The final walk with preptin

Lucie Mrázková; Marta Lubos; Jan Voldřich; Erika Kužmová; Denisa Zrubecká; Petra Gwozdiaková; Miloš Buděšínský; Seiya Asai; Aleš Marek; Jan Pícha; Michaela Tencerová; Michaela Ferenčáková; Glenda Alquicer Barrera; Jakub Kaminský; Jiří Jiráček; Lenka Žáková

doi:10.1371/journal.pone.0309726

Peer Review History

Original SubmissionMay 9, 2024
21 Jun 2024 Decision Letter - Haim Werner, Editor PONE-D-24-18330THE FINAL WALK WITH PREPTIN?PLOS ONE Dear Dr. Žáková, 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 05 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. 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, Haim Werner 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. Thank you for stating the following financial disclosure: “The work was supported by the Czech Science Foundation (grant No. 19-14069S (to LZ) and 22-12243S (to MT)), by the National Institute for Research of Metabolic and Cardiovascular Diseases (Program EXCELES, ID Project No. LX22NPO5104) - Funded by the European Union – Next Generation EU and by the Academy of Sciences of the Czech Republic (Research Project RVO:6138963, support to the Institute of Organic Chemistry and Biochemistry).” 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. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: “The work was supported by the Czech Science Foundation (grant No. 19-14069S (to LZ) and 22-12243S (to MT)), by the National Institute for Research of Metabolic and Cardiovascular Diseases (Program EXCELES, ID Project No. LX22NPO5104) - Funded by the European Union – Next Generation EU and by the Academy of Sciences of the Czech Republic (Research Project RVO:6138963, support to the Institute of Organic Chemistry and Biochemistry).” We note that you have provided funding information that is 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: “The work was supported by the Czech Science Foundation (grant No. 19-14069S (to LZ) and 22-12243S (to MT)), by the National Institute for Research of Metabolic and Cardiovascular Diseases (Program EXCELES, ID Project No. LX22NPO5104) - Funded by the European Union – Next Generation EU and by the Academy of Sciences of the Czech Republic (Research Project RVO:6138963, support to the Institute of Organic Chemistry and Biochemistry).” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 6. 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: 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: 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: No ****** 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: Preptin previously was thought to have specific biological activities, such as stimulating bone growth, but the authors have done a thorough analysis and revealed no significant biological activity associated with preptin or its analogs, suggesting the associated functions of preptin may be influenced by factors, such as higher levels of IGF2 or IGF2 prohormones present in tissues. The authors claim has taken in the context of the previous literature, which when analysing their interesting and thorough data, I support their assumptions. The data is clearly reported and the manuscript written very clearly. The authors synthesized 16 different forms of preptin, including human and rodent analogues or fragments and they assayed them to test the interaction with the receptors to which IGF2 can bind and cells from target tissues previously associated with preptin activity, such as osteoblasts. Contrary to previous published indications no significant binding or cellular effects of preptin were seen. The explanation that as preptin is separated from pro-IGF2, then higher levels of preptin will also mean higher levels of IGF2 so that some of the symptoms contributed to actions of preptin may be due to higher levels of IGF2 is certainly feasible. This is backed up by the lack of phenotypic changes that were observed in preptin KO mice. Reviewer #2: This manuscript describes the chemical synthesis of preptin and a series of preptin analogues and analysis of their in vitro biological activities. There is some controversy in the literature as to the biological action of preptin, which arises during proteolytic maturation of the pro-IGF2 peptide. This study sought to clarify some of these controversies. The peptides were successfully chemically synthesised. Subsequent in vitro analyses, which were appropriate for gaining an understanding of action via the IGF/insulin receptors and biological processes downstream, essentially failed to demonstrate any significant activities for all of the peptides. The conclusions are sound and it is commendable for the authors to submit this for publication such that some further clarity is provided to the field. Comments/suggestions: The last two sentences of the introduction are vague and could be written more concisely. Page 3 please clarify “its levels negatively correlate with bone mineral density and osteoporosis” – correlate with lower or higher bone mineral density?? Page 3 please clarify “to approach?? the receptor for preptin, we performed several binding experiments” Page 10 last paragraph “after overnight starvation in a clean culture medium without serum” – not “cultivated” Page 14 first paragraph of results. “confirmed” – not “conformed” It is noteworthy that some of the peptide treatments result in a lower Akt phosphorylation than the control (Fig 3b). What is added to the control – is it the same vehicle as used for the peptides? Does this mean there is something inhibitory in the vehicle control? What was the rationale for using 10-8M peptide and how does this relate to the binding affinities derived in Fig 2? General comments on immunoblots: Was a positive control used to demonstrate the ability to activate Akt, Erk and PI3K? Were responses normalised to demonstrate that no changes in expression of Akt, Erk and PI3K have occurred during stimulation? No blots are provided and at minimum representative blots should be included (perhaps in the supplementary section) In the insulin secretion assay, it does not appear that the cells are producing significant amounts of insulin in response to the positive control (10mM glucose). Is this the case and if so how is it possible to comment then on the activity of the preptin peptides? Page 20 last sentence “(REF)” Figure 8 legend the English is not clear “treated with the adjacent? concentration of the compound” I am not sure if conclusions can be drawn from the alizarin S staining as there is considerable variability at the 0 ng/ml concentration treatments. It is hard to see how any statistical differences could be derived with this variability. ****** 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: Jillian Cornish Reviewer #2: Yes: Briony Forbes ******** [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. https://doi.org/10.1371/journal.pone.0309726.r001
