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: APPLICATION OF PROBLEM-BASED LEARNING AND CASE-BASED LEARNING INTEGRATED METHOD IN THE TEACHING OF MAXILLARY SINUS FLOOR AUGMENTATION IN IMPLANT DENTISTRY
Review round: 1
Reviewer: 1

Output:
Basic reporting: overall, the writing is clear and well-written, with professional English used throughout.  The descriptions of PBL and CBL (and distinctions between the two) are ambiguous and not entirely correct or consistent with some of the literature.

I provide the authors with the following comments and suggestions:
– I believe that the description of PBL is mischaracterized, to some extent.  The claim in line 49 on page 1 - “this teaching method is based on questions”  is not precisely true. The socratic method is based on asking and answering questions.  Problem based learning is a student-centered approach to problem solving. Asking questions may or may not be part of the methodology.  Barrows, 1996 provides the definition of problem-based learning and outlines the main characteristics of the methodology. Barrow points to authentic problems as the basis for PBL, not necessarily questions. PBL is also, in its strictest form, self-directed and student driven, with students working together in small groups. In your introduction the suggestion that the teacher is leading students to examine and answer questions in a step by step manner is actually contrary to the characteristics of PBL outlined by Barrows.  There are numerous modified versions of PBL out there (published) but if you are using a definition of PBL that is different from that of Barrow you should provide the citation and explicitly state how it is different from Barrow’s.   You should also explain what you mean by “the traditional approach” (line 55) as this could be interpreted many different ways.

You should also make a stronger comparison between PBL and CBL in the introduction. These are two different methodologies. Currently, the description you include of CBL is very much like the description of PBL. It would be helpful to indicate how they are different and mention some contexts in which one is used over the other. Nadershani et al. 2013 provides a good example of how to do this (in the context of dental education) and includes many sources that you might find relevant. Nadershahi, N. A., Bender, D. J., Beck, L., Lyon, C., & Blaseio, A. (2013). An Overview of Case-Based and Problem-Based Learning Methodologies for Dental Education. Journal of Dental Education, 77(10), 1300–1305.

Some sentences could be rephrased for clarity: for example, the paragraph starting at line 137 needs to be reworded.  The second sentence beginning with “Besides, the difference of theory test…..”   is unclear and I’m not sure if you are trying to say that the results for the case analysis were also significantly different?
Experimental design: I appreciate the sample size in the study and that gender of participants was included.  Gender should also be included in Table 1.  If there are data on level of experience of participants that should also be included.  Currently there is no assessment of baseline knowledge for either control or experimental group. The authors also do not explain why some of the choices in study design were made.  For example, I am unsure why you chose to combine PBL and CBL into a new methodology rather than selecting to use one or the other. I don’t think there is anything wrong with using a modified version or a combined version but you need to provide justification for it, describe how the new method is different from both original methods, and include a discussion of why the new method you use is appropriate.

The authors also could provide more detail about the rationale for selecting maxillary sinus floor augmentation for their case: what is it about maxillary sinus floor augmentation that makes learning these skills particularly challenging?  Why is this more challenging than learning other skills in implant dentistry? What evidence exists to support your claim that this is more challenging than learning other skills?

Some other comments about the study design and reporting of the study design:
Line 99 – teaching methods.  You should include a description of the control group activities. If they didn’t participate in discussions did they simply attend lectures? Were these lectures in person or online? Did the experimental group also attend lectures, in addition to the discussion sessions or  did they attend discussion groups only?

Sections 2.2.1, 2.2.2, 2.2.3  -  it is not clear whether ALL students engaged in these activities or if these were just activities in which the experimental group participated. You should clarify the differences in learning experience between the control and experimental group.  A chart or table would be a nice way to demonstrate this.

In your description of these activities, it also seems that the teacher is doing a larger percentage of the instruction and directing and that there is very little self-directed learning on the part of the students.  If you are claiming that this methodology is a blend of PBL and CBL you should highlight the components of these activities that reflect the PBL and CBL experiences.

Line 129 -  I’m not sure what you mean by “the final exam, which was made of the theory test and case analysis”.  Can you describe the exam?  How many questions, what were the formats for the questions, what content was covered, is this a validated exam, was it piloted with any other group or is it an exam that has been given previously to other cohorts?

Line 134  - more information should be provided about the questionnaire given at the end of the training. How many questions, what were the formats for the questions, what content was covered, is this a validated questionnaire, was it piloted with any other group? It would be helpful to include a copy of the questionnaire as supplemental material with the manuscript
Validity of the findings: Line 142 – you need to include a summary of the results from the questionnaire rather than just directing the reader to the table. What were the major results? Did you perform any sort of analysis on these results?  If you did not analyze them quantitatively or qualitatively, then you should not report or mention them in this manuscript.

Line 146 – I think that the section 3.3 is referring to data obtained from the questionnaire but this is unclear.  You should specify from where these conclusions are being drawn. It also seems that you are reading quite a bit into the results of the survey.  Was any sort of analysis conducted?  It is possible to conduct quite robust analyses of qualitative data.

Discussion – line 151 – is there evidence to support your claim that implant dentistry is growing rapidly?  You should include statistics to support this, as well as other statements you make about the presence of absence of implant clinics.

In line 189 you claim that students were more likely to actively go about clinical work with questions in mind and were motivated to solve questions etc.. but this was never specifically tested in your study and there are no data to support these claims. Either make it clear that this is anecdotal and that it has not been tested so you do not know whether this is truly the case, or provide evidence to support these claims.
Additional comments: in addition to comments above, you need to make clear what you mean by “traditional method” hroughout the manuscript. Also - the discussion section largely reiterates many points from the introduction.  The entire section in the discussion on PBL and CBL (lines 160-180) could be moved and combined with the introduction to make that more robust. You discussion section should discuss the significance of your results, the limitations of your study (this is currently not included), and what your results mean in the context of other existing literature on the topic.


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: APPLICATION OF PROBLEM-BASED LEARNING AND CASE-BASED LEARNING INTEGRATED METHOD IN THE TEACHING OF MAXILLARY SINUS FLOOR AUGMENTATION IN IMPLANT DENTISTRY
Review round: 1
Reviewer: 2

Output:
Basic reporting: a.	Clear and unambiguous, professional English
i.	Line 26: Ninety-two clinicians who were receiving training between 2015 and 2017…should be Ninety-two clinicians who received training…
ii.	Line 31: After three months of training, teaching performance of both groups was evaluated through a theory test, case analysis and a…what do you mean by teaching performance?  This implies that the participants
iii.	Line 45:  instead of “by now”, use “currently”
iv.	Line 70: typo: toadvances should be to advances
v.	Line 72: “Implant dentistry is an emerging independent branch of dental science and it involves knowledge of many other dental fields such as” may be better stated as “implant dentistry is an emerging independent branch of dental science which involves
vi.	Line 75: what do you mean by social recognition?  Perhaps social is the wrong word here, perhaps “greater recognition in the field of dentistry”?
vii.	Line 76: training in implant dentistry
viii.	Line 77: why is it of great urgency to find out how to enhance theoretical knowledge and basic skills of clinicians?
ix.	Line 91:  should be: ninety-two clinicians who received training…were divided randomly and equally…
x.	Line 103:  discussions were held every other week and lasted 3 class sessions every time.  Do you mean 3 class sessions every other week?  How long was each class session, or how long on average did each class session occur?
xi.	Line 107:  This clinical case was a case on an adaptation syndrome…
xii.	Line 110:  spell out Computed Tomography (CT)
xiii.	Line 116: leaving some, not leavingsome
xiv.	Line 118: explore the, not explorethe
xv.	Line 118:  The students would then begin to search…
xvi.	Line 126: largely encouraging so as to
xvii.	Line 127: students develop
xviii.	Line 127:  thinking.  Not thinking,
xix.	Line 129:  final exam, which…not final exam,which (space after comma)
xx.	Line 138: control group, respectively.  Not control group , respectively
xxi.	Line 156: clinical practice.  Not clinicalpractice
xxii.	Line 162: over-reliance on.  Not over-relianceon
xxiii.	Line 169: develop an
xxiv.	Line 186: literature and to propose.  Not literatureand topropose
b.	Introduction and Background
i.	Introduction
1.	More detail is needed on the benefits and theories of PBL.  For example…” many schools desired to follow McMaster University, where the model for student-centered, problem-based, small-group learning took shape.  The faculty and administration at McMaster University had found that the PBL approach to learning encourages students to direct their own learning and focus on what they need to know to solve real-life problems, to interact with others and work as a team to solve problems, to describe knowledge and ideas in a manner that can be understood by other team members and to use an existing knowledge base to solve new problems in a creative manner (Schmidt, H., Hermans, H., Venekamp, R.  The Development of Diagnostic Competence: Comparison of a Problem-based, an Integrated, and a Conventional Medical Curriculum.  Academic Medicine 71(6):658-664, 1996.)
2.	Line 52:  What is the step by step manner to examine and answer these questions?  How does this aid in learning the subject content?
3.	Line 55:  there are many other important comparisons to the benefits and advantages of PBL over a traditional didactic teaching approach.  For example…” A study was performed to elucidate the goals of PBL programs in different institutions.  Goals common to all programs include the desire to: stimulate development of communication skills; emphasize concepts and interdisciplinary biomedical principles required for the understanding and management of health problems; provide for learning in context for the development and practice of reasoning skills that will be used throughout life; develop skills for management of information; foster active, self-directed learning, and cultivate the personal qualities required for a career in the health professions including a sensitivity to the needs of others and responsiveness to ethical and moral issues.  (Herron, M., Whitney, M., Weeks, B.  Preclinical Curricular Alternatives: Goals and Methods of Problem-Based Learning.  Journal of Veterinary Medical Education 20(1):14-18, 1993.”…and “The comparison of an innovative pedagogy in an institution and its former conventional program or a separate conventional didactic program seems like a natural one, assessing the merits of a new curriculum against those of the old format.  There is evidence that PBL students retain knowledge much longer than students taught conventionally.   In studies requiring integration of basic and clinical knowledge, problem-based students tend to do better in providing causal explanations of pathophysiologic processes underlying disease (Camp. G.  Problem-Based Learning: A Paradigm Shift or a Passing Fad?  Medical Education Online 1:2, 1996.)”
4.	Line 58:  Both PBL and CBL are described separately.  What are the similarities and what are the differences?  PBL is case based, but CBL is not necessarily problem based learning.
5.	Line 75:  What do you mean by social recognition?  Why does implant dentistry need to gain greater social recognition?  Do you mean that implant dentistry is gaining greater recognition as a legitimate dental specialty?
6.	What is the traditional teaching approach at this program?  A brief but detailed explanation is necessary to compare to PBL-CBL

c.	Figures: Table 1:  in the description of the table, briefly describe each group ie, the two groups (experimental group exposed to the PBL-CBL method; control group in the traditional didactic curriculum)
i.	In the top row, the Score of Theory test should be relabeled as Average Score of Theory Test.  Same for Score of case analysis
Experimental design: 2.	Experimental Design:
a.	Line 111:  What comprises a comprehensive analysis? What does this mean?
b.	Line 123:  how were group leaders determined?  What were their roles?
c.	Line 123:  What are some examples of how the group leaders summarized their discussions and presented summaries?
d.	Line 127:  how does this relate to learning?
e.	Line 127:  what was the other method of teaching and learning? Compare and contrast
f.	The experimental design is basic, but appropriate for comparing the experimental group and control group.
g.	Line 129: more explanation of the exam.  What do you mean by “it was made of the theory and case analysis”?
h.	Line 143:  summary of questionnaire, examples of questions and responses
i.	Line 147:  what roles were shifted of the teacher in the classroom as a result of participating in this method?  Where is the questionnaire for the teachers?
Validity of the findings: 3.	Validity of the Findings:
a.	Line 153: reference for dentists attending specialized training, or is this the authors’ opinion?
b.	Line 156: reference for this statement?
c.	Line 188: example of the developing and use of a clinical case? Examples of a main logical thread, key theoretical knowledge, examples of reasonable treatment plans and teacher summaries?
d.	Line 187: where did the teacher summarize the plans from different groups?  In a lecture format?  And why did the teacher do this?  What were the benefits of this?
e.	Line 189:  students were more likely to actively go about their clinical work with questions in mind:  what is the evidence of this?  How was this evaluated?
f.	Line 190: they were motivated to solve these questions in their clinical work instead of conducting clinical operations passively; they put theories into practice in a more flexible way…where is the evidence of this?  How was this evaluated?  Was it the teachers’ observations?  Students’ self-perceptions?  How could one tell the difference in clinical work between students that had gone through PBL-CBL and those that had gone through traditional teaching and learning methods?
g.	Conclusion: students exhibited increased level of theoretical knowledge and higher ability of case analysis:  the increased level of theoretical knowledge was exhibited by higher test scores, but how can you conclude that these students had a higher ability for case analysis?  There were no research questions on this topic, and no data to support it.
h.	In Table 2 the evaluation of PBL-CBL method by the experimental group, several of the items surveyed seem a bit ambiguous in exactly what was being asked and answered.  For example, the item “this approach enhances my ability to analyze and solve problems”…what methods were employed such that the students actually knows what this means?  Were the guided in ways to analyze and solve problems? By which criteria?
i.	Also in the table of items surveyed, students voted yes or no on “this approach helps me master theoretical knowledge”…the students probably should have been asked about ability to master theoretical knowledge compared to a traditional didactic curriculum, such as efficiency, ability to recall in the long term, etc.
j.	And, a Likert Scale would have been much better to use, rather than Yes/No
k.	In the discussion, limitations of the study need to be discussed.  Such as that one course in PBL-CBL may not be enough to fully have students realize the benefits of PBL.  Also, the lack of a Likert Scale.

l.	Conclusion:  what are the implications for this research, future directions, benefits, other applications, etc.  This conclusion is too brief.  How can you conclude that the students have a higher ability of case analysis?  How was this measured?  There is no evidence of this in your data.  The only research question that was answered was the comparison of theoretical knowledge between the two groups via examination.
Additional comments: 4.	Overall, while I also have the opinion that students learn theoretical knowledge better and have better long-term recall, have developed critical thinking and problem solving skills, do better in group learning situations and develop teamwork and communication skills, learn how to analyze and process cases better, in this paper there doesn’t seem to be the evidence and data to support anything other than that the experimental group performed better on one exam in theoretical knowledge than the control group.  While that in itself has much validity, any conclusions drawn from that are speculative.  This paper does however discuss applying PBL-CBL to a specific part of a curriculum in which it has not been done before, so there are benefits in discussing that in this paper.


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

Input:
Title: APPLICATION OF PROBLEM-BASED LEARNING AND CASE-BASED LEARNING INTEGRATED METHOD IN THE TEACHING OF MAXILLARY SINUS FLOOR AUGMENTATION IN IMPLANT DENTISTRY
Review round: 1
Reviewer: 3

Output:
Basic reporting: There are some minor improvements that can be made to the document to improve the language for the reader's understanding. Specifically, the sentence on lines 138 through 140 that begins with the word "besides' is ambiguous in meaning. Can you please clarify this sentence, and verify that "Besides" is the appropriate word usage?
Experimental design: In the Introduction, a clearer distinction between the CBL and PBL approaches to learning is need to identify how the two approaches differ and how they are similar. In addition, how are these two approaches integrated? Is it that both are utilized in their entirety, or is there some hybrid combination of the approaches that only uses certain aspects of each? Please clarify.

In the section on Teaching Methods, can you please provide data concerning the amount of time that the control group met in oder to add verification that the differences in learning outcome are not just due to the amount of time that was spent by each group interacting with the material?

In the Materials and Methods, can you please provide more information about the content and structure of the Theory test and the Case analysis so give the reader a clearer view of how your data on performance was collected?

Some additional discussion of the results from the questionnaire on line 142 is needed. Was the control group given a questionnaire as well to gauge their response to traditional teaching methods?
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: APPLICATION OF PROBLEM-BASED LEARNING AND CASE-BASED LEARNING INTEGRATED METHOD IN THE TEACHING OF MAXILLARY SINUS FLOOR AUGMENTATION IN IMPLANT DENTISTRY
Review round: 2
Reviewer: 1

Output:
Basic reporting: There are several minor edits to English grammar and phrasing needed throughout; this would help with overall flow and clarity (for example, check the tense used throughout the paper - within sections or paragraphs this should be consistent). Some citations are not in English - be sure that all citations are translated.
Line 81 in the introduction "problem-based students" should probably be changed to "students in problem-based learning classrooms/activities"... something similar.

Line 90 - the description of CBL is a little confusing to me. CBL does not necessarily require that students simulate doctors' sessions with patients.

The last paragraph of the introduction (on Implant Dentistry) seems out of place or it seems that there is a clunky transition to this paragraph. Consider adding subheadings or sections to the introduction to help with the flow of the narrative.
Line 118 - it would be helpful to include a sentence explaining why maxillary sinus augmentation is particularly difficult to teach (compared to other procedures) - does it require greater background knowledge than other procedures? is it just a more complicated procedure?  are there novel technical skills that must be mastered?
Experimental design: Line 129 - what do you mean by "there were no differences in learning experiences"?  It's highly unlikely that all participants have identical learning experiences unless they all attended the same institutions throughout their entire educational career- surely there are some differences. it might be better to state how they are similar - for example, all participants hold a DDS or DMD degree or have participated in a residency program, etc.

Line 138 - What is "matters that warrant special attention"?  is this the name of a course?
I have a much better idea of the differences between the control and experimental groups but I'm still confused about some aspects of the study design:
lines 142-144 - you state that the amount of time spent by each group in training is equivalent. do you mean 40 min/session or do you mean the number of sessions (or both).  how many total hours of instruction time/training team did each group get? it is not clear from the current text whether this is truly equivalent or not.

it is still not clear to me which aspects of the activity draw from PBL and which draw from CBL (in fact, it seems to most closely align with CBL) - the authors should call these out specifically since they claim that the training method is an integration of both methodologies.  Instructors at other institutions who may wish to replicate such a training process should be able to understand exactly how this activity integrates PBL and CBL.

the results section indicates that the final exams consisted of short answer questions - how were these graded in order to ensure consistency in grading and how were points determined? is there a rubric that was used?

for the case analysis - was this a novel case or one that the students saw previously? again - how were students graded? is there a rubric?

line 196-198 - I'm not sure what you mean by "the difference of theory test (Degrees of freedom: 91, F=38.432, p=0.001<0.05) and case analysis (Degrees of freedom: 91, F=45.443, p=0.001<0.05) was statistically significant."

