PONE-D-22-09375Trypanosoma cruzi PARP is enriched in the nucleolus and is present in a thread connecting nuclei during mitosis.PLOS ONE

Dear Dr. FERNANDEZ,

Thank you for submitting your manuscript to PLOS ONE. After careful review, we feel that it has merit but does not fully meet the criteria for publication in PLOS ONE in its current form. Therefore, we invite you to submit a revised version of the manuscript that addresses all the points raised by both reviewers..

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We look forward to receiving your revised manuscript.

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Claude Prigent

Academic Editor

PLOS ONE

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2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://www.cambridge.org/core/journals/microscopy-and-microanalysis/article/abs/nucleologenesis-in-trypanosoma-cruzi/1D537BAAFF5DD76FC04E13C85D41377C

The text that needs to be addressed involves lines 68-73 in your Introduction.

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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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

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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

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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

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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: I really enjoyed reading the manuscript of Kevorkian et al on PARP in T. cruzi. In my opinion, the authors presented a comprehensive story definitely worth publishing. I have one suggestion and one cosmetic critique.

Suggestion: please extend info on PARP in other trypanosomatids

Cosmetic critique: please fix references for consistency and compliance with PLoS One style (word capitalization)

Reviewer #2: See uploaded document for additional comments.

I thank the authors for their work, which I enjoyed reading.

I think overall the study shows promise and will open up new interest into PARPs in NTDs, but I feel there is a lack of quantitative data to support conclusions as most of the work is observational at this point.

That said, many of the core issues could be addressed by re-analysing images and clarifying aspects of certain experiments which I feel would vastly, and quickly improve this dataset.

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Reviewer #1: Yes: Vyacheslav Yurchenko

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Review_comments.docx

PONE-D-22-09375R1Trypanosoma cruzi PARP is enriched in the nucleolus and is present in a thread connecting nuclei during mitosis.PLOS ONE

Dear Dr. FERNANDEZ,

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 check reviewer2 comments, which seem relatively minor to me.

Please submit your revised manuscript by Dec 02 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,

Claude Prigent

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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: (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 #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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

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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

**********

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: All my concerns have been properly addressed. I think the revised version of the manuscript is an improvement over R0.

Reviewer #2: Reviewer 2 comments:

This reviewer wishes to thank the authors for their improvements to the manuscript -

the article has been significantly improved to accommodate suggestions by both reviewers.

1) Figure 1 in manuscript

I personally think it would be worth including the data under stress just so people are aware that there is no effect under these conditions – would be fine as a supplementary figure if preferred.

2) Also, why not use the inhibitor to look at spindle formation/cytokinetic defeats etc? Why not carefully profile the spindle progression across mitosis and the formation of the thread structure?

Reviewer response to author’s comment:

Suggestions that may help, though not necessary for this particular study. Perhaps the authors could consider employing expansion microscopy to help visualise the spindle thread better or even TEM could assist. Perhaps FISH of the telomeres to help define mitosis?

Whilst I do also completely agree with the authors it is challenging in parasites of this size, I also work exclusively on kinetoplastids and know it is very possible to examine the spindle and more miniature structures in this organism.

There are papers tracking spindle dynamics/cytokinesis etc in Leishmania and T. brucei. For example, Ambit et al. 2011 is a good example of this type of analysis (though in Leishmania) https://journals.asm.org/doi/10.1128/EC.05118-11

I also appreciate that defining the mitotic stages is not easy but perhaps looking through single cell transcriptomics/ cell cycle specific transcriptomics studies (re cell cycle) may help find potential markers of different cell cycle regulated proteins which could be tagged, for example. Though lacking in T. cruzi, profiles from related parasites would no doubt be useful in guiding some decisions.

Minor point 1:

Overall, regarding controls – it is fine to keep the main figure clear, but I would still put the corresponding controls in a supplementary figure to reassure readers that they have been done. Just because it doesn’t look pretty or fit neatly in a figure doesn’t mean they do not belong in a manuscript. Thus I would strong urge the authors to include all controls performed.

Even an image of a ponceau stained gel would be helpful. The comparison is not part of the questions, I understand, but if you already did the western blot then it would have been possible to do this without additional work. For additional bands on a western blot – perhaps indicating you are aware of them, and you are unsure of what they maybe would be helpful.

Reviewer comment:

What if you are not seeing phenotypes simply due to poor overexpression of one truncated piece Vs another – looks like from the WB that the expression of the recombinant version of TcPARP is different in each cell line.

Reviewer response to author’s comments:

Though the authors are saying that it is not the focus of the study,

you are comparing how cells have grown relative to WT without any indication of how well your experimental system is performing. As said by the authors – you cannot control the level of expression, so it is possible that comparing the different construct expressing strains to the WT is not showing a phenotype because that construct is not expressing the protein at a high enough level when compared to the WT level?

Perhaps worth considering this as a possibility irrespective of the focus of the paper for future works.

Minor points 2:

Line 62: have shown only one PARP and PARG ---- perhaps “posses (?) only one PARP and PARG”

Line 63 – reference for this statement

Lines 91-97 – please add reference (s) for these statements

Line 235: Did or did not (sorry in my version it reads “did observe significant…” but I presume you mean didn’t!

Line 241: I would suggest “but since we worked with cultures during exponential growth, the reason for the nucleolar marker de- localization in our model remains unknown. “

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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.

Trypanosoma cruzi PARP is enriched in the nucleolus and is present in a thread connecting nuclei during mitosis.

PONE-D-22-09375R2

Dear Dr. FERNANDEZ,

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.

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Claude Prigent

Academic Editor

PLOS ONE

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