PONE-D-23-03975Bortezomib is more effective than carfilzomib in reactivating mutant human cystathionine beta-synthase  in micePLOS ONE

Dear Dr. Kruger,

Thank you for submitting your interesting manuscript to PLOS ONE. As you will note from the following/attached reviews, your work was critiqued by two experts in the field who both saw value in the study but also raised some valid concerns. In many cases, these can be addressed textually, but I would urge you to perform the controls noted by Reviewer #2. Because Reviewer #1 was concerned by the study power, which would require additional mice/experiments, here I would suggest--as advised by both reviewers--that you soften your conclusions as needed. A further discussion of key points is also needed in places, as is additional information on the study design/experimental procedures. Should you choose to resubmit your work, please note that the revised study would be re-reviewed by one or possibly both original reviewers, so please carefully address how the text and data have been revised. In addition, please submit your revised manuscript by May 19 2023 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.

Thank you again, and we look forward to receiving your revised manuscript.

Sincerely yours,

Jeffrey L Brodsky

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please make sure that all information entered in the 'Ethics Statement' section regarding ethics approval is also included in the Methods section of the manuscript.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. 

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. Thank you for stating the following financial disclosure: 

"This work was funded by grants from the National Institutes of Health (DK101404 to WK and CA006927 to Fox Chase Cancer Center)." 

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." 

If this statement is not correct you must amend it as needed. 

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

"This work was funded by grants from the National Institutes of Health (DK101404 and CA006927).  We thank Cynthia Myers for the synthesis of bimoclomol in the FCCC Organic Synthesis Facility.  We thank Xiang Hua and the FCCC Transgenic Mouse Facility for production of the Tg-T191M transgenic mouse, and the FCCC Laboratory Animal Facility for mouse husbandry.  We also thank Rita Michielli for mouse tail genotyping. "

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

"This work was funded by grants from the National Institutes of Health (DK101404 to WK and CA006927 to Fox Chase Cancer Center)." 

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

6. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

7. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

8. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. 

9. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. 

  

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

[Note: HTML markup is below. Please do not edit.]

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: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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

**********

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 is a well-written manuscript and the underlying work advances our understanding of the therapeutic potential of proteosome inhibitors. However, the authors should temper their conclusions with regard to the relative efficacy of the proteosome inhibitors, which at times oversimplify and overstate the findings. There are several specific areas that should be addressed. Please see attached:

Reviewer #2: In this paper, the authors continue their long-standing work treating transgenic mice with proteasome inhibitors to examine therapeutic potential. They have previously shown for (I believe) all of the mutants except for the T191M that bortezomib (BZ) rescues HCy clearance and CBS expression and activity.

The new contribution of this paper is comparing the effects of carfilzomib (CZ) to BZ. They find that CZ impairs hepatic proteasome activity but is largely ineffective at rescuing serum HCy and restoring CBS expression and activity with the exception of the S466L and I278T mutations (more on this later). The authors also perform some low doze BZ and CZ experiments, which are not very enlightening but probably merit inclusion nonetheless.

Largely as a description of comparative results I think the paper comes to appropriate conclusions, although I think the authors need to be even more explicitly cautious in their discussion. Not only does the dosing and route of administration potentially affect their outcomes, but so too does the fact that they are selecting only one time point for examination, and I could easily believe that the pharmacokinetics of the drug, combined with the synthesis and turnover of CBS, might yield different results if a more systematic examination of time points were conducted. The larger point of the paper, though, is that, despite the fact that it seems to inhibit the proteasome fairly robustly, CZ is not at least in these assays nearly as protective as BZ, which implies at least at face value that BZ works through proteasome-independent mechanisms. The authors mention this in the discussion, but I am a bit surprised that conclusion is not more front-and-center in the abstract.

Points for attention:

1. For CZ-treated animals were control mice injected and/or pumped (with water)? The methods do not indicate this, though they explicitly state that the BZ controls were pumped with diluent. If the controls for CZ were not so-treated, that would be in my opinion a pretty serious problem of rigor.

2. Related to the above, the experiment in Fig. 5 lacks (at least for the serum HCy and body weight) the control of animals injected with diluent on the same schedule. It is hard to interpret especially 5B without such a control. I can imagine that the stress of being repeatedly handled and jabbed might affect any number of physiological parameters. If that control wasn’t done, it either needs to be done or the experiment should not be included.

3. Figure 6—it isn’t clear that the Bimo had any effects on the system at all. A simple explanation is that the compound is not active as intended. Absent a way to confirm that it is doing something in vivo, it’s a really difficult experiment to interpret and I would take it out.

4. The I278T animals are strange. CZ diminishes serum HCy but without any increase in CBS expression or activity. How do the authors account for this?

5. Gels need migration standards.

6. Label font sizes in Fig. 2 are discrepant and the correlation graphs in Fig. 4 look like they were dropped straight from Excel. Showing treated animals to the left in 5E is a bit unconventional, and I would recommend keeping color designations (UT, BZ, CZ) consistent in Fig. 3. Overall for aesthetics I think the figures should be cleaned up.

**********

6. 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

**********

[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.

Attachments

Attachment

Submitted filename: Bortezomib vs. carfilzomib in CBS homocystinuria Review.docx

PONE-D-23-03975R1Examination of two different proteasome inhibitors in reactivating mutant human cystathionine beta-synthase in micePLOS ONE

Dear Dr. Kruger,

Thank you for re-submitting your manuscript to PLOS ONE. I have read carefully over the manuscript in light of the reviewers' comments, and prior to acceptance would suggest that the following minor issues are addressed. 1. Please edit/clarify the following added text, "in inducing in inducing", and "that the lowering..."2. Please add some additional details to the construction of the mouse strain in the Methods, with regard to Reviewer 1's comment on the background.3. Please be specific in the text with regard to "almost all the mice"...numbers would be helpful here.4. Based on the suggested title change, please add a sentence or two placing your work in the context of a prior paper on the effects of different proteasome inhibitors in another conformational disease (PMID: 18775310) Please submit your revised manuscript by Jun 09 2023 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.

Thank you again for submitting this interesting study to PLOS ONE, and I trust that these final changes will not be too laborious.

Sincerely yours,

Jeffrey L Brodsky

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.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[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.

Examination of two different proteasome inhibitors in reactivating mutant human cystathionine β-synthase in mice

PONE-D-23-03975R2

Dear Dr. Kruger,

Thank you for making these final edits to your paper--we are delighted to accept the paper for publication.

Within one week, you’ll receive an e-mail from the journal detailing any required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jeffrey L Brodsky

Academic Editor

PLOS ONE