PONE-D-19-27855

Non-invasive evaluation of the equine gastrointestinal mucosal transcriptome

PLOS ONE

Dear Dr. Whitfield-Cargile,

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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ACADEMIC EDITOR:

The authors have provided some valuable RNA seq data obtained using exfoliate and GI mucosal samples from healthy horses. They have compared these RNA seq and report a relatively strong correlation between them. The authors try to establish a non-invaisive methodology to obtain global gene expression pattern unique to GI tract. As suggested by reviewers 2 and 3, the correlation does not appear to be strong. This is a well-written manuscript and I would suggest the authors to respond to reviewers comments to increase the impact of the manuscript.

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We would appreciate receiving your revised manuscript by Jan 18 2020 11:59PM. When you are 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Sripathi M Sureban, Ph.D.

Academic Editor

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: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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: This study provides a very logical and detailed evaluation of how the exfoliome relates to the transcriptome. I have no substantial edits or comments to revise the paper and I am in support of its publication.

1. Line 38: Typo “healthy” horses.

2. Line 109: Typo “Irvine,” CA.

Reviewer #2: Overall this reviewer believes that this is a well written manuscript on a timely and interesting topic. However, there appears to be confusion as to the most recently accepted terminology for epithelial cell identity, particularly within the crypt base. A point by point review can be found below.

Abstract

Line 37-39: Please revise sentence. As written is appears that feces was directly collected from all sites in addition to mucosal samples. Line 38 misspelling ‘four healthy’ not ‘health’ horses.

Introduction

Line 63-64: Vague statement, I would recommend just stating what the main limitation(s) of ultrasound in the horse are. This is not an equine journal and it cannot be assumed that the average reader would know.

Lines 67-77: I found these lines implicit not explicitly written. Furthermore, if a sentence starts with ‘first’ then a following sentence should start with ‘second’. It is not obvious why endoscopic biopsies are inferior to “other” types of biopsies. This is inferred in the following sentences and this reviewer agrees with this statement however I am not sure the average reader would know the difference. I believe that this entire section should be re-written to make the description of limitations of each biopsy technique more clear and more concise.

Line 82-84: It is not clear what the author means by ‘mechanistic’? Identifying intestinal inflammation can be of diagnostic value and important for direct clinical interventions particularly because current clinical therapies for intestinal disease are limited. However, I would agree that diagnosing dysfunction more specifically may help direct future therapies.

Line 94-95: “global” is used twice in this sentence, please consider using another term

Line 96: The word transcriptome should be changed to exfoliome or mucosal transcriptome? Or both?

Line 98: The term exfoliome should be defined within the manuscript earlier and then used throughout the introduction.

Results

Table 1: I don’t believe the abbreviation for right dorsal colon (RDC) is stated within the text prior to use within the table. Please be consistent throughout using either RD or RDC for right dorsal colon. Rectal should be changed to Rectum.

Table 4: Exfoliome doesn’t appear to be very well correlated with mucosa from any section of the intestine although I appreciate that the values are positive? This is, however, stated very strongly throughout the manuscript.

Line 245-247: Stem cells, goblet cells and Paneth cells are all examples of intestinal epithelial cells, along with enterocytes, enteroendocrine and tuft cells. This sentence is written as if the author is not aware of all of the cell types that make up the intestinal epithelium? Please clarify.

Line 246: How is the author defining ‘crypt cells’? Do they mean transit amplifying cells? The crypt arguably consists of stem, Paneth/Paneth-like cells, goblet cells, and transit amplifying cells (small intestine versus colon).

Line 251: Should reference Gonzalez et al AJVR, particularly because this manuscript specifically identifies biomarkers in equine GI derived tissues.

Line 252: See above. Not sure what the author is meaning to represent with IECs? Furthermore, intestinal epithelial stem cells are commonly called crypt based columnar stem cells. Again, not sure what cell types the authors are trying to distinguish. Please clarify, as written there appears to be confusion as to all of the cell types and the nomenclature used for the types of intestinal epithelial cell types.

Line 265: Be consistent through manuscript. RD or RDC to represent the right dorsal colon.

Discussion:

There is a general lack of discussion on the pathways listed in Table 2. There doesn’t appear to be a point in listing the specific Canonical pathways. Although the reviewer finds these pathways to be interesting there is no discussion as to the significance of these findings.

The authors have failed to fully describe other limitations of this technique. Many times in disease GI motility is decreased or absent. How would this could further delay/impact the quality of RNA collected? Does the author propose a way to distinguish different segments of the bowel? It is unfortunate that the authors only chose to sample from the ileum as opposed to other segments of the small intestine such as the duodenum and jejunum where many inflammatory forms of enteritis arise. Similar comment with regard to the different segments of the colon.

Figure 1 Legend: Please better define what the yellow squares with associated error bars represent?

Supplemental Figure 2 Legend: Please include what the labeling on the x axis represents. Every figure and figure legend should stand alone and be defined.

Figure 4: Grouping of cells types is confusing on right side of figure. Normally the acronym CBC is used for crypt based columnar stem cells. If the author is generally grouping all crypt based columnar cells (not just stem cells) those would include intestinal stem cells, Paneth cells and transit amplifying cells (if small intestine). I would argue that hallmark biomarkers for crypt based columnar stem cells include Lgr5, Ascl2, Olfm4 to name a few (but these are the major ones). There is overlap between biomarker expression throughout the crypt and between active and reserve (quiescent) stem cells. Please review the literature for the most recent accepted nomenclature for the cells comprising the intestinal epithelial crypt.

With regard to the data represented in Fig 4B, there appears to be low expression of Paneth cell markers in the small intestinal samples and higher expression in the right dorsal colon and even the rectum in one horse? This is strange considering Paneth cells aren’t thought to exist in the colon? A comment on this is recommended.

Reviewer #3: The study is the first to compare the tissue and exfoliome transcriptomes in horses. The paper flows quite nicely, using appropriate lab techniques and statistical methods for data analysis. Conclusions are supported by their data.

Major comments:

1. Small sample size (n=4) and heterogeneous study subjects. Although all called "healthy" horses, they differ in terms of age and reasons for euthanasia. With such a small sample size, it is difficult to generalize the study findings to healthy horses at large.

2. The Spearman correlation coefficients between exfoliome and each tissue source range from 0.155 to 0.186. These are only considered week associations, albeit statistical significance. Also, the corresponding scatter plots did not show a clear positive correlation. Therefore, potential clinical usage of exfoliome is not very convincing. It would have been a much stronger study if the authors included both healthy and GI diseased horses and demonstrated difference between the groups using exfoliome.

Minor comments:

1. All the figures are of low resolution and very hard to see.

2. line 38: change 'health' to 'healthy'

3. line 214: define 'TMM'

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-19-27855R1

Non-invasive evaluation of the equine gastrointestinal mucosal transcriptome

PLOS ONE

Dear Dr. Whitfield-Cargile,

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.

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

ACADEMIC EDITOR: This is a very good study and the revised manuscript is significantly better than the previous versions. Please address the minor concerns raised by the review #2.

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

We would appreciate receiving your revised manuscript by Mar 21 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Sripathi M Sureban, Ph.D.

Academic Editor

PLOS ONE

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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: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: 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: Overall, this is a significantly improved manuscript. The authors have addressed almost all of this reviewer's concerns.

A few points that need to be addressed.

Abstract:

Line 41: There was a coorelation, questionable if this could be stated as 'strong', please revise.

Intro: Overall, a much nicer read!

Results:

Line 223: This statement is what is mis-represented in the abstract. As written, “There was a strong and significant coorelation of all tissues…an, although of lesser magnitude, significant correlation between the exfoliome and each tissue source”.

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 #1: No

Reviewer #2: No

Reviewer #3: 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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

Non-invasive evaluation of the equine gastrointestinal mucosal transcriptome

PONE-D-19-27855R2

Dear Dr. Whitfield-Cargile,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Sripathi M Sureban, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: