Aripiprazole reduces liver cell division

Tinkara Pirc Marolt; Barbara Kramar; Klara Bulc Rozman; Dušan Šuput; Irina Milisav

doi:10.1371/journal.pone.0240754

Peer Review History

Original SubmissionJune 20, 2020
2 Aug 2020 Decision Letter - Ezio Laconi, Editor PONE-D-20-18998 Aripiprazole reduces liver cell division PLOS ONE Dear Dr. Milisav, 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. ============================== Both reviewers and AE feel there is merit in the content of the Ms. However major issues need to be addressed before it can be re-considered for publication Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact. ============================== Please submit your revised manuscript by Sep 16 2020 11:59PM. 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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. In table 1, please provide the actual numerical results, including fold change and p-values. 3. We note that in line 71 you refer to schizophrenic vs. healthy populations. Please be more specific about what "healthy" population means. For example, does this mean non-schizophrenic and/or no other major underlying health conditions? 4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. [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: No 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: No 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: Comments to the Authors The manuscript from Tinkara Pirc Marolt et al. entitled “Aripiprazole reduces liver cell division” (Manuscript Number: PONE-D-20-18998) seems to be an interesting manuscript. There are points which need to be addressed. 1. Abstract appears so long. It can be short and concise. 2. Lines 194-, figures do not appear in order: Figs 1a, b, 1d, 1e, 1c, and 1f. 3. Lines 212-, the word “Untr” is not seen in the figure. 4. Table 1 seems difficult to be understood by readers. It has quite a bit of room for improvement. 5. In Table 1, cell counts are less than the control in both 2.23 and 6 uM ARI. There may be difference comparing the result of Fig. 1b. 6. In terms of statistics, authors used ANOVA to compare groups, but a test for normality seems lacking. Reviewer #2: In this paper, it is interesting that aripiprazole reduces the number of hepatocytes by inhibiting the division of hepatocytes. However, there are still some problems in the experimental design and discussion. Majoy modification suggestions: 1. It is suggested that primary hepatocytes of aripiprazole induced liver injury animal model be used to verify the conclusion that the number of hepatocytes decreased but the activity remained unchanged. 2. To increase the research on signal pathway of aripiprazole inhibiting hepatocyte division. Minor modification comments: 1. Flow cytometry results on cell cycle should be displayed, not just statistical charts. 2. The morphological changes of aging hepatocytes should be observed. 3. Line197-198：The conclusion of Figure1c should be added. For example: ‘this indicates that the cytotoxicity is the same after long-term treatment’ should be added later. Meanwhile, since Figure1d-e are described first in this paper, Figure1c should be placed after Figure1d-e (i.e., the Ordinal Numbers of Figure1d-e should be changed to Figure 1c and 1d, and the Ordinal Numbers of Figure1c should be changed to Figure1e). 4. Line 198-199：The method used here is the ‘capillary flow cytometry’ but the method described in line 210 is the ‘microcapillary cytometry. 5. Line 190-199：The results of this section should be summarized in a sentence at the end of the paragraph. 6. Line251-253：The description of Figure4 is too simple and should be more detailed. 7. In the discussion part, the experimental contents should be summarized and analyzed in depth. 8. Subheadings should be added to each section of the results to summarize what the results reveal so that the reader can better frame the text. ****** 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. https://doi.org/10.1371/journal.pone.0240754.r001
Revision 1
16 Sep 2020 Author Response Dear Dr Laconi, dear Reviewers, thank you for your valuable comments towards the manuscript improvement. We have addressed all raised points and hope that our manuscript is now suitable for publication. 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 Done. 2. In table 1, please provide the actual numerical results, including fold change and p-values. Provided. 3. We note that in line 71 you refer to schizophrenic vs. healthy populations. Please be more specific about what "healthy" population means. For example, does this mean non-schizophrenic and/or no other major underlying health conditions? Non-schizophrenic population (line 64 of the manuscript was modified accordingly). The text now reads:” Alcohol or illicit drug dependence is estimated in up to 50 % of patients with schizophrenia [6, 7], and is 4.6-times higher than in the population without this disease [8]” Review Comments to the Author Reviewer #1: Comments to the Authors The manuscript from Tinkara Pirc Marolt et al. entitled “Aripiprazole reduces liver cell division” (Manuscript Number: PONE-D-20-18998) seems to be an interesting manuscript. There are points which need to be addressed. 1. Abstract appears so long. It can be short and concise. The abstract was shortened. 2. Lines 194-, figures do not appear in order: Figs 1a, b, 1d, 1e, 1c, and 1f. The figures were re-numbered. 