PONE-D-24-17362Effect of prenatal alcohol consumption on dental enamel formation in offspring – an animal study protocolPLOS ONE

Dear Dr. de Paula-Silva,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Boyen Huang, DDS, MHA, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Partly

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PONE-D-24-17362

The article entitled “Effect of prenatal alcohol consumption on dental enamel formation in offspring – an animal study protocol" by Leme et al. proposed to analyze the rat tooth enamel development with the embryonic alcohol exposure. This is a novel research area which tried to investigate the effect of alcohol on causing developmental defects of the enamel.

They are using various methods to investigate the structure of the enamel and tried to identify the underlying protein gene activity in causing these enamel defects.

I really enjoyed reading the proposed project and I have following comments to improve the content of this protocol.

1. Under the animals and experimental procedures

Exposure to alcohol will be carried out following a binge drinking model, in which the animals will receive doses of ethanol via gavage (3g/Kg, [30% w/v]) for 3 consecutive days followed by 4 days of rest per week [33, 34]. The animals of the control group will receive distilled water. After one week of ethanol exposure ….

Explain the rationale of using this alcohol concentration. The cited two references have similar concentrations what is the rationale for using the (30% W/V) versus 1- 5% of alcohol concentrations.

2. The figure 2 has a confusion on left hemi mandible – Are you going to use the incisors and molars both for Rt-PCR and micro-CT? How you are going to do that? Please explain.

3. In the histological evaluations under histometric analysis “Microscope will be operated in fluorescence mode” Why you need the fluorescence mode as these sections will be H &E sections.

4. Please indicate the data availability according to the journal policy.

Reviewer #2: Roberta Duarte Leme and co-workers propose a binge drinking experimental model to study the effects of 3g/Kg alcohol [30% w/v] in pregnant rats on dental development of the pups.

The introduction part should be re-organized:

1- rationale of the study

2- State-of-the-art of alcohol effects on dental development

The paragraph on general effects of alcohol on embryogenesis should be reduced as it is out of the scope

References should be up-dated by avoiding papers written in non-English language

Authors should distinguish studies on humans and on animals

Figure 1: authors should add the time of exposure to alcohol

Animals: Doses, time and duration of exposure should be justified

Overall, investigations of 10-days-old rats (PND 10) should be revised to the fact that neither incisors nor molars are fully erupted

Photography: not sure that authors could see some defects on incisal enamel due to the very short time of adversity and incomplete development at PND 10

Knoop microhardness test: it would be more appropriate to investigate enamel hardness by nano-indentation rather micro-indentation due to the small size of samples

Histology: Authors should justify why “bone blocks containing incisives and molars will be removed from the previously dissected mandibles and maxillae.”

Real time reverse transcription-polymerase chain reaction (RT-PCR): not sure that authors will have enough material for RT-PCR analysis especially at PND 10

Target genes and proteins: authors should add MMP2 and MMP9 to be able to compare their data with zymography

Figure 2 should be corrected in accordance with modifications of the protocol

Is it possible to add a paragraph on expected data?

Abstract should be revised according to modifications of the paper

Reference list should be completely revised. Main studies reporting alcohol effects on dental development are quiet old, authors should up-date the reference list and include recent papers (de Carvalho P, et al., 2022; Blanck-Lubarsch M et al., 2019; etc…). They should add the important review of Silva MJ et al., 2016 reporting an association between alcohol consumption and MIH.

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Reviewer #1: Yes: Dr. Devi Atukorallaya

Reviewer #2: Yes: Sylvie Babajko

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PONE-D-24-17362R1Effect of prenatal alcohol consumption on dental enamel formation in offspring – an animal study protocolPLOS ONE

Dear Dr. de Paula-Silva,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 04 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Boyen Huang, DDS, MHA, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: No

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Despite we may still discuss the feasibility of all investigations at PND10, authors replied to most of the comments, did preliminary pilot studies for feasibility and improved their manuscript.

Reviewer #3: This is a study protocol to examine the effect of prenatal alcohol consumption on dental enamel formation using rats as the model.

The study's scope is highly relevant to understanding the effect of prenatal maternal alcohol consumption on dental enamel formation. In humans, the enamel matrix of the primary teeth is laid down in utero, and matrix maturation also starts in utero. Unlike primary teeth, the majority of permanent teeth mineralization happens postnatally. Therefore, the effect of prenatal alcohol drinking is studied on populations with primary dentition or mixed dentition.

In rodents like rats and mice, the first molar and incisor enamel organs are formed prenatally; however, unlike human teeth, the enamel matrix mineralization happens postnatally in all teeth.

Therefore, this study design raises a fundamental question regarding the period of alcohol treatment for the mother mice: Is the prenatal alcohol treatment reasonable enough to observe the effect on the enamel matrix formation and mineralization, which happens postnatally? Further, how can the outcomes be applied to human tooth formation?

Overall comments on the methodology:

• The detail level is inconsistent method by method.

• Too many sites refer to previous reports about the methodology. The authors need to present all the steps of the procedure.

• All the experiments need to contain fundamental information, i.e., what types of specimens were used and how they were prepared, the number of samples, etc.

• There is no mention of the sample size in each experiment.

• The resolution of the two figures is too low to read.

The following are the section-by-section comments.

Animal and experimental procedure:

• There is no mention of controlling and monitoring water and chow consumption.

• The rationale for the sample size is not clear. How it's determined?

• The sex of the offspring should be accounted for.

Micro-computed tomography:

• The meaning of the sentence “ The region of the molars and surrounding bone will be removed for analysis” is unclear. Does it mean bone and molars will be dissected out from the mandible? If so, why is it necessary? Analyzing software can extract the preferred target region on the microCT image.

Scanning electron microscopy-energy dispersive X-ray

• What kind of instrument will be used to cut the tooth?

• How can artifacts caused by the cutting process be avoided?

• Any plan to etch the surface?

Histometric analysis

• The meaning of the sentence, “Bone blocks containing incisives (incisors?) and molars will be removed from the previously dissected mandibles and maxillae.”, is unclear.

• There is no description of what kind of structure will be observed after H&E staining.

• Magnification x10 would be too small to observe cells and matrix structures.

In situ zymography

• The procedure how to obtained the section was missing

Real time reverse transcription-polymerase chain reaction

• The collection of the ameloblasts is not described well enough.

• The methods in reference #46 are not for the pure ameloblasts collection but for enamel organs enriched with ameloblasts. Authors need to be aware of it and note it.

Expected results

• Compared to the amount of the experiment information, expected results are too limited. Some reports use a similar method to this protocol (reference #29, 30, and DOI: 10.2485/jhtb.16.61, etc.). Therefore, the authors should be able to present the expected results of each experiment based on the previous reports.

Reviewer #4: This paper highlights an important area of research addressing the effects of maternal alcohol consumption on the development of enamel. Thank you to the authors for putting this paper together and working towards helping us better understand the impact of prenatal alcohol consumption on developing dentition. Some overall concerns raised by this paper are listed below:

There are multiple tests completed in this study, with many outcome evaluations completed—photography, micro-computed tomography, Knoop microhardness test, scanning electron microscopy-energy dispersive x-ray, Raman spectroscopy, histometric analysis, immunohistochemistry, in situ zymography, real time reverse transcription-polymerase chain reaction, and statistical analysis also. It is inferred that the developmental defects of enamel (DDE) will be found in offspring from rats exposed to alcohol consumption compared to those without. With 10 different DDE analysis techniques employed, no hypothesis was provided for any of the 10 tests. Without clarity on the reasoning and anticipated findings of these tests, the study lacks focus and does not have clearly defined outcomes. It seems that the tests have been assembled in an exploratory manner without a specific, well-defined objective. A ‘Study Protocol’ should have clearly defined outcomes for the physical properties (volume, thickness and density) of enamel and cellular changes associated with alcohol consumption.

Another concern is that the proposal does not have any content on standardising the tests or assessing the error. Each test described in the paper needs more detailed elaboration.

Power calculation should be an essential part of ‘Study Protocol’.

Statistical testing: The description on statistical testing is inadequate. Each quantitative method should contain adequate description for statistical testing. The authors should also discuss what they will do if the underlying assumptions of ANOVA or similar tests are violated.

Additional comments:

Animals and experimental procedures:

- How long is the mating period? Are female rats exposed to alcohol throughout this period?

- How long will the rats be exposed to alcohol in total? What is done if the female rat does not become pregnant in the expected mating period?

- “Binge drinking is characterised by bringing BAC up to 0.08 grams % or above, having the potential to generate signs of intoxication” – what are the signs of intoxication?

- Please clarify for a general reader why was a binge drinking model chosen and not a lower alcohol consumption percentage.

- P7: 3g/kg (i.e., 3gm/1,000gm x 100%) yields a value of 0.3%, and it is difficult to determine how this equates to 30% w/v.

The specific issues with some of the methods are provided as examples below.

Photography:

- Some of the key data are missing, e.g., camera angulation, room lighting, magnification, and quantitative vs qualitative analysis.

Micro-computed tomography:

- How will the authors ensure the density measurement is accurate and standardised? Will the molars and surrounding bone be removed from the micro-CT images or from the specimens before imaging?

- “… the collected incisors and molars will be mesiodistally hemisected and one of those sections will be prepared for SEM” – was there uniformity with the selected hemisection? How will this be selected and how will the orientation be standardised?

Knoop microhardness test:

- Will this test be carried out on flat or curved surfaces? What will be the orientation of the specimens/sections?

- Figure 2: For 10d offspring, microhardness testing will only occur for incisors and not molars. Although it is noted in the text that the molars are still under development, the figure should stand independent to the text, and this is not made clear for the general reader. There are similar issues with other tests.

Scanning electron microscopy-energy dispersive x-ray:

- What many spots will be examined from each specimen? Will there be outputs for other elements?

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-24-17362R2Effect of prenatal alcohol consumption on dental enamel formation in offspring – an animal study protocolPLOS ONE

Dear Dr. de Paula-Silva,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jan 06 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Boyen Huang, DDS, MHA, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #3: Yes

Reviewer #4: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #3: Partly

Reviewer #4: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: The revision addressed most of the reviewers' concerns. However, some minor points are still required for revision.

1. Resolution of Figures: Although the new figures are saved at 600 dpi, the letters in each figure are still too blurry and unreadable. All contents and text in the Figures need to be readable.

2. Collection of ameloblasts for real-time RT-PCR: As the author is aware now, the collected sample will not be homogenous with ameloblasts but enamel organs rich in ameloblasts. Therefore, it is not appropriate to describe the "collection of ameloblasts" here, even with the remark "not specifically designed for the collection of pure ameloblasts." This remark actually reduces the reliability of the sample collection. Instead of adding the remark, it is recommended to state that the collected sample will be enamel organs rich in ameloblasts. This way, it is more accurate in describing the sample collection.

3. The model of SEM is missing. This information is necessary to be consistent with other sections including information on all instruments' makes and models.

Reviewer #4: Overall the paper has greatly improved in quality since the previous review, thank you to the authors for their hard work.

There are still areas in which can be improved, mostly around grammar. The paper requires a few more read-throughs by the authors and minor edits for comprehension. For example “having the potential to

generate signs of intoxication, impairing performance on tasks related to timing, response

inhibition, and position discrimination” on page 6, and “… we will take photographs of right hemimandibles from both groups (n=22), along with incisors and molars (n=22) from the left hemimandible in each group” on page 10. The wording of both these examples can be further improved.

Please also review paper for finer details – for example on page 6, “It differs from the chronic alcoholism model, which is less common today” – no definition of the chronic alcoholism model is provided, this comparison is consequently unclear. Additionally on page 14, “… secondary antibody for 1 hour, washed in PBS” – The acronym for PBS has not been introduced. Please review full article to further improve readability.

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Reviewer #3: No

Reviewer #4: No

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Effect of prenatal alcohol consumption on dental enamel formation in offspring – an animal study protocol

PONE-D-24-17362R3

Dear Dr. de Paula-Silva,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Boyen Huang, DDS, MHA, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #3: Partly

Reviewer #4: Yes

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #3: Partly

Reviewer #4: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #3: Yes

Reviewer #4: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: This revision addressed most of the comments from the reviewer. However, the texts in the figures are still not readable enough on my end in the review package.

Reviewer #4: All comments noted in previous reviews have been addressed by the authors. Thank you again for your hard work in this paper.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

Reviewer #4: No

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