Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma

Fani Karagianni; Christina Piperi; Vassiliki Mpakou; Aris Spathis; Periklis G. Foukas; Maria Dalamaga; Vasiliki Pappa; Evangelia Papadavid

doi:10.1371/journal.pone.0248298

Peer Review History

Original SubmissionJuly 14, 2020
11 Aug 2020 Decision Letter - Salvatore Papa, Editor PONE-D-20-21841 Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma PLOS ONE Dear Dr. Karagianni, 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 revise the use of MTT assay as a cell proliferation assay. As correctly stated by the reviewer this assay is a cytotoxicity assay rather than a proliferative assay. To determine the effect on cell proliferation the authors are encouraged to preform cell cycle analyses with PI and BrdU staining and analyses of cyclin expression by immunoblots WB analyses. Please submit your revised manuscript by Sep 25 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. We noticed minor instances of text overlap with the following previous publication(s), which need to be addressed: (1) https://onlinelibrary.wiley.com/doi/full/10.1111/jdv.16593 The text that needs to be addressed involves the Introduction section (lines 48-56). In your revision please ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [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: Partly ******** 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: The author here shows an interesting finding of synergistic effect of ruxolitinib and resminostat to inhibit proliferation and induce apoptosis on Cutaneous T-cell lymphomas. Although there are certain concerns I would like the author to address: 1. Figure 1B shows cell survival of SeAx cells around 60% at 24h whereas 80% at 48h for combination treatment with 1uM resm/15 uM rux This is surprising as mostly in case for any anticancer drug, cell death should be increasing with increase in time point, or we may say cell survival should be decreasing. Also, its contradicting to the result obtained from Apoptosis study in Figure 3, where percentage of apoptotic cells (early + late) are more in 48h (almost 60%) compared to 24 h (35%). The author should explain this discrepancy. 2. Although the author have written a detailed discussion of their finding, but it would be better for the manuscript to have a short (conclusion section to summarize the manuscript for ease of an reader) separately following the discussion and before author contribution. Not just as a part of abstract section. 3. I would prefer the author to mention in full before using abbreviations like WGS for Whole genome sequencing or NGS for Next-Generation Sequencing, if they mean so. (Introduction section, page 11 line 68). 4. The figures representations could be improved (eg. In fig 3B and fig 3D the asterisk marks does not have consistent font size within or in comparison as well). 5. Each figure caption could start with a title in bold (to explain what this data/figure is about in short) then followed by the legend. This helps the reader to quick glance through. (eg., Fig 1. Cell viability assay of resminostat and/or ruxolitinib on CTCL. Effect of resminostat and/or ruxolitinib in cell proliferation in Myla (A) and SeAx (B) cell lines at 24h and 48h. Values are the mean � standard deviation of three experiments. p <0.05, p<0.001 statistically significant differences versus untreated and single drugs or combination. Reviewer #2: This manuscript describes an interesting study, reporting the induction of apoptosis in 2 lymphoma cell lines following treatment with specific concentrations of resminostat and ruxolitinib alone and in combination. However, the manuscript is lacking 3 independent repeats of all data and despite a clear increase in efficacy when resminostat and ruxolitinib are employed in combination as opposed to their use as individual compounds, to describe the result as synergistic is incorrect as this has not been investigated in the study. Describing the MTT as a proliferation assay is also incorrect. However, the assessment of cell cycle using annexin v/PI staining and the status of proteins in a variety of cell signalling pathways, using the MILLIPLEX MAP Multi-Pathway Magnetic Bead is sound. The authors refer to a poor survival rate (line 57) for those with lymphoma (MF). Please can the authors include