Measuring the rate of NADPH consumption by glutathione reductase in the cytosol and mitochondria

Kenneth K. Y. Ting; Eric Floro; Riley Dow; Jenny Jongstra-Bilen; Myron I. Cybulsky; Jonathan V. Rocheleau

doi:10.1371/journal.pone.0309886

Peer Review History

Original SubmissionMay 10, 2024
18 Jul 2024 Decision Letter - Chen-Guang Liu, Editor PONE-D-24-18553Measuring the rate of NADPH consumption by glutathione reductase in the cytosol and mitochondriaPLOS ONE Dear Dr. Cybulsky, 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 submit your revised manuscript by Aug 30 2024 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. 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Additional Editor Comments: Dear Dr. Myron Cybulsky Thank you for sending the manuscript "Measuring the rate of NADPH consumption by glutathione reductase in the cytosol and mitochondria" to PLOS ONE for publication. Given the reviewer's comments, minor issues still need to be solved before acceptance. I have consolidated the review comments below, hoping they will help you modify the paper. Reviewer 1: I read with great interest the authors' manuscript entitled "Measuring the Rate of NADPH Consumption by Glutathione Reductase in the Cytosol and Mitochondria," which is intended for submission to PLOS ONE. Congratulations on this publication; it is a very thorough and valuable contribution. Since this is a laboratory protocol, my research team and I reviewed it to identify any questions we might have if we were to perform this measurement. We have only a few technical questions, which I have noted in the attached PDF. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes ****** 4. If the manuscript contains new data, have the authors made this data 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: N/A ****** 5. Is the article 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 highlight any specific errors that need correcting in the box below. Reviewer #1: 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: I read with great interest the authors' manuscript entitled "Measuring the Rate of NADPH Consumption by Glutathione Reductase in the Cytosol and Mitochondria," which is intended for submission to PLOS ONE. Congratulations on this publication; it is a very thorough and valuable contribution. Since this is a laboratory protocol, my research team and I reviewed it to identify any questions we might have if we were to perform this measurement. We have only a few technical questions, which I have noted in the attached PDF. Thank you for the opportunity to review the article. ****** 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: Yes: Petra Hartmann ******** [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: PONE-D-24-18553 reviewer.pdf https://doi.org/10.1371/journal.pone.0309886.r001
Revision 1
31 Jul 2024 Author Response RESPONSE TO THE REVIEWERS’ COMMENTS I would like to thank reviewer #1 for their insightful comments (copied below in bold font). Each point that was raised is addressed below. Changes to the text in the revised manuscript are highlighted in yellow. 1. Based on what did you use 10 ml of trypsin (usually a lot for a flask of this size)? Please describe this. What is the final concentration of trypsin? Is it necessary to cool the fugue to this cell line? We agree with the reviewer that 10 ml of trypsin is more than the typical recommended volume for a T75 flask. We have now adjusted the volume of both trypsin and DMEM, which deactivates the trypsin, to 3 ml (LINE 164 – 165). This volume is based on the recommendations from StemCell Technologies as we purchased the T75 flasks from this company (https://cdn.stemcell.com/media/files/pis/29301-PIS_1_0_2.pdf). We believe that changing the volume of trypsin will not change the results generated in our study. The final concentration of trypsin is 0.25% Trypsin/ 0.1% EDTA prior to deactivation with 10% FBS in DMEM. We are unsure of the last question. We assume that the reviewer is asking if it is necessary to cool the centrifuge for this cell line. Our response is that centrifugation was performed at room temperature or 22 degrees C (LINE 165). 2. What conditions were used during this time? We have now revised the manuscript and provided the recommended antibody concentration dilution in our method section, as well as the temperature of primary antibody incubation with PVDF membrane overnight (LINE 330 – 333). 3. Have you used a standard protocol for QPCR ? If not, please provide details of the final concentrations. Yes. We have used a standard protocol for QPCR. We have now revised the manuscript to provide more details of the final concentration and the QPCR protocol (LINE 356 - 358). 4. Please clarify the item numbers of groups. Only one group has an item number description. Please specify the item numbers of the groups. What statistical software used to? Please indicate this in the main text. We have now revised the manuscript and provided a paragraph in the method section, titled as “statistical analysis”, to clarify the definition of ‘n’ and the description of statistical software used to calculate the provided statistics (LINE 360 – 364). Attachments Attachment Submitted filename: Rebuttal Letter_MIC.docx https://doi.org/10.1371/journal.pone.0309886.r002
21 Aug 2024 Decision Letter - Chen-Guang Liu, Editor Measuring the rate of NADPH consumption by glutathione reductase in the cytosol and mitochondria PONE-D-24-18553R1 Dear Dr. Cybulsky, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. 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Kind regards, Chen-Guang Liu, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Dear Dr. Myron Cybulsky I am glad to inform you that the manuscript "Measuring the rate of NADPH consumption by glutathione reductase in the cytosol and mitochondria" could be accepted by PLOS ONE for publication. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes ****** 4. If the manuscript contains new data, have the authors made this data 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 ****** 5. Is the article 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 highlight any specific errors that need correcting in the box below. Reviewer #1: 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: The authors have answered the questions raised and I have no objections to the publication of this manuscript. ****** 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: Yes: Petra Hartmann ******** https://doi.org/10.1371/journal.pone.0309886.r003
Formally Accepted
28 Aug 2024 Acceptance Letter - Chen-Guang Liu, Editor PONE-D-24-18553R1 PLOS ONE Dear Dr. Cybulsky, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Chen-Guang Liu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0309886.r004

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