3. Lines 212-, the word “Untr” is not seen in the figure. Thank you, removed. 4. Table 1 seems difficult to be understood by readers. It has quite a bit of room for improvement. The actual numerical results, including fold change and p-values were added. 5. In Table 1, cell counts are less than the control in both 2.23 and 6 uM ARI. There may be difference comparing the result of Fig. 1b. There is no discrepancy between the data in Table1 and Fig 1b (now left panel). The number of long-term treated cells was set equal at the day 0 (100 %). The numbers of cells in each sample were counted 2 and 4 days later and the numbers of ARI treated cells in every biological replicate were lower than that of the cells in any other sample in the case of the 6 uM ARI treatment in Fao and immortalized hepatocytes. 6. In terms of statistics, authors used ANOVA to compare groups, but a test for normality seems lacking. Data were analysed with GraphPad Prism, which has in built algorithms that test the equality of variances from medians, the Brown–Forsythe test. The data were added as a supplement S5. Reviewer #2: In this paper, it is interesting that aripiprazole reduces the number of hepatocytes by inhibiting the division of hepatocytes. However, there are still some problems in the experimental design and discussion. Major modification suggestions: 1. It is suggested that primary hepatocytes of aripiprazole induced liver injury animal model be used to verify the conclusion that the number of hepatocytes decreased but the activity remained unchanged. We agree that the primary hepatocytes are the best model, however, they do not proliferate in cell cultures. An animal experiment would require an application to the ethics committee to perform an invasive procedure, liver regeneration after hepatectomy. ARI and OLA are hydrophobic and one would need to feed the animals by gavage. We have additionally confirmed the reduced growth upon the ARI treatment on the neonatal immortalized hepatocytes (Figure 1b, right panel). 2. To increase the research on signal pathway of aripiprazole inhibiting hepatocyte division. Gene expression of DNA-damage induced genes (p53 and Gadd45a) and cell cycle inhibitors (p16, p21 and p27) was determined. Minor modification comments: 1. Flow cytometry results on cell cycle should be displayed, not just statistical charts. These are in supplemental figures S1-S4. 2. The morphological changes of aging hepatocytes should be observed. Morphology of long-term treated cells is depicted in Fig 1a. We have not observed nor measured any increase in cell demise. 3. Line197-198：The conclusion of Figure1c should be added. For example: ‘this indicates that the cytotoxicity is the same after long-term treatment’ should be added later. Meanwhile, since Figure1d-e are described first in this paper, Figure1c should be placed after Figure1d-e (i.e., the Ordinal Numbers of Figure1d-e should be changed to Figure 1c and 1d, and the Ordinal Numbers of Figure1c should be changed to Figure1e). The Figures 1c-1e were re-numbered and the conclusion was added: “This indicates that cell survival is the same in all treated cells during the long-term treatment.” 4. Line 198-199：The method used here is the ‘capillary flow cytometry’ but the method described in line 210 is the ‘microcapillary cytometry. The term ‘microcapillary flow cytometry’ is now used. 5. Line 190-199：The results of this section should be summarized in a sentence at the end of the paragraph. “This indicates that cell survival is the same in all treated cells during the long-term treatment.” 6. Line251-253：The description of Figure4 is too simple and should be more detailed. The figure and its legend are supplemented. 7. In the discussion part, the experimental contents should be summarized and analyzed in depth. New text was added. 8. Subheadings should be added to each section of the results to summarize what the results reveal so that the reader can better frame the text. Subheadings are added. Attachments Attachment Submitted filename: Pirc-Marolt-Response to reviewers.docx https://doi.org/10.1371/journal.pone.0240754.r002
2 Oct 2020 Decision Letter - Ezio Laconi, Editor Aripiprazole reduces liver cell division PONE-D-20-18998R1 Dear Dr. Milisav, 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, Ezio Laconi, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions 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: Comments to the Authors According to the comments from the Reviewers, the Authors responded adequately and conducted several modifications appropriately. This seems a quite well-written and reshaped manuscript. Therefore, this can be suitable for publication in the journal. Reviewer #2: After the revision, the structure of the paper is reasonable and logical. The results are illustrative and the statistical analysis is performed properly and strictly. The manuscript is easy to understand and written in standard English. In general, the conclusions are presented in an appropriate manner and supported by data. Therefore, my recommendation is acceptance. ****** 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.0240754.r003
Formally Accepted
16 Oct 2020 Acceptance Letter - Ezio Laconi, Editor PONE-D-20-18998R1 Aripiprazole reduces liver cell division Dear Dr. Milisav: 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 Dr. Ezio Laconi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0240754.r004

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