PONE-D-24-24759Roscovitine does not protect cystic fibrosis bronchial epithelium against Pseudomonas aeruginosa infectionPLOS ONE

Dear Dr. Guillard,

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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Thank you for submitting your manuscript addressing the therapeutic potential of roscovitine against Pseudomonas aeruginosa in cystic fibrosis (CF). This work tackles an important area of CF research, particularly given the challenges posed by multidrug-resistant pathogens. Your use of a 3D model of fully differentiated F508del-CFTR human bronchial epithelium is a notable strength, providing a robust experimental platform. However, there are certain areas, indicated below, where the manuscript can be refined to improve clarity, accuracy, and alignment with the presented data.

1) Your data presentation is generally thorough, but incorporating more direct references to numerical findings and statistical significance within the main text would enhance clarity. For instance, the observed improvement in ASL bactericidal activity at 25 µM should be quantified and discussed in greater detail. Ensuring that all conclusions are explicitly tied to the data while avoiding speculative extrapolations will improve the manuscript’s rigor and focus.

2) The manuscript would also benefit from a more explicit discussion of its limitations. The small sample size (n=6) and the in vitro nature of the experimental model limit the generalizability of the findings. Additionally, the absence of mechanistic insights into why roscovitine enhances ASL bactericidal activity at 25 µM but fails to protect epithelial integrity leaves unanswered questions. Addressing these issues in a dedicated section would provide important context for readers and help position your work within the broader research landscape.

3) The conclusions currently overstate the findings, particularly the assertion that roscovitine does not protect CF bronchial epithelium against P. aeruginosa . While the data clearly demonstrate the lack of epithelial protection, they also reveal enhanced bactericidal activity of the airway surface liquid (ASL) at 25 µM. This is an important observation that should be reflected more prominently. A more balanced presentation of these results, emphasizing their complexity, would strengthen the overall narrative. Revisions to the abstract, results, and discussion to reflect this duality are recommended.

4) The figures, though informative, could be made more intuitive. Figures 1 and 3, in particular, would benefit from additional annotations or more detailed captions to improve their interpretability. This would allow readers to better grasp the key findings at a glance without needing to refer back to the main text.

Overall, this manuscript has potential but requires significant revision. Specifically, the conclusions should be rephrased to reflect the nuanced findings, limitations must be discussed more thoroughly, and the presentation of data should be refined for clarity and precision. By addressing these issues, the study will make a valuable contribution to CF research and meet the standards expected for publication in PLOS One .

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Please submit your revised manuscript by Jan 30 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:

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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,

Kevin Looi, Ph.D

Academic Editor

PLOS ONE

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

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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

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“The author(s) received no specific funding for this work.”

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Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. 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.

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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

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

Reviewer #1: Yes

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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

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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

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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: The manuscript titled "Roscovitine does not protect cystic fibrosis bronchial epithelium against Pseudomonas aeruginosa infection" showcases a well-presented research on the effects of roscovitie - a cyclin-dependent kinase inhibitor, in cystic fibrosis - psedomonas aeruginosa co-infections. As people living with Cystic Fibrosis are prone to co-infections which may worsen their conditions. It is imperative to understand the metabolic function of treatments available for pwCF.

In this manuscript, the author argues that roscovitine, a treatment regimen for pwCF does not protect the bronchial epithelium of CF disease model against P. aeruginosa infection. From their data, they conclude that the medication only does not pose a significant antibacterial effects and previously reported effects could rather be the result of the anti-inflammatory property of roscovitine combined with ceftriaxone administered to mice.

From the data presented, the manuscript is technically sound and the data presented by the authors supports their conclusions. The statistical analysis was arguably performed accurately and with rigor.

I commend the authors for putting up this interesting research as it sheds more light into the important details as to intertwines between inflamatory response and antibiotic responses for systematic diseases.

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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

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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-24-24759R1Roscovitine enhances the bactericidal activity of the airway surface liquid of the cystic fibrosis bronchial epithelium but does not protect against Pseudomonas aeruginosa infectionPLOS ONE

Dear Dr. Gullard,

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.

==============================

Throughout the manuscript, be sure to correct the pwCF represents "people with CF" and not "patients with CF"

Scale bars on images should be made larger for reference.

In the discussion - it would be helpful to elaborate on some potential limitations of incorporating Roscovitine into a regimen for chronic infection, Specifically the authors provide evidence in the literature that Mycobacterium spp. infections get worse during treatment. As an emerging pathogen in CF, it is important to note that in some individuals, Pa and Mycobacteria may both be in the lung at the same time.

==============================

Please submit your revised manuscript by Apr 18 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,

Subhra Mohapatra, MS 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. 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 #2: All comments have been addressed

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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 #2: Yes

**********

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

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 #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 #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 #2: Throughout the manuscript, be sure to correct the pwCF represents "people with CF" and not "patients with CF"

Scale bars on images should be made larger for reference.

In the discussion - it would be helpful to elaborate on some potential limitations of incorporating Roscovitine into a regimen for chronic infection, Specifically the authors provide evidence in the literature that Mycobacterium spp. infections get worse during treatment. As an emerging pathogen in CF, it is important to note that in some individuals, Pa and Mycobacteria may both be in the lung at the same time.

**********

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

**********

[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.

Roscovitine enhances the bactericidal activity of the airway surface liquid of the cystic fibrosis bronchial epithelium but does not protect against Pseudomonas aeruginosa infection

PONE-D-24-24759R2

Dear Dr. Guillard,

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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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,

Subhra Mohapatra, MS PhD

Academic Editor

PLOS ONE

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