are tumours with a very different progression risk. Subsequently, several searches with different platforms are performed. Which has been the criteria used?

Results: L204 Table 2. Please, group miRNA by families, there are several from the same family.
Fig1. Are the miRNA differentially expressed between tumour and healthy samples? Please, clarify.
L236 This figure is not understood
L247 Fig 6 is irrelevant. Please skip it.
L252  Need to talk about RECK gene?
Why is decided to analyze selected genes in cell lines and not in patients samples?It is not clear to me if there are 10 or 20 samples.

Discussion: Why the authors refer to progression? It is difficult to extrapolate the results of a in vitro study with a reduced number of samples.
Conclusion: It should not refer to progression because of the study design.


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

Input:
Title: IDENTIFICATION OF POTENTIAL GENES IN UPPER TRACT UROTHELIAL CARCINOMA USING NEXT-GENERATION SEQUENCING WITH BIOINFORMATICS AND IN VITRO ANALYSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The language needs to be polished. There are some repetitions. The method should be included in the result part, at least the way of expression should be changed, For example, In result "we analysed two pairs of patient  specimens to compare renal pelvis tumours and adjacent nontumour urothelium tissues through NGS". It is unnecessary.
2. There are some misspellings, such as in Fig.9 levels of localisation, it should be localization. Furthermore, the text seems unrelated to the localization.
Experimental design: 1. In the Immunohistochemical staining part: incubated with an antibody against PDE5A,
190 CYBRD1, and ZEB2 at 37°Cfor 1 hour. The detailed information of the antibodies should be provided as clone No, dilution, and company.
2.For the Next-generation sequencing, what are the sequencing depth and coverage?
3.Several urothelial cancer cell lines (RT4, BFTC905, J82, T24, UMUC3, BFTC 909 ) were used in this study. It seems that they are all bladder urothelial cancer cell lines. UTUC cell line would be best for the validation.
Validity of the findings: 1.In the result part, NGS analysis identified 86 upregulated genes and 115 putative genes by using miRmap analysis. It showed that no potential candidates for upregulated genes were identified after interaction. The Fig. 2A should be revised since the interaction number is zero. It is kind of strange that there is no potential upregulated gene. Please provide more evidence.
2.Fig 9 of histopathological analysis of different levels of localization for PDE5A, CYBRD1, and ZEB2 staining in human UTUC samples. The quality of pictures are bad, please provide better pictures in higher magnification. The signal of Zeb2 should be in the nuclei of tumor cells, while it is hard to discern the location of signals.
3.The discussion is kind of lacking focus. Since the main result was derived from the mRNA  NGS, it should be compared with the other NGS study in urothelial carcinoma.
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: IDENTIFICATION OF POTENTIAL GENES IN UPPER TRACT UROTHELIAL CARCINOMA USING NEXT-GENERATION SEQUENCING WITH BIOINFORMATICS AND IN VITRO ANALYSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: There are some statements not accurate.
1. In the abstract, "lower PDE5A expression in tumor specimens was also demonstrated in paired UTUC tissue" should be by using paired UTUC and normal tissues, lower PDE5A expression was also demonstrated in tumor specimens.
2. "Our findings suggest these candidate genes may play crucial roles in UTUC progression". The results only suggest these genes may play some roles in UTUC tumorgenesis. Furthermore the statement "may be potential targets both as prognostic factors and for therapeutic strategies" is lacking powerful evidence. There is no survival information.
3. In the discussion part, "combining expression profiling with potential candidate genes, including our NGS dataset, may increase the accuracy rate when diagnosing UTUC". What is the point of using these genes in diagnosing UTUC? how? by blood or urine test? If they are available, this kind of description should be avoided.
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: VARIATION IN PURPLE SEA URCHIN (STRONGYLOCENTROTUS PURPURATUS) MORPHOLOGICAL TRAITS IN RELATION TO RESOURCE AVAILABILITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The Literature Cited is poorly formatted and inconsistent. There also are errors in the Literature Cited. For example,
Station, B. M., Columbia, B. & Tg 1991. The title of the paper actually is for Levitan 1991. I checked the journal Ecology for 1991 with pages starting at 431 and there is no paper by Station et al.
Experimental design: Line 142. The lantern index. Why use an index? I looked at the data file and made a couple of graphs. It seems more appropriate to use an ANCOVA with test size as a covariate both for lanterns vs diameter and gonad vs weight. The limited range of measurements makes the results look linear and so analysis would be simple and, very important, graphs would be easier to visualize because actual data would be presented rather than index values.
Validity of the findings: no comment
Additional comments: Studies that are intensive or extensive and examined gonad yield need to be included. For example,
Schroeter, S. C., N. L. Gutiérrez, M. Robinson, R. Hilborn and Peter Halmay 2009. Moving from Data Poor to Data Rich: A Case Study of Community-Based Data Collection for the San Diego Red Sea Urchin Fishery, Marine and Coastal Fisheries: Dynamics, Management, and Ecosystem Science 1(1): 230-243.

Rogers-Bennett, L., W. A. Bennett, H. C. Fastenau and C. M. Dewees 1995. Spatial variation in red sea urchin reproduction and morphology: implications for harvest refugia. Ecol. Appl. 5:1171-1180.

Ebert, T. A., J. C. Hernández, and M. P. Russell 2012. Ocean conditions and bottom-up modifications of gonad development in the sea urchin Strongylocentrotus purpuratus over space and time. Marine Ecology Progress Series 467: 147–166.
Line 61-64. Change in jaw size associated with changes in food availability should include:
deVries, M. S., S. J. Webb, and J. R. A. Taylor 2019. Re‑examination of the effects of food abundance on jaw plasticity in purple sea urchins. Marine Biology 166:141 doi.org/10.1007/s00227-019-3586-1
deVries et al. argue that jaws are plastic but changes in relative jaw size are not adaptive.
Line 64. Check Dix (1972); he did not report that urchins could decrease in size. Also, decrease in body size has been reported as an artefact of measurement. See
Ebert, T. A. 2020. Growth and survival of postsettlement sea urchins. pp 95-145  In: J. M. Lawrence [ed.] Sea Urchins: Biology and Ecology 4rd edition (Developments in Aquaculture and Fisheries Science 38) Academic Press, San Diego, California.

Line 218. “reallocate” suggests that materials are taken from the gonads and used to build the lantern. A different view would be that when food is scarce gonads don’t grow and lanterns grow a bit better than the rest of the body.

Line 236. Urchins with larger lanterns scrape more algae off rocks is very interesting in light of the publication by DeVries et al. (2019) cited above. If there are quantitative data, and so more than just personal observation, this would make a very useful contribution because the only other works that actually measured scraping ability are those of Black et al. back in the ‘80s.


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: VARIATION IN PURPLE SEA URCHIN (STRONGYLOCENTROTUS PURPURATUS) MORPHOLOGICAL TRAITS IN RELATION TO RESOURCE AVAILABILITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall the manuscript is clearly written, however several consistency related issues need to be addressed:

- The use of P values for minor statistics is not consistent e.g. a P value is used when describing the results for sea urchin densities on line 157-158 but not for following results describing mean test diameter or body shape in lines 159-162. Similarly line 122 doesn’t not have an associated P value.

- The use of sea urchin and urchin is interchangeable throughout the manuscript. Suggest that sea urchin is used universally throughout.

- The use of barren, urchin barren and sea urchin barren is used throughout. Although all three are accepted terms it would be useful to decide upon one and use it consistently.

- Check that all species names are italisied in the Literature Cited section e.g. Line 283 and 293.

- Choose and or & between author names in text and in the Literature Cited section e.g. lines 324 and 334.

-Some journals titles are written in abbreviated form and some are not. Please be consistent throughout e.g. Lines 283 and 334.

- An M is used to denote mean values through the results section but is not used consistently e.g. it is used on Line 157 and 158 but not Line 159 and 160. Please also specify if the ± refers to standard error or standard deviation.

There are four in text references that do not appear in the Literature Cited section. Please either add to the Literature Cited section or remove if not relevant to the paper.
- Harrold and Pearse 1987 - Line 59.
- Ebert 1996 - Line 204.
- Leighton 1966 - Line 209.
- Lewis et al. 1990 - Line 222.

Line 29 - 31: This statement needs referencing.

Line 185: ...encrusting [algae] was negatively….

Line 202: You state that the barrens developed one year prior to the study. Should this not be three years? Lines 80 - 83 suggest that the barrens formed in 2014 and the experiments were conducted in 2017.

Figure 1: The right hand border for the inset box needs to be shifted slightly so it is obscured behind the main graph's border.
Experimental design: The research questions appear to fit within the primary research aims and scopes of the journal are well defined.

The most interesting aspect of this research to me is that it reports on a system that has so recently changed from kelp forest to urchin barren, therefore providing insight into what can be expected in terms of sea urchin phenotypic plasticity over the relatively short term. I think more emphasis needs to be placed on this point in the final section of the introduction as a mosaic landscape containing kelp forest and urchin barrens is not in itself a unique situation.

The field and laboratory investigations have been well designed and executed and the methods section contains sufficient detail to replicate.
Validity of the findings: Line 83 states that 89 sites were surveyed however the associated csv file only contains data for 83 sites (assuming each site corresponds to a single row). Is the csv file missing the other six sites and were these included in the analysis or excluded for some reason?

Line 165: The mean lantern index value for both kelp forests and urchin barrens is the same (0.32) thought the statement above says they are significantly different. Is  one of these values a typo?

Figure 2: The statistical values in this figure are somewhat confusing and are not elaborated on in the figure legend. The P values in the figure do not correspond to the P values that are reported between Line 168 and 171.  The in figure values suggest a significant effect (P<0.001) on lantern index for both the density of Brown Algae and Folised Red Algae whereas both P values in the manuscript for these algae types show non-significance. Could you please explain what this difference is and why two seemingly different statistical outcomes are being reported?

Line 173: I don’t think a post-hoc Tukey Test is necessary here seeming that there are only two levels of the one factor (Zone = Barren or Kelp Forest). It would suffice to say that the gonad index is significantly higher in kelp forests (index value) than in urchin barrens (index value, F/P values).
Additional comments: An interesting read and tidy little experiment which shows how sea urchins can adapt to changing food availability in the relatively short term after kelp forests have changed to urchin barrens.


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: VARIATION IN PURPLE SEA URCHIN (STRONGYLOCENTROTUS PURPURATUS) MORPHOLOGICAL TRAITS IN RELATION TO RESOURCE AVAILABILITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thank you for sending through a revised manuscript and taking the time to go through each of the comments made in the initial review. The revisions made have rounded out the paper well.


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: A NOVEL ONE-STEP QUICK ASSAY FOR DETECTION OF SARS-COV2 ANTIBODIES ACROSS MAMMALIAN SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well-written and clear to read.

It is recommended that the author discusses the rationale for study design and explains why the assay was developed against the nucleocaspsid region. Some additional background on the nucleocapsid vs spike region will add value to the general readers.

The author is suggested to include all the images as a supplementary file. Only representative images are currently included.
Experimental design: Some additional details regarding assay design can be included.
Validity of the findings: It is suggested to submit all the underlying data with regard to fluorescence images of negative controls and patient cohort.
Additional comments: In the manuscript submitted by Dr. Zhou, he describes the development of a non-amplification, solution based-testing strategy for antibodies against the SARS-CoV2 N nucleocapsid. This one-step rapid test is based on an agglutination reaction between antibodies in the patient sera with recombinant antigen fused to GFP, aided by protein A/G/L. This protein aggregates can be then visualized using a fluorescence microscope. The test was able to predict SARS-CoV2 infection in 39/40 patient samples and produced no false positivity among 50 pre-pandemic samples. The merits of this test surely is the time and ease of the testing. However, listed below are a few concerns regarding the manuscript in its current form.
1.	The author is suggested to provide all the results from the pre-pandemic samples as well as the SARS-CoV2 patient cohort as supplemental data. It is recommended to show fluorescence images and a summary table.
2.	Please discuss the time of blood draw in the methods and results section. The details of the time delay for the blood draw is mentioned in the supplemental table but not in the main manuscript. This is an important piece of information to suggest that the one-step rapid test can detect antibodies even after viral clearance. It is also suggested that the author discusses this point.
3.	Is this assay amenable to quantification? Can the results provide more information than positive/negative status?
Here are a few minor suggestions for consideration and discussion:
1.	The nucleocapsid region of SARS-CoV2 shares 90% homology with SARS-CoV1. Will this one-step test produce false positive results in patients who previously contracted SARS-CoV1?
2.	Will the test produce false positive results if patients present with high antibody levels against another infection (non-SARS-CoV2 pathogen)? Will these other antibodies cause crosslinking among the protein A/G/L? The current negative samples from the San Diego Blood Bank may not help address this issue.


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: A NOVEL ONE-STEP QUICK ASSAY FOR DETECTION OF SARS-COV2 ANTIBODIES ACROSS MAMMALIAN SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: The focus of current manuscript by Dr. Xianjin Zhou is to develop a relatively quick and specific qualitative serological test to detect antibodies against SARS-CoV2. Currently multiple serological tests are available to detect antibodies against SARS-CoV2 that vary in specificity, sensitivity and the time. The author used GFP fused-antigen-antibody complex aggregation to visualize the test result by a fluorescence microscope. Following are the concerns..
1)	Did the author try one-step method using only serum, or any other biological fluid?
2)	Why did author incubate negative control mix for 5-days but not 5mins at room temp (Fig 1C) ?
3)	Visualization of GFP signal is challenging because of autofluorescence. Can this test be replicated with any other fluorescence tag?
4)	It was mentioned that 30µg/µl Antigen-GFP  protein was used to test (Fig 1B). Is 30µg minimum amount needed? Did the author try varying concentrations of Ag-GFP mix with SARS-CoV2 positive sera / Rabbit polyclonal abs against SARS-CoV2 (in presence of A/G/L )? This should be tested as it helps to rule out the false positive outcome.
5)	It is mentioned that the test volume is 5-6µl, what concentration of A/G/L was used? Does the test sensitivity depend on concentration of Ag-GFP and A/G/L  ? or the amount of Abs present in sera? Varying signal intensities are present among patient samples in Fig2.
6)	Relevant citations were missing in most part of the introduction.
7)	Author should avoid text redundancy in methods and main text.


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: A NOVEL ONE-STEP QUICK ASSAY FOR DETECTION OF SARS-COV2 ANTIBODIES ACROSS MAMMALIAN SPECIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: 1. Author is requested to mention the materials and methods section in detail. The present information seems to be incomplete in assessing the reproducibility of the assay.
2. One-step test should be described in detail describing the concentration of antibodies, antigens used eliminating any assumptions during peer -review, and discussion.
Validity of the findings: 1. It will be interesting to have the titer results shown with the antibody across a broad range in patients with varying infection severity.
2. Author should report when were the samples collected to detect the time point applicability of the assay.
3. Comparison should be made with rapid antibody detection tests available in the market or it should be highlighted in the discussion that how this test differs from the existing ones with respect to novelty, sensitivity, and specificity.
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: A NOVEL ONE-STEP QUICK ASSAY FOR DETECTION OF SARS-COV2 ANTIBODIES ACROSS MAMMALIAN SPECIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The author has systematically addressed the concerns raised in the first review report.

1.	The author has updated the introduction to include the requested background information and rationale for study design.
2.	The author has now provided image results for all the agglutination reactions as supporting data in supplemental files.
3.	The methods and results section has been revised to include the blood draw details.
4.	The author has discussed the issues of cross reactivity in the discussion section.

Overall, the author provided satisfactory responses to address the critiques.


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: A NOVEL ONE-STEP QUICK ASSAY FOR DETECTION OF SARS-COV2 ANTIBODIES ACROSS MAMMALIAN SPECIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: The author addressed all the concerns. However, I want to add the following comment.

It is not true that only formalin fixed tissues exhibit auto-fluorescence. Various substances, metabolites, and protein complexes of blood too exhibit auto-fluorescence (the auto-fluorescence properties of blood is being studied to use as diagnostic purposes). The author should discuss this aspect as a limitation or caution to avoid false positive result of the test (this is important as fluorescence intensity is the outcome of the test).


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: A NOVEL ONE-STEP QUICK ASSAY FOR DETECTION OF SARS-COV2 ANTIBODIES ACROSS MAMMALIAN SPECIES
Review round: 2
Reviewer: 3

Output:
Basic reporting: NA
Experimental design: The author has addressed all the concerns.
Validity of the findings: The author has addressed all the concerns.
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: HICENTERPRISE: IDENTIFYING LONG RANGE CHROMOSOMAL CONTACTS IN HI-C DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Im not sure that this is the first freely available tool for long range chromosome contacts between domains, is that true? I think this is capable in juicebox, which is also free. What should be advertised is that this is a step above current free software by injecting statistical vigor to what is currently available.  The authors provide a novel tool to attach P and Q values to HiC interactions, something that I have not seen. The software has a long list of software dependencies, but it is provided through a python installer

Figure 1b needs a label for the distance illustrated in the y and x axis.
Line 67-73 The enhancer atlas needs defined more thoroughly, (what is it?, usefulness?)  This paragraph needs one or two more sentences to be clearer.
Figure S1 needs to have a label for the axes.  How does it display p-value?

Line 46 The distinction between region and domain is not clear.
Line 68: Most will not know numpy format, but many will know binary.
Line 85-93  Domain level is not clear to the general reader.
Line 95: “Chromatin has no native structure” seems speculative and needs references.
Line 102: HUVEC needs to be defined.
This line in the supplemental “proposed much more statistically
important and hypothetically interesting contacts” seems speculative, how are they more important?
Experimental design: no comment
Validity of the findings: no comment
Additional comments: While the supplemental tutorial is nice, it is more to understand how the software works, rather than a tutorial. The best source for tutorials is their online presence, both tutorials, and a github repository to display their code.
Figure S2 could also be a misassembled inversion, or an inversion between the genome accession and the HiC DNA-seq.
The HiC-DC software errors are likely due to a version error in R or in an R package.


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: HICENTERPRISE: IDENTIFYING LONG RANGE CHROMOSOMAL CONTACTS IN HI-C DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please provide some form of graphical summary of the results of your cross-comparison with Homer and HiCCUPS, ideally by plotting the results of each tool as you do in figure 1.

Fig 1: Please add units to axes of Fig1B. Is it 0 to ~550kbp?  Please also add some details of the genome and location within genome of the data you display in the figure caption.
Experimental design: I feel the manuscript could use a more detailed discussion of the exact problem HiCEnterprise is trying to solve. In particular, an explanation of the two modes of operation (region vs domain), how they differ, and what biological problems each attempts to solve would be very useful.

Similarly, the discussion could use a brief overview of how the tool performs relative to others, and when a biologist should use this tool instead of others.

the example dataset is not discussed in sufficient detail. please provide a few sentences

While not required for publication, I suggest the authors submit the HiCEnterprise python package to pypi and bioconda to ease installation.
Validity of the findings: In multiple places the manuscript refers to HiCEnterprise being the only tool which can identify long-range contacts from HiC data. While I am not very familiar with this specific area of bioinformatics, a cursory literature search found at least one other tool which seems to be targeted at the same problems as HiCEnterprise (e.g. FitHiC2; Kaul et al. Nature Protocols 2020; doi: 10.1038/s41596-019-0273-0). Please update the manuscript to either describe that HiCEnterprise addresses a subtly different problem and therefore is unique, or perform experiments that compare HiCEnterprise to existing tools and show how  HiCEnterprise outperforms these tools.

I would also be interested to see how HiCEnterprise, Homer, and HICCUPPS performed on data consistent with a null hypothesis of random interaction/"negative control". The most obvious approach to this would be to shuffle the coordinates of one end of the paired association such that "ends" of the hiC read data seem randomly associated, however the authors may have more intelligent and realistic ideas that explore the null distribution of HiC data.
Additional comments: There are many missing spaces in the abstract where it seems text was copy-pasted from a PDF, removing spaces between lines. please double check the text in the peerj website.


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: HICENTERPRISE: IDENTIFYING LONG RANGE CHROMOSOMAL CONTACTS IN HI-C DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is mostly well written with few typos and grammatical errors.

Literature is well referenced, although there is a few more tools available for annotation of long-range interactions (for example, cLoops doi: 10.1093/bioinformatics/btz651 and chromosight doi:10.1101/2020.03.08.981910).

The structure of the article slightly deviates from the PeerJ standard, however it is well justified by a purely computational nature of the presented work. Code and data used for this paper is available. However the Results section only references Supplementary figures, and in my opinion there should be a main figure with key findings.

The figures could benefit from more detailed legends, and annotations (e.g. text from lines 62-65 should be in the legend, not main text, and plots need to have their axes labelled, et cetera).
Experimental design: The purpose of this work was to implement previously described approaches to discovery of regions with significantly enriched interaction frequency in Hi-C data in one unified framework, and make them more accessible to use. The work appears to be performed well and the produced software will be useful for the field.

However the paper would benefit from more detailed explanation of how the analysis of interactions between extended domains could be useful. Does it identify anything different from finding compartments? Additionally, the article would benefit from more examples of identified interactions, and some analysis about them, e.g. their size distribution, or distance separations between interacting regions.

It would be great if the authors could implement input of Hi-C data based on more standard and flexible formats, in particular cooler from Mirny lab (https://github.com/mirnylab/cooler), or .hic from the Lieberman Aiden Lab.
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: HEMATOLOGICAL AND CYTOCHEMICAL CHARACTERISTICS OF PERIPHERAL BLOOD CELLS IN THE ARGUS SNAKEHEAD (OPHIOCEPHALUS ARGUS CANTOR)
Review round: 1
Reviewer: 1

Output:
Basic reporting: I found this article interesting, properly structured and well written
Experimental design: This article is an original work about hematological and cytochemical analysis of peripheral blood cells in argus snakehead.
The research question is well defined but in my opinion is incomplete. In general rules, the standard parameters in the vast majority of hematological analysis of peripheral Blood are: hemoglobin (Hb), red blood cell (RBC) count, hematocrit(Hct), white blood cell (WBC) count, thrombocyte count (TC), erythrocyte sedimentation rate (ESR), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular volume (MCV). In this manuscript some blood parameters are analyzed but there are some that have not been performed . I recommend the authors to complete the analysis to obtain full data to compare with other fish species.
Validity of the findings: I found the discussion too long, I recommend to shorten a little bit. Maybe some comparisons could be summarized in a table and attached as supplementary figure. For example, the number of erythrocytes in different species, the morphological characteristics of mature erythrocytes….
On the other hand, in the discussion when authors discuss the staining results obtained they speculate about of the possible explanation when cells are positives or negatives. In my opinion I have found too many speculations and I believe that authors can look for bibliography that support their results or perform experiments to probe that suggestion.
Additional comments: I have  minor comments for the authors:
•	In the materials and methods, I do not found commercial references in many reactives and in the microscope. Please add them.
•	In the materials and methods, in the section: blood cell count and measurements, authors should include the formula that they used to calculate the number of blood cells.
•	In Table 1 you should indicate the N number, mean, SD, indicate and the statistical analysis in the legend. In addition, in my opinion I think you should indicate the count of neutrophil, monocytes, lymphocyte in cell/mm3 as the rest of cells. I have found in some papers  for example https://doi.org/10.1080/24750263.2019.1705647 that they usually use the same units and I think it is better to compare between cell types.
•	In Figure 1, you should indicate the magnification of the image in the legend.
•	In Figure 2, you should indicate the magnification of the image and the scale bar in the legend. Moreover, I found some trouble to know which staining belongs to every image so I recommend to indicate in the edge of the image the initials of the staining used in order to clarify the figure. Also, I found some images blurred for example G2, D2, D4, D6, I recommend to improve them.
•	I do not found match with table 3 and Figure 2 images. For example, neutrophils and monocytes have ++ in table3 for α-NAE but in the Figure 2 G2 seems less positive than G3. In the same way, for AS-D neutrophils and thrombocytes have ++ but in figure 2 F6 is more similar to F3 and F2 seems the highest. I don’t know if maybe there is a mistake or these differences are due to the image quality?


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: HEMATOLOGICAL AND CYTOCHEMICAL CHARACTERISTICS OF PERIPHERAL BLOOD CELLS IN THE ARGUS SNAKEHEAD (OPHIOCEPHALUS ARGUS CANTOR)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Reviewer’s Comments and Suggestions-
1.	Criticism- SEM  of Blood cells would have given detailed information of the morphology.
2.	Suggestion- SEM of blood cells should be done.
3.	Introduction on economic importance (with reference) of the fish and the relevance to the current study is not emphasized.
4.	Comments on language or grammar issue- The language of the manuscript is clear and reasonably good. However, grammar regarding sentence construction and use of words need some improvement as suggested by the reviewer.

Corrections-
Introduction-
Line 63-7A: A few more references as detailed below should be included.  B. Massar, Sudip Dey, R. Barua and K Dutta (2012): Microscopy and Microanalysis of hematological parameters in common carp, Cyprinus carpio L inhibiting a polluted lake in North East India, Micros, Microscopy and microanalysis,18; (1077-1087)

Lines 106-107: Check the English grammar. (“smears should be prepared ………………”) is to be corrected.

Line 117: Mention the make and model of the light microscope.
Line 128: ……………min at 370C, “then” …is the correct grammar.
Line 149: “After rinsing in distilled water for 3 times….” Change into “After three times rinsing….”
Lines 155-56: “After rinsing in distilled water ……” may be changed as “After 3 minutes rinsing in distilled water……”
Line 162: The sentence may be changed into “After 3 times rinsing………”
Lines 186-188: Scanning electron microscope may be used to have detailed information on morphology and size of cells to improve the standard of the manuscript.
Line 265- “………showed weakly positive staining…………”
Line 276: “Neutrophils exhibited strongly positive reaction for……….”
Line 278: “……………weakly positive reaction for……….”
Line 282: “……..showed positive reaction….”
Line 419: “……..strongly positive reaction…..”
Line 448: “……………positive reactions………..”
Line 458:……………….”showed” may be replaced with were……
Line 466: replace “exhibited” by “were”
Experimental design: Adequate. However, SEM study may improve the standard of the manuscript.

Suggestion: Few SEM study may be done.
Validity of the findings: Findings are valid and satisfactory except SEM study.

Suggestion: Few SEM study may be done.
Additional comments: Importance of the issue-

1.	Aims and objectives are clear.
2.	The manuscript is written clearly with nice micrograph and relevant tables
3.	References are adequate and up-to-date.

Strengths as well as weakness of the manuscript-

The authors made a nice attempt and deserve appreciation for their extensive data. The manuscript is clearly written in professional language. If there is weakness, it is in the absence of SEM works. The SEM analysis should be done or at least the need for Scanning electron Microscopy (SEM) should be emphasized in this type of work.
	If SEM analysis cannot be done, the reason(s) for the same should be mentioned clearly before ACCEPTANCE.


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: HEMATOLOGICAL AND CYTOCHEMICAL CHARACTERISTICS OF PERIPHERAL BLOOD CELLS IN THE ARGUS SNAKEHEAD (OPHIOCEPHALUS ARGUS CANTOR)
Review round: 2
Reviewer: 1

Output:
Basic reporting: I found this article interesting, properly structured and well written
Experimental design: This article is an original work about hematological and cytochemical analysis of peripheral blood cells in argus snakehead.The research question is well defined and the methods are described with suficient detail.
Validity of the findings: The authors have addressed all of the corrections suggested, and now the manuscript has substantially improved
Additional comments: The authors have addressed all of the corrections suggested, and now the manuscript has substantially improved. However, there are some issues that it should be corrected that I detailed bellow:
•	As the table 3 has been changed now in the discussion there are some sentences that are not update such as:
- Line 768” The strongly positive reactions for POX and SBB” and they should add ACP.
-Line794” were positive for PAS and AS-D staining” and they should add ACP.
•	In the table 4, I recommend to include in the legend the meaning of the acronyms (Hb, HCT and MCV ) to clarify the information.
•	I found some mistakes that I detail  bellow:
-Line 535,541,743: rection, I think is reaction please change it.
-Line 739:whoes , I think is whose please change it.


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: INTEGRATED METABOLOME AND TRANSCRIPTOME REVEALED THE FLAVONOID BIOSYNTHETIC PATHWAY IN DEVELOPING VERNONIA AMYGDALINA LEAVES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some English language corrections needed, especially in the Introduction See Minor Corrections:

P. 5 line 17 : what do you mean by "herb"? I think you mean "medicinal herb" instead

P. 5, l 31-34. Make a single phrase separated by comma.
By Illumina sequencing, the obtained over 200 million valid reads were assembled into 60,422 genes, among which 31,872 genes were annotated at least in one of public databases. Greatly increasing molecular resources devoted to V. amygdalina that lack genome sequence information.

P. 6, l.44 rapidly regenerating

Materials and Methods,
P. 7, l. 106. how were the time collection 10,20, 30 DAG points determined? Please explain in context of the plant’s life history.

P. 7, l. 105, Five plants located in

P. 7, l. 111, remove “The”, start the phrase “ 5g of …

The article is well structured.

The study is quite descriptive, the hypothesis that different flavonoids are present in different development stages is implied throughout the paper.

Caption Figure 6, correct please: "Proposed pathways of lavonoid"  -> flavonoid
Experimental design: The data are original.

The question is relevant and fills a knowledge gap.

The investigation lacks some standards used in the RNAseq community. I point them out here:

Materials and Methods.

Why did the authors not use BUSCO for transcriptome completeness analysis?
This method is now standard, and is important to know the representation of the genes in the transcriptomes. It gives an idea how redundant their annotations are. A possible reference genome is sunflower.

Why do they use FPKM for transcript quantification, RSEM also gives out TPM, the latter is a better measure of expression for comparing samples. See : https://rna-seqblog.com/rpkm-fpkm-and-tpm-clearly-explained/

Why did they use actin as reference gene ? Please cite reference or else add the expression stability as a separate supplemental table/figure.

I always suggest to plot primer efficiency before calculating relative expression levels, as the base (number of amplification cycles needed) may not be 2 depending on primer binding efficiency. Specially for non-model species with unknown genomes, this  base number cannot be just assumed and needs to be estimated for each primer.

P. 8, l.172. the Duncan method is a multiple rate test, therefore should be called : “Duncan MRT

P. 10, l. 223- 227. which statistical test was performed to determine DEG?
l. 227 : Venn diagrams are representations.
The analysis DE with multiple test correction: either Bonferroni, Benjamini-Hochberg, others see: https://nodepit.com/node/de.helmholtz_muenchen.ibis.ngs.edgeR.EdgeRNodeFactory
Validity of the findings: The other statistics are well performed.

Young leafs are better defended, therefore they show more flavonoid content and diversity.

I have suggestions for data representation and analyses to support their findings:

Figure 6 :  I suggest showing the annotated unigenes that are of interest in panel b
only (yellow and red colors, i.e. p<0.05 and p< 0.01) and replacing unigenes by their annotated gene name. Move the large picture to supplemental files.
The same for panel C:  replace unigenes by FMT-like , FGT-like etc. these names are more informative.

As a supplemental figure: it might be interesting to see alignments of the Unigenes identified in Table 1 (PAL), as multiple were mapped as the same Arabidopsis gene. Else are these assembly errors ?

Alignments might also allow a phylogenetic analysis for the multiple genes identified. The mapped genes to a sequenced Asteraceae species might be used for phylogenetic reconstruction, as for instance for Helianthus.

Else the presence of multiple copies for gene families that are usually single copy in other plants could be an indication of polyploidy.
Is there anything known about the plants’ ploidy level?
Additional comments: Dear author,

Your paper "Integrated metabolome and transcriptome revealed the flavonoid biosynthetic pathway in developing Vernonia amygdalina leaves"contains useful resources and information. Please consider the suggested corrections, for improvement.

Best regards!


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: INTEGRATED METABOLOME AND TRANSCRIPTOME REVEALED THE FLAVONOID BIOSYNTHETIC PATHWAY IN DEVELOPING VERNONIA AMYGDALINA LEAVES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The MS needs careful language revision so that statements are clear for their meaning and the draft is corrected for numerous grammatical errors.
More in-depth analysis is needed so that better biological inferences could be drawn, otherwise, the study is very superficial in spite of availability of all the raw data.
Experimental design: More information on statistical analysis is needed.
Validity of the findings: Statistical analysis is poorly explained. Details are missing in the text and in legends. Often it is not clear how many biological replicates were used, specially in RNA-Seq.
Additional comments: Review for the MS #55707 by Shui et al. submitted to PeerJ

The MS needs careful language revision so that statements are clear for their meaning and the draft is corrected for numerous grammatical errors.
The number of biological replicates used for RNA-seq and qRT-PCR needs to be specified clearly.

The authors claim that a change from 2.2% to 2.5% was a “significant” change in accumulation of flavonoids (line 181), but the statistics I Figure 1b does not support this claim. Moreover, the description of test statistic (which test was used, how many biological replicates were used, whether the P-value significance was post-hoc corrected; if so which tests, if not why) is completely missing.

I wonder the rational, why only 3 metabolites are specifically described (lines 184-188) and presented in Figure 2? This gives a misleading impression that the authors overlooked overall profiles. I find this description like putting the cart ahead of the horse. A better rational is required, or the description should be reorganised keeping in mind the next section.
In the abstract, the authors claim that they identified 42 flavonoids. In the results section, the authors describe the patterns of their accumulation in lines 189-210. This component forms an important part of the study of the authors. I wonder why the authors did not choose to present their results in the main body in forms of figures such as heatmaps, Venn diagrams etc, as they do for the genes in the next figures.  Figure 2 should better represent the “dynamic” patterns of change of metabolites over time.
The overarching claim that “Our LC-MS analysis provides a metabolic basis for further exploration on the flavonoid biosynthetic pathway in V. amygdalina leaves.” should be supported by relevant figures and the data could be better analyzed and represented to justify this claim.
	Moreover, as the authors introduce the importance of secondary metabolites of this plant, including its several medicinal usages, the authors should describe which unique metabolites they identified in their profiling study. The authors should also describe unique genes. The results of RNA-Seq mostly focuses on few very well known genes/ gene families. I presume, the motivation of such description was to show that they were able to identified well characterised genes. But then the study should be extended to new/novel intermediate metabolites and associated genes. This would only add value to the study.

Overall, I think that there is significant amount of data generated in this study. The data might be better represented and analysed to utilise its full power. The MS might further benefit from some reanalysis and reorganisation as well as careful English editing.


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: INTEGRATED METABOLOME AND TRANSCRIPTOME REVEALED THE FLAVONOID BIOSYNTHETIC PATHWAY IN DEVELOPING VERNONIA AMYGDALINA LEAVES
Review round: 2
Reviewer: 1

Output:
Basic reporting: English was largely improved.

Little corrections needed, as indicated in their submitted word document.

There's room for improvement in Figure 2. Comments below.

All else is well.
Experimental design: Improvements to previous version were made.

In the "Response to reviewers documents" they have some new information in the form of a table and two Figures, labeled Table A1, Figure A1, Figure A2. This is useful information, which should be included in the supplemental Figures.

All technical standards were met.
Validity of the findings: Ok.
Additional comments: Dear Author,

Your manuscript was greatly improved from the previous version.

The only suggestion for correction is the title and an improvement for Figure 2:

Title should read: « Heatmap of flavonoid profiles in Vernonia amygdalina leaves. The data were calculated as Log10(Peak area+0.01). »

Please add a hierarchical clustering below the heat map.

Please place names horizontal and use abbreviations if needed- as names are too long. Explanations of the abbreviations can be included as Figure foot.

Else, nice work!

Best wishes!


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: EXPERIENCING FEAR DURING THE PANDEMIC: VALIDATION OF THE FEAR OF COVID-19 SCALE IN POLISH
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the authors have done a good work. However, they may have to read through the manuscript once more for few typos and grammatical errors.
Also, authors may stick to the use of a research term. For instance, they may use either "participants" or "subjects" but not both. Further, they may revise the use of "sex" and "gender" in their manuscript.

Lines 274-278: Authors may try and make Table 1 more comprehensive by including the demographic characteristics and Cronbach's alpha.

At line 281, authors stated "...RMSEA=0.067, 90% CI [0.059, 0.094]..." Can authors explain why they used 90%CI as the others were all 95%?

Lines 309 - 322: Authors may state the r values related to the relationships.
Experimental design: Line 66: Participants:- this paragraph needs revision. Specifically,
Authors may need to justify the use of two different group of samples in this study.

Start new sentences with words (Seventy-eight persons) and not figures (78 persons) as in lines 168 and 174.

The sentences in lines 168-169 and 174-175 are not clear. Specifically, "...declared adherence to the group of people at high..." is not comprehensible.

Procedure
Authors stated "Some other data not related to this study and yet unpublished were included in the surveys...". Authors may have to explain why this data was added to this study especially as they were not collected together with the data of this study.
Validity of the findings: The conclusion is too lengthy. Authors may further summarize it into a paragraph.
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: EXPERIENCING FEAR DURING THE PANDEMIC: VALIDATION OF THE FEAR OF COVID-19 SCALE IN POLISH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is clear, references, context and lierature analysis is exaurient.
Experimental design: The research has conducted with rigurous investigations and Statistical Analysis. Technical & Ethical Standards were rispected.
Validity of the findings: The results are intersting and abolutly adapt to polish population. O
Additional comments: Very intersting work


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: EXPERIENCING FEAR DURING THE PANDEMIC: VALIDATION OF THE FEAR OF COVID-19 SCALE IN POLISH
Review round: 1
Reviewer: 3

Output:
Basic reporting: it is acceptable
Experimental design: it is acceptable
Validity of the findings: it is acceptable
Additional comments: I have read the topic with interest.
I think that the study has some strengths: Considerable sample size, and working on hot topic. However, the manuscript needs some clarifications/modifications:
3-	Abstract: please report sampling procedure and study time. I would see a clear message for implications for public.
4-	Introduction: I think that it will be great if the authors can provide some information regarding how the Poland react to the COVID-19 (e.g., government' policies, infected cases, deaths). I believe that this will strengthen the manuscript. Please report study aims and
hypothesis. please update your references on the fear validations:
For example:
Chi, X., Chen, S., Chen, Y., Chen, D., Yu, Q., Guo, T., ... & Zou, L. (2020). Psychometric evaluation of the fear of COVID-19 scale among Chinese population.

Stănculescu, E. Fear of COVID-19 in Romania: Validation of the Romanian Version of the Fear of COVID-19 Scale Using Graded Response Model Analysis. International Journal of Mental Health and Addiction, 1-16.

Pakpour, A. H., Griffiths, M. D., & Lin, C. Y. (2020). Assessing psychological response to the COVID-19: the fear of COVID-19 Scale and the COVID Stress Scales. International Journal of Mental Health and Addiction.

Ahorsu, D. K., Imani, V., Lin, C. Y., Timpka, T., Broström, A., Updegraff, J. A., ... & Pakpour, A. H. (2020). Associations Between Fear of COVID-19, Mental Health, and Preventive Behaviours Across Pregnant Women and Husbands: An Actor-Partner Interdependence Modelling. International Journal of Mental Health and Addiction, 1-15.

Chang, K. C., Hou, W. L., Pakpour, A. H., Lin, C. Y., & Griffiths, M. D. (2020). Psychometric testing of three COVID-19-related scales among people with mental illness. International Journal of Mental Health and Addiction, 1-13.

5-	Method: sampling procedure is still unclear to me. I would see more information on COVID-19 condition during data collection period.


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: EXPERIENCING FEAR DURING THE PANDEMIC: VALIDATION OF THE FEAR OF COVID-19 SCALE IN POLISH
Review round: 2
Reviewer: 1

Output:
Basic reporting: NOTHING
Experimental design: NOTHING
Validity of the findings: Comments to the Author
It is an interesting research that contributes to understanding the role of fear in Poland population during the time of quarantine in COVID-19 pandemic. There is needed some valid and reliable instruments in order to assess the fear in this kind of studies about the self-perception of emotions, anxiety, fear and mental health problems.

The paper has been written well. It is very important that authors had obteined an ethical approval of the University of Silesia in Katowice. The paper is appropriate, the sample is adequate and it has current references. The authors confirm the good psychometric properties of the questionnaire as well as the original version and in other validations, traslate, countries and samples, for instance, university students.

 I have minor revision. I have reported my comments in the following:
- Please add some information on mortality and morbidity rates on COVID-19 during data collection time.
- Why are there two samples? Both from the general Polish population? I don´t understand the objective of these.
- Which is the median score of fear in FCV-19S?
- Add more authors and studies in discussion about the psychometric properties in the different adaptation in all languages because now the paper has not much references and how do you explain this results?. Discussion has only 3 references, it is so poor. Please, add more and more. It is the most important step to find validity of the findings or not but authors should add more evidence about your results
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: EXPERIENCING FEAR DURING THE PANDEMIC: VALIDATION OF THE FEAR OF COVID-19 SCALE IN POLISH
Review round: 2
Reviewer: 2

Output:
Basic reporting: Professional article structure, figures, tables. Raw data shared.
Experimental design: Rigorous investigation performed to a high technical & ethical standard.
Validity of the findings: Local validation of an existing tool
Additional comments: This psychometric study evaluated the characteristics of the questionnaire to assess the fear of the epidemic. The questionnaire is already translated and used in many countries, so this study adds only one national version to the many that exist. The procedure adopted by the authors is the standard one. the results are in line with expectations. The authors reported and discussed the limitations of the study.


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

Input:
Title: NFIB PROMOTES THE MIGRATION AND PROGRESSION OF KIDNEY RENAL CLEAR CELL CARCINOMA BY REGULATING PINK1 TRANSCRIPTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) The abstract contains acronyms. Please define before using acronyms.
2) Introduction (line 33: use more recent statistics)
3) Page 2, Line 40, Kim et al 2011 is an incorrect citation. This paper reports 29.1% deaths and deals with RCC, not KIRC
4) Page 2, Line 44, Hanahan 2008 doesn't mention KIRC
5) Metastasis is in an organism. The authors do not demonstrate metastasis in mice etc. Hence, the change of title is required from metastasis to migration.
Experimental design: Materials and methods:
1) Under transfections, please provide the siRNA targetting sequence for NFIB and PINK1
2) Please include treatments/experimental groups under colony-forming assay
3) Please write the full form of IHC and ChIP on page 4 line 137 and 145 respectively
4) Please include scale bars in Fig 2D,E, I, J
Validity of the findings: This study demonstrates the role of NFIB in promoting KIRC migration and proliferation through the transcriptional activation of PINK1. ChIP experiments confirmed the binding sequence on the PINK1 gene promoter. This study is important and of significance since this molecular interaction could result in the activation of pro-oncogenic pathways in cancer cells.
Additional comments: I propose the following experiments for this paper to be accepted for publication.
1) In vitro migration does not necessarily mimic in vivo metastasis. I suggest the authors perform an invasion assay at the minimum.
2) I suggest the authors knockout PINK1 (using CRISPR-Cas9) in the NFIB silenced cells that are being used in the study and express exogenous PINK1 under a constitutively expressing promoter looking at the effect of KIRC cell migration, invasion, and proliferation. If PINK1 overexpression in NFIB silenced cells do not rescue the phenotype, it would possibly suggest an alternate pathway mediated by NFIB in KIRC cell migration., invasion, and proliferation.


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: NFIB PROMOTES THE MIGRATION AND PROGRESSION OF KIDNEY RENAL CLEAR CELL CARCINOMA BY REGULATING PINK1 TRANSCRIPTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the writing is clear. Certain sections have been flagged in my comments below.
Experimental design: No comment.
Validity of the findings: Some conclusions have been overstated and in some cases, the effect size is exaggerated in the text. These sections have been flagged in my comments below.
Additional comments: In this manuscript, Wang and colleagues find expression of a DNA-binding protein, NFIB, to be upregulated in kidney renal clear cell carcinoma (KIRC). They find NFIB to bind the promoter sequence of PINK1 to regulate its transcription. While the authors present convincing data for these conclusions, some of their other inferences are premature. There is no clear data showing that the tumorigenic functions of NFIB are in fact mediated by PINK1. The recue experiments conducted by the authors are poorly designed and do not address this question. Also, the authors claim NFIB/ PINK1 to be regulators of metastasis with no supporting data. These areas must be address in the form of simple new experiments and re-writing of the text.

Major comments:
1.	The rationale behind the design of rescue experiments conducted by authors in Figure 4 is unclear to me. Why are PINK1 expression levels (Fig. 4a, b) measured when the cells are treated with a PINK1 siRNA? What is the conclusion drawn by these experiments?
2.	The authors do not show effects of PINK1 overexpression or knockdown (in the absence of NFIB overexpression). These experiments are important to further implicate PINK1 in KIRC.
3.	The authors show images from colony formation, invasion, and migration assays in Figure 2 and 4 to test tumorigenic properties of NFIB and PINK1. Yet, they provide no quantification for these assays. These end points of these assays can be easily quantified and must be included in the manuscript.
4.	Western blots confirming the knockdown or overexpression of NFIB/ PINK1 must be included.
5.	The images chosen for transwell invasion assay comparing NC and NFIB overexpression vectors in LoMet-ccRCC cell line do not seem to show appreciable differences. Similarly, the authors must comment in the results section that neither overexpression nor knockdown of NFIB seems to have an effect on the migration (wound healing assay) of 786-0 cells.
6.	Throughout the entire manuscript (abstract through discussion), the authors state that both NFIB and PINK1 regulate KIRC metastasis. While the in vitro studies performed in this manuscript implicate these proteins in promoting aspects of tumorigenesis, their role in metastasis is unexplored in this study. The authors should rewrite portions of the text that overstate their conclusions.
7.	What is the scoring criteria that authors use for their IHC data? There is no information provided in the methods section. Additionally, NFIB is a nuclear protein and it must be clarified if protein localization to the nucleus is taken into account for scoring purposes.
8.	The authors should state the statistical test used for determining p-values in the figure legends.

Minor comments:
1.	Line 40: Vague phrasing of statistic. Unclear what the 51% of cases corresponds to. The original statistic in Kim et al. 2011 is that the median survival of patients that die from renal cell carcinoma (29.1%) is 1.9 years.
2.	Line 67” “In cell experiments” should be written as “in vitro” or “in cell lines”
3.	Line 191: I recommend authors chose between GEPIA or KMPLOT plots. They show similar things from similar datasets and having two panels is redundant.
4.	Line 209: It is not clear what comparison was made in obtaining the DE gene list. I presume it is tumor vs normal adjacent tissue in KIRC but this should be explicitly stated here.
5.	Line 217: I presume the authors identified targets whose promoters have the NFIB binding consensus sequence. The analysis methodology must be described more clearly.
6.	Line 269: “KIRC may facilitate the development of KIRC.” Two problems- the first KIRC is a typo and should be NFIB. Also, there is no evidence in this study that implicated NFIB in the development of KIRC, rather it supports tumor progression.


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: NFIB PROMOTES THE MIGRATION AND PROGRESSION OF KIDNEY RENAL CLEAR CELL CARCINOMA BY REGULATING PINK1 TRANSCRIPTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript identifies that NFIB promotes tumor proliferation and metastasis by up-regulating the transcriptional expression of PINK1. The topic of the manuscript is important as the identification and characterization of tumorigenesis and metastasis is key to understand cancer biology, which ultimately may improve diagnostics and therapeutics. The role of NFIB in tumor invasion and progression has been reported and studied in multiple cancer types and the authors report it for the first time in KIRC. The authors analyze gene expression data to identify differentially expressed genes and ultimately identify PINK1 as a NFIB target. Overall the manuscript is well structured and not many grammatical or syntax mistakes were found
Experimental design: The main issues in this paper is the lack of clear methods. It is not clear how the authors did the RNA-seq analysis to identify the  differentially expressed genes. In addition, it is not clear how the authors decided to focus on PINK1 after the gene promoter analysis. The research question is well defined and it is well stated. It is also important to note that the authors  should adjust their p-values throughout the manuscript.
Validity of the findings: The findings seem to be valid. However, clear methodology will help support the conclusions of the manuscript. It should also be noted that the results obtained in this paper for the role of NFIB/PINK1 is based on cell line data, which not always represents exactly what happens in tumorigenesis and cancer biology. Therefore, the authors should emphasize the limitations of using cell lines in the study. It will also enrich the manuscript if the authors analyze the mutational landscape of these tumors, as PINK1 interacts with the oncogenic PI3K/Akt/mTOR and these genes could be altered in some tumors. Could the NFIB or PINK1 over-expression be present in a subset of samples with a specific type of alterations? Could somatic copy number variation play a role in NFIB/PINK1 over-expression? Do the samples  analyzed have structural alterations in these genomic regions?
Additional comments: -	The RNA-seq analysis is not clearly explained in methods as it only references a previous publication. It should be stated what data was acquired (fastq, TPM expression?) for each of the studies used. In addition, was all the expression data processed uniformly? What software, version and parameters were used to obtain the differentially expressed genes?
-	What are the units shown on Figure 1B?
-	Is the y-axis correct in Figure 1C? I believe adjusted p-values should be used
-	Are the * in Figure 1D adjusted p-values? Is it also over-expressed in other renal cancer cell lines or is this a KIRK specific?
-	Table 1 should use adjusted p-values and results should be reported accordingly
-	Show hazard ratio and its 95% confidence interval, and the N for low and high NFIB expression. Shouldn’t the authors adjust their p-value in between 1H-J. Color scale should be consistent between the three plots. In addition, tables with demographic and clinical data should be provided for the three cases, to show similar patient populations on low vs high expression. Are there other differentially expressed genes also associated with prognosis for KIRK? Is NFIB one of the top prognostic related genes?
-	Show median overall survival for high vs low NFIB expression
-	Is NFIB an essential gene? Is it associated with synthetic lethality based on CRISPR screens such as DepMap or ProjectScore
-	Line 218 How was the potential TF binding site predicted? Was this analysis only done for PINK1 or all differentially expressed genes? It is not clear how the authors determine to only study PINK1 based on NFIB analysis.


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

Input:
Title: NFIB PROMOTES THE MIGRATION AND PROGRESSION OF KIDNEY RENAL CLEAR CELL CARCINOMA BY REGULATING PINK1 TRANSCRIPTION
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: NFIB PROMOTES THE MIGRATION AND PROGRESSION OF KIDNEY RENAL CLEAR CELL CARCINOMA BY REGULATING PINK1 TRANSCRIPTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: Abstract:
1. Line 15: explicitly list the databases used for the bioinformatic analysis.
2. Line 15: The bioinformatic analysis provided rationale for the authors to investigate NFIB. Yet, they say that they use this analysis to "confirm" it. Rephrasing is recommended strongly.
3. Line 23: Experiment"s"
4. Line 25: The authors do not provide any data to implicate NFIB/ PINK1 in the development of KIRC. This must be rewritten as KIRC tumor progression.

Main text:
1. Line 203: "worse" prognosis
2. As I mentioned in my original comments, it is unclear if the authors show protein level changes in the expression of NFIB and PINK1. In their response they said that this data is included. I presume they are referring to panel A in these figures. It is unclear if this is WB or qPCR data. This should be clarified in the main text and also the figure legends.
3. Line 235-236: no data for "associated with tumor cell metastasis". This must be deleted for accuracy.
4. Line 238: Delete "nuclear". All transcription factors are nuclear proteins.
5. Line 260: Tumor progression, not metastasis.
6. Line 265: Tumor progression, not metastasis.
7. Recommend moving lines 278-282 (and corresponding figure panel) to Fig 4. It flows a lot better in the manuscript.

Figure legends:
1. The figure legends have not been updated since the revision. This is true for all the figures.
2. Legend for Figure 1: Data in G can not be from three independent experiments. I presume each dot is a separate tissue specimen.

Figures:
1. Fig 1H: Why does the Kaplan Meyer plot say RAB17 on top? These curves should be for NFIB, not RAB17.
2. Fig 2F, 3G, 5H, 6F: Why is the axis labelled "metastasis number." The authors are counting the number of cells invaded in a transwell assay.
3. Fig 2J, 3M, 5L, 6J: Axis label should be "relative" wound size
4. Fig 5K, 6I: The authors should flip the orientation of the images to match those of similar assays in earlier figures. This is important so the differences between the different treatment groups can be readily compared.
5. All figures need scale bars.
6. General stylistic comment: The authors frequently use circles/ squares in dot plots/ curves to differentiate 2 conditions. I highly recommend the use of different colors so that these figures can be interpreted more easily.
Experimental design: No additional comments.
Validity of the findings: No additional comments. Few instances where authors overstate conclusions have been flagged in the "basic reporting" comment section.
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: NFIB PROMOTES THE MIGRATION AND PROGRESSION OF KIDNEY RENAL CLEAR CELL CARCINOMA BY REGULATING PINK1 TRANSCRIPTION
Review round: 3
Reviewer: 1

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