PONE-D-22-04805

Hmong Microbiome ANd Gout, Obesity, Vitamin C (HMANGO-C): A phase II clinical study protocol

PLOS ONE

Dear Dr. Straka,

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 Sep 18 2022 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Cristina Vassalle

Academic Editor

PLOS ONE

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2. Thank you for stating the following in the Funding Section of your manuscript:

“Minnesota Partnership for Biotechnology and Medical Genomics grant #18.08, Targeting the gut microbiome to prevent the increasing incidence of obesity in immigrant populations. This funding source had no role in the design of this study and will not have any role during its execution, analyses, interpretation of the data or decision to submit results.”

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

 “D.K received funding from  Minnesota Partnership for Biotechnology and Medical Genomics grant #18.08, Targeting the gut microbiome to prevent the increasing incidence of obesity in immigrant populations. This funding source had no role in the design of this study and will not have any role during its execution, analyses, interpretation of the data or decision to submit results.”

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

3. Thank you for stating the following in the Competing Interests section:

“D.K. serves as CEO and holds equity in CoreBiome, a company involved in the commercialization of microbiome analysis. The University of Minnesota also has financial interests in CoreBiome under the terms of a license agreement with CoreBiome. These interests have been reviewed and managed by the University of Minnesota in accordance with its Conflict-of-Interest policies.”

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4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

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5. We note that the original protocol file you uploaded contains a confidentiality notice indicating that the protocol may not be shared publicly or be published. Please note, however, that the PLOS Editorial Policy requires that the original protocol be published alongside your manuscript in the event of acceptance. Please note that should your paper be accepted, all content including the protocol will be published under the Creative Commons Attribution (CC BY) 4.0 license, which means that it will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution.

Therefore, we ask that you please seek permission from the study sponsor or body imposing the restriction on sharing this document to publish this protocol under CC BY 4.0 if your work is accepted. We kindly ask that you upload a formal statement signed by an institutional representative clarifying whether you will be able to comply with this policy. Additionally, please upload a clean copy of the protocol with the confidentiality notice (and any copyrighted institutional logos or signatures) removed.

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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: 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 #1: Yes

Reviewer #2: 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 #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: Yes

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: This is an interesting and well written paper; the protocol is carefully described.

Some comments.

In the introduction the possible role of Vit C on gut microbiota should be shortly added to better define point 1 of the secondary objective, e.g. in a healthy population high doses of Vit C have shown a positive shift of bacteria populations in the gut (Otten 2021).

Similarly, in the introduction when dealing with metabolic diseases, published data on the presence of dysbiosis in gout patients and the role of specific urate-reducing drugs in modifying gut microbiota should be provided.

Are pts under diuretic treatment allowed in the study?

"To assess study medication adherence, we used the medication diary sheet recorded by participants at Visit 2". It should be discussed that the assessment of blood levels of ascorbic acid after treatment is not planned.

Reviewer #2: This study protocol is well structured and well elaborated., although there are some critical points that the authors should remedy.

The authors should better explain the rationale for the administration of vitamin C to subjects without gout/hyperuricaemia.

How were the doses of vitamin C to be administered chosen?

Among the primary objectives, the authors should also introduce placebo especially in the patient group.

In my opinion, when analysing the data, the authors should also take gender into account.

Fig. 1 is actually a table

As far as blood and urine tests are concerned, the authors want to study the urate-creatinine trend.

It would also be good to analyse markers of oxidative stress, such as ROS, or indices of biological oxidative damage such as malondialdehyde and isoprostanes.

Vitamin C at high plasma concentrations may act as a pro-oxidant (1) Have the authors ever tested this effect or is there any data for the treatment they want to perform? Any treatment with vitamin C in patients genetically predisposed to high uric acid levels should be performed for life. According to the authors, would this not lead to an increased oxidative risk in these subjects? (2)

1) Julia Kaźmierczak-Barańska , Karolina Boguszewska, Angelika Adamus-Grabicka

Bolesław T. Karwowski. Two Faces of Vitamin C—Antioxidative and

Pro-Oxidative Agent.Nutrients 2020, 12, 1501; doi:10.3390/nu12051501

2) Ian D. Podmore, Helen R. Griffiths, Karl E. Herbert, Nalini Mistry, Pratibha Mistry & Joseph Lunec. Vitamin C exhibits pro-oxidant properties. NATURE | VOL 392 | 9 APRIL 1998

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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 #1: No

Reviewer #2: 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-22-04805R1Hmong Microbiome ANd Gout, Obesity, Vitamin C (HMANGO-C): A phase II clinical study protocolPLOS ONE

Dear Dr. Straka,

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 Dec 04 2022 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,

Cristina Vassalle

Academic Editor

PLOS ONE

Additional Editor Comments:

Please, answer to reviewer 2

Most of the comments made were not taken into account. The authors state that they have difficulties in introducing other markers or the placebo due to their limited budget.

In my opinion, there are aspects not to be underestimated in this study, such as the production of oxygen free radicals in patients subjected to high doses of vitamin C for long periods.

Solving the problem of Fig/Tab. 1

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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

**********

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: 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 #1: Yes

Reviewer #2: 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 #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 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: I thank the AA for their responses. For me, the paper is ready for publication in its present form since all my questions have been answered in a satisfactory manner

Reviewer #2: Most of the comments made were not taken into account. The authors state that they have difficulties in introducing other markers or the placebo due to their limited budget.

In my opinion, there are aspects not to be underestimated in this study, such as the production of oxygen free radicals in patients subjected to high doses of vitamin C for long periods.

Solving the problem of Fig/Tab. 1

**********

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

**********

[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.

Hmong Microbiome ANd Gout, Obesity, Vitamin C (HMANGO-C): A phase II clinical study protocol

PONE-D-22-04805R2

Dear Dr. Straka,

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,

Cristina Vassalle

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: