PONE-D-23-38789Age and Sex-related Variations in Murine Laryngeal MicrobiotaPLOS ONE

Dear Dr. Thibeault,

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 Mar 01 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,

Peter Gyarmati

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Note from Emily Chenette, Editor in Chief of PLOS ONE, and Iain Hrynaszkiewicz, Director of Open Research Solutions at PLOS: Did you know that depositing data in a repository is associated with up to a 25% citation advantage (https://doi.org/10.1371/journal.pone.0230416)? If you’ve not already done so, consider depositing your raw data in a repository to ensure your work is read, appreciated and cited by the largest possible audience. You’ll also earn an Accessible Data icon on your published paper if you deposit your data in any participating repository (https://plos.org/open-science/open-data/#accessible-data).

3. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

4. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. 

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

5. Thank you for stating the following financial disclosure: 

"SLT

R01DC012773-10 

NIDCD NIH

https://www.nidcd.nih.gov/

No"

  

Please state what role the funders took in the study.  If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" 

If this statement is not correct you must amend it as needed. 

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

6. Please expand the acronym “NIDCD NIH” (as indicated in your financial disclosure) so that it states the name of your funders in full.

This information should be included in your cover letter; we will change the online submission form on your behalf.

7. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

8. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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: Yes

Reviewer #2: Yes

**********

4. 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

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript by An et al. describe the results of a study aiming to identify age- and sex-based differences in murine laryngeal microbiota. The authors used 16s rRNA sequencing to compare laryngeal microbial profiles between sexes and across four age cohorts. They compared relative abundances at the phyla and genera levels as well as measured a series of diversity indices and found differences across age, but not sex.

I found this study interesting and well designed. The methods were described in appropriate detail. Most of my comments are about how the study is presented and with significant revisions, I think that this study is publishable. Below I describe my overall concerns and then list some detailed comments.

1. Expanding and clarifying the results and figures

The figure legends were quite short and missing some information that would help readers. Across all the figures, it would be helpful if the authors included the age category names that associate with each group (e.g., 6 wk = adolescent) since they mainly refer to these names in the main text. It would also be helpful if there were a consistent color palate across figures for the age categories, but this is pretty minor and optional. Adding significance labels to the relevant figures (e.g., figure 4) would also help readers quickly identify the pattern the authors are pointing out in the text. Figure 5 and 6 would definitely benefit from further explanation. For example, in figure 6, what does the thick red vs thick black vs thin black lines mean on the plot? The results in Figure 6B are also not mentioned in the main text of the results (although I see they are discussed in the discussion). These data should be reported in the results section. I also find the reporting of the statistics in the results to be a little sparse. For example, on L204 the authors should mention what type of statistical test they used. I also noticed that the figure 1 labels are incorrect and do not match the description in the main text.

2. detecting sex-based differences

As the authors mention in the discussion, their ability to detect sex-based differences in laryngeal microbiota is likely confounded by age. Was there an attempt to account for this in the analyses? I imagine it is possible to fit a linear model to the data and remove the component that is due to the age effect. I believe there are functions within the limma R package that do this.

3. Adding to the discussion

The authors’ discussion focuses on contextualizing their results with what is known about gut microbiota and aging, but what about other studied organs? The oral cavity for example is connected to the larynx via the pharynx. Do the authors expect that certain organs might have more similar microbiomes? Also, I suggest adding a paragraph on the drawback of this study and/or future directions arising from the results of the authors’ work here.

4. Revising manuscript language to improve readability

I found this manuscript a little hard to follow and noticed some consistent typos throughout. I’ll provide a few examples here and highlight a couple of line edits below to use as a guide for revision. In the introduction the authors’ discussion of what is known about the gut microbiome (L86-90) was confusing. I found the methods generally very easy to follow. The results and discussion had a few typos including double parenthesis, using periods instead of commas, and sentences with missing words (e.g., L296 for a sentence with a missing word).

Detailed comments:

L96-97 – I don’t know that this is likely to be universally true across species. Be more specific here (murine models + humans?)

Methods – include versions for any software used where relevant.

L167-168 – Authors report 2 samples removed from adolescent group, but in the figures in the results it looks like 1 adolescent and 1 mature adult (6 wk and 12 mo) were removed. Please update whichever is incorrect.

L184 – write out amplicon specific variant fully at the first mention in this new section.

L186-188 – double parentheses here.

Results – Age groups should be referenced in a consistent manner. In paragraph 1 of results starting on L181 the authors call the same group “middle-aged” and then “older adult.”

L189-190 – move to discussion.

L203-204 – Is the significant decreased referring specifically to Corynebacterium?

L205 – Does “groups” refer to sex here?

L207-208 – stats are vague here.

L211 – write out what LEFSe is.

L248 – this is interpretation and should be in the discussion.

L318 – I wouldn’t say confirmed here. Why would we assume the same in the larynx?

L367 – A citation for a manuscript in review should not include the journal name.

Data availability statement should be written out in the manuscript by the acknowledgements.

Reviewer #2: - The authors state that that age related microbial variation may potentially underlie biological mechanisms and leave the larynx more susceptible to disease pathogenesis. The authors go on to discuss age related pathology, presumably presbylarynx. It would be beneficial to better understand how microbial changes could potentially drive the atrophy seem in this condition.

- While gut and skin changes are briefly introduced, the introduction would benefit from description of whether there has been studied age related changes in microbiota composition in other parts of the airway.

- While I understand the need to parse out individual microbial mechanisms, is is possible that the stability of the laryngeal microbiota was related to the chosen model and not actually reflected in humans? For example, the environment, mechanical stresses of vibration are needed to potentially interact and produce age related findings. There are some model limitations that should be discussed.

- Several proofreading errors were present in the manuscript.

**********

6. 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 #1: No

Reviewer #2: No

**********

[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.

Age and Sex-related Variations in Murine Laryngeal Microbiota

PONE-D-23-38789R1

Dear Dr. Thibeault,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Peter Gyarmati

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The present study explored potential age and sex-related changes in the laryngeal microbiota across the lifespan in a murine model. The reviewer comments have been addressed and are incorporated ito the revised paper.

**********

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 #1: No

Reviewer #2: No

**********