Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides

Alexander P. Schwarz; Veronika A. Nikitina; Darya U. Krytskaya; Ksenia P. Shcherbakova; Alexander N. Trofimov

doi:10.1371/journal.pone.0273224

Peer Review History

Original SubmissionJanuary 26, 2022
14 Jul 2022 Decision Letter - Dragan Hrncic, Editor PONE-D-22-02598 Reference gene selection within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides PLOS ONE Dear Dr. Schwarz, 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:- please do follow directions of reviewers to improve your manuscript ============================== Please submit your revised manuscript by Aug 26 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. 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Reviewer #1: Yes Reviewer #2: Yes ******** 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: In the present manuscript PONE-D-22-02598 authors investigated the effects of expression stability of 9 housekeeping genes widely used as reference and selected suitable reference genes for RT-qPCR analysis within the brain structures in rat model of mild ketosis. They used contemporary methodology and generated a large number of interesting results with potential interest to genetics-oriented readers. The manuscript should be improved for clarity according to provided comments and directions. Minor points Title - Title should be changed to better reflect a full content of the manuscript. - Maybe, one form of the title could be:” Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides” General - English should be checked with corrections of some typos. Abstract - Please, introduce all abbreviations in appropriate places. - Avoid starting this section with an abbreviation on the first place of the sentence. - Address the impact of model of mild ketosis induced by medium-chain triglyceride supplementation and its relationship to genes expression. Introduction - Add a paragraph and explain the importance of selected genes, and describe some other studies which investigated the similar problematic. - At the end of this section should be placed the aim of this study. Material and methods - In Animals and study design subsection, please add the number of ethical permission. - Also, your manuscript will benefit by adding of one new figure with experimental timeline, which should be placed in Animals and study design subsection. Results - In Figures 3-5, for more clarity, colors could be additionally added, but it is not mandatory Discussion - Some typos should be corrected. Reviewer #2: Positive comments 1. The paper is well written 2. The rationale behind executing the project is sound 3. The selection of initial reference genes for testing was based on the role of different genes in cell functions and also based on available literature Revision Needed 1. Authors need to explain the PCR efficiency of the primers in the manuscript 2. Why were the authors using 2 mL/kg daily? Please give some supportive references. 3. Line no. : 154: "RefFinder® online tool (https://www.heartcure.com.au/reffinder/)". Delete the website address as it was mentioned once in line 129. 4. In general, the authors should be writing medium-chain triglycerides (MCT) as either ‘MCT’ or ‘MCT oil’ in whole article. 5. Though gene expression was normalized with the three reference genes selected, this data still needs to be validated for gene expression with the bottom genes. This further proves the validity of the reference genes selected (Sahu., et al 2018) ******* 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: Yes: Nikola Šutulović 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. 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Revision 1
3 Aug 2022 Author Response Prof. Dragan Hrncic, MD, PhD Academic Editor PLOS ONE Dear Dr. Hrncic, Thank you and the reviewers for working on our manuscript and all the valuable comments and suggestions. We have prepared a revised version of the article with the changes marked. Briefly, we have corrected some typos throughout the manuscript and changed the title according to the reviewer's suggestion. Please find the detailed answers to the reviewers’ comments below. Reviewer 1 «Title - Title should be changed to better reflect a full content of the manuscript. - Maybe, one form of the title could be:” Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides”». We changed the Manuscript title according to the reviewer’s suggestion. «Abstract - Please, introduce all abbreviations in appropriate places. - Avoid starting this section with an abbreviation on the first place of the sentence. - Address the impact of model of mild ketosis induced by medium-chain triglyceride supplementation and its relationship to genes expression.» We edited the abstract to mention the full name of the method and added abbreviations’ disambiguation whereby needed (e.g. MCT, RT-qPCR). We also added two sentences describing our model of mild ketosis: this approach allows to reproduce certain neuroprotective effects of the classical ketogenic diet while avoiding its adverse effects. Ketogenic treatment targets multiple metabolic pathways, which may affect the reference gene expression. «Introduction - Add a paragraph and explain the importance of selected genes, and describe some other studies which investigated the similar problematic. - At the end of this section should be placed the aim of this study.» We rearranged this section, moving the paragraph about the aim of the study to the end of the section. We also added information about the functional properties of the analyzed gene products. «Material and methods - In Animals and study design subsection, please add the number of ethical permission. - Also, your manuscript will benefit by adding of one new figure with experimental timeline, which should be placed in Animals and study design subsection.» We added the permission number and created a figure with the experimental timeline (Figure 1 in the revised version). «Results - In Figures 3-5, for more clarity, colors could be additionally added, but it is not mandatory» We made colored versions of the graphs (figures 4-6 in the revised manuscript). Reviewer 2. 1. «Authors need to explain the PCR efficiency of the primers in the manuscript». We added the information about the range of measured PCR efficiencies to the Method section. The full information is presented in the Supplementary figures S4-S6. 2. «Why were the authors using 2 mL/kg daily? Please give some supportive references.». We used this dosage to mimic typical low doses used in human studies. We added the supporting references in the Materials and Methods section. 3. «Line no. : 154: "RefFinder® online tool (https://www.heartcure.com.au/reffinder/)". Delete the website address as it was mentioned once in line 129.» We deleted the repeated mention of the URL. 4. In general, the authors should be writing medium-chain triglycerides (MCT) as either ‘MCT’ or ‘MCT oil’ in whole article. We chose MCT for the whole article in the revised version. 5. Though gene expression was normalized with the three reference genes selected, this data still needs to be validated for gene expression with the bottom genes. This further proves the validity of the reference genes selected (Sahu., et al 2018) We would like to thank Reviewer 2 for this suggestion, but we decided not to change our article in this way. Of course, the calculations for the genes of interest to the unstable genes could be used as a vivid illustration of the biases resulting from incorrect normalization. However, it would be a good approach if we were working with «marker» genes that moderately changed under the analyzed experimental conditions. In our case, we work with a new model, which is not well described in rats. So, we feel that the calculation of custom gene of interest expression normalized to different references would make our article more complicated and would not clarify the results. We are looking forward for your review of the revised manuscript. Alexander Schwarz Ph.D., Research Fellow Laboratory of Molecular Mechanisms of Neuronal Interactions I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry St. Petersburg, Russia Aleksandr.Pavlovich.Schwarz@gmail.com Attachments Attachment Submitted filename: Schwarz_et_al_Response_to_Reviewers.docx* https://doi.org/10.1371/journal.pone.0273224.r002
5 Aug 2022 Decision Letter - Dragan Hrncic, Editor Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides PONE-D-22-02598R1 Dear Dr. Schwarz, 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 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, Prof. Dragan Hrncic, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0273224.r003
Formally Accepted
26 Sep 2022 Acceptance Letter - Dragan Hrncic, Editor PONE-D-22-02598R1 Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides Dear Dr. Schwarz: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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 Professor Dragan Hrncic Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0273224.r004

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