Revision 1
2 Aug 2024 Author Response We would like to thank you and the reviewers for the careful assessment of our work and detailed comments. These comments were very valuable, helping us to clarify the major points of the paper and to remove ambiguities. We hope that we have addressed all concerns raised by the reviewers and implemented their suggestions effectively. Our point-by-point responses to the reviewers’ comments are as follows. All changes, which we made in the original manuscript are highlighted with track changes. Reviewers' comments: Reviewer #1: We thank Jillian Cornish, as one of the first authors working on preptin, for her positive review of our manuscript. We found no specific comments in her review that needed to be responded to. Reviewer #2: Thanks to Briony Forbes for her excellent and detailed review of our manuscript, and here are the answers to her questions: The last two sentences of the introduction are vague and could be written more concisely. We've rewritten it. Page 3 please clarify “its levels negatively correlate with bone mineral density and osteoporosis” – correlate with lower or higher bone mineral density?? We have edited the text into a more readable version. Page 3 please clarify “to approach?? the receptor for preptin, we performed several binding experiments” We've corrected it. Page 10 last paragraph “after overnight starvation in a clean culture medium without serum” – not “cultivated” It has been corrected. Page 14 first paragraph of results. “confirmed” – not “conformed” It has been corrected. It is noteworthy that some of the peptide treatments result in a lower Akt phosphorylation than the control (Fig 3b). What is added to the control – is it the same vehicle as used for the peptides? The reviewer is right here, sometimes the phosphorylation of Akt by several of our peptides does not seem to reach control values. This is due to the fact that the signal was very weak and there was a relatively large range of values for these weak signals, so the average values can fall below the control values. The vehicle was the same for all peptides and controls. The only values that seem inhibitory are for peptides 6-13, which are very short or stapled analogues of preptin, which are really significantly lower, and we discuss their low effect in the Discussion section. What was the rationale for using 10-8M peptide and how does this relate to the binding affinities derived in Fig 2? The 10-8M concentration was used for three reasons. First, to get closer to physiological conditions. Besides, we did signaling at higher concentrations, but the results were the same. As the reviewer correctly points out the second reason for using the 10 nM concentration results from the usual Kd values of insulin-like peptides (insulin and IGFs), which are not very different from 10 nM The third reason was the sensitivity of signal detection in Western blots, which is not sufficient for efficient detection of hormone activities below 10 nM. Was a positive control used to demonstrate the ability to activate Akt, Erk and PI3K? Were responses normalised to demonstrate that no changes in expression of Akt, Erk and PI3K have occurred during stimulation? In our first experiments, we used insulin and IGFs as a positive control and they signaled highly. Compared to these controls, our signals were very weak and when we repeated the experiments already with respective analogues, we did not use these positive controls and only looked at any effect compared to control unstimulated cells. No blots are provided and at minimum representative blots should be included (perhaps in the supplementary section) Representative blots were added (Figure S46) to the supplemental material. In the insulin secretion assay, it does not appear that the cells are producing significant amounts of insulin in response to the positive control (10mM glucose). Is this the case and if so how is it possible to comment then on the activity of the preptin peptides? It is true that 10 mM glucose does not come out significantly in our reported RIA essay, although the graph shows an increase. We do the RIA test with and with other secretagogues quite routinely and normally insulin secretion is significantly increased after stimulation with 10 mM glucose. Here, the non-significant behaviour is given by the ANOVA statistical test we used. In this test, the results are influenced by the whole evaluation group and not just two columns. If we used a t-test between only 0 mM and 10 mM glucose, the result would be significant (P = 0.0253 (i.e. P < 0.05)). Page 20 last sentence “(REF)” It has been corrected. Figure 8 legend the English is not clear “treated with the adjacent? concentration of the compound” It has been corrected. I am not sure if conclusions can be drawn from the alizarin S staining as there is considerable variability at the 0 ng/ml concentration treatments. It is hard to see how any statistical differences could be derived with this variability. Yes, we agree that Alizarin staining is difficult to evaluate statistically. In our case, Alizarin staining serves as a confirmation of ALP measurement, which, like Alizarin staining, shows the highest effect for IGF1, an effect also known from the literature. Neither IGF2 nor preptin achieve this effect and statistical increase in ALP. The paragraph related to funding has been removed from the manuscript and information on funding has been added to the Funding Statement section of the online submission form. We hope that the response together with changes and corrections will fulfill the reviewer’ as well as your requirements, and that this revised manuscript could be considered for publication in the PloS One. Please do not hesitate to contact me in case of any further queries. Attachments Attachment Submitted filename: Mrazkova_Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0309726.r002
19 Aug 2024 Decision Letter - Haim Werner, Editor THE FINAL WALK WITH PREPTIN? PONE-D-24-18330R1 Dear Dr. Žáková, 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. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Haim Werner Academic Editor PLOS ONE Additional Editor Comments (optional): Authors have satisfactorily addressed reviewer's comments. Reviewers' comments: https://doi.org/10.1371/journal.pone.0309726.r003
Formally Accepted
4 Sep 2024 Acceptance Letter - Haim Werner, Editor PONE-D-24-18330R1 PLOS ONE Dear Dr. Žáková, 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. Haim Werner Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0309726.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .