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: TRANSCRIPTION FACTORS INVOLVED IN ABIOTIC STRESS RESPONSES IN MAIZE (ZEA MAYS L.) AND THEIR ROLES IN ENHANCED PRODUCTIVITY IN THE POST GENOMICS ERA
Review round: 1
Reviewer: 1

Output:
Basic reporting: ----------------------
Experimental design: ---------------------------
Validity of the findings: ----------------------
Additional comments: Commend the authors for their extensive data set, compiled over of many researches. In addition the manuscript is clearly written in most parts. If there is a weakness, it is in the 1 and 2 which should be improved upon before Acceptance.
1-The English language should be improved to ensure that an International audience can clearly understand your text. Some examples where the language could be improved include lines 167, 454, 456– the current phrasing makes comprehension difficult.
In line 167(intricate) ~ it is possible instead of this word (complicated).
In line 454 subjugated ~ it is possible instead of this word (submit or subject).
In line 456 immense ~ it is possible instead of this word (enormous) and so on.
2-In Figure (1) Abbreviations meaning must be written below
For example:-
Abscisic acid (ABA).
Reactive oxygen species (ROS).
myeloblastosis oncogene (MYB).
myelocytomatosis oncogene (MYC)
ABA-responsive element binding protein (AREB).  ABA-binding factor (ABF).
ABA- independent regulons include; the NAC (CUC, NAM and ATAF)
The zinc-finger homeodomain (ZF-HD) regulon
The dehydration responsive element binding proteins (DREBs)
The cis-acting element (DRE)
The cis-acting element (CRE)
C-repeat (CRT) and so on.

Thank you very much for this wonderful opportunity to participate in directing this work.

Dr. Salwa El-Sayed Morsy Mohamed.
Molecular Cell Biology Lecturer
Department of Molecular Biology
Genetic Engineering & Biotechnology Research Institute (GEBRI),
University of Sadat City-Sadat City, Egypt. P.O. Box 22857/79
         Tel:  +2 01095215769
Email: salwa3eg@yahoo.com
          salwa.khedr@gebri.usc.edu.eg


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: TRANSCRIPTION FACTORS INVOLVED IN ABIOTIC STRESS RESPONSES IN MAIZE (ZEA MAYS L.) AND THEIR ROLES IN ENHANCED PRODUCTIVITY IN THE POST GENOMICS ERA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Langauge may be improved and typos need to be corrected.
Experimental design: Some of the paragraphs need to be restructured to have more clairity.
Validity of the findings: Authors need to add stress-specific role of TFs and the cross-talks of TFs during stress.
Additional comments: The review titled “Transcription Factors involved in Abiotic stress responses in Maize (Zea mays L.) and their roles in Enhanced Productivity in the Post Genomics Era” compiled various experiments and approaches. I appreciate the efforts put up by the authors.

Following are the major comments:
Introduction:
Line 85 to 105: I think you have to define the content of a paragraph. For example, this paragraph provides information about many subjects. Arrange something like “uses of maize in one paragraph (economical, genetical, functional, etc. uses), types of economically important abiotic stresses in maize in the next paragraph and types of different molecular mechanisms identified in maize in another paragraph”.
Line 106 to 112: Wondering why do you want focus only on ROS when several pathways are involved in stress tolerance? Better provide complete review of literature about different mechanisms.
Line 106 to 112: Please simplify the content.
Line 116 to 118: Please rewrite as there is no meaning in the sentence.

Transcription factors:
Please follow some pattern while compiling the experiments, like stress-wise or function-wise to have a better clarity on the role of TFs.
Line 157 to 237: The whole page is about TFs in different crops but not from maize except one or two references. Instead, you might want to provide a generic information on different types of TFs, number of TF identified in maize, structural characterisation etc.
From the TFs described in the review, we have come to know that they are involved in drought/salt/cold/etc tolerance. It would be nice to get to know the actual functional mechanisms of those TF in stress tolerance.
Line 743 to 747: “Li et al. (2018) analyzed the expression of ZmDREB4.1 and found that overexpression of this gene in transgenic tobacco led to repressed stem elongation and petiole, hypocotyl and leaf extension. In maize, Overexpression of this gene suppressed growth and regeneration of the calli. However, ZmDREB4.1 was not induced by any abiotic or biotic stress treatments.”

This para doesn’t add any value to the draft. I might suggest that avoid information that are not going to add any value to the draft.

Tables and figures/Additional information:
Additional tables explaining the functional and trait-specific role of TFs would provide useful information to the researchers. In the title, it is mentioned “productivity”. Are any TFs associated with agronomically important traits?
As you mentioned in the introduction, the role of TFs during stress is complex and the tolerance mechanisms involve cross-talks of many pathways. Adding figures to explain the cross-talk of TFs and explain their role in abiotic stress tolerance would be useful.
You also want split the Figure 1 for specific stresses rather providing generic and simplified overview of stress tolerant pathways.

Minor comments:
I could casually find a lot of typo with complex sentence formation. Please get a professional copy-editing support.
Line no 80: Change “yields” in to “yield”.  Throughout the text use yield.
Line no 83: “at diverse times during the crop growth stages”. You mean to say “during the different crop growth stages”?
Line no 88: Change “This” in to “The”
Line no 89: Use current year statistics on yield.
Line 442: “Sevens” to “seven”


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: TRANSCRIPTION FACTORS INVOLVED IN ABIOTIC STRESS RESPONSES IN MAIZE (ZEA MAYS L.) AND THEIR ROLES IN ENHANCED PRODUCTIVITY IN THE POST GENOMICS ERA
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1-  The review is covered most published articles in the field of review and within the scope of the journal.

2- Based on my knowledge, there is no the same review in maize. I think this is a useful review.

3- In the introduction, the subject was clearly introduced.
Experimental design: 1- The survey methodology section must be extended.

2- The sources are enough and quoted appreciate.

3- The review organized logically.
Validity of the findings: 1- The introduction was prepared very well, and it appears that the authors used enough references and could make a logical relation between them and their conclusion is valid, but it is necessary the conclusion will be rewritten and cover their findings.

2- The review includes a useful section as “future outlook” that give a future direction.
Additional comments: Conclusion section must be rewritten and cover the findings.


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

Input:
Title: TRANSCRIPTION FACTORS INVOLVED IN ABIOTIC STRESS RESPONSES IN MAIZE (ZEA MAYS L.) AND THEIR ROLES IN ENHANCED PRODUCTIVITY IN THE POST GENOMICS ERA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Met all the criteria.
Experimental design: Met all the criteria.
Validity of the findings: Met all the criteria.
Additional comments: The authors addressed all the comments very 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: PROGNOSTIC VALUE OF MEAN PLATELET VOLUME/PLATELET COUNT RATIO IN PATIENTS WITH RESECTABLE ESOPHAGEAL SQUAMOUS CELL CARCINOMA: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a retrospective study that can clearly illustrate the prognostic value of MPV/PC for esophageal squamous cell carcinoma. However, for this study, the sample size is not large enough, and the selected statistical methods appear still questionable.
Experimental design: 1.	Did the researchers set up a validation group to verify the conclusion?
2.	Line 121-122, The levels of MPV/PC ratio were significantly correlated with the CRP levels (P =0.029). CRP is an inflammatory marker just like MPV/PC. These two markers would interact with each other in the Cox regression model to influence the final conclusion.
Validity of the findings: 1.	CEA is closely related to the occurrence, recurrence and metastasis of ESCC. Why didn’t researchers consider CEA as one of the prognostic factors of CSS?
2.	Why did the researchers use the X-tile program instead of the ROC to calculate the optimal cut-off values? What are the advantages of this method relative to the ROC?
3.	There are some grammatical errors/writing style issues. e.g in the abstract, “respectively” is needed at the end of the first sentence in the results 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: PROGNOSTIC VALUE OF MEAN PLATELET VOLUME/PLATELET COUNT RATIO IN PATIENTS WITH RESECTABLE ESOPHAGEAL SQUAMOUS CELL CARCINOMA: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: İntroduction is too short and literature review is not satisfactory.
Experimental design: Feng et. al used X-tile program to find the cut-off vaules. I prefer to find these valueas with ROC analyses. they should make the statistics again. The results are not consşistent with the other reports in the literature. How do the author explain that MPV is not an independent prognostic factor but MPV/PC is a prognostic factor. İs it a numerical chance?
Validity of the findings: statistical analyses are not satisfactory and results are not consistent with the literature. I dont know the spesifity and sensitivity of the cut -off values.
Additional comments: In this retrospective study you should find and mention the specifity and sensitivity of the cut-off values. why you did not use ROC analyses?
Why the results are not consistent with the literature? exempale: a prognostic factor must be in relation with other prognostic factor like TNM,...


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

Input:
Title: PROGNOSTIC VALUE OF MEAN PLATELET VOLUME/PLATELET COUNT RATIO IN PATIENTS WITH RESECTABLE ESOPHAGEAL SQUAMOUS CELL CARCINOMA: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Some grammatical errors exist in the manu.Please exam carefully and correct.
Experimental design: 1.The cut-off value of MPV/PC ratio was obtained by X-tile plot.Please compare the method X-tile plot and ROC curve.
2.Please give more details in Figure 4.
Validity of the findings: The prognosis of ESCC is determined by many factors. The inflammation is important in the development of cancer.Please analysis the impact of neutrophil/ platelet ratio on prognosis and discuss it.
Additional comments: The author invested the prognostic role of MPV/PC ratio in ESCC patients which was an interesting topic.
A low MPV/PC ratio level (≤0.04) was associated with poor CSS in ESCC.But we all konw, the prognosis of ESCC is determined by many factors. The inflammation is important in the development of cancer.Please analysis the impact of neutrophil/ platelet ratio on prognosis and discuss it.Besides,.The cut-off value of MPV/PC ratio was obtained by X-tile plot.Please compare the method X-tile plot and ROC curve.Some grammatical errors exist in the manu.Please exam carefully and correct.


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

Input:
Title: PROGNOSTIC VALUE OF MEAN PLATELET VOLUME/PLATELET COUNT RATIO IN PATIENTS WITH RESECTABLE ESOPHAGEAL SQUAMOUS CELL CARCINOMA: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 4

Output:
Basic reporting: Your introduction needs some improvements.
1.In line 62, what's the 8th incidence for EC, is it in China? or all over the world?
2.Please explain why you introduced MPV,PC and cardiovascular disease, in this study about esophageal cancer, in line 69-71.
3.In line74 "However, the role for MPV/PC ratio in ESCC has not yet been evaluated." Actually, there were several studies about MPV, COP-MPV and MPV/PC and EC, as you cited in the discussion  part line 156-168. And also another study : Reduced mean platelet volume is associated with poor prognosis in esophageal cancer. Shen W, Cui MM, Wang X, Wang RT. Cancer Biomark. 2018;22(3):559-563. doi: 10.3233/CBM-181231.
So, what's the novelty of this study need more detail discussion.
Experimental design: The MPV (PC) and CRP detection methods need more sufficient details. What about the CRP detection? Is it serum CRP or whole blood CRP? And how about the detailed methods.
Validity of the findings: The conclusions should be supported by the data. But the novelty of this study need more detail discussion.
Additional comments: There are some grammar issues should be improved , for example lines 87, and so on.


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

Input:
Title: PROGNOSTIC VALUE OF MEAN PLATELET VOLUME/PLATELET COUNT RATIO IN PATIENTS WITH RESECTABLE ESOPHAGEAL SQUAMOUS CELL CARCINOMA: A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a retrospective study that can clearly illustrate the prognostic value of MPV/PC for esophageal squamous cell carcinoma. However, for this study, the sample size is not large enough, and the selected statistical methods appear still questionable.
 It has been addressed.
Experimental design: 1.Did the researchers set up a validation group to verify the conclusion?
 It has been addressed.

2.Line 121-122, the levels of MPV/PC ratio were significantly correlated with the CRP levels (P =0.029). CRP is an inflammatory marker just like MPV/PC. These two markers would interact with each other in the Cox regression model to influence the final conclusion.
 It has been addressed.
Validity of the findings: 1.CEA is closely related to the occurrence, recurrence and metastasis of ESCC. Why didn’t researchers consider CEA as one of the prognostic factors of CSS?
 It has been addressed.

2.	Why did the researchers use the X-tile program instead of the ROC to calculate the optimal cut-off values? What are the advantages of this method relative to the ROC?
 It has been addressed.

3.	There are some grammatical errors/writing style issues. e.g in the abstract, “respectively” is needed at the end of the first sentence in the results section.
 It has been addressed.
Additional comments: No additional comments


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

Input:
Title: PROGNOSTIC VALUE OF MEAN PLATELET VOLUME/PLATELET COUNT RATIO IN PATIENTS WITH RESECTABLE ESOPHAGEAL SQUAMOUS CELL CARCINOMA: A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 2

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


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

Input:
Title: A PROPOSED UPDATE FOR THE CLASSIFICATION AND DESCRIPTION OF BACTERIAL LIPOLYTIC ENZYMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The Manuscript tile "A proposed update for the classification and
description of bacterial lipolytic enzymes"  Article ID(#37278)

Thomas C A Hitch Corresp and  Thomas Clavel reporting the classification of lipolytic enzymes.-
-This study very interesting and easily understandable
-language good well written-
-figure and tables enough
-references good
Experimental design: -experimental genomic study well analysis
Validity of the findings: -over all the well presentation
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 PROPOSED UPDATE FOR THE CLASSIFICATION AND DESCRIPTION OF BACTERIAL LIPOLYTIC ENZYMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The manuscript was well-structured. The authors provided clear background for the research and defined the necessity to expand the classification system of bacterial lipolytic enzymes. With more enzymes discovered, the current system is not sufficient and various “novel” families were claimed without further analysis and confirmation. With careful research, the authors proposed an improvement of the current system based on pairwise sequence alignment, and new families and subfamilies were added. Type proteins for each of the families and subfamilies were also described with details such as conserved sequence motif and effect of metal ions. Table 1 was clear and informative, but I would suggest the authors could summarize here the consensus sequence and the metal-dependency or other representative features for easier reference.


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 PROPOSED UPDATE FOR THE CLASSIFICATION AND DESCRIPTION OF BACTERIAL LIPOLYTIC ENZYMES
Review round: 1
Reviewer: 3

Output:
Basic reporting: NO comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript entitled “A proposed update for the classification and description of bacterial lipolytic enzymes” by Thomas Hitch and Thomas Clavel provides expanded families and subfamilies of lipolytic enzymes. At the same time, the authors also provided methods and tools to group further newly identified enzymes into the present classification system. The authors did pairwise sequence comparison to confirm or exclude the novelty of the already claimed new families. The authors not only expanded the classification system published by Kovacic et al (2019) but also corrected some errors in that system. The authors provided the most up to date and most reasonable approaches to classify the lipolytic enzymes. I only have few comments below:
1.	The authors are trying to incorporate their classification system into the already published and used widely system which has been updated recently by Kovacic et al (2019). Although the authors try to be clear and not causing confusing by giving a summary section about the changes in section 3.1 Overview part, it is not clear enough. I recommend the authors only describe the expanded subfamilies/families and the families got changed in the existing system. That means in the range of family IX to XIX, the authors should only list family X, XI, XIII, and XVIII which already exist but changed by the authors.
2.	In the summary table of bacterial lipolytic enzyme families, the authors need to highlight the changes they made over the existing system to make it more clear.
3.	The authors should also include the bacteria origin for the enzymes listed on the table, although they did this in the text part. This is important since some of the enzymes are isolated from metagenomic libraries.
4.	The protocol for origin check of metagenomics enzymes sounds very arbitrary. Can the authors provide paper evidence that this protocol has been reasonably used before or some other evidence this protocol is appreciated?


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

Input:
Title: THREE-DIMENSIONAL PRINTING WITH BIOMATERIALS IN CRANIOFACIAL AND DENTAL TISSUE ENGINEERING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The abstract is well written.
The introduction is well written.
The survey methodology section is well written.
The body text/results/discussion sections are clear.
The conclusions are fine.
Experimental design: Not applicable, this is a review.
Validity of the findings: Not applicable, this is a review.
Additional comments: There are a number of cases where there is no space before the reference, see: "disease(Rai et al., 2017).", "developing(He et al., 2015)", "shaping(Torres et al., 2011)"; or indeed figures: "layer(Fig.1B)"

The authors should use the correct symbol for micro instead of the u, e.g.: "50um". Please correct ALL instances.

Please change "The control of porosity’s" to read "The control of porosity"

The authors correctly state "SLS was developed by Carl Deckard" but fail to cite the thesis - please correct this. Also include other research output from Deckard and coworkers.

The authors correctly state "SLA printing dates back to 1986, when Charles Hull, ‘father of 3D print’, first promoted the 60 term ‘stereolithography’ in his U.S. patent, Apparatus for production of three-dimensional 61 objects by stereolithography" but fail to cite the patent - please correct this. Also include other research output from Hull and coworkers.

The authors cortectly state "Binder jetting is a technology developed at almost the same period with FDM. Its first development is in the early 1990s. In 2010, the first binder jetting machine was commercially obtained." but fail to cite the patents/papers - please correct this.

Figure 1 legend needs to explain what A-D are.

Figure 2: legend needs correcting as it appears the image is reproduced from another journal, so the authors need the copyright permission from the publishers of those images (supply this to PeerJ with the revised manuscript), and to ensure the legend is the same as in the original manuscript, and to cite 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: THREE-DIMENSIONAL PRINTING WITH BIOMATERIALS IN CRANIOFACIAL AND DENTAL TISSUE ENGINEERING
Review round: 1
Reviewer: 2

Output:
Basic reporting: the article is a nice article but does not do very much to the literature as there are many such review articles. also the article hasn't spoken about cell electrospining and bio-electrospraying.
Experimental design: the aims are very mundane as there are many such review articles.
Validity of the findings: the review should also talk about cell electrospinning and bio-electrospraying
Additional comments: the review is incomplete as all techniques are not discussed.


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

Input:
Title: THREE-DIMENSIONAL PRINTING WITH BIOMATERIALS IN CRANIOFACIAL AND DENTAL TISSUE ENGINEERING
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: 1. How many articles were searched for by the keywords described? Does table1 list all the articles and  How did authors filter these articles?
2. For the articles searched, how did evaluate the authenticity and reliability of the results?
3. could the authors describe the advantages and disadvantages of different cell lines and different animal models?
Validity of the findings: The article  provides a comprehensive and detailed reference about AM techniques, and  evokes thinking.
Additional comments: The pictures lacked necessary legends, such as fig1, please mark  the main part of AM printers.


