n-Butylidenephthalide recovered calcium homeostasis to ameliorate neurodegeneration of motor neurons derived from amyotrophic lateral sclerosis iPSCs

Yu-Chen Deng; Jen-Wei Liu; Hsiao-Chien Ting; Tzu-Chen Kuo; Chia-Hung Chiang; En-Yi Lin; Horng-Jyh Harn; Shinn-Zong Lin; Chia-Yu Chang; Tzyy-Wen Chiou

doi:10.1371/journal.pone.0311573

Peer Review History

Original SubmissionMay 16, 2024
14 Jun 2024 Decision Letter - Stephen D. Ginsberg, Editor PONE-D-24-19706n-Butylidenephthalide recovered calcium homeostasis to ameliorate neurodegeneration of motor neurons derived from amyotrophic lateral sclerosis iPSCsPLOS ONE Dear Dr. Deng, 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. ============================== After careful consideration by 2 Reviewers and an Academic Editor, all of the critiques of both Reviewers must be addressed in detail in a revision to determine publication status. If you are prepared to undertake the work required, I would be pleased to reconsider my decision, but revision of the original submission without directly addressing the critiques of the Reviewers does not guarantee acceptance for publication in PLOS ONE. If the authors do not feel that the queries can be addressed, please consider submitting to another publication medium. A revised submission will be sent out for re-review. The authors are urged to have the manuscript given a hard copyedit for syntax and grammar. ============================== Please submit your revised manuscript by Jul 29 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, Stephen D. Ginsberg Section Editor PLOS ONE Journal 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 in the Acknowledgments Section of your manuscript: 'The authors are grateful to the funding from National Science and Technology Council in Taiwan (MOST 111-2314-B-303-027-MY2) and the support from the core facilities provided by the Instrument Center of Department of Medicine Research, Buddhist Tzu-Chi General Hospital and Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan.' 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: 'Funder: National Science and Technology Council Award Number: MOST 111-2314-B-303-027-MY2 Recipient: Chia-Yu Chang This funder provides study design, data collection, and analysis. They also make decisions regarding publication.' Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A 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: The authors follow an interesting strategy and show possible targets for a neurodegenerative disease such as ALS, for which there is currently no cure.The structure is very easy to understand and clearly arranged with a very nice summarizing figure 6, even if the introduction almost fails to mention more recent publications/studies, especially in the context of ALS mutations and Ca-imaging. (e.g. PMID 37595581 or PMID 31105804). Furthermore, the authors should revise the results and discussion section again and make sure that there is not already too much discussion in the results section. Besides that the limitations of this study should be mentioned in the discussion, especially with regard to the treatment or the treatment concentrations/ treatment time, cell culture effects, etc. In addition, clarification is needed on the experiments carried out, particularly in relation to the n-number: 1.) What does the sentence in line 321 “Mean ± SD of n = 4 independently treated cultures per treatment group” mean? Normally at least 2-3 biological replicates with 2-3 technical replicates should be made in the cell culture to exclude possible passages/aging effects in the cell culture. Thus, at least 2+ different passages should have been differentiated to MNs. In addition, where do the concentrations of the BP treatment come from, has an MTT-/LDH-assay been carried out with regard to the tolerability of the treatment to figure out the best concentration. 