PONE-D-20-07569

Impact of processing method on donated human breast milk microRNA content

PLOS ONE

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PLOS ONE

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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: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: Yes

Reviewer #2: No

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: First I want to appreciate the investigators for the great work done. This study is an incredible assessment for miRNAs and the functioning in infants. This study highlights great advances in breast milk donation practices and much required knowledge in this field.

My review is based on the questions above;

1. The manuscript is generally sound but there is a great need for increment in sample size, the analysis proved that need but ultimately the knowledge has been well explained.

2. The interplay of analysis between function and composition based on the processing method was well elaborated. This was one of my strongest points.

3. The data was made available. More analysis on function in relation to miRNAs especially on the processes can be generated if possible.

4. Standard English was used. I believe they however meant "revealed" and not revelled in the discussion concerning HPP on line 328. This can be clarified please.

I would love to encourage the authors to explore more information on the composition of breast milk in relation to other products after the preservation method used.

Thank you

Reviewer #2: This manuscript by Smyczynska et al describes the differences of two different processing methods on donated human breast milk on microRNA level. This subject is of high interest and the analysis of influence of HoP and HPP on micro RNAs is unique although many publications already claimed that HPP preserves proteins and other nutrients better than HoP.

The paper is well structured and has a good readability although I would recommend a native English reviewer. The main weakness in this study I see in the sample size. Only three samples were analyzed and one sample (B) was additionally excluded. At least I want to see a critical discussion about this fact. Nevertheless, they supported the theory for using HPP processing in a well structured and clear way and supported it with nice and clear figures.

Other comments:

L28: et al. instead of at al.

L43: Recommendations From, no capital letters

L117: six instead of 6

L167 and L328: revealed instead of reveled

Reviewer #3: The authors of the manuscript compared the total and exosome-bound content of small noncoding RNAs (miRNAs) in human milk depending on two preservation methods (HoP and HPP). Authors showed that HPP is less detrimental to human milk miRNAs than HoP and thus has a potential as a processing procedures for human milk banks.

In general, the comparative analysis is very essential because could have an impact on the miRNA level in infants.

However, I have only small concerns about the study design, and believe that some additional experiments or data are required to support the conclusions.

Unfortunately, I was not able to find the access the sequences of the study. Please submit the .fastq data from miRNA to https://www.ebi.ac.uk/ena.

Authors used milk samples, obtained on the 50th day of lactation. Why not the 90th day or colostrum samples? Authors should explain the selection.

Do you have any information about the bacterial composition of the investigated milk (unprocessed vs HPP samples)? If yes, please provide or discuss. Latest studies as reviewed by Simpson et al, 2015 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4682386/ have highlighted that the expression of miRNAs is profoundly impacted by a variety of bacterial pathogens and that likewise miRNAs impose strong pressure to the invading microorganisms.

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Reviewer #1: Yes: Dr Okurut Emmanuel

Reviewer #2: No

Reviewer #3: No

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Impact of processing method on donated human breast milk microRNA content

PONE-D-20-07569R1

Dear Dr. Smyczynska,

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,

Tao Huang

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 #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 #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

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 #2: Yes

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 #2: Yes

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 #2: The authors addressed my comments and changed inaccuracies carefully. The argument on the low sample-size was highlighted and spelling mistakes were corrected. Also the comments of the other authors were addressed. Great work was done on an important topic.

Reviewer #3: (No Response)

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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: Yes: Christian Robben

Reviewer #3: No