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

Input:
Title: THREE-DIMENSIONAL PRINTING WITH BIOMATERIALS IN CRANIOFACIAL AND DENTAL TISSUE ENGINEERING
Review round: 1
Reviewer: 4

Output:
Basic reporting: The manuscript by Liao and colleagues is interesting and well presented. The topic is relevant to the field of tissue engineering and can be of interest for the readership of Peer J. The introduction well matches with the subject presentation.
Experimental design: The study is well designed and the methodology is consistent as well as the bibliography section. Figures are properly organized in order to be easy to understand and representative for the readers. The overall organization of the work is adequate.
Validity of the findings: Each work described is analyzed considering its strenghts and weaknesses.
Additional comments: The manuscript by Liao and colleagues is valuable.
Some minor revisions concerning the language has to be addressed prior to publication:
- there are mistakes in lines 135, 161, 186, 188, 298,... In general some careless mistakes can be found throughout the manuscript, please double check the work and fix all of them

- TMJ acronym has to be defined in extenso at least once

- on line 273 esophagus is wrongly reported as an organ containing cartilages!! If the authors intend cricoid cartilage close which the esophagus starts they should consider that cricoid is anatomically included in larynx.


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

Input:
Title: THREE-DIMENSIONAL PRINTING WITH BIOMATERIALS IN CRANIOFACIAL AND DENTAL TISSUE ENGINEERING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The abstract is well written.
The introduction is well written.
The survey methodology section is well written.
The body text/results/discussion sections are clear.
The conclusions are fine.
Experimental design: Not applicable, this is a review.
Validity of the findings: Not applicable, this is a review.
Additional comments: The authors have revised the manuscript in line with the reviewers suggestions and the manuscript is improved.


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

Input:
Title: THREE-DIMENSIONAL PRINTING WITH BIOMATERIALS IN CRANIOFACIAL AND DENTAL TISSUE ENGINEERING
Review round: 2
Reviewer: 2

Output:
Basic reporting: the article is a good article but has ignored the work most important to this article. therefore it should discuss the work of bio-electrospraying and cell electro spinning. the following article should also be cited,

Small, 2(2006)216-219.
Analyst, 136(2011)878-890.
Biomacromolecules, 7(2006)3364-3369.
Analyst, 138(2013)2215-2223.
Experimental design: the study seems sound
Validity of the findings: the results are valid
Additional comments: the additional information meaning the introduction should contain the works citing the bio-electrospraying and cell electrospimnong.


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

Input:
Title: THREE-DIMENSIONAL PRINTING WITH BIOMATERIALS IN CRANIOFACIAL AND DENTAL TISSUE ENGINEERING
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript currently meets the publishing criterions and agrees to publish.


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

Input:
Title: THREE-DIMENSIONAL PRINTING WITH BIOMATERIALS IN CRANIOFACIAL AND DENTAL TISSUE ENGINEERING
Review round: 2
Reviewer: 4

Output:
Basic reporting: The manuscript has been properly integrated according to reviewers' comments and it is now suitable for publication in PeerJ. I would like to suggest the Authors to double check again the whole work since some careless mistakes can still be found.
Experimental design: see above
Validity of the findings: see above
Additional comments: see above


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

Input:
Title: FACTORS ENCOURAGING MOBILE INSTANT MESSAGING SERVICE USE IN MEDICAL EDUCATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Literature review is not sufficient enough. Please see the general comments.
Experimental design: The analysis is a little superficial. Please see the general comments.
Validity of the findings: The findings themselves are fine, but the discussion does not suggest any theoretical or practical implication of the current study. Please see the general comments.
Additional comments: This study investigated the use of Kakao Talk in clinical education, by examining 39 groups of medical students’ interactive records during a one-week clinical education. It statistically analyzed the relationship between the number of posts during the first two days and the last three days. The results showed the more students posted, the more likely they would particulate academically in the discussion. Students were more likely to post academic related messages in the later half of the week. In addition, the number of posts in the first two days was positively correlated with that of the last three days.

This study deals with an interesting and relevant topic. It could be a valuable addition to the literature regarding using mobile-instant-messaging in educational settings, particularly in medical education. However, there are many issues to be addressed before the manuscript can be considered at a publishable level.
1.	My biggest concern about this research is its significance. To me, the results achieved by the study is pretty obvious and self-explanatory. For instance, the more students used the service, the more likely they would share more academic information. And once they became familiar with the service, the learning environment and the content, it is predictable that students would share more academically related posts. Therefore, in addition to the analysis of the number of posts, I would personally want to see more interesting and sophisticated content analysis results. For instance, how about students’ collaborative learning, the level of thinking? Just some random thoughts.
2.	Another problem I have with the manuscript is the literature review. The authors hardly reviewed any literature regarding this topic substantially, which fails to provide a solid academic context to situate the current study. Without an up-to-date and critical review of related literature, this study is hardly situated in the scholarship of this field of study.
3.	What is the theoretical and practical implication of the current study? What theories did the authors refer to structure the study? After presenting the results, how would the authors interpret the achievement theoretically? Any practical suggestions?
4.	I also have some questions regarding the details of the manuscript. Some are listed below as examples. The authors can address them but my biggest concerns have been stated above in points 1-3.
a)	71. More explanation is needed on “the influences on student educational performances vary”. How did it vary and why?
b)	77. What is the other media?
c)	80. Any reference for this statistic result?
d)	87. On what basis did you assume that “Kakao Talk would be used often when it is considered helpful for academic purposes?” (87)
e)	90. Where did the hypothesis that “an increase in student activity on Kakao Talk would influence its later use for academic purposes?” come from?


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: FACTORS ENCOURAGING MOBILE INSTANT MESSAGING SERVICE USE IN MEDICAL EDUCATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: A.	Clear and unambiguous, professional English used throughout.
i.	Good
B.	Literature references, sufficient field background/context provided.
i.	Good
C.	Professional article structure, figures, tables. Raw data shared.
i.	Good
D.	Self-contained with relevant results to hypotheses.
i.	Moderate
Experimental design: A.	Original primary research within Aims and Scope of the journal.
i.	Good
B.	Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
i.	Not bad
C.	Rigorous investigation performed to a high technical & ethical standard.
i.	Good
D.	Methods described with sufficient detail & information to replicate.
i.	Good
Validity of the findings: A.	Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
i.	Good
B.	Data is robust, statistically sound, & controlled.
i.	Good
C.	Conclusions are well stated, linked to original research question & limited to supporting results.
i.	Moderate
D.	Speculation is welcome, but should be identified as such.
i.	Good
Additional comments: A.	Overall
i.	This study is not a confirmation of causality (“Kakao talk might be useful to medical students’ educations”), but a relevance problem (“MIM service use during the early period of clinical education might related to use for academic
ii.	purposes during later periods.”). Therefore, causal expressions throughout the thesis must be replaced with relevance expressions in whole manuscript.
iii.	The reference format of the introduction section (line 71; 1-9) and discussion section (line 200; [7]) is different.

B.	Abstract
i.	Line 49; Please replace “influence” to “relation”

C.	Introduction
i.	Line 80; spelling error (aka Ka Talk, Kakao Corp)
ii.	Line 86; Please, remove the following hypothesis.
1.	“Moreover, there might be factors that encourage the use of Kakao Talk for academic purposes and increase students’ familiarity with its educational use.”
iii.	Line 86; Please, remove the following hypothesis.
1.	 “We hypothesized that an increase in student activity on Kakao Talk would influence its later use for academic purposes. Therefore, this study investigated whether, in our hospital, student activity on Kakao Talk early in the clinical education period influenced Kakao Talk activities for academic purposes later in the clinical period.”

D.	Discussion
i.	Line 216~219; I understand that early Kakao talk usage is related to late Kakao talks usage for academic purposes. Why does this result lead to Kakao talk boosting students' educational outcome? It can be interpreted that students with a tendency to use messenger in early period more often use messenger for academic purposes in late period. To assert that Kakao talk has increased students' educational outcome, you should compare the academic achievement between the groups with and without Kakao talk. Therefore, the conclusion should be changed as follows. “Early Kakao talk usage is related to late Kakao talks usage for academic purposes.”


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: FACTORS ENCOURAGING MOBILE INSTANT MESSAGING SERVICE USE IN MEDICAL EDUCATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article must be expressed clearly in English.
1. Abstract should be arranged clearly. Introduction needs to be added with solid grounds on why this study can be explored by using references.
2. Mehtods also should be written simply to make readers understand clearly. The extended sentences listing all variables can make the article confused.
3. The structure of the article should be rearranged. In particular, in Methods, subheadings are needed.
4. Reference should conform to format.
Experimental design: 1. Study design: to make it clear, Figure on the process of the experiment can be helpful.
2. The number of participant including students and teachers (professors and fellows) can be written as (n= ), which can make the extended sentences simple and clear.
3. Research questions should be added.
4. The data were analyzed rigorously. However, the article emphasizes just the statistical findings (number) from the collected data. The statement on the detailed information which could cause students' chats on virtual space should be presented.
Validity of the findings: The discussion should be appropriately stated as some parts don't show the consistency of the results. The article can be better if dividing the section into 2 parts, Discussion and conclusion
Additional comments: I appreciate your raw data and effort
However, overall structure and statement should be modified.


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: EVOLUTIONARY COUPLINGS DETECT SIDE-CHAIN INTERACTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript presents a somewhat refreshing perspective to the question of a high current relevance, namely, how to measure the performance of contact-prediction sequence-based methods. The authors show a broad knowledge of the field, give a sufficient background, and generally the manuscript is well-written and comprehensible.
Experimental design: The experimental design is sound to the problem stated. My only concern is the introduction of the minimum distance between heavy atoms as a contact measure very late into the results section. The authors (quite rightfully) state that this measure is perhaps physically most relevant, so in my opinion it should feature prominently throughout the results section right from the beginning. I would recommend restructuring this section so that it is compared to C-alpha-based distances and geometric center-based distances in Figs. 2-5. Then I would appreciate a comment on why center-based distance might be preferred (computational efficiency, I presume?).

Another way to detect contacts that could be interesting in this context are resudue interaction networks (e.g. https://academic.oup.com/nar/article/44/W1/W367/2499329, https://bmcproc.biomedcentral.com/articles/10.1186/1753-6561-8-S2-S2). The authors might want to look into these tools.

Minor comment: is residue center a center of mass or geometric center? I understand that they are spatially close, but this should be clearly stated.

Another minor comment: It should be clearly explained how the distance cutoffs are chosen. It is mentioned somewhere in passing that they are chosen to pick the same number of contacts as the 8A cutoff for C-alpha's, however, I would appreciate if that was stated somewhere around the reference to Fig. S1.

Third minor comment: In the figure legend to Fig. S1, 150 proteins are mentioned, which have not been introduced to this point. Please fix the legend.
Validity of the findings: The obtained results are perfectly sound and supported by the data.
Additional comments: I have some additional minor comments to the presentation:

1. SI Fig. S7 seems important to me and should be oved to the main text.
2. Neural networks (mentioned on p. 9) are a type of machine-learning algorithms. Please fix the wording.


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: EVOLUTIONARY COUPLINGS DETECT SIDE-CHAIN INTERACTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is written unambiguously and organized well. The introduction is detailed, relevant and well referenced. Figures and tables are comprehensive and helpful.

There are some minor comments:
1) Line 29: A reader may expect here supporting references to recent relevant reviews on X-ray crystallography, cryo-electron microscopy, and other listed methods.
2) Line 28: “...primary amino acid sequence...”. Here, the word “primary” has to be omitted (there are no secondary or tertiary sequences). Or, alternatively, the authors can use the term “primary structure”.
3) Lines 93, 137, 195, 323: “...primary chain...”. Again, the word “primary” has to be omitted (there is no secondary or tertiary chains).
4) Line 65: “... can be chosen to use as a reference point … “ The words “to use” can be omitted.
5) Line 200: “ … large fraction of the these couplings ...” The word “the” should be removed.
6) Legend to SI Fig. S3, line 5: “... and found that couplings fail to fully recover C-alpha defined contacts...” Should be “C-beta” instead of “C-alpha”.
Experimental design: The experimental design was clear. The question is clearly stated and the authors answer it unambiguously.

The authors investigate the relationship between the residue-residue covariation revealed by couplings analysis methods and the type of defining the contacts between residues. In other words, they ask the question, what type of contacts are found by coupling analysis methods? The answer is that these are contacts that are formed between geometric-center of side-chains. This is expected because it is side-chains (not C-alpha atoms) that mutate coordinately in the course of evolution. But the value of the study is that the authors confirmed this expectation explicitly and quantified the found effect.

The only small concern is a requirement that the number of contacts made by side-chain centers was the same as the number of C-alpha contacts. As a result, the authors use different values of the threshold for different proteins, which looks artificial from the physical point of view (lines 89-92). If the way of calculation of the contacts does not influence the results, why then not to show results for a more general method of detecting contacts in 3D structure, with the same threshold for all proteins?  I don't see a problem that the number of C-beta or geometric-center contacts will be different from C-alpha ones. If the authors want to compare histograms they can normalize the histograms before comparison or compare cumulative distribution like in Kolmogorov-Smirnov test.
Validity of the findings: Data are robust and controlled. Conclusions are well stated and relevant.
Additional comments: None


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: STRUCTURAL AND EVOLUTIONARY CHARACTERISTICS OF DYNAMIN-RELATED GTPASE OPA1
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors with this papers aimed at elucidating the molecular architecture of OPA1 to better understand the IMM fusion, by modeling the structure of human OPA1 and analyzing the protein-protein interaction and the sequence/structure characteristic through bioinformatics approach.

The English is professional and mostly clear, with some little errors.
Line 24: I'm not sure I understood correctly what the authors mean writing “threading approach”.
Line 51: reduction instead of reducation
Line 141: “there was a hydrophobic region was found just before site S2”. Too much “was” in the phrase.
Line 162: “The exons 23-25 encoded peptide”, an “a” is missing after encoded.
Line 202: “In additional”, probably “In addition” or “Additionally” will sound better.
Lines 220: “However, the PH domain was less conserved, it was speculated that its function might vary from species to species”, probably “However, being the PH domain less conserved, it was speculated that its function might vary from species to species” could sound better. Or something likes this.
Line 219: linkers instead of linker

Sufficient field background is provided with adequate literature. Only some suggestions:
Lines 41-44 “In mammalian cells, the conserved dynamin-related GTPase optic
atrophy 1 (OPA1) is indispensable for both cristae morphology and the inner mitochondrial
membrane (IMM) fusion (Friedman & Nunnari 2014; McNew et al. 2013; Pernas & Scorrano
2016)” The references Friedman & Nunnari 2014 and McNew et al. 2013 are really nice reviews but do not exactly treat the topic of the sentence. Friedman & Nunnari is very focused on fission rather than fusion. McNew is focused on GTP-dependent membrane fusion rather then OPA1.

Lines 59-60: “Biochemical studies indicated that OPA1 disease alleles associated with DOA displayed a variety of mitochondrial defects in several activities, involving GTP hydrolysis, cardiolipin association, and membrane tubulation (Ban et al. 2010; Belenguer & Pellegrini 2013)” I suggest to add a reference here regarding the different degree of mitochondrial dysfunctions observed in patients’ cells.

Line 63-64: regarding the DOA+ phenotypes, Ban 2010 is not the correct reference. For a short summary about all the possible phenotypes caused by OPA1 mutations I suggest Del Dotto et al. 2018 Pharm Res. For a more in-depth knowledge I suggest the experts in the field: Yu-Wai-Man, Chinnery, Carelli, Amati-Bonneau. For example Yu-Wai-Man 2010 Brain described that DOA+ phenotypes could be generated by mutations in dynamin domain or other domains, and not only in GTPase domanin, and that those mutations could be missense, but also non sense or deletion or splice. Furthermore, DOA+ phenotypes could be given also by mutations that generate haploinsufficiency. Thus the phrase “The DOA+ phenotypes are missense mutations, relating to the highly conserved amino acid residue positions and exerting a dominant negative effect on multi-systemic disease” should be revised.

The structure of the article conforms to an acceptable format of ‘standard sections’.

Figures are relevant to the content of the article and of sufficient resolution. They are described quite appropriately in text, but the legends should be detailed better. For example in figure legend 2 the authors should detail what N or C does mean.
The legend of the supplementary table 1 is missing. For a better understanding by readers, the authors should detail which OPA1 variant are using (variant 8 or 1) and in the first column specify if is aminoacid position or nucleotide position.

The raw data has been made available.

The results are not completely relevant to the hypotheses.
Already in the title and in the abstract the authors declare that their results reveal something more about the mechanism of mitochondrial fusion OPA1 dependent, but in my opinion it is not so. Some data are of interest but do not allow to make new assumptions about the fusion mechanism.
For example, concerning the fusion Mitofusins dependent, Qi, 2016 JCB and Mattie 2018 JCB, combining bioinformatic predictions from the Mitofusins structure with biochemical and functional experiments, really provide structural insights into the mechanisms of mitochondrial outer membrane fusion. The work by Qi et al. further implicates the GTPase domain as the site of mitochondrial tethering, rather than the C-terminal HR2 domain as previously proposed. The work of Mattie et al. shows that metazoan Mitofusins contain only a single transmembrane domain and leads to a revised understanding of Mfns as single-spanning outer membrane proteins with an N(out)–C(in) orientation. Furthermore Mattie et al. provide functional insight into the IMS contribution to redox-regulated fusion events.
The authors, by bioinformatics analysis of the OPA1 structure, here presented some new information, as the possible mode of OPA1 dimerization clearly indicating the involved aminoacids. On the other hand much of the information reported is already known.
In figure 8 the authors proposed a model for cristae formation and membrane fusion that it is not well supported by functional experiments already present in literature and not well described/discussed by authors themselves.
Experimental design: Research question will be relevant and meaningful. Discovering something new about the structure of OPA1 that could help the understanding of the IMM fusion mechanism would really fill an existing knowledge gap. Unfortunately the results of the authors do not cover up this gap.

The investigations performed are rigorous.

Methods are described with sufficient detail and information to replicate. Only some errors and some suggestions:
Lines 112-113 “Blue native page was performed as described previously with slight modifications
(Wittig et al. 2006)” the authors should describe the modifications.
Line 112 1 mM GTP instead of 1mm GTP
Fig 5A showed an SDS-PAGE and this technique is not reported in Methods.
Lines 178-186 this part could be moved to Methods. The authors should detail better the purification conditions, as the amount of IPTG used, the elution conditions, temperature necessary for the expression of the protein….
Validity of the findings: Results section should be slightly reorganized.

Lines 121-138 are not results. This information is already present in literature. This part should be moved to the introduction section.

Lines 135 References Anand et al. 2014; MacVicar & Langer 2016 concern only the processing of OPA1 and not all the other notions. The authors should add some other references.

Figure 1 should be part of the introduction.

Lines 144-147: Concerning the different OPA1 functions, what could be the biological meaning of the different pI?

The paragraph on the dimerization of OPA1 is really interesting.
Lines 187-189: in figure 5B the authors show that OPA1 formed stable dimers and multiple oligomers independently on GTP/GDP binding, suggesting that OPA1 itself could interact and form oligomers. This result is really surprising, also because contradicts the literature knowledge.
Yamaguci 2008 Mol Cell reported that the addition of 1 mM GTP enhanced self-binding of Opa1, whereas 1 mM GDP completely blocked complex formation. Thus GTP promoted in vitro assembly of Opa1, whereas GDP favored the dissociated state.
Ban 2017 Mol Cell reported that in native-PAGE, a major dimer band at a steady state was moved to form higher oligomers of approximately 10 molecules after incubation with GTP or GTPγS, suggesting that GTP binding stimulates oligomerization on membranes.
Mitofusin 1, a dynamin-like GTPase as OPA1, forms dimers in the presence of GTP but not of GDP.
Qi 2016 JCB reported that MFN1 forms a dimer when GTP or GDP/BeF3−, but not GDP or other analogs, is added.
Ishihara 2004 JCS reported that in cell lysates, the cytosolic domains of MFN self-associate only when GTP is added at an enzyme-active temperature.
Which is the authors’ explanation? Since these results contradict the present literature, I think the authors should support better these data and not liquidate them in 3 lines without an appropriate discussion. I think other experiments are necessary.

Lines 190-204: this analysis is really interesting.
I have a doubt: the aminoacids shown in Figure 6 do not coincide with those described in the text.
In mode 1 there are 15AA instead of the 12 described. In mode 2 there are 18AA instead of the 17 described. Could the authors explain me better?
It is really interesting that, unlike mitofusins that dimerize by GTPase domains, for OPA1 neither of these two dimeric interfaces involved the GTPase domain or the PH domain. These differences should be reported and this part should be expanded and discussed better.

Lines 202-205: “In additional, only a small number of sites in the two dimeric interfaces overlapped, suggesting that the human OPA1 protein could further form a multimer on the basis of dimerization”. What does it mean? It would be interesting to explain this sentence better, perhaps adding some hypotheses.

Lines 219-221: which could be the function of the linkers encoded by exons 17-18 and exons 6-8? Could the authors write some hypothesis? Some speculations? The same for the PH domain.

The last paragraph of the results session, Disease-related sites in human OPA1 protein, is very confusing and has many inaccuracies.
Line 223: why the authors reported only 65 mutations?
On the locus-specific database dedicated to OPA1 (http://opa1.mitodyn.org/) at least 414 OPA1 variants are listed, more than 60% are considered pathogenic and two-thirds of them are in the coding sequence, mainly located in the dynamin and GTPase domains.

Lines 224-233 and Supplementary table 1: I don’t understand the diseases nomenclature that the authors use. In literature, usually, there are non-syndromic DOA, DOA and neurosensory deafness (DOAD) and syndromic-DOA (DOAplus). Since 2010 OPA1 mutations have been linked to an expanding spectrum of neurodegenerative phenotypes, such as spastic paraplegia, multiple sclerosis-like syndrome, Behr-like syndrome and most recently syndromic parkinsonism and dementia. These are the monoallenic condition, where the patients have 1 allele WT and 1 allele mutated. Then there are the bi-allelic conditions, where the patients have 2 alleles mutated with 2 different mutations associated with complex and severe phenotypes.
In the table what is DOA+ and OPA1? What does it means? What is OPA1 and BEHRS? Which is the differences between (DOA+ and OPA1) and (OPA1 and DOA+)?
I think the authors should conform to the nomenclature commonly used in literature. To get a summary overview on the mutations OPA1 and related diseases I recommend reading the review Del Dotto 2018 Pharm Res and to deepen my advice is to read the papers of experts in the field as Yu-Wai-Man, Amati-Bonneau, Carelli, Chinnery….

Line 234: “Within the 65 of disease-related variants, we discovered…”, it is not a discovery, but an observation.
This paragraph does not present any result but only observations, most of which have been known for a long time. Moreover, these observations are based on 65 mutations, when today more than 400 are listed in the database indicated above. I do not understand what the end of this paragraph is. At least the analysis should include the whole mutation panel. This paper is not a review. The last paragraph should be revised.

The discussion section is too hasty and pulled away. There is no real discussion of what the authors observe in the models they generate and proposed in figure 8.

Figure 8A: the cristae formation by OPA1 l-form. Cristae are not formed solely with long forms. Del Dotto 2017 Cell Rep and Lee 2017 JBC showed that long and short have the same ability to organize cristae. Thus, the authors should revise this part of the proposed model.
Figure 8B bottom: membrane fusion model. “s-form should support a bridge between l-form and cardiolipin”, I don’t understand the meaning of this phrase.
If short forms facilitated the IMM fusion, then the fusion rate of the long + short together should be greater than that of the long ones alone. But it is not so. Long forms have fusogenic capacity but are not able to interconnect the mitochondrial reticulum which remains therefore fragmented to 100% in cells. While long and short together influence the morphology of the reticulum that becomes filamentous and interconnected. The authors should explain better the proposed model, keeping in mind also the functional experiments already present in the literature.
Lines 263-264: “interaction between PH domain and cardiolipin promoted binding and then facilitated double-membrane fusion”, even the short form have the PH domain and should have the same fusogenic capacity as the long ones, but they are very less efficient. What explanation do the authors give?

Lines 267: “these findings have deepened our understanding of OPA1 and likely will ultimately lead to improvements in human health”, I think this sentence is a little bit exaggerated. The results reported by the authors are not particularly innovative. There are quite interesting data that could be useful to better understand the IMM tethering by OPA1 and how the fusion process can take place. Although the authors need to broaden and better discuss various aspects. But these results certainly do not lead to improvements in human health.
Additional comments: The paper is not particularly innovative, but has some interesting and new ideas that need to be developed better.
My advice is to implement the paper following the above suggestions.


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

Input:
Title: STRUCTURAL AND EVOLUTIONARY CHARACTERISTICS OF DYNAMIN-RELATED GTPASE OPA1
Review round: 1
Reviewer: 2

Output:
Basic reporting: Introduction:

The introduction needs more details.
I suggest the authors to develop a little more the general context of the study. In their introduction the authors indeed soon focus on OPA1. Only a short sentence describes mitochondrial dynamics; the main proteins that control mitochondrial fission or outer membrane fusion are not quoted. Furthermore, it would also be interesting to add few informations regarding the function of mitochondrial dynamics.
The description of the structure (and the mechanisms) of the Dynamin Super Family, well reviewed in the recent publication of Jimah & Hinshaw Trends in Cell biology 2019, also need to be expanded.

I recommend to the authors to check carefully the bibliography. I suggest to add more recent reviews on mitochondrial dynamics than “Chan 2012” and “Van Der Blieck 2013” (lines 40-41). These reviews are very interesting but significant advances have been made with regard to this topic in the last past years (Del Dotto 2018 only concerns OPA1). The same remark can be made for “Lenaers 2012” for optic nerve atrophies (line 58). Furthermore, some references are not well quoted: “Friedman & Nunari 2014” (line 48) does not focus on OPA1, in “Song 2007 and 2009” (lines 48-51) the experiments were made in Mouse Embryonic Fibroblast and not “humans”, “Praefcke & MacMahon 2014” (line 55) concerns Dynamins and not OPA1 ….

There it appears to be some confusions and some missing informations:
-line 53: Cristae-tightening-related OPA1 function is important not only for apoptosis (add Yamaguchi Mol Cell 2008) but also for mitochondrial energetics (Patten Embo 2016).
-line 58: Does  OPA1 mutations really causes  blindness in most cases” ?
-line 63: mtDNA deletion also occurs in isolated DOA and not only in DOA+ (Yu-Wai-Man Brain 2009).
-line 64: DOA+ phenotype are proposed to exert a dominant negative effect dominant.

Methods :

I recommend to the authors to move the paragraph “lines 178-185” from results to material and methods and to describe more carefully their structural analysis (see below)

Results and Figures :

I suggest the authors to add more details on OPA1 proteolysis (in the introduction).

More than 300 mutations have been described in OPA1 (not only 65). I suggest the authors to enlarge their analysis and to add the reference “https://databases.lovd.nl/shared/genes/OPA1”.

Figures are relevant and well described.
Does OPA1 really contain a PH domain as shown in Fig. 1 ?
Is OPA1 really purified to homogeneity (Figure 2A is a result of Comassie blue staining and has to be moved in Supp data) ?
A Figure comparing the schematic representation of dynamin super family members (classical dynamin and Dynamin Related protein) would be very informative.

Discussion :

I recommend to the authors to integrate to their model recent data showing in other that s-OPA1 alone can sustain cristae structure (Del Dotto et al Cell Report 2017,  Lee et al JBC 2017) and to develop the description of the works concerning the various functions/properties of OPA1 isoforms.
Experimental design: The model proposed for OPA1 sounds good for the core domains 9-16 to 26-28 due to the large number of corresponding proteins probably included in the template but no explanation was given to justify the accuracy of the model.
However, the model proposed for the elongated form seems suspicious at least in its N-terminal extremity because of the succession of short alpha helices covering the region corresponding to exons 3 to 5b giving rise to a long helical N terminal domain.  A random sequence will always give rise to a small alpha helix by this approach. I suggest you to provide sequence alignments especially in support of Figure 7, which leaves anyway to suppose that the model cannot be reliable for exons 3 to 5b.
Validity of the findings: The conclusions given on the structure of the elongated form of OPA1 need deeper critical discussion and can’t be supported in absence of experimental evidences supporting this model. For instance, experiments on the elongated form could be realized by SAXS.
Additional comments: Using a threading approach the authors model the structure of the human OPA1 protein. This study could have be interesting because the structure of this protein has not been experimentally solved and since this information could help to better understand its functions. However major bibliographic and experimental concerns have to be solved before publication.


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

Input:
Title: STRUCTURAL AND EVOLUTIONARY CHARACTERISTICS OF DYNAMIN-RELATED GTPASE OPA1
Review round: 2
Reviewer: 1

Output:
Basic reporting: Title and Introduction
I appreciate the changes in the title, which now better reflects the contents of the paper, and in the introduction, which now is more complete.
Only some little errors:
Line 42 “while Drp1 involves in fission of OMM” should be changed in “while Drp1 is involved in fission of OMM”. Furthermore, Drp1 is involved in OMM and IMM fission and not only in OMM. It oligomerizes to generate a constriction ring around mitochondria.

Line 50-51 the references Song 2007 and Song 2009 regard mitochondrial network and cristae morphology but not mtDNA copy number or respiratory capacity of MEF OPA1 null.

Line 96 “the four isoforms including exon 4b completely processed into s-forms” should be “the four isoforms including exon 4b are completely processed into s-forms”
Experimental design: Methods
Lines 150-151 SDS-Page is only mentioned but not described.  At least a reference should be indicated.
Validity of the findings: Results
Lines 165-166 “We discovered that exon 4b-encoded peptide was hydrophobic while the others around were hydrophilic”.
This was already demonstrated by Elachouri 2011 Genome Research. In that paper the authors showed that NT-OPA1-exon4b is tightly bound to the mitochondrial membrane fraction and it is embedded in the IMM (by a protease protection assay on permeated mitochondria). Elachouri et al. reported “NT-OPA1-exon4b structure is composed of two transmembrane (TM) domains, one common to all OPA1 isoforms, and one encoded by exon 4b, plus a 35-aminoacid intermediate domain without signature motif….In vertebrates, the second TM domain consisting in a 15-amino-acid-long hydrophobic sequence is encoded by exon 4b, whereas in yeasts and Drosophila, the second TM domain is systematically present in the unique isoform”.
Thus, the authors should change the sentence “We discovered that exon 4b-encoded peptide was hydrophobic while the others around were hydrophilic” with “We confirm that ….” adding also the reference Elachouri 2011 Genome Res.

Lines 201   “Compared the structure of the C-terminal region..” should be changed in “Comparing the structure…”

Line 205-206 “the TM-score was all more than 0.5, although these proteins did not mediate membrane fusion like OPA1” should be write more clearly. “the TM-score of all was higher than 0.5…”could be correct?

Line 206-207 “Compared OPA1 with mitofusin 1, atlastin 1 and altastin 3, which were involved in membrane fusion, the TM-score was all less than 0.3” should be changed in “Comparing…. the TM-score of all was minor than 0.3”

Line 208 “OPA1 had a PH domain, while other proteins did not have” which proteins? Probably the sentence should be “OPA1 had a PH domain, while other proteins involved in fusion process did not have”.
Line 215-216 “From the peak position of the OPA1 (exons 6-28) elution profiles of a S200 size-exclusion column, the result indicated the molecular weight…” should be changed in “From the peak position of the OPA1 (exons 6-28) elution profiles of a S200 size-exclusion column, the result indicated that the…”

Line 218-220 It’s a pity that the authors decided to remove the experiment regarding OPA1 oligomerization independently by the presence of GTP or GTP, instead of going into more detail on this point. It was a really new and interesting result.

Line 225 and 230 “Between 15 AA…” and “Between 18 AA…” should be changed with among.

Line 235 “which was different from mitofusins that mediate membrane fusion” this sentence is not complete. It could be better “which was different from mitofusins that mediate membrane fusion by GTPase domains”. Add also a reference about mitofusins dimerization.

Lines 235-239 “Additionally, interactions between GTPase-GTPase domains had been reported to be essential for the function of dynamins, but our results showed neither of these two dimeric interfaces of nucleotide-free OPA1 proteins involved in the GTPase domain. We speculated that GTP binding and self-assembly may promoted OPA1 GTPase-GTPase domains dimer formation to mediate inner membrane fusion”. I'm not sure I understood what the authors mean. This sentence should be better explained.

Lines 262 “In human OPA1, mutations have been found to cause diseases” should be changed in “Human OPA1 mutations have been associated to a large spectrum of neurodegenerations”

Line 278 “The disease-related variant sites in the N-terminal region are concentrated..” the authors used the pass form in all the manuscript.

Lines 261-285 despite the changes that the authors have made following my suggestions, I still think that this paragraph does not add anything new.
The authors performed this long analysis to conclude that “Therefore, most of the disease-related mutations in the OPA1 protein were located in the C-terminal region, while in the alternative splicing region, almost no disease-related mutation occurred”. Nothing of new. The same massage was already reported in Amati-Bonneau 2009 IJBCB and Ferrè 2009 Hum Mut.

Discussion
Line 321 “What’s more, a deletion mutant assay suggested the domain next to the GTPase domain is necessary for cardiolipin binding” should be changed in “Furthermore, a deletion mutant assay suggested that the domain next to the GTPase domain is necessary for cardiolipin binding”.

Line 322 The reference Del Dotto 2018a is not included in the references list.

Lines 317-319 “By using an in vitro membrane fusion assay, the experiments unveiled the molecular basis of most minimal IMM fusion that OPA1 l-form and cardiolipin on each side of the membrane separately are sufficient for fusion (Ban et al. 2017; Liu & Chan 2017)” Write better this sentence.

Lines 315-325 This part remains confused, unconvincing and pulled away.
In the first my revision I underline that even the short forms have the PH domain and should have the same fusogenic capacity as the long ones, but they are very less efficient.
In the rebuttal letter the authors answer was” The s-forms have the PH domain, so the s-forms may also interact with cardiolipin on the membrane. However, there is an obvious difference between the l-forms and s-forms, the s-forms do not include the transmembrane region. In addition, the inner membrane fusion is a complex process. The interaction may promote binding, but it does not mean that the s-forms should have the fusogenic capacity”.
My experience on OPA1 derives from 18 years of studies on OPA1 ko or mutated cells and I really know that the obvious difference between the l-forms and s-forms is that the s-forms do not include the transmembrane region. Furthermore I quantified the fusion rate in OPA1 mutant cells and I really know that inner membrane fusion is a complex process.
In the revised manuscript the authors write that "Combined with our work, we speculated that the charge interaction between PH domain and cardiolipin promoted binding and then accelerated double-membrane fusion” and nothing else.
I repeat the same observation: this speculation is not supported by in vivo experiments. s-forms have the PH domain and can bind cardiolipin but they have not fusogenic capacity (Del Dotto 2017 Cell Report). Furthermore, l-forms alone have the same fusion rate of the l- and s-forms together (Del Dotto 2017 Cell Report). Therefore the role of the PH domain in the fusion process does not seem convincing to me. It could have a more structural role.
The authors should ameliorate this part.

Figures
Figure 2 reported OPA1 isoform 8 in its long and short forms. But isoform 8 is completely processed in s-forms and the l-form is not present in vivo.
Additional comments: the manuscript requires other small revisions


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

Input:
Title: THE EFFECTS OF GLUTEAL SQUEEZES COMPARED TO BILATERAL BRIDGES ON GLUTEAL STRENGTH, POWER, ENDURANCE, AND GIRTH
Review round: 1
Reviewer: 1

Output:
Basic reporting: Make the citations like this (Contreras et al., 2015). The parentheses go before the period.

Every time you say, "in previous study," instead say "in a previous study."
Experimental design: I see why you chose the seated position for the squeeze group (to increase compliance), but this position of hip extension leads to lower glute activation compared to full hip extension (see Worrell et al., 2001). In addition, a narrow stance likely leads to reduced glute activation compared to a wider stance. This could mean that better results could have been achieved with standing glute squeezes if compliance was similar. Might want to mention this.

In addition, you didn't instruct the bridge group to squeeze their glutes maximally at the lockout portion of the movement. This may have evened out the results had you done so. May want to mention this as well.
Validity of the findings: Interestingly, squeezes which were in hip flexion transferred better to dynamometer strength measured in hip hyperextension compared to bridges which reach full hip extension. Might want to mention this.

Why would glute girth decrease at the measurements above and below the greater trochanter? It couldn't be muscle atrophy. But fat loss seems unlikely. Please offer some explanation.
Additional comments: The squeezes you mention in the intro were from a standing position which would involve a higher glute activation compared to a seated squeeze. Might want to mention this.

Page 6, line 53, change to: Gluteal girth is a less researched feature of the muscles compared to their strength, power, and endurance, but noteworthy nonetheless.

Page 7, line 92, change to: Hip girth measured similarly with a tape measure demonstrated high reliability (ICC = 0.96-0.97) in a previous study.

Page 8, line 117: I was under the impression that if someone was right handed, their left hip hip extensor is stronger while their right hip flexor was usually stronger (since they kick with their dominant leg). So you might have used the nondominant leg for testing. Could be wrong though. Just thought you might want to look into this.

Page 8, line 119: you mean as opposed to 90 degrees of knee flexion? If so, please state this.

 Page 10, line 201, change to: For example, one 12-week study of progressive hip strengthening and other exercises

Page 11, line 255, change to: be of clinical importance

Page 12, line 275, please revise...I can't make sense of 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: THE EFFECTS OF GLUTEAL SQUEEZES COMPARED TO BILATERAL BRIDGES ON GLUTEAL STRENGTH, POWER, ENDURANCE, AND GIRTH
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see entire review in comments
Experimental design: Please see entire review in comments
Validity of the findings: Please see entire review in comments
Additional comments: Congratulations to the authors on the production of this work.  While this is a relatively simple study there is value in the answering of simple research questions.  I have a few minor suggestions and questions.  Below, I have quoted a part of your manuscript and made my comment.

Abstract

“Hip extension weakness is correlated with low back”

This is minor but it should be noted that hip extension weakness is only sometimes correlated with pathology/painful states.  There are a number of papers where increased hip extension strength is correlated with pathology.  This can be easily seen in the SR by Neal et al (2019) where hip Abduction strength was not related to PFPS and where increased hip strength was related to increased PFPS risk (https://www.ncbi.nlm.nih.gov/pubmed/30242107)

Introduction

Line 38-41 “For example, gluteal strength (hip abduction, external rotation, and extension strength) has independently predicted non-contact anterior cruciate ligament injuries and been associated with knee overuse injuries. (Khayambashi, 2016; Kollock, 2016)”

I realize the authors used the term “predicted” but this is again a bit strong.  They calculated ORs after the fact.  They didn’t identify people before hand and “predict” who got injured.  Again, the point here is that we should be cautious in how certain we are when making the case for the relevance of hip strength to pathology.  Yes, there are cases where there is an association but there are many papers where it is not associated.

Lines 93-95

We need a bit more information here.  What was the make of the dynamometer?  Was it peak force that was found? Was it the average force over 1 second etc?  Can you add a picture of the set up.  The methods should allow for the reproduction of this paper.

Line 104

Was the experimenter blinded to what stage the participant was in the study (e.g. pre or post) and blinded to group when using My Jump.  My Jump requires a subjective determination of leaving the ground and landing.  If you use this in future work I would suggest this blinding and perhaps having multiple assessors.

Line 126

I am actually having trouble seeing how many gluteal squeezes were prescribed here.  The contraction lasted 5 seconds, they the rested briefly and were supposed to do this for 15 minutes?  But, the 15 minutes could be split throughout the day?  If someone did 5 seconds on and 2 seconds off they might perform around 8 repetitions per minute and could theoretically perform 15 minutes of this?  Sixty 5 second glute squeezes in 15 minutes?

Was any of this tracked?

Last, pictures of both exercises would be helpful.

Line 167

How was compliance measured?  Is this a percentage of days performed, percentage of the potential maximum repetitions, percentage of participants who did at least one set?

Line 251

The largest limitation of this study is the lack of a control group who did nothing.  Both peak strength and endurance can be influenced by many factors.  This should be noted.

It might also be relevant to note how poorly hip extension strength when tested with dyno poorly translates to functional tasks.  One interpretation of your findings is not just that a Glute Squeeze performs similarly to a Glute Bridge but how poor of an exercise a Glute Bridge might be.  Especially considering the prescription dosage of the exercise.  Daily performance of low force, high repetitions (60) would not be the ideal way to increase strength.

Perhaps, adding a discussion on this can put your findings into context for the reader.


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

Input:
Title: THE EFFECTS OF GLUTEAL SQUEEZES COMPARED TO BILATERAL BRIDGES ON GLUTEAL STRENGTH, POWER, ENDURANCE, AND GIRTH
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: 129 the text reads that each group could perform the 15 minutes/day however they wanted which describes the "duration" of the daily exercise.
144  Text states that the bridge group did the same "frequency" as the squeeze group but that was never defined and was self selected in the squeeze group.

138 The squeeze group was instructed to do a MIVC of the glutes
144-151  Were the bridge subjects instructed to also use a maximal glute squeeze while doing the bridges or only whatever it took to lift the pelvis off the ground?
Validity of the findings: 169-177   was there a statistically significant difference between the 11.3% increase in the bridge group and the 15.56% in the squeeze group?

178-183  do the authors have any theories as to why the girth increased with the squeeze group and not in the bridge group despite strength increases in both groups?  How would they explain the strength increases without the hypertrophy found in the squeeze group?

222-226  Were these differences between groups statistically significant?

245-253  was there a follow-up questionnaire of the study subjects asking them about specific barriers they faced that resulted in the lower compliance in the bridge group.  Was it the change of position required to complete the exercise or other circumstances?
Additional comments: Nice study, very well thought out.


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: AN ARTIFICIAL-VISION- AND STATISTICAL-LEARNING-BASED METHOD FOR STUDYING THE BIODEGRADATION OF TYPE I COLLAGEN SCAFFOLDS IN BONE REGENERATION SYSTEMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English is ambiguous, the introduction statement needs references for justification.

The English is ambiguous, for example, line 33-38, on the other hand, ..., on the other hand line 94: The computational tool used ... has been the statistical software R (R Core Team, 2017). line 108-109, line 141-144, 254 etc.

In sufficient literature reference, line 51-64, general statements without reference.
line 68, image segmentation methods are listed, but no further introduction, pros/cons.

line 91, the R squared definition equation in a paragraph, do we really need this?

line 108-112 general statements no reference.

line 125-126: the above ... procedures, even though all are valid, are not usually applicable for all cases. This is not a professional description.

line 407: the linear models are the most simple and usual parametric models, but in chemistry and biology domains, the nonlinear models are also very important and useful. Any reference, example to justify this general statement?

Equation numbers are missing: line 390, 394, 401, 412, etc.

Figure 7, label small letter inconsistent with the capital letters labelled in the figure.
Experimental design: The overall experimental design is proper, from image preprocessing, to feature analysis, such as correlation and time dependency modeling/analysis among features, and the  regression model fitting for collagen degradation.

The procedure to obtain the image data is clear, the paper needs more introduction and justification on using the RandomForest, the pros and cons in comparison with other machine learning methods.

The paper modeled and examined time dependency of collagen mass loss, mean area and mean circularity in a systematical way. Some interesting patten was found, such as the two sigma models for collagen mass loss (Figure 6).

The paper modeled the degradation feature with the extracellular features, mean area, and mean circularity.

The paper modeled the collagen degradation with respect to area, circularity and grayscale histogram mode.
Validity of the findings: In terms of the methodology, the pipeline of processing, analysis, modeling is general.

The time dependency model, the overall degradation model are properly modeled. Given the relatively small data set of 60 images, the 3-folder cross validation result is reasonable.

The findings by feature correlation analysis and modeling is novel.
Additional comments: The literature review and description needs to be revised.

The modeling and time dependency findings are interesting, application/practical usability of needs to be justified.


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: AN ARTIFICIAL-VISION- AND STATISTICAL-LEARNING-BASED METHOD FOR STUDYING THE BIODEGRADATION OF TYPE I COLLAGEN SCAFFOLDS IN BONE REGENERATION SYSTEMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1, The English has to be improved to make it easier to understand. Some examples:
(1) Line 255-256: To achieve this goal, the classes of ROI were first defined. Based on the identification
of these classes, a model is estimated from the training sample and. It associates the ROI of
each pixel with its…
→ separate clearly into two setenses.
(2) Line 629: This methodology could be
also useful to study de degradation of other scaffolds appart from collagen type I.
→ de degradation. appart -> apart

(3) Line 728: Moreover, by
tudying the parameters of the first and second logistic components of the model,
…
→ tudying is a typo
(4) Line 48: It's not clear what the authors mean with 'since it is mainly of a qualitative nature'.
(5) Line 25, Line 148 and Line 187 sentences are hard to understand.

2, In both the article and the figures, the authors have to check carefully with names of the three cell groups (CCO, CCT and CO). Currently the mix-and-match is confusing.
(1) In the article I see four: CCO, COO (Line 498), CO, CCT.
(2) Line 642: Differences between fittings corresponding to CCT and CCT groups have
643 been found studying the first and second logistic functions, ...
→ CCT and ?

2, In the whole literature, although the authors mentioned multiple times, I failed to find a reference mentioning why collagen is a key scaffold to study bone regeneration. It'd add great value to this study, it the authors can find support information for (1) this is important, (2) this is similar to some other important biological questions that might use this method.

3, Although "a random forest classification model" has been mentioned in the abstract, introduction, and keywords, I failed to find details of the model is provided in the manuscript.
Line 169: Proposal of an automatic procedure, based on the combination of image segmentation techniques and supervised classification Random Forest, to identify the different regions of interest in the images obtained by optical microscopy. It is important to emphasize that the Random Forest classification model is trained taking into account the information pro- vided by qualified laboratory personnel in the qualitative study of this type of images. We present, therefore, a model that is capable of automating a complex and time consuming task in the context of this type of analysis.
→ However, no data/model/significance were provided for raw features definition by experts group or the random forest model. As a key part of the study, the authors have to put that part into the manuscript.

4, In the introduction part, (Line 61)the authors expressed their concerns on method based on human-decision. In Line 122-129, they mentioned multiple methods with concern that none can fit all cases. The authors have to make it more clear if their method can concur both points with method comparison, otherwise remove these points.
Experimental design: 1, The authors provided here a case study. Without providing raw information of how their expert team selected features at the first place, and include in article/script how they built random forest tree model to select features, their results cannot be repeated.
Moreover, the authors didn't not provide information on whether their trained model work on other dataset of the same kind, or other biological question.

2, The authors raised the question of automatic and object method to measure bone regeneration in an in vitro system. However, without description of current method undertaken to fulfill the same target, or comparison with certain methods mentioned in the script, it's hard to evaluate how meaningful the question is.
Validity of the findings: The authors included only one dataset as their training set. Although a cross-validation has been provided in Figure9, it's not a comprehensive cross-validation as the features (the non-mentioned random forest tree model from experts provided features) were not selected only based on a subset of samples.
The authors didn't provide a golden-standard of mass loss in their experiments across CCO, CCT and CO. It'd be very helpful to evaluate their results if they can add these information.
Additional comments: The authors built a novel pipeline with public algorithms/packages for a case study, with (1) expert provide measurement of features; (2) use random forest tree to select feature from (1);  (3) use the classifier on the four features to process image segmentation; (4)  modeling with GAM and two-factor logistic model to look into biological insight of the experiment.

1, The authors mentioned one improvement of their method is to use classifier (machine learning) instead of Threshold to process segmentation. In Figure2 and 3, it's hard for human-eyes to directly judge which method is better, a more detailed comparison is required to support the authors' point. Other than that, please show the results if we feed the threshold-based segmentation into down-stream analysis, would it provide a significantly worse model in either GAM or logistic mixed model.

2, The authors mentioned other methods in Line 624, any possibility to provide a comparison?

3, Line 628: while the first logistic component accounts for the 50% of collagen mass, it'd be more careful to think the model can explain the degradation process by calling it "other degradation factor". As the authors also mentioned that 'experimental error' is included. More insight from the biological or medical side to interpret the output might be a huge plus of the understanding.

4, Fig 4 and 5 provide similar information. A log-transform of area and circularity values would help to show the pattern with other factors.

 5, Figure 6 panel E and F share the same title, I believe F is the 'Comparison between the second logistic component of CCO and CCT treatments'. Please edit that.


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

Input:
Title: MORPHOLOGY AND DEVELOPMENT OF A NOVEL MURINE SKELETAL DYSPLASIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript entitled “Morphology and development of a novel murine skeletal dysplasia”. The goals of the study are clearly defined at the outset of the study and the authors organized the work to clearly achieve these goals. Data in figures is organized, easy to follow and relevant to the presented hypothesis.
Experimental design: The authors describe the phenotype of a novel mutant that arose naturally in the lab from a related ongoing study of the Longshanks mouse line. The materials and methods are thorough in describing the evolution of this project; important to the background for the study. The extensive skeletal analyses via microCT and other measurements shows a clear shortened limb phenotype of these mice that may allow for novel insight once the mutation has been identified in future studies. I commend the authors on this careful analysis through the postnatal growth of the animals.
Validity of the findings: Based on the data presented, the authors describe a mutation that negatively impacts endochondral ossification, rather than intramembranous ossification. These data are presented in a compelling manner by comparing the size and structure of bones generated by these two processes. The study, however, falls short of more detailed studies that would provide more substantial evidence that this is the case and that will be critical in future studies of these animals. The following points should be addressed prior to publication:

1.	Based on histologic evidence in Figure 5, it is clear that chondrocytes are severely affected by the Nabo mutation during postnatal skeletal development. The authors also describe shortened limbs during embryonic development in Figure 3 and Table 2. Given that proper establishment of the cartilage anlagen at embryonic stages is critical to formation of the postnatal growth plate, the authors should also include histologic images of the developing skeleton at embryonic stages (similar to those provided in Figure 5).

2.	The combined data of this report strongly suggests that chondrocytes are most affected by the Nabo mouse mutation, however the authors fall short in providing more mechanistic evidence that this is the case, especially at embryonic stages of development. Further histologic studies would allow the authors to conclude more clearly that chondrocytes (and endochondral ossification) are chiefly affected. Possible analyses could include in situ or immunohistochemistry for CollagenX, Collagen2, and Aggrecan.

3.	In line 373 and 374, the authors suggest that the growth plates of Nabo mice “show irregularities in bone and cartilage matrix at the chondrosseous junction.”  A few mechanistic studies should be provided to support this point further; the only evidence provided is histologic images of these regions. In situ or immunostaining for CollagenX, Collagen2, MMP13, or similar would provide clearer evidence that matrix is affected in the junction (i.e., lack of collagenX expression would suggest defects in chondrocyte maturation which lead to the disruption of the chondro-osseous junction). TRAP staining for osteoclasts may also provide the authors with evidence that matrix is being degraded properly for endochondral ossification.
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: MORPHOLOGY AND DEVELOPMENT OF A NOVEL MURINE SKELETAL DYSPLASIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: Please provide a rationale for the ages and sample sizes chosen for each analysis. For example, skulls of N= 5 mice per genotype were examined by microCT at postnatal day 75, but growth plate histology was done on only N=3 Nabo mice but N=8 wild type at postnatal day 14 (lines 132 and 174). Why not examine growth plates of older mice (5 weeks and/or 8 weeks age) and why not examine more than N=3 of the Nabo? Please provide justification. Was a power analysis conducted to determine sample sizes?
Validity of the findings: Minor comment: figures 2 and 3 are referred to out of order
Additional comments: This manuscript thoroughly characterizes a skeletal dysplasia-like phenotype in a new mouse mutation. The paper is organized and well-written. Although it does not appear to mimic any known (published) human skeletal condition, this new model may have important implications for understanding mechanisms of endochondral ossification during normal and aberrant growth. My only substantive concerns relate to the ages and sample sizes as described in the Experimental Design section of this review. There needs to be better justification for why each time point was selected and why sample sizes vary between analyses.

I also recommend changing all instances of growth plate "width" to "height" to match more customary terminology since it is measured in the direction of longitudinal growth.


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

Input:
Title: MOLECULAR CHARACTERIZATION OF NEARSHORE BAITFISH POPULATIONS IN BERMUDA TO INFORM MANAGEMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is clearly written and largely error-free. The introduction and discussion mostly appropriately review the literature, although I have a few suggested changes/additions (see general comments below).
Experimental design: Methods were clearly and comprehensively explained. Research is original and fits into the aims and scope of this journal. Molecular methods were not cutting edge, but sufficient to explore the questions posed by this study.
Validity of the findings: I think this study’s findings are valid and conclusions and are clearly stated (but see comments below for a few specific suggestions).
Additional comments: I really appreciated this study and thee clear writing and high quality condition of this manuscript! It is great to see such economically and ecologically important fishes getting some much needed attention!

Comment 1: You mention misidentification of some individuals (Line 227). Can you comment on whether or not these were small individuals and thus harder to identify?

Comment 2: The Lavoue et al. 2014 reference is somewhat outdated in the context in which it appears on Line 237. If you are going to discuss clupeiform phylogenetics it might be more appropriate to reference this paper, which is more comprehensive and up to date: Egan et al. (2018) Phylogenetic analysis of trophic niche evolution reveals a latitudinal herbivory gradient in Clupeoidei (herrings, anchovies, and allies).

Comment 3: You state that your findings about the relationships between Anchoa and Jenkinsia are congruent with recent research (line 236). This is not the case, although this is likely due to taxon sampling and the small quantity of genetic data used in this study. Contradictory results are reported in Lavoué et al. 2013 & 2014 references in your manuscript and several other recent studies including these:

Egan et al. (2018) Phylogenetic analysis of trophic niche evolution reveals a latitudinal herbivory gradient in Clupeoidei (herrings, anchovies, and allies).

Bloom DD & Lovejoy NR (2014) The evolutionary origins of diadromy inferred from a time-calibrated phylogeny for Clupeiformes (herring and allies).

Comment 4: Wording in Line 248 is not quite right in my opinion. It would be more appropriate to say something like “phylogenetic analyses based-upon morphology suggest it Is more closely related to….”

Comment 5: The paragraph starting at line 289 needs to be revised. I am not quite sure what the authors are trying to get across. Please add a topic sentence and edit to make the take home message of this paragraph clear.

Comment 5: It is not clear to me how the statement that “baitfish families typically have global distributions” relates to the findings of this study (line 289).

Comment 6: The claims in lines 292-295 need additional citation and need to be elaborated upon because right now they are an oversimplification. Lavoué et al. 2013 simply study the biogeographic history of Clupeoidei and do not compare rates of baitfish endemism in tropical versus temperate regions. Furthermore, there are many small, tropical clupeoidei with very large distributions (e.g. Encrasicholina spp.) as is also the case with some other tropical baitfish species (e.g. Atherinidae spp.). It might be worthwhile to point out some specific examples of endemism (e.g. Stolephorus nelsonii or Nematalosa vlaminghi) or reference some studies describing rates of endemism in small marine fishes to make your points more convincing.