2.) The number of MNs tested in calcium imaging with only 9 individual MNs per group is too low. Under "normal lab" conditions, this is just the minimum number of cells that should be examined in a single Ca imaging measurement. Therefore, more MNs would be necessary, e.g. through further technical replicates AND biological through further differentiations. --> It would have been nice to show the possibly different Ca baselines between the lines first, especially with the isogenic control line (=analysis of basal intracellular Ca2+ levels and spontaneous Ca2+ signals is missing). --> Furthermore, the authors discuss several times that treatment with BP also has an effect on different receptors. The question then arises as to why this was not also tested with S-AMPA or NDMA, for example, in order to detect their effects on the release of Ca. 3.) Figures 3-5 lack information on the amount of loaded protein and the n-number of analyses for all Western blots. If the blot was only done once, where do the error bars in the graphs come from? If several blots were made, then point out to the reader that the blot shown is a “representative blot” and show all blots for the respective figures in the Supp section. Finally, all graphs should be displayed as box and whiskers plots including “show all points” to make the results clearer for the reader. Reviewer #2: In the manuscript “n-Butylidenephthalide recovered calcium homeostasis to ameliorate neurodegeneration of motor neurons derived from amyotrophic lateral sclerosis iPSCs” the authors tested the protective efficacy of n-Butylidenephthalide (BP) on the survival of motor neurons (MN) derived from patients with Amyotrophic lateral sclerosis (ALS). They found that BP treatment restored the “normal” channels function and activated autophagic pathways that attenuated neuron degeneration. They suggest that BP is a promising candidate for future treatment of ALS. While the data is well-organized, and experiments were conducted properly, there are some concerns that must be addressed before is achieved a suitable version of the manuscript for publication. Main concern The scheme depicted in figure 6 does not clearly illustrate the obtained results and include elements not studied in the research performed (i.e. NCX, PMCA). Furthermore, no synapse-specific tests were performed in the conducted experiments, all data is related to “post-synaptic” effects. Please clarify the concepts outlined in the figure and redraw the diagram. Minor comments Line 68 mistyped word: “antagonized” change to antagonize. Figure 2. It would be helpful to add the cell type in labels where the BP was applied (i.e. BP-G85R) ****** 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: Yes: Adán Dagnino Acosta ******** [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.0311573.r001
Revision 1
13 Sep 2024 Author Response Dear reviewers, We followed Oriel Jerome Delas Alas Vida’s suggestion and incorporated raw data, and shared the "minimal data set" for the manuscript to reflect the suggestions that provided on 8/28. The content includes: � Figure 1E: Original immunofluorescent images and quantified data of HB9/ DAPI. � Figure 3B (NMDAR1), 3D (GluR3), 5D (activated caspase 3), 6B (LC3BII), 6D (p62): Original blotting images and quantified data of Western Blot. � Figure 5B: Original immunofluorescent images and quantified data of neurofilament/ DAPI. � Figure 2C: The quantified data of basal calcium content. � Figure 2G, 2H: The quantified data of G85R, BP-G85R, and G85G under KCl and L-glutamate explored. � Figure 4D, 4E, 4F: The quantified data of G85R, BP-G85R, and G85G under agonist (S-AMPA, NMDA and S-AMPA+NMDA). We appreciate the time and effort that you have dedicated to providing insightful feedback to strengthen our paper. The following is a point-by-point response to the questions and comments has been raised. Comments from reviewers and the responses: Reviewer 1: Major revisions 1. The authors follow an interesting strategy and show possible targets for a neurodegenerative disease such as ALS, for which there is currently no cure. The structure is very easy to understand and clearly arranged with a very nice summarizing figure 6, even if the introduction almost fails to mention more recent publications/studies, especially in the context of ALS mutations and Ca-imaging. (e.g. PMID 37595581 or PMID 31105804). [RESPONSE]: Thank you very much for reminding us and making our content better. The introduction has been updated to include the recent publications. In response to your suggestion, PMID 37595581 was included to introduce that ALS-associated mutations exhibit impaired calcium homeostasis, leading to changes in excitability, failure of action potential conduction, and activation of various calcium-dependent degenerative pathways (Lines 51 (Yang et al., 2023 (14)). However, PMID 31105804 primarily discusses the Thrifty Food Plan recommendations, which do not appear to be related to ALS or motor neurons. Additionally, we reviewed the publications by the authors of that article but were unable to find any that are related to ALS mutations and calcium imaging. Therefore, a reference reviewing the excitotoxicity caused by calcium dysregulation in ALS has been cited instead (Line 51, Verma et al., 2022 (15); PMID: 35078537). 2. Furthermore, the authors should revise the results and discussion section again and make sure that there is not already too much discussion in the results section. [RESPONSE]: Thank you for your suggestions to improve our content and structure. Thank you for your suggestions to improve our content and structure. The results section has been revised to avoid excessive discussion (lines 172-174, 183-186, 202-203, 212-215, 222-224 of the original manuscript). Additionally, redundant introductory statements that were already provided in the introduction have been deleted. 3. Besides that the limitations of this study should be mentioned in the discussion, especially with regard to the treatment or the treatment concentrations/ treatment time, cell culture effects, etc. [RESPONSE]: Thank you for your suggestions to enrich our content and make it more comprehensive. We have addressed the following limitations in the discussion section: � Drug’s concentration and treatment duration: We did not conduct extensive testing of BP’s effective concentration and treatment duration. However, BP has been previously applied in neurological models, including both animal and cell models, which may allow us to predict the potential effective concentration and treatment duration in our model based on prior reports (Line 285-287). The results, including the effective dose (ranging from 10 to 530 μM) and the effective treatment duration (ranging from 6 to 72h) in cell models, were revealed in this study as expected. � Comprehensive study on the mechanism: In this study, we focused on the mechanisms by which BP regulates calcium influx, calcium-associated receptors, and improves the survival of neuron cells. Systematic approaches, such as RNA sequencing or proteomic analysis, to further uncover the detailed mechanisms of BP remain to be explored in future research. (Lines 287-290). � Relevance of cell model: We utilized SOD1G85R mutant MNs as the cell model in this study, which limits the findings to a specific type of familial ALS. The applicability of our findings to multiple genetic mutations and sporadic ALS needs further investigation (Lines 290-292). Moreover, a comprehensive 3D organoid or animal model may better simulate the ALS disease condition than using a single cell type when studying the treatment potential of a drug, and this approach can be further improved in future studies. (Lines 292-294) 4. In addition, clarification is needed on the experiments carried out, particularly in relation to the n-number: 1.) What does the sentence in Line 321 “Mean ± SD of n = 4 independently treated cultures per treatment group” mean? Normally at least 2-3 biological replicates with 2-3 technical replicates should be made in the cell culture to exclude possible passages/aging effects in the cell culture. Thus, at least 2+ different passages should have been differentiated to MNs. [RESPONSE]: Thank you for your suggestions so that we can better clarify the content. Originally, two biological replicates and two technical replicates were performed to conduct statistical analysis. Although two different passages were tested in the original submitted version, we have included three biological replicates (G85R: passage numbers 40, 32 and 25; G85G: passage numbers 44, 35 and 29) with three technical replicates in the revised manuscript to mark our statements more convincing. The updates are on Figure 1E. 5. In addition, where do the concentrations of the BP treatment come from, has an MTT-/LDH-assay been carried out with regard to the tolerability of the treatment to figure out the best concentration. [RESPONSE]: Thank you very much for your suggestion. The concentrations of BP treatment were based on a previously published iPSC model (Chang, 2014) from our group. MTT assays were used to determine the cytotoxicity of BP on DS-iPSC-derived neurons (concentrations tested: 0, 10, 50, and 100 μM). The results showed that 100 μM BP caused significant cell death in DS-iPSC-derived neurons, while 50 μM BP resulted in 80% cell viability. Based on these results, 10 μM and 20 μM were selected for our study. We added the reference on Line 155. 6. 2.) The number of MNs tested in calcium imaging with only 9 individual MNs per group is too low. Under "normal lab" conditions, this is just the minimum number of cells that should be examined in a single Ca imaging measurement. Therefore, more MNs would be necessary, e.g. through further technical replicates AND biological through further differentiations. [RESPONSE]: Thank you for the constructive opinion. We have repeated the study to further improve this figure. MNs (D42-D46) were subjected to three replicate experiments, and the AMPAR and NMDAR agonists were introduced to present the overactive calcium influx situation. Additionally, Fluo-4 was used instead of Fura-2 because of the repair and replacement of the detection instruments. The number of evaluated MNs was also increased to n = 95, 55, and 49 for the G85R, BP-G85R, and G85G groups induced with KCl and L-glutamate, respectively, and to n = 30, 41, and 32 for the G85R, BP-G85R, and G85G groups induced with agonists, respectively. In brief, compared with the G85G group (control), the G85R group showed distinct difference when stimulated with KCl and L-glutamate, respectively. In G85R group, it showed a significant decrease in calcium influx (△F/F0) under KCl (0.518 ± 0.3356) and a greatly increase in calcium signaling under L-glutamate (3.130 ± 1.3304). Moreover, BP treated-group (KCl: 1.063 ± 1.4433; L-glutamate: -0.044 ± 0.3722) has the same trend as G85G (KCl: 1.258 ± 0.8873; L-glutamate: 0.265 ± 0.1904). In addition, stimulating three groups (G85R, BP-G85R and G85G) with glutamate receptor agonist also showed that G85R (S-AMPA: 0.491 ± 0.4262, NMDA: 0.147± 0.1312; S-AMPA + NMDA: 0.596 ± 0.5328) had a higher calcium signal, while BP-G85R (S-AMPA: 0.130 ± 0.0617; NMDA: 0.055 ± 0.0773; S-AMPA+NMDA: 0.247 ± 0.1486) and G85G (S-AMPA: 0.044 ± 0.0724; NMDA: 0.093 ± 0.0508; S-AMPA+NMDA: 0.017 ± 0.0888) had a significant downward trend. These findings suggest that BP treatment can restore voltage-gated influx and prevent glutamate related transmitter-induced excitotoxicity in MNs carrying an ALS-associated SOD1 mutation to a healthy state. These results are presented in Figures 2G, 2H, 4D, 4E, and 4F, and are described in the figure legends and in the manuscript (Lines 189 to 199; 212 to 222). 7. It would have been nice to show the possibly different Ca baseLines between the Lines first, especially with the isogenic control Line (=analysis of basal intracellular Ca2+ levels and spontaneous Ca2+ signals is missing). [RESPONSE]: Thank you very much for pointing out what we need to improve to make our content more complete. The figures have been revised to present the Ca2+ baselines (Figure 2C) and spontaneous Ca2+ signals (Figure 2A and 2B) at the beginning in G85R and G85G group. The results showed no significant difference in basal intracellular calcium levels and spontaneous calcium transients of MNs among these two groups (Fig 2A, 2B and 2C, G85R: 0.196 ± 0.1251, G85G: 0.154 ± 0.1035). The conclusion is described in Line 185 to 187. 8. Furthermore, the authors discuss several times that treatment with BP also has an effect on different receptors. The question then arises as to why this was not also tested with S-AMPA or NDMA, for example, in order to detect their effects on the release of Ca. [RESPONSE]: Thank you for your suggestions to make our content more complete. To assess the impact of BP on different glutamate receptors, we utilized specific agonists for NMDA and AMPA (S-AMPA, NMDA, S-AMPA+NMDA) to stimulate three groups of cells (SOD1G85R, SOD1G85R+BP, SOD1G85G; Figure 4A, 4B and 4C). The results, as shown in Figures 4A, 4B, and 4C, with statistical analysis of the ΔF/F0 ratio presented in Figure 4D (S-AMPA), 4E (NMDA), and 4F (S-AMPA+NMDA). Our calcium image results reflected the findings from the Western blot results in Figure 3. These data indicated that BP not only reduces the abnormal overexpression of NMDAR1 and GluR3 in SOD1G85R MNs but also decreases the calcium flux in response to NMDA and AMPA-specific agonists. The description of these results is provided in Lines 208 to 224. 9. Figures 3-5 lack information on the amount of loaded protein and the n-number of analyses for all Western blots. If the blot was only done once, where do the error bars in the graphs come from? If several blots were made, then point out to the reader that the blot shown is a “representative blot” and show all blots for the respective figures in the Supp section. [RESPONSE]: Thank you very much for your reminder to improve our content. We have updated the original data of triplicate blotting in Figures 3-5 (have been convert to Figure 3, 5 and 6) in the supporting data. We have meticulously and thoroughly re-identified all the data. The results of the analysis do not affect the statistical trend, and we have also confirmed their accuracy. 10. Finally, all graphs should be displayed as box and whiskers plots including “show all points” to make the results clearer for the reader. [RESPONSE]: Thank you very much for your reminder to make our content more complete and clearer. In fig 1E, 2C, 2G, 2H, 3B, 3D, 4D, 4E, 4F, 5B, 5D, 6B, 6D, we have displayed the data as box and whisker plots to enhance clarity and credibility. Reviewer 2: Major revisions 1. In the manuscript “n-Butylidenephthalide recovered calcium homeostasis to ameliorate neurodegeneration of motor neurons derived from amyotrophic lateral sclerosis iPSCs” the authors tested the protective efficacy of n-Butylidenephthalide (BP) on the survival of motor neurons (MN) derived from patients with Amyotrophic lateral sclerosis (ALS). They found that BP treatment restored the “normal” channels function and activated autophagic pathways that attenuated neuron degeneration. They suggest that BP is a promising candidate for future treatment of ALS. While the data is well-organized, and experiments were conducted properly, there are some concerns that must be addressed before is achieved a suitable version of the manuscript for publication. The scheme depicted in figure 6 does not clearly illustrate the obtained results and include elements not studied in the research performed (i.e. NCX, PMCA). Furthermore, no synapse-specific tests were performed in the conducted experiments, all data is related to “post-synaptic” effects. Please clarify the concepts outLined in the figure and redraw the diagram. [RESPONSE]: Thank you for your suggestions to make our content clearer and more complete. We have updated the concept of Figure 7 (Figure 6→Figure 7) and redrawn the diagram. Since all the data relates to post-synaptic effects, we removed the description of the pre-synaptic gateway. In addition, we deleted the description of calcium-related channels such as PMCA, NCX, and VGCC, and focused on AMPAR and NMDAR, which are the main subjects of this article. Minor revisions 2. Line 68 mistyped word: “antagonized” change to antagonize. [RESPONSE]: Thank you very much for your advice, we have made the corrections accordingly (Line 69). 3. Figure 2. It would be helpful to add the cell type in labels where the BP was applied (i.e. BP-G85R) [RESPONSE]: Thank you for your suggestions to make the article clearer and easier to understand. We have replaced the BP conditions in the figure with BP-G85R (see Figure 2E, 2G, 2H, 4B, 4D, 4E, 4F, 5A, 5B for details). Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0311573.r002
23 Sep 2024 Decision Letter - Stephen D. Ginsberg, Editor n-Butylidenephthalide recovered calcium homeostasis to ameliorate neurodegeneration of motor neurons derived from amyotrophic lateral sclerosis iPSCs PONE-D-24-19706R1 Dear Dr. Deng, 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, Stephen D. Ginsberg, Ph.D. Section Editor PLOS ONE 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: All comments have been addressed ******** 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 ****** 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 ****** 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: The authors answered and revised all the points raised to my complete satisfaction and also made further improvements to validate their data. Besides that die authors revised the content of their manuscript, so that it now reads much better and feels more coherent overall. Sorry for the error in my part in citing the wrong PMID number. Reviewer #2: The authors have thoroughly reviewed and significantly enhanced the manuscript. Every point raised during the initial round of the review process has been carefully addressed and incorporated. ****** 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 https://doi.org/10.1371/journal.pone.0311573.r003
Formally Accepted
29 Oct 2024 Acceptance Letter - Stephen D. Ginsberg, Editor PONE-D-24-19706R1 PLOS ONE Dear Dr. Deng, 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. Stephen D. Ginsberg Section Editor PLOS ONE https://doi.org/10.1371/journal.pone.0311573.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 .