PONE-D-20-02190

The production profile of lipid metabolites in urine of marmoset with wasting syndrome

PLOS ONE

Dear Prof. Murata,

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"This work was supported by the Cooperative Research Program of Primate Research Institute, Kyoto University, the Japan Society for the Promotion of Science, the Takeda Science Foundation, the Nipponham Foundation, Hoyu Science Foundation, and the Futaba Foundation to T.M.."

We note that one or more of the authors are employed by a commercial company: CLEA Japan.

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Reviewers' comments:

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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: Partly

Reviewer #3: No

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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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: No

Reviewer #3: No

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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 work deals with PUFA selected metabolites in Marmosets suffering MWS. Because the cause is still unknown the subject should be deemed quite interesting. By comparison to the attached comments to the previous reviews I find the work almost acceptable for PLoS ONE. Unfortunately, we still do not know what is the real cause of MWS but the authors provided some mediators and mechanism for the development of that illness.

Some minor remarks:

- Please add clearly in the conclusion that the cause of MWS remains unknown but the paper revealed some mechanisms and mediators of MWS

- Lines 79-83: the authors provide information about different types of cages used in the experiment. Please make it clear for the reader when and why such cages were used. I suppose that an individual cage is ideal for urine sampling. If another cages were used, how the urine was sampled? Add appropriate info sentence.

Reviewer #2: Thank you for your interest in MWS, not enough researches are focused on this to find a solution.

As much as I enjoyed your paper, I have a number of reservations that need to be addressed before I can recommend it for publishing. Find the notes added to the manuscript for details.

In large:

1) Diarrhoea itself does not equal MWS. You need to add a section on how you identified MWS. From the read of your paper, it sounds like you can't say you were looking at MWS but maybe focus on inflammation or chronic diarrhoea. If they do have MWS maybe you can present weight loss data.

2) You could do a better literature search, recent papers on MWS have not been included - see text for reference.

3) Can you give us the nutrient concentrations of their diet? How much AA, DHA and EPA are they actually ingesting?

4) as it stands, all your results support is that there is inflammation in animals with diarrhoea. You need the diet information to link with AA. If they eat the same diet and same amount of fatty acids, then causation will be somewhere else. You need the diet information to tie this all together, or change the scope of your paper to inflammation and darrhoea.

Reviewer #3: I commend the author's efforts to try and shed light on to disease like WMS which remains a mystery even after so many attempts, however I do think there are some major flaws in the design. I do understand that sample sizes can be challenging but in a prevalent colony as described by the owners, a n of 7 for both groups do significantly limit the conclusions that can be drawn.

The title says production profile - I would remove production.

I do think there are many authors, actually more authors than study subjects and it would be good to know if they satisfied the criteria for authorship.

Ln24: It is a syndrome and hence more symptoms are needed to justify the syndrome.

Ln 34: I do not think given the low number of subjects, a substantial conclusion can be got from this.

Ln 42: I do think it is redundant to have steroid and glucocorticoids in the same line

Ln 72: Sampling method should be elaborated. LN 104; Were there liners? How did you clean them in between. If feces and urine fall on the same surface,how does one confirm there is no residual effect of the feces. How do you know these inflammatory lipids were not from a biofilm on the floor? The trays needs to be described?

ln 84 onwards: Diet is a huge component of serum and urine lipid profiles. They should have looked at animals on the same diet to see how they compared.

Ln 104: Urines should be replaced with urine samples. What was the duration between voiding and urine collection and storage. Volatile lipids can potentially be lost.Marmosets in certain facilities are trained to urinate, was an attempt for this made

LN 111 onwards:What was the normalization technique used?Was the quantity of metabolites normalized to USG or creatinine.

There should also be some way to look into if there was active sediment or not, or at least clinical signs or something to substantiate that these mediators were not produced within the urinary tract as a result of inflammation or infection. WMS is syndrome and hence this is important.

ln 146: This is an over stretch and this conclusion is not substantiated.

