PONE-D-24-05687Identification of microbial antigens in liver tissues involved in the pathogenesis of primary biliary cholangitis using 16S rRNA metagenome analysisPLOS ONE

Dear Dr. Katsumi,

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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

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: N/A

Reviewer #2: No

Reviewer #3: 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

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #2: Yes

Reviewer #3: 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 collection details of the liver sample have not been mentioned, such as the number of patients from whom the samples were obtained, the hospitals from which the samples were collected, how these samples were obtained from pateint, and other relevant details.

Please use more recent references, as out of the 34 references, 14 are older than 5 years.

Statistical analysis is not available; therefore, it is necessary for you to include statistical analysis in your research, especially when comparing samples and when you have a control group.

Additionally, no information has been provided regarding age and gender differences in the results.

Reviewer #2: GENERAL COMMENTS

The paper is commendably well-written, providing lucid explanations of both the disease itself and the objectives of the study, thereby ensuring accessibility for readers unacquainted with the subject matter. The authors adeptly contextualize their research within the existing literature, delineating the gaps they seek to address. The research's objective is articulated with clarity, underscoring its significance for enhancing understanding of the disease and hinting at its potential implications for designing novel therapeutic approaches. The selection and description of materials and methods are appropriate, facilitating replication. While the results are effectively presented and supplemented with figures and tables for enhanced comprehension, minor issues merit attention.

The findings convincingly demonstrate the presence of exogenous pathogen-associated antigens, particularly bacteria, in both tissue and serum samples obtained from PBC patients participating in the study. Importantly, the identified species align with previous reports, reinforcing the theory of environmental factors' involvement in the disease's pathogenesis. The ensuing discussion is well-informed, seamlessly integrating insights gleaned from the literature with the study's results. The authors conscientiously acknowledge the study's limitations, recognizing them as areas for improvement in future investigations.

Overall, the study successfully achieves its aim of identifying exogenous pathogens, presenting their results, and contributing to the understanding of the disease and its pathogenesis. Furthermore, it paves the way for further experimentation, underlining the imperative for continued research in this domain. While there are minor issues that warrant review and refinement in the final draft, the paper's substantive contributions to the field of PBC research warrant its publication, pending resolution of these issues, as it promises to catalyze advancements in future studies of PBC.

MINOR ISSUES AND RECOMMENDATIONS

*Materials and methods:

1. Remember always clarifying the significance of initials the first time they are mentioned, even if it sounds unnecessary (e.g., stained with HE, what does HE stand for?) (page 12, final line)

2. Regarding the 16S rRNA analysis (page 14, lines 1 to 3), is there any information on why those specific regions were selected or past studies that have used them? It would be good to give a little more context on why those regions are used for the analysis and references to past, similar studies that used them.

3. For the validation study (page 14, line 17), I would suggest adding a little bit on how the authors designed the primers. Software used and other details.

4. About statistics analysis. Did the authors use any kind of statistics? Even just descriptive statistics? If so, it should have been mentioned.

*Results:

1. About the table for the clinical characteristics of the patients (page 29), in the legend it says that the parameters are presented as the median for continuous variables, then I assume some statistics were used. As said before, all kinds of statistics should be mentioned.

2. Did the authors have patients fill in questionnaires? If so, it also should be mentioned.

3. DNA library in PBC sera paragraph (page 16), I think grammar can be improved in this section, for some reason I find it hard to understand some phrases (e.g., fig 1B shows that reads peak histogram in PBC samples?)

4. 16S rRNA metagenome analysis in sera (page 17, lines 7 to 9), there are two phrases with the same meaning in this section, it turns repetitive.

*Tables and figures:

1. This detail was something that I found mostly in tables and legends, but I will advise the authors to check the entire document for the style regarding the writing of species names, as in some parts of the document the species are written in italics, but in some they are not in italics. It may sound like a small thing but being consistent throughout the text is important.

2. Also in table 4, the writing for Sphingomonas panacis should be checked as the species (i.e., panacis) should always go all lower case.

3. Legend for figure 3 (Page 28, line 6), there is a phrase that sounds loose, like some information is missing or the phrase is incomplete.

4. In figure 2B, where is the before image for the hepatocyte area? It is mentioned in the legend that it is supposed to be a before and after image, but the figure only shows the after. Also, the scale bar is missing in this figure 2B.

5. In figure 3, do the samples have any kind of ID that could be put in the graphic to indicate to which sample each line corresponds? If so, it should be added to the image for better interpretation.

Reviewer #3: Following suggestions are recommended to further improve your manuscript;

1) kindly check out some recent plos one publications and format your manuscript as per journal guidelines.

2) kindly add line numbers.

3) kindly arrange all the intext and end bibliography as per journal guidelines.

4) it is recommended to arrange all the manuscript sections as per journal format.

5) kindly make a separate table for primer sequences instead of discussing them in paragraph.

6) it is suggested to correct the manuscript spacing as per journal format.

7) kindly arrange all the figures and figure legends as per journal guidelines, as they should be mentioned after the paragraph they are discussed in.

8) what does (mean 2578 reads) means throughout the manuscript.

9) kindly revise all the tables of manuscript to be in uniform format.

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Reviewer #1: Yes: Souzan H Eassa

Reviewer #2: No

Reviewer #3: No

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Identification of microbial antigens in liver tissues involved in the pathogenesis of primary biliary cholangitis using 16S rRNA metagenome analysis

PONE-D-24-05687R1

Dear Dr. Tomohiro Katsumi,

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,

Hin Fung Tsang

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

Reviewer #3: 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 #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

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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: (No Response)

Reviewer #3: 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: (No Response)

Reviewer #3: Yes

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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: (No Response)

Reviewer #3: All the comments have been addressed. I would suggest to accept the manuscript. I must appreciate the author's findings and hard work.

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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: Yes: Souzan H Eassa

Reviewer #2: No

Reviewer #3: No

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