Lines 217-224 need to be rewritten for better flow and clarity. I would include a subheading for "limitations of the study"
Validity of the findings: line 189-192 - was a t-test or ANOVA used?  why were both tests used and which variables were subjected to each statistical test? a more robust description of the statistical analysis is required.

again - it is never explained in the manuscript how this approach integrates PBL and CBL.  the authors should specifically call out which characteristics of PBL are reflected in their approach and which characteristics of CBL are reflected in their approach. this could be done easily with a chart or table.  This is essential as one of their main points in the discussion is that this is "this study was the first to apply a teaching method integrating both PBL and CBL in a specific curriculum..."

Conclusions can also include a statement about how this might be relevant for other educators or implemented at other institutions.
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: APPLICATION OF PROBLEM-BASED LEARNING AND CASE-BASED LEARNING INTEGRATED METHOD IN THE TEACHING OF MAXILLARY SINUS FLOOR AUGMENTATION IN IMPLANT DENTISTRY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: i have reviewed the changes made to this manuscript based on my suggestions from my first review.  I am satisfied with the improvements made by the authors.


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: APPLICATION OF PROBLEM-BASED LEARNING AND CASE-BASED LEARNING INTEGRATED METHOD IN THE TEACHING OF MAXILLARY SINUS FLOOR AUGMENTATION IN IMPLANT DENTISTRY
Review round: 2
Reviewer: 3

Output:
Basic reporting: 1)	Discussion Notes (Section 4):
a.	Lines 220-224 with the sentence beginning “Besides…” does not read smoothly and should be reworked to be clearer in their intent to convey information
b.	Line 227 beginning with “More…” Could be better stated as “Further research is needed to confirm this”

2)	Materials and Methods Notes (Section 2)
a.	Line 129 “There were no differences in learning experience…” Do you mean that there was no difference in the educational background between the control and the experimental groups?
b.	Line 139 In place of “Totally”, I believe you mean “In total”
c.	Line 139 Do you mean to say that every session lasted 40 minutes?
d.	Line 140 “Beyond that…” Does “that” refer to the class sessions?
e.	Line 143 Do you mean to say that every session lasted 40 minutes?
f.	Line 144 The sentence “In addition…” could just be changed to “The experimental group did not attend any lectures”

3)	Introduction Notes (Section 1)
a.	Line 81 The citation is not in English
b.	Line 87 The sentence beginning “On the other hand…” is unclear in its meaning and should be reworded
c.	Line 117 Why is teaching maxillary sinus floor augmentation particularly challenging? Please include an explanation in the text.
Experimental design: 2)	Materials and Methods Notes (Section 2)Lines
a. 146-168 In your description of the experimental group, it would be helpful to explicitly state which portions of their class design is taken from PBL and which is derived from CBL
b.	Lines 147-154 and 176-181 are identical, and it appears that the first describes the discussion design and that the second describes the clinical exam that the students were given
i.	Was the clinical exam written or oral?
ii.	Are the two exactly the same? Is the case the same that had already been discussed in the class, or was the exam a novel clinical scenario?
iii.	On line 175, it would be helpful to add “exam” to the subject heading for clarification
Validity of the findings: The discussion still lacks an analysis of the results within the context of existing literature on the topic.
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: APPLICATION OF PROBLEM-BASED LEARNING AND CASE-BASED LEARNING INTEGRATED METHOD IN THE TEACHING OF MAXILLARY SINUS FLOOR AUGMENTATION IN IMPLANT DENTISTRY
Review round: 3
Reviewer: 1

Output:
Basic reporting: Please use consistent tense throughout the manuscript – currently needs more editing for grammar and English language.

Be consistent with use of either PBL-CBL or CBL-PBL throughout the manuscript. Both are used in various places – pick one and stick with it.

The discussion of MSFA and the particular challenges of teaching this topic is much improved.

The discussion of PBL and CBL, their similarities and differences is much improved.

The last paragraph of the Introduction (right before Materials and Methods) needs to be rewritten for better English grammar and wording.

Throughout the manuscript you mention that you are evaluating the “efficiency” of this teaching method.  Do you mean “efficacy” instead of “efficiency”?   if you mean efficiency is an outcome, you should have some measurement of time on task or time for completion.

ABSTRACT: There is not a real hypothesis in the abstract.  This may be okay as the research may be exploratory, in nature, however surely the authors went into the study with a hypothesis about the method – it would be good to include it. Also – in the results section of the abstract you don’t indicate what you were testing with the theory test and case analysis.  Was this all satisfaction with the method?  In the conclusion for the abstract, as well – the application of the methods shows positive results for what? Learner satisfaction? For improved learning?  Be clear about what the findings mean for the reader. The abstract needs to give a reader a condensed version of the study so that they can quickly determine what you did and why you did it.  Please revise the abstract to include the essential details of the study.

I see that in the Materials and methods section you attempt to indicate which elements are drawn from PBL and CBL by using (CBL) and (PBL) but you do not indicate in the manuscript that this what you are doing.  You could provide general statements about the ways in which your method draws from each of these. If you want to identify the specific parts of each method and label them as (CBL) or (PBL) you need to indicate this in some sort of introductory sentence or two before you do it, otherwise readers will not know what the parenthetical abbreviations are supposed to indicate. My personal preference would be to simply indicate all this in a chart or diagram but if you do it in text, be sure you clearly explain what you are doing to the reader.

Description of statistical analysis needs to be rewritten for English language and grammar.
Experimental design: Did the traditional group not complete a questionnaire soliciting their opinions on that approach? If not – why not?  I’m not sure the questionnaire results are meaningful if you only have satisfaction scores from the experimental group.  It’s possible the traditional group would also have revealed a high satisfaction rate, too. You also make the claim in your discussion that PBL-CBL method gained popularity among the students but if you only allowed the experimental group to express their thoughts about teaching and learning methods you haven’t actually captured the perceptions of the rest of the class.

This sentence before the Discussion section is inaccurate:  The participants in the experimental group present a generally better understanding of the background knowledge and the key points when performing MSFA.  Unless the learners were observed performing MSFA and there are data on observed performance, you don’t know that this is true.  Your results may show that the experimental group performed better on exam questions about MSFA but you don’t have data on how this translates to actually performing the procedure.

also – in the discussion, the data may support your hypothesis that this method was more effective but one study doesn’t prove that.  Be careful with your wording.

Also- I still don’t understand why teaching efficiency is your outcome.  Are you suggesting that the method lets you reduce the number of teaching hours for the topic so that it is more efficient?  Or that learners learn to perform the procedure more quickly so it is more efficient?  “efficient” implies that there is a more rapid rate of productivity.  If you mean “effective” of “efficacious” than please change this throughout the manuscript.

Is there a rubric for grading the case analysis? If so – please include it.  It would also be helpful to see some of the questions that are included in the case analysis.
Validity of the findings: This sentence in the methods: “These comments were largely encouraging so as to help the students develop a scientific way of thinking.”  This is an assumption unless you cite literature to support that providing encouraging words helps promote scientific thought.  Please provide citations.

The discussion section still has many paragraphs that would be more appropriate in the introduction section.  The discussion should simply contextualize your results in the existing literature.  What do your findings contribute to this body of work?  How do your results support or refute what is already known?  What is significant about what you found?

I appreciate that a section on limitations is added but you should include some text describing why the limitations you mention are limitations. Currently, it feels like it was tagged on at the end without consideration for how readers might be able to take these points as suggestions for future work.

Conclusion – again, as in the abstract, the application showed positive results – for what?  For student satisfaction? For retention of knowledge? For application of knowledge? Your hypothesis is not clear.
Additional comments: All comments are indicated in the areas, 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: APPLICATION OF PROBLEM-BASED LEARNING AND CASE-BASED LEARNING INTEGRATED METHOD IN THE TEACHING OF MAXILLARY SINUS FLOOR AUGMENTATION IN IMPLANT DENTISTRY
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors have done well in addressing many of the issues that were pointed out in the previous reviews. The justification for focusing on Maxillary Sinus Floor Augmentation is included. Also, the explanation of CBL versus PBL, and the way in which they were combined for this study, provides much more clarity for their experimental design.  However, some issues remain and are listed below.
1.	“Traditional” teaching approach is mentioned throughout the paper (Lines 23, 99, 101, 174, 186, 196) but is not clarified until line 196 as “teacher-centered”. A clarification/explanation of “traditional” teaching approaches needs to be introduced at the onset of the paper.
2.	Lines 43-49 discuss different anatomical factors involved in MSFA and the prevalence of two of the factors, but not the third (maxillary sinus walls). Also, the name of one of the anatomical factors changes from one line to the next (maxillary septum to septa).
3.	The grammatical wording in the following lines contain misused words, spelling errors, incorrect tenses or awkward phrasing: Lines 35, 49, 58,69, 71, 83, 84, 122, 152, 156, 157, 167, 185, 228
Experimental design: 4.     Line 75 asserts “Such problems are best rooted in cases”. What is the basis of this assertion? Are their citations to back this claim up?
5.	Line 106 states that the participants in the control group had “no discussion in or beyond each session” suggesting that the students had no interaction outside of class. I believe that the authors mean that there was no scheduled discussion time inside or outside of class, but I feel that this sentence asserts something different.
6.	Who were the people involved in grading the exams? Were the exams graded blindly, or were the graders aware of which group was being graded?
Validity of the findings: 7.         Line 184 states that that this study was the first to apply a teaching method integrating PBL and CBL in a specific curriculum. This is inaccurate as other disciplines have combined both methodologies for teaching. For example, the citation below which is not included in the review article references.