I do agree with the other reviewers that the CV and the study design and conclusions have severe limitations,this along with small number of marmosets make me question the validity of these findings. While I do understand how difficult it is to work with marmosets, and samples can be scarce, I do not think the current study can be published as isin good faith.Sorry

182- urines should be replaced with urine samples.

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Reviewer #1: No

Reviewer #2: Yes: Francis Cabana, PhD

Reviewer #3: Yes: Joseph Cyrus Parambeth

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Attachments

Attachment

Submitted filename: PONE-D-20-02190_reviewerFC.pdf

PONE-D-20-02190R1

The profile of lipid metabolites in urine of marmoset with wasting syndrome

PLOS ONE

Dear Prof. Murata,

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.

We would appreciate receiving your revised manuscript by Jun 19 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,

Petr Heneberg

Academic Editor

PLOS ONE

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 #3: (No Response)

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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 #3: Yes

**********

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

Reviewer #1: 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 #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 #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: In my opinion the authors improved the text according to the questions raised by the reviewers. It is acceptable for publication.

Reviewer #3: Good job on the revisions. Thank you.

I do thing this needs some language editing and proof checking.

Please check the manuscript for language errors.

- 45 MWS. There is also no definitive diagnosis method for MWS.

insert 'antemortem'

89 Vitamin D. In CLEA, they were fed on 40-50 g CMS-1M (CLEA japan,

- J should be uppercase in Japan

95 such as fecal condition, appetite, hair condition and movement, and if they were suspected *any

96 problems, they would be **consulted with veterinarians.

- Add *to have, ** evaluated by the veterinarian

105 and/or antibiotics, and persistent weight loss [2]. The veterinarians in each institution assessed the

106 animals and *included in this study.

-*Prior to inclusion in the study

157 We comprehensively measured

- delete comprehensively

257 *multifactorial disease and the cause of it remains unclear,

- please add a

In all of the below line numbers, I do think they should say, marmosets with MWS, rather than MWS

109 however, that of MWS were around or less than 300 g at urine samples collection. Frequent and

110 recurrent diarrhea, and persistent weight loss for over six months were observed in MWS

116 as healthy (N = 7) in primary care or MWS (N = 7).

172 acid (DHA) in heathy (N = 7) and MWS (N = 7)

175 The urinary levels of arachidonic acid (AA)-derived catalyzed metabolites in heathy (N = 7) and

176 MWS (N = 7). di. dihydro; te, tetranor; COX, Cyclooxygenase; CYP, CYP450 epoxygenase; LOX,

177 lipoxygenase. *p<0.05, **p<0.01 compared to healthy urines.

180 The urinary levels of arachidonic acid (AA)-derived isoprostanes produced by non-enzymatic

181 oxidation in healthy (N = 7) and MWS (N = 7). **p<0.01 compared to healthy urines.

186 metabolite in healthy (N = 7) and MWS (N = 7). *p<0.05 compared to healthy urines. LOX,

187 lipoxygenase; COX, cyclooxygenase; CYP, cytochrome P450.

218 urinary lipid metabolites in MWS. These results may reflect that MWS marmosets may have

222 in plasma, 13,14-dihydro-15-keto derivatives, were increased in the urines of MWS [22]

231 amounts of PGE2, PGF2α and a metabolite or TXA2 were markedly increased in the urines of MWS.

234 ameliorated dextran sodium sulfate–induced colitis in mice [26]. Collins D et al. showed in an in vitro

240 In this study, we found that F2-IsoPs were increased in the MWS urine samples

250 this study, only 5 types of n-3 PUFAs-derived metabolites were constantly detected in MWS urines

The abstract has many language errors

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Reviewer #1: Yes: Jerzy Juskiewicz

Reviewer #3: Yes: Joseph Cyrus Parambeth

[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.]

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The profile of lipid metabolites in urine of marmoset wasting syndrome

PONE-D-20-02190R2

Dear Dr. Murata,

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