Dong, Junhong & Zeng, Ping. (2017). The Application of CBL Teaching Combined with PBL Teaching Method in Biochemistry Experiment Teaching. 10.2991/meici-17.2017.126.

8.  The discussion of study limitations beginning Line 225 to 228 reads like a bullet list of things that could have been done differently in the study, and should be expanded to involve an actual discussion of the study limitations and the ramifications therein.
9.	Along with the above citation, it appears that the references are not as exhaustive of the available literature on PBL and CBL used in dental education as they could be. The following citation is an example of a pertinent publication that may further inform the current study.

An Overview of Case-Based and Problem-Based Learning Methodologies for Dental Education Nader A. Nadershahi, Daniel J. Bender, Lynn Beck, Cindy Lyon, Alexander Blaseio
Journal of Dental Education Oct 2013, 77 (10) 1300-1305
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: APPLICATION OF PROBLEM-BASED LEARNING AND CASE-BASED LEARNING INTEGRATED METHOD IN THE TEACHING OF MAXILLARY SINUS FLOOR AUGMENTATION IN IMPLANT DENTISTRY
Review round: 4
Reviewer: 1

Output:
Basic reporting: Manuscript still needs editing for English language as there still remain some typos and grammatical errors.
Experimental design: Please include the results of the questionnaire for the control group. Currently, you only indicate satisfaction for the experimental group but do not provide the data for the control group even though both groups were provided with the questionnaire.  A table would be appropriate so that readers could compare differences between experimental and control group.  If no statistical analyses were conducted on these data please explain why.  If you do not have data to report for both groups please omit the discussion and results of the questionnaire for the experimental group.
Validity of the findings: see notes on design.
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: APPLICATION OF PROBLEM-BASED LEARNING AND CASE-BASED LEARNING INTEGRATED METHOD IN THE TEACHING OF MAXILLARY SINUS FLOOR AUGMENTATION IN IMPLANT DENTISTRY
Review round: 4
Reviewer: 2

Output:
Basic reporting: This article has greatly improved, and the authors have made most of the necessary changes to present their research clearly and concisely. However, some awkward sentence construction and word usage remain,  and a few additions to the text are still needed for clarity. I am attaching an annotated pdf that contains my comments and suggestions.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSIS REVEALED GENES INVOLVED IN THE FRUITING BODY DEVELOPMENT OF OPHIOCORDYCEPS SINENSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: The transcriptome sequencing and analysis at different developmental stages of the same sample are very important for studying the dynamic development of Ophiocordyceps sinensis. On the basis of transcriptome, some genes related to the development of fruiting bodies were analyzed, which is of guiding significance for future research.
Additional comments: 1, As the authors provide, since “Reads of CM, DF and FB were aligned to the O. sinensis CO18 genome using HISAT2”, the authors need to provide matching rate of RNA-seq data on genome, which is helpful for evaluating the quality of sequencing data.

2, Line 90-96, According to the author, RNA data were first compared to the reference genome, and then assembled by stringTie software, resulting in 7518 protein coding genes and 233 putative novel genes. But then, why were 11,497 genes used for comparative analysis with literature data? These data did not match the Table S1. The best way to evaluate the integrity of transcriptome data was to use conservative gene database comparisons, such as BUSCO, which also needed in transcriptome analysis.
And then, now that the author had made a new assembly and produced some new gene, the author should submit the new assembly results to a public database.

3, Line 100-101, Line 166-168 had been described in the materials and methods and can be deleted.

4, In the “2.3. Clustering of gene expression profiles across three growth stages”, The authors should analyze the different gene expression patterns with the phenotypes from hyphae to fruiting body development. Different developmental stages have different physiological functions. Theoretically speaking, such as, the mycelial stage is dominated by vegetative growth and basic physiological metabolism, while the fruiting body stage will have more new protein synthesis, energy consumption, secondary metabolites, morphogenesis, sexual development and so on.

5, Since the reliability of RNA sequencing data were evaluated by qPCR, the results of qPCR and RNA-seq (TPM) should be displayed simultaneously on two coordinate axes to replace Fig5.

6, Line 355-360, What is the difference between DEGs and DETs? In the manuscript, the analysis description of DETs is not presented.

The English quality of the manuscript is not well, especially in the tense problems of different chapters. Usually, the general description of the material method and the result discussion section needs to use the general past tense. It is suggested that the author look for the native English colleagues to polish the manuscript.
What follows is some obvious inappropriateness:
Line 38, “spp.” need not be italicized.
Line 56, “Neurospora. crassa, Aspergillus. Nidulans,” should be “Neurospora crassa, Aspergillus nidulans,”.
Line 66-68 should be given corresponding references.
Line 74, “reveals” should be “revealed”
Line 104 “There are 19” should be “There were 19”
Line 135 “There are seven” should be “There were seven”
Line 278 “2.8. Validation oftTranscriptome” should be “2.8. Validation of transcriptome”
Line 345-346, Is reference 45 referred correctly?


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

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSIS REVEALED GENES INVOLVED IN THE FRUITING BODY DEVELOPMENT OF OPHIOCORDYCEPS SINENSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: See my recommendations to authors.
Experimental design: See my recommendations to authors.
Validity of the findings: See my recommendations to authors.
Additional comments: Tong et al generated and analyzed RNA-Seq data for Ophiocordyceps sinensis, a medicinal ascomycete with poorly known development. The data generated are useful and will certainly be of interest to people working on fruiting body development in general or on Ophiocordyceps. However, the paper needs a very thorough revision. I’m offering general and more specific suggestions below.

General comments
- Please present read mapping statistics - what % of reads mapped to the reference genome? Also, please clarify if the genome strain or a different strain was used for RNA-Seq. I suspect the latter, in which case, read mapping % could be low due to the divergence of the used strain from the reference genome’s strain.

- I have reservations about the appropriateness of ASProfile method for fungi. This was developed for gene-poor animal genomes and may not perform well in fungi. Fungi need dedicated (e.g. region-restricted probabilistic modeling) pipelines for splicing reconstructions. The authors should examine whether the surprising results they got might have been caused by this.

- The discussion aspect of the paper could be improved. The authors make some interesting observations, but these are hardly placed in a wider or even Ascomycota-specific context. They could take advantage of the close relationship of Ophiocordyceps to Sordaria, Neurospora and Podospora - species with abundant information on fruiting body development.

- The co-expression analyses are neither described in the method nor seem to be correctly done. It seems the analysis had the mature fruiting body stage before the younger stage in the time series (although I should say I find the nomenclature of developmental stages confusing, so I might be wrong here).

- The english should be improved. This is too often said, but the quality of the english sometimes interferes with understanding.

- The conclusions section is hardly a conclusion, rather, its merely a bullet-point listing of main findings. In its current form it does not add any context or synthesis. This is true to the paper in general - it lacks depth, short of exciting discussions or informed speculations.

Specific comments:
l38 - protruding of —> development of
l40 - cleisthothecium —> perithecium
l43 - TCM contrasts with ‘some asian countries’
l49 - ‘favorable’
l49 - costs —> takes
l52 - replace ease by ‘reduce’
l58 - maybe worth mentioning that fruiting body development in Cordyceps species has also been studied extensively, and that fruiting bodies represent a state of complex multicellularity that evolved by unique mechanisms in fungi (see recent reviews).
l66-70 - a better summary of the findings from previous fruiting body transcriptomes would be useful. Why is RNA-Seq from the same stages  as before warranted?
l85 - just ‘during development, …’
l87 - delete ‘were submitted…’ because its redundant
l88 - please supply a table that shows read mapping statistics for each sample (# of reads, # of reads after QC, % of mapped reads). % of mapped reads is particularly crucial in this case, if RNA-Seq was not done on the strain for which the genome was sequenced.
l95 - where do the 11497 genes come from? Above you state 7518 genes were detected.
l98 - ‘genes’
l101 - no need to repeat the definition of DEGs here - this is stated in the methods.
l107 - ‘ribosomal structure protein’ is an imprecise term, please specify what this gene exactly is.
l109 - express —> expression …..upregulated —> higher
l111 - no need to state the year of the study.
l112 - this conclusion is too general. there are >200 cyp450s in fungal genomes, it is unlikely that all of them are associated with vegetative growth. Please remove.
l114 - why p<0.005 here? It was p<0.001 above.
l118 - Figure 2: label for the horizontal axis missing. Given the lack of this information, my guess is that the figure shows the counts of genes with the given GO term. This is not enrichment per se, just the counts, so if this is the case, then the figure is not too informative. Consider plotting p-values instead.
l124 - if I understand right, the GO terms mentioned on lines120-123 belonged to genes upregulated in fruiting bodies vs mycelium. If so, why do you conclude these processes are more active in the vegetative mycelium? The opposite would make sense to me. Carbohydrate metabolism could also refer to cell wall modofication and/or synthesys processes.
l130 - a major question for me in all co-expression analyses is why DF follows FB? FB is the mature fruiting body, whereas DF is the developing fruiting body (confusingly called ‘developed’ throughout, would be better to call it young fruiting body).
l135 - no mention of gene co-expression in the methods, so the reader doesnt know how these co-expression modules were inferred. Please specify.
l143 - sentence sounds like an enrichment analysis is in the background, but no test is specified. Please state what test the p-value is coming from.
l146 - delete ‘patterns’ (expression patterns can’t increase)
l152 - starch is also a reserve material for many species, which gets mobilized upon fruiting body growth. E.g. there is extensive literature on starch metabolism in the fruiting bodies of Agaricus bisporus and Coprinopsis. This should be discussed. Also worth discussion is the upregulation of ribosomal constituents - this could suggest increased demand for proteins associated with fruiting body growth.
l156 - this is a stretch and not supported by the data. Please remove.
l159 what is ‘sexual maturation’? Please rephrase.
l162 - cluster7 is the most interesting to me - constantly higher expression in both fruiting body stages than in mycelium. I think the authors could devote more attention to this cluster. E.g. what previously described fruiting body genes are in this cluster? Any Neurospora/Podospora orthologs in this cluster that are involved in sexual development?
l166 - this might not be true for fungi, where the dominance of intron retention events suggests posttranscriptional regulation via the nonsense mediated decay pathway.
l169-174 - the contradiction between alternative splice sites vs intron retention being the most numerous AS event vs most abundant type of affected gene bothers me. I don’t have an explanation for this, but the results is suspiciously surprising. Intron retention is unversally the most common AS event in fungi. I’m wondering if this result was caused by the software used, which is developed for gene-poor animal systems and not for fungi.
l178 - specify the test used for calculating p-value
l226 - ‘anserina’ is consitently misspelled as ‘anserine’
l229 - ‘reveal’ is an overstatement here. Please rephrase.
l238 - ‘homology’ —> homolog
l242 - cite those few studies
l255 - rephrase sentence (yeast ste11 can’t regulate conidiophore development)
l268 - what is relatively? If expression difference wasn’t significant please state so.
l288 - the conclusions section is a mere listing of main findings, there is little discussion or synthesis here.

l307 - please define developmental stages. What is considered developing and mature fruiting body?
l311 - ‘ascus forming’ —> without asci. Also, if you refer to young fruiting bodies, then ‘developing fruiting body’ as the name of the stage would be better.
l312 - perithecium —> perithecia
l314 - please state how many biological replicates were analyzed.
l338 - these perl-scripte might need to be provided as supplemental material by journal policy.
l341-347 - this entire paragraph is not clear. What is the aim here?
l359 - ‘statistical enrichment’ makes little sense here. please provide details on how hierarchical clustering was done.
l377 - ‘was —> ‘were’


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

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSIS REVEALED GENES INVOLVED IN THE FRUITING BODY DEVELOPMENT OF OPHIOCORDYCEPS SINENSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: Solid.
Additional comments: The manuscript by Tong et al. investigated transcriptomic dynamic changes in Ophiocordyceps sinensis during development of fruiting body formation at three stages. By comparing the expression and splicing profile among different tissues, authors identified a set of genes responsive to the transition of different development stages and validated their expression pattern. The experimental design was solid and the conclusions are well supported by the sequencing data. While their data are of apparent interest to the community, I do have some concerns regarding the results presentation and materials and methods description.
(1)	The title of section 2.1 (line 84) is the same as section 2.2 (line 99). May consider to make changes to the title of section 2.1.
(2)	After re-assembly of transcriptome data, authors identified 7518 protein coding genes and 233 putative novel genes (line 90), however, the number is 11497 in line 96. Which number is correct?
(3)	The software used for the identification of differentially expressed genes (DEGs), should be given. Are they DESeq, EdgeR, EBseq or other softwares? I could find the information any section of the ms.
(4)	Statement is improper in line 122. Because asexual mycelium (CM) is the initial stage, may change it to “percentage of down-regulated genes were higher than up-regulated genes”. Please also correct corresponding statements in other places (e.g. line 108, line 274, line 285).
(5)	The developmental order is from CM to DF to FB. In line 137, the statement “transition from CM to FB” is improper. Similarly, figure 3B is wrong in development order. Please check the data carefully and make changes accordingly to results of this part (line 135-162).
(6)	Statement is not clear in line 173-174. Please define what AS gene is. What does “different” mean here? Does it mean different splicing gene (DSG)? Moreover, how many different alternative splicing (DAS) events related to 937, 946 and 652 DSGs? In figure 4B, the number of IR-related DSG (figure 4B) is similar to corresponding AS event number (figure 4A). Does this mean nearly all AS events are differential spliced? Please recheck the data.
(7)	Section 4.2 is ambiguous. Please re-order the analysis steps. For example, lines 343-347 should be before line 339, lines 352-353 should be after line 355, and so on.
(8)	Lines 341-343. Please explain why this analysis was done? The results section did not mention this part.
(9)	Lines 355-369. The results section do not contain the content related to differentially expressed transcripts (DETs). Please remove it.
(10)	 Line 369-370. Hierarchical clustering is used for classifying samples.
(11)	 Line 371-372. What does “Q value” mean? Which method was used? The method of this part is not clear. Also, if differential splicing analysis was done here, please add the corresponding content.
(12)	 Line 373. What’s the difference between TopGO and the previous method used for GO enrichment analysis? Please use standard method.

Minor:
(1)	Lines 55-56. “O.sinensis like fungi” to “Ascomycetes fungi”.
(2)	Line 81. change “aids” to a suitable word.
(3)	Line 85. “duplicate samples” to “samples with two biological replicates”.
(4)	Line 88-89. “more than 63 million paired-end, reads per replicate, were generated with Illumina HiSeq” to “more than 63 million paired-end sequence data from each replicate were generated with Illumina HiSeq 2000 platform”.
(5)	Line 89-90. Please cite the original one paper of HISAT and Stringtie.
(6)	Line 107. Remove “, p < 0.001” and do it in other places.
(7)	Line 115. Please confirm your criteria of DEG and enrichment analysis: p-value or FDR? 0.01 or 0.001 or 0.005? They variated in different places (including supplementary tables).
(8)	Line 124. “during” to “in”?
(9)	 Line 131-132. Which figure or table supports your result?
(10)	 Line 133-134. Only abbreviation of development stage is enough.
(11)	 Line 176. “AS genes” to “DSG”.
(12)	 Line 214-215. Remove “, FDR < 0.001” and do it in other places.
(13)	 Line 248. “MAPK ” to “MAPK”.
(14)	 Line 253. Please give exact gene number.
(15)	 Line 269. “Transcript Factors” to “transcript factors”
(16)	 Line 280. “FB and CM stages” to “three development stages”.
(17)	 Line 296. “that” to “than”.
(18)	 Line 303. “enriches” to “supports”.
(19)	 Line 340. “scaffold” to “transcripts”.


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

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSIS REVEALED GENES INVOLVED IN THE FRUITING BODY DEVELOPMENT OF OPHIOCORDYCEPS SINENSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Please reconfirm the data of Table_1_Mapping_results_of_O._sinensis_transcriptomes and provide detailed and valuable information as much as possible.
One of the two data (Raw Reads and Clean Reads in Table_1.....) seems to be mistake.


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

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSIS REVEALED GENES INVOLVED IN THE FRUITING BODY DEVELOPMENT OF OPHIOCORDYCEPS SINENSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Ok.
Experimental design: Ok.
Validity of the findings: Ok.
Additional comments: In general, I supportpublication of thsi manuscript. However, authors only addressed some of the issues I raised for hte previous version. I think these thould be done. I have the following additonal comemnts:
(1)	Importantly, authors did not do as I suggested previously reorganizing the order of comparisons among three stages (CM-DF-FB). The order should be: DF vs.CM, then FB vs. DF and the last FB vs.CM in the main text. Please also re-organize the figures based on the right order (such as Figure 1A and 1B, Figure 2, Figure 4 and Table 1).

(2)	Please add the method of clustering analysis of gene expression in Materials and Methods section.

(3)	Line 151-152. The definition of A5SS and A3SS are wrong. So are in line 430-431.

(4)	Statement is still improper in line 202-203. Maybe it could be that “103 and 58 genes were up-regulated and down-regulated, respectively, in FB” because DF is a former stage and FB is a latter stage. Please also correct corresponding statements in all other places (e.g. line 214-215, line 220-221 and so on).

(5)	Line 236, “transcripts” or “genes”? (Also, in line 238, 242, 244 and etc.).

(6)	Line 271. Please give the full name of different AS events (exon skip, ES; intron retention, IR; alternative donor, A5SS and alternative acceptor A3SS and so on).

(7)	In Figure 4, the x-lab of A doesn’t match to that of B and C. Please give the full information of different types of AS events. Or only give the number of AS events among A3SS, A5SS, IR and ES in Figure 4A. Meanwhile, please correct corresponding content in main text (including Materials and Methods and Results).

(8)	Legend of Figure 5. FDR < 0.05 or 0.001?

(9) Theerare new papers on the topic, and these shoudl be referred. For exmaple, Wen et al. (2019). Transcriptome changes during major developmental transitions accompanied with little alteration of DNA methylome in two Pleurotus species. Genes 10 : art no. 465


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

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSIS REVEALED GENES INVOLVED IN THE FRUITING BODY DEVELOPMENT OF OPHIOCORDYCEPS SINENSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: Sound.
Experimental design: Sound.
Validity of the findings: Sound.
Additional comments: After a careful check, I consider authors have properly addressed all concerns that I have raised for the precious 2 versions. I have no additonal comments, and would like to endorse its acceptance for publication by the Peer Journal.


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: GENETIC ARCHITECTURE STUDY OF RHEUMATOID ARTHRITIS AND JUVENILE IDIOPATHIC ARTHRITIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: To date, the largest study in the genetics of RA (Okada et al., Nature, 2014) has identified 101 loci associated to the disease. However, the authors report 789 loci. In the case of JIA, around 30 loci have been associated to the disease at the genome-wide significance level, but the authors report 129 loci. What is the origin of these discordant observations? This is an important point to clarify.
Experimental design: no comment
Validity of the findings: No comment
Additional comments: Jia et al. have performed a comparative analysis at gene- and pathway-levels between two related autoimmune conditions, rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). They have used recently developed methods for the study of complex traits. While applying the new methodologies is interesting, the work has some concerns that have to be addressed:

1.	The authors performed a gene-based association analysis using fastBAT method and identified a huge numbers of genes -431- associated in the RA dataset. However, it seems that the authors do not pay special attention to these results: are the 431 genes identified already known genes for RA? Are there new genes for RA between them?

2. Some of the descriptions are presented ambiguously: in the abstract, the authors stated that ´we conducted analyses at the SNP, gene and pathway levels´; however, they actually did not perform the SNP-level analysis. This is an important issue that should be avoided through all the manuscript.

3. The last sentence of Introduction section says that the ‘results suggest that some genetic variants can significantly increase the susceptibility of both diseases’; however, the present work does not formally assess (through a statistical test) this question. The work is limited to a comparison between SNPs and genes for RA and JIA. Therefore, the author should be more rigorous when reporting the conclusions from their actual approach and results.

Minor point: there are some typos on lines 164-165, 192-193.


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: GENETIC ARCHITECTURE STUDY OF RHEUMATOID ARTHRITIS AND JUVENILE IDIOPATHIC ARTHRITIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: It is an interesting article that needs some more clinical and biological concepts, in order to make it more practical.
Experimental design: The experimental design is clear, but I suggested the authors to retrieve some more data if possible.
Validity of the findings: no comment
Additional comments: The authors addressed several issues I had previously raised, but the manuscript still needs significant changes, although it has been improved by the authors. Moreover, the inclusion of two additional cases increased the scientific value of this paper.

INTRODUCTION
- in the first two paragraphs, although the JIA definition is appropriate, it’s important to provide the reader (who may not be an experienced pediatric rheumatologist) with some additional information about JIA, especially as regards the classification in 7 forms according to EULAR classification, which is quite different from AR or, more in general, chronic arthritis in adults.

-some concepts about genetic predisposition to JIA and AR are completely lacking in the introduction, especially as regards HLA association. I recommend the authors to fill this gap by taking advantage of some specific references that consider and discuss this aspect under the double point of view of JIA and AR (HLA-DRB1 alleles and juvenile idiopathic arthritis: Diagnostic clues emerging from a meta-analysis. Autoimmun Rev. 2017 Dec;16(12):1230-1236. doi: 10.1016/j.autrev.2017.10.007; & Genetics and the Classification of Arthritis in Adults and Children. Arthritis Rheumatol. 2018 Jan;70(1):7-17. doi: 10.1002/art.40350)

MATERIALS and METHODS/RESULTS

-did the authors perform their research taking in account also the different subtypes of chronic arthritis, especially as regards JIA (with its different subtypes)? Indeed, systemic forms are increasingly considered autoinflammatory syndromes (as well as AOSD for AR) and some specific forms (e.g. psoriatic) are considered separately from AR in adults. If they have not done that, is it possible to retrieve this data as well? If so, how are the results? Anyway, this is a point that deserve some discussion whatever is the answer or results, as emphasized also by the references recommended above.

DISCUSSION
-in addition to address the observation right above, I would suggest the authors to provide some immunological concepts about their main observation (“We observed significant enrichment of NO2-dependent IL12 pathway and IL27 pathway for both RA and JIA”). It would be important to emphasize the potential biological (and practical) significance of their research.
-please, separate discussion and conclusion.


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

Input:
Title: GENETIC ARCHITECTURE STUDY OF RHEUMATOID ARTHRITIS AND JUVENILE IDIOPATHIC ARTHRITIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: In response to:

'Response: We agree with the reviewer and have revised our manuscript accordingly.
The revised introduction section: “Our results suggest that some genetic variants are associated with both diseases and their unique genetic risk loci may be related to their distinct clinical features .” ' -->

Again, they are not doing any SNP association test. They did not discover any new associated variant to both diseases. Please limit the conclusions to the actual results of the manuscript.
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: GENETIC ARCHITECTURE STUDY OF RHEUMATOID ARTHRITIS AND JUVENILE IDIOPATHIC ARTHRITIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is well-written: there are a few minor grammar inconsistencies that the authors can easily fix by proof correction. Tables, figures and references are appropriate.
Experimental design: Experimental design and aims are clearly explained.
Validity of the findings: Both the discussion and conclusion improved with authors' revision. I have no additional major concerns.
Additional comments: The authors addressed appropriately my main observations.


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: COMPARISON OF BACTERIAL DIVERSITY AND ABUNDANCE BETWEEN SEXES OF LEPTOCYBE INVASA FISHER & LA SALLE (HYMENOPTERA: EULOPHIDAE) FROM CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	The article uses clear and unambiguous text and is conform to professional standards, but sometimes the sentences resulted to be very short and as a list of examples and respective references.
•	The state of art, although brief, is enough complete.
Experimental design: •	The experimental design resulted incomplete about the following several aspects:
•	Although authors known the existence of different populations of L. invasa (authors cited Nugnes et al, 2015 – where the two putative species were infected by different Rickettsia bacteria) they do not characterize the studied population. This aspect could be fundamental to define the insect-symbiont system. Furthermore, Nugnes et al, 2015 speculated about the rickettsia involvement in the manipulation of the sex, but authors did not give any information about the sex-ration of their studied population.
•	-In parallel, I think it’s crucial for the completeness of the manuscript that authors compare their studied population and other populations with different sex ratio (population where no males were recorded, from Italy, Tunisia, Brasil). Adding other samples with different sex-ratio could significantly make the manuscript more complete.
•	In m&m paragraph, references are often not complete: in lines 118-119 there are no mention about the reference of the used primers as in lines 140-149 about used softwares.
•	In results paragraph a phylogenetic tree was reported but in m&m paragraph there are no mentions about the methodology used to obtain it.
Validity of the findings: •	Results paragraph should be shorter, often the same results are in the text and in the tables. I would suggest summarize the paragraph avoiding the lists of the found bacteria (order, families etc.) that are already reported in the tables, but to focus on the main bacteria.
•	Taking in account that the main bacteria found in females were Rickettsia, authors should have evaluated the phylogenetic placement and the genetic diversity of these bacteria since other studies were previously carried out (Nugnes et al.,2015)
•	The discussion paragraph tends to generalize too much the results found in the work. Often the authors referred to host-symbiont systems involving all the insect orders, I would suggest to be restricted to Hymenoptera order, and to refer to other orders in case no example about wasps exists.
•	In the discussion paragraph, there are sometimes statements non supported by tests or reference. For example in lines 265-266: how do the authors assert the effect of bacteria in L. invasa? this statement should be supported by tests carried out with infected and uninfected strains of L. invasa.
-	In lines 316-317 authors declare: “we cannot rule out the possibility that male-Rickettsia is obtained through horizontal transmission in some way”. Authors should take in account that these males represent the offspring of an infected female, hence a low titre of bacteria in males could be present because bacteria could have been transmitted from the mother itself, but the density was too low that had no effects on sex determination. Therefore, the density of the bacteria could play a crucial role in the determination of the sex, as in Giorgini et al.,2010, where Pnigalio putative males eggs obtained from cured females showed a low bacterial density, but the presence of Rickettsia was not excluded. The environimental factors could influence the density of the bacteria and hence decrease or increase their effect on the host.
Hence I would suggest to take in account the following papers:
-	Jaenike J. Spontaneous emergence of a new Wolbachia phenotype. Evolution. 2007;61: 2244–2252.
-	Bordenstein SR, Bordenstein SR. Temperature affects the tripartite interactions between bacteriophage WO, Wolbachia, and cytoplasmic incompatibility. PLoS ONE. 2011;6: e29106.
-	Giorgini M. Induction of males in thelytokous populations of Encarsia meritoria and Encarsia protransvena: a systematic tool. BioControl. 2001;46: 427–438.
Additional comments: •	Based on my previous statements, I think the work presents some gaps that need to be filled as they are essential for an appropriate study of the Leptocybe invasa bacterial pattern. Therefore, I think that this work does not deserve publication in this form and requires major 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: COMPARISON OF BACTERIAL DIVERSITY AND ABUNDANCE BETWEEN SEXES OF LEPTOCYBE INVASA FISHER & LA SALLE (HYMENOPTERA: EULOPHIDAE) FROM CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript requires the input of a language editor to improve the grammar and sentence structure so that it is easily understandable by readers and the English used is of a professional standard.

Important literature has not been included and the information which is included on the background and context is sparse. The introduction should be expanded (as indicated in the MS) and the discussion could benefit from more reference to other literature. Most of the discussion is almost a repetition of the results section with very general statements made but not linked back to the study and highlighting the relevance of these findings to the organism of interest.
Experimental design: This study substantially elaborates on work which has been started on the genus Leptocybe, investigating bacteria present within the species. The MS could benefit from a very clearly defined research question (which will become relevant and more meaningful if more background information and context is provided - as mentioned in 1. above, this is lacking and needs substantial improvement).

A major area of concern is that the specimens used for this study have not been identified either morphologically or at a molecular level. Literature has indicated the presence of at least two haplogroups within what has been called Leptocybe invasa. Molecular confirmation of identity would be preferable as the morphological taxonomy is not fully resolved. The two haplogroups have very different global distributions and host preferences. Furthermore, the study could benefit from identifying the predominant bacteria to species level thereby agreeing with other similar literature on Leptocybe and allowing more concrete speculation (based on literature) as to the role these bacterial species could be playing in the biology of the organism.

The materials and methods section requires substantial more detail and explanation of why certain methods were chosen and implemented.
Validity of the findings: The conclusions to some extent address the results obtained but also discuss other areas which have not been addressed in the introduction or discussion in adequate detail.
For example the below sentence "These results enrich the information of microbial information of L. invasa, help research the reproductive strategy, sex control and invasive mechanism, and lay the foundation for further studies on the excavation and utilization of microbes for the biological control of L. invasa." is very broad and includes important aspects which can be further investigated and addressed with the results of this study, but  no clear links have been made between the results and the potential application for control.
Additional comments: A very interesting study with interesting results. Identification of the specimens used for this study, as well as identification of the predominant bacteria to species level, would greatly increase the importance of this work. Incorporation of specimens from the two different haplogroups would improve this study even further and be of large interest and relevance to the community of individuals working with this insect pest.

Detailed comments and suggestions have been included in the manuscript, using track changes.


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: COMPARISON OF BACTERIAL DIVERSITY AND ABUNDANCE BETWEEN SEXES OF LEPTOCYBE INVASA FISHER & LA SALLE (HYMENOPTERA: EULOPHIDAE) FROM CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors improved the manuscript, taking in account the comments suggested by the second referee directly in the pdf of the first version of the manuscript. However, some important information in the introduction and discussion should be treated better, sometimes in the discussions some results are not sufficiently treated in connection with L. invasa biology or management.
Experimental design: The experimental design shows the same problems highlighted by referees in their previous reviews.  Authors did not take in account all the suggestions made by the referees, but they only based on the comments reported directly on the manuscript by the second referee. Thus, the manuscript has improved as regard the form and the grammar, but the main themes still show lacking argumentation and development. Furthermore, although authors added some information about the L. invasa characterization, they completely overlooked the methods they used and, in my opinion, obtained sequences should be published on sequence database. The sex ratio was not calculated on the studied population, but referred to a previous work (Zheng et al., 2018). Based on the literature about endosymbiotic bacteria, the titre of bacteria (especially those manipulating the reproduction) could change due to environmental characteristics (temperature) that could lead a difference in the sex ratio.
Validity of the findings: In this new version of the manuscript, results did not change. Authors try to streamline the paragraph avoiding the list of found bacteria but, on the other side, authors did not identify the predominant bacteria to species level as previously request by the two referees.
I can not find any crucial change respect to the previous version of the manuscript.
Furthermore, the last part of the conclusions is completely confused about the use of sterile males in a partially thelytokous population (where males are not crucial for the reproduction) and, about the use of antibiotics in the change of offspring.
Additional comments: As already mentioned, the authors modified the manuscript only based on comments on pdf. They completely did not consider the main suggestions by the two referees to improve their research.

Therefore, I think that this work does not deserve publication in this form and requires again major 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: COMPARISON OF BACTERIAL DIVERSITY AND ABUNDANCE BETWEEN SEXES OF LEPTOCYBE INVASA FISHER & LA SALLE (HYMENOPTERA: EULOPHIDAE) FROM CHINA
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript needs a language editor update, some sentences are not clear. It could improve the whole MS.
Experimental design: Most of aspects were improved by authors.It is a pity authors could not find other populations (from other countries - or other lineage) to compare them. Please see my suggestions at the end.
Validity of the findings: Results  and Discussion were clearly summarized and improved.
Please read my general comments.
Additional comments: Authors followed the most of the suggestions of the reviewers. Nevertheless I need to make some considerations:
- in the first revision it was suggested to compare the Chinese L.invasa with those of other countries, I imagine that the authors failed to obtain these samples. It is therefore important to stress, already from the title, that the research is focused on L. invaded from China. I suggest to change the title.
- In my opinion, the phylogenetic reconstructions  trees without outgroup, without the most important RIckettisia species, without the information in M&m paragraph (i.e. number of bootstrap or generations) is not befitting fo PeerJ journal. I would suggest the authors to make a simple blast against genebank database and take in account the similarity with lineages (for L. invasa) and rickettsia group.
- molecular characterization: the PCR program refers to a single genes or both of them? (COI-16S)
- line 99: “…fast growth of L. invasa” please add populations.
- line 128. The sentence “In this research, a total of 656 females and 51 males were collected (Table S1). “ should be in “results” paragraph.
- 224: transitional family: please change in transitional group.
- from line 332 to 336: please rephrase, some sentences are not clear.

In conclusion paragraph:
-  “bacterial endosymbionts” please change with “endosymbiotic bacteria”;
- “Some bacterial” please change with “some bacteria”
- i think 340-342 is ripetitive period. The same concept with different sentences. Please rephrase


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

Input:
Title: EFFECTS OF INDIUM EXPOSURE ON RESPIRATORY SYMPTOMS: A RETROSPECTIVE COHORT STUDY IN JAPANESE WORKERS USING HEALTH CHECKUP DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: To the Authors:
The authors conduct a retrospective cohort study of workers from 22 companies in one region of Japan that handle some form of indium, which seems to automatically enroll workers from these companies in a form of medical surveillance required by Japanese law that includes at least annual health checkup or ‘indium medical checkups’ every six months based on likelihood of exposure that seems to include blood draws for serum indium and KL-6 monitoring. Workers were considered exposed if they ‘handle indium’ and unexposed if they do not ‘handle indium.’ The outcome of interest is self-reported new respiratory symptoms during a health checkup. The authors calculate Hazard Ratios using several modeling approaches to conclude indium-exposed workers are more likely to develop new respiratory symptoms during the study compared with unexposed workers, despite very low serum indium and KL-6. Study design and statistical methods were applied to reduce potential biases, which strengthens conclusions.
Some of the conclusions might benefit from more explanation or clarification. For example, the success of the 2013 regulatory changes are questioned, based on the biomarkers presented, it would seem the regulations/prevention strategies are largely successful and should be celebrated. Perhaps, there are aspects that could be improved, but more specifics might help clarify. Also, the authors suggest the prevalence of symptoms in indium-exposed workers has increased or at least ‘were not reduced,’ and there don’t seem to be any prevalence or incidence data presented to draw this conclusion. I also wonder if the dusty work environments reported by a higher proportion of indium-exposed workers should be included in the limitations. I know the modelling included this as a covariate, but perhaps you can clarify how the new respiratory symptoms can be attributed to indium rather than dust. Lastly, it would be useful to include additional information on what is considered ‘indium handling’ as it defines the exposure and is not necessarily intuitive. I hope you find these general comments useful and provide a few more specific comments below, but commend you on a well designed study and well written manuscript.
Abstract: Line 23: Does non-handling = non-exposed? Less-exposed perhaps, but unlikely non-exposed.
Line 33: ‘respiratory symptoms…were not reduced’ Can you conclude this? There is no comparison to pre-2013 regulations to make this comparison.
Introduction: Line 45: Does global consumption equal production?
Line 55: Define ‘indium-handling workers’
Line 57: symptom > symptoms
Line 58-59: Last sentence of paragraph is confusing and should be reworked.
Line 67: Expand on ‘indium medical examination’ – what it involves and who gets it and why? Lung function testing included?
Line 75: have reduced > have been reduced
Materials and Methods: Line 91: any more information on the range of ‘indium-handling companies?’
Line 124: Can voice wither be attributed to indium?
Line 126: Is a palpitation a respiratory symptom?
Line 132: Define ‘KL-6.’
Results: Line 196: Consistent use of commas in numbers, ie 6,033 – likely based on journal specifications
Line 202+: How can respiratory symptoms be attributed to indium when indium-exposed workers were more likely to work in dusty environments?
Line 212: These are very low biomarkers and that should be discussed at some point in manuscript.
Discussion: Line 237: ‘regulatory strengthening did not reduce the prevalence of subjective respiratory symptoms’ seems too strong of a conclusion based on findings presented in manuscript.
Line 264: Define ‘standard values.’
Conclusions: Line 301: no pre-regulations/2013 data presented, so symptom prevalence cannot be tied to regulations?
Figure 1: is there a component of time from left to right that should be labeled?
Figure 3: Label Y axis.
Table 1: Statistical test and P values for demographic comparison of exposed and non-exposed? Also, probably shouldn’t be considered non-exposed, perhaps less exposed?
Tables 3 and 4: Females more likely to report respiratory symptom, is this expected? Should it be discussed in 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: EFFECTS OF INDIUM EXPOSURE ON RESPIRATORY SYMPTOMS: A RETROSPECTIVE COHORT STUDY IN JAPANESE WORKERS USING HEALTH CHECKUP DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comments
Experimental design: The author analyzed using data from three years, however, the onset of worker's respiratory symptoms should be analyzed the data with the work conditions or  the situation of exposure through the questionnaire at the annual special health check ups. The persuasiveness of the whole paper was reduced in that respect because the author analyzed only using the data of the presence or absence of respiratory symptom symptoms as output.
Validity of the findings: Scientific evidence can be found in the epidemiological calculations, however, it seems that the rationale is not sufficient to conclude that strengthening legislation did not reduce the incidence of respiratory symptoms in indium workers.
The author concludes that the use of protective equipment should be strengthen, however according to the results of this paper, 92.6% of indium workers are using protective equipment. This is a contradiction, and it will be necessary to discussed more carefully.
Additional comments: This is an interesting paper on the development of respiratory symptoms in indium workers.
However, there are points that should be revisited overall.


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

Input:
Title: EFFECTS OF INDIUM EXPOSURE ON RESPIRATORY SYMPTOMS: A RETROSPECTIVE COHORT STUDY IN JAPANESE WORKERS USING HEALTH CHECKUP DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: Indium compounds, especially indium-tin oxide, are used to make liquid crystal displays and have recently been recognized to evoke pulmonary toxicity when inhaled as respirable particles.  Far east Asia including Japan produces the major part of the indium compounds; in 2010 the Japanese MHLW issued a technical guideline for preventing health hazards of ITO workers and in 2013 it was enacted.  This paper compared the subjective respiratory symptoms in Japanese indium workers before and after the legislation; the author measured HR of respiratory symptoms scores of indium workers relative to that of non-indium workers retrospectively.  HR did not decrease after the legislation and the author concluded that indium workers’ symptoms did not decrease after the introduction of strict regulations. The aim of the study is important and clearly stated, however, there is a few questions that remains unanswered.
Validity of the findings: no comment
Additional comments: Major Comments

1.	It has been reported that respired indium stays for a long time in the body including the lung; the biological half -life of the serum indium(sIn) is estimated to be 8.09 years (Thorax, 2015,V70, pp 1040), therefore, two to three years in between the samplings cannot separate the exposure levels sharply. Longer intervals would be better.  Alternately, how about focusing the two groups of newly engaged workers naïve to indium processing, before and after the new rules. That would give us clearer data. Would such comparison available in your data?

2.	Symptomatic changes are important; however, they may not be a sensitive marker to detect the effects of tightening regulations. According to the guideline of MHLW, employers are required to measure the workers’ sIn every six months, and it has been reported that sIn is correlated with the degree of radiological (interstitial or emphysematous changes), functional (diffusing capacity of the lung for carbon monoxide) and serum biomarkers of interstitial pneumonia (KL-6 and SP-D), therefore, sIn, rather than symptoms, appears to be a sensitive marker in assessing the effects of implementing new regulations.

Minor Comments

L16 (Abstract)
 I think the author should add emphysema, alveolar proteinosis and cholesterol granuloma as they are characteristic, although not specific, findings of indium lung (Tohoku J. Exp. Med. 2019, V248, pp143).
L94 exclusion criteria
It appears that the exclusion criteria adopted in this study could omit some heavily exposed workers, who had engaged in indium processing for long durations.


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

Input:
Title: EFFECTS OF INDIUM EXPOSURE ON RESPIRATORY SYMPTOMS: A RETROSPECTIVE COHORT STUDY IN JAPANESE WORKERS USING HEALTH CHECKUP DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript is much improved following revisions and I commend the author for his efforts. The manuscript warrants publication and contributes meaningfully to the field.


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: ISOLATION AND IDENTIFICATION OF L-ASPARAGINASE-PRODUCING ENDOPHYTIC FUNGI FROM THE ASTERACEAE FAMILY PLANT SPECIES OF IRAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: I have reviewed the manuscript by Hatamzadeh et al. The premise behind the manuscript is nice, however, some points need to be addressed. The main revisions which need to be made and other queries are listed below. Other minor issues, which the authors could utilize to improve the manuscript, are indicated on the annotated manuscript which I have attached to this review.

Comments to be addressed:
What was the control while isolating fungal endophytes?
Have authors submitted a voucher specimen(s) of L-asparaginase-producing endophytic fungi in any repository?
Line 137: Identification of Fungal Endophytes “No results have been presented regarding the identification of endophytic fungi in the text” What was identity % of the isolated strains with NCBI strain(s)? It should be mentioned in the text.
The discussion needs to be strengthened.
Table 3: Nothing has been described about the data presented in table 3. Please elaborate.


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: ISOLATION AND IDENTIFICATION OF L-ASPARAGINASE-PRODUCING ENDOPHYTIC FUNGI FROM THE ASTERACEAE FAMILY PLANT SPECIES OF IRAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors described the potential of endophytic fungi from seven medicinal plants to produce L-Asparaginase, an enzyme used for industrial and pharmaceutical purposes, particularly as anticancer agents. It is obvious while reading the manuscript that an intensive work was done by the authors. However, many points in the manuscript need to be carefully addressed to improve not only the quality of the research but also its scientific value (check the main manuscript for more comments)
1.	Overall, the English language needs to be significantly improved.
2.	Abstract
The abstract is not a reflexion of the manuscript, please this need to be carefully addressed. For instance while reading the abstract you may think that only 104 isolates were obtained but that is not the case in the main document. Another point, isolates were first screened using a qualitative method before quantification. There is no such indication in the abstract. For the form, there are too many repeated sentences in the abstract; some data are missing, etc.. (See the reviewed manuscript version for more comments).
3.	Introduction
In the introduction, please give the appropriate definition of endophytes. Many references are not appropriately cited (this is valuable for other parts of the document), while many claims do not have references. This can be seen as plagiarism. Please this issue needs to be carefully addressed. The introduction have some repeated sentences as well. More importantly, the rationale for investigating these plants species is not mentioned (more comments in the main manuscript).

4.	Results
Identification of fungi: a proper description of the colonization frequency is needed. In fact, the experiment was performed and data collected so the presentation of results should be as clear as possible for better appreciation of the quality and amount of work conducted.

The activity was measured qualitatively by measuring diameter on agar plates. There is a need here to show the range of diameters obtained and the most potent according to the method used.   Thereafter, the criteria used to select those used in Nessterization assay should be mentioned to create fluidity in the text. Moreover, how many endophytes were screened qualitatively? How many showed potency?? What was the higher diameter of production???? Many questions need to be addressed in this section.
Overall, the presentation of data needs careful reconsideration. Details information are needed to better appreciate the scientific values.
5.	Discussion
In the discussion section only six plants are mentioned and while reading the results section, there is no indication of the inactivity of endophytes from some plants and not others. This highlights the lack of clarity in the writing. Issue to be addressed.
The misuse of references is noticeable in this section as well as redundant sentences.
The most noticeable problem in this section is the fact that no discussion is provided on the isolation frequency, endophytes colonization per plants, the dominant species etc. Overall, many questions need clear answers. For instance, which plant had the most endophytes colonization? which organ per plant was the most colonized? in general which organs, why? could the environment, time of sampling had any influence on that colonization frequency? ........
6.	Conclusion
No indication from this study revealed that endophytes from Asteraceae produced L-asparaginase with superior activity, since no activity was actually tested.
The authors projected to perform the clinical trial on pure asparaginase, and not the actual preclinical study. In general, the conclusion needs carefully editing.
Experimental design: The protocol of isolation needs to be carefully amended (my comments are included in the manuscript)
Validity of the findings: Acceptable
Additional comments: I encourage the authors to revise their manuscript following the comments and appended edits in the main text.


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

Input:
Title: ISOLATION AND IDENTIFICATION OF L-ASPARAGINASE-PRODUCING ENDOPHYTIC FUNGI FROM THE ASTERACEAE FAMILY PLANT SPECIES OF IRAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: I have reviewed the manuscript by Hatamzadeh et al. The manuscript still needs a major revision. I would suggest that authors should carefully read what they are writing as there are a lot of typographical mistakes. A lot of information is missing in the material and methods section. A sound rationale for their study is still needed. A proper explanation is needed in some parts of the discussion and the conclusion needs to be strengthened. Authors must recheck the results they have presented particularly in Table 3. The manuscript still needs attention on the English at some places. I would like to suggest some improvements to make the manuscript more clear and focused. The other issues, which the authors could utilize to improve the manuscript, are indicated on the attached file.
Experimental design: The authors need to focus on the material and methods section as a lot of information and details are missing.
Validity of the findings: The authors need to validate some results which I have pointed in the attached file.
Additional comments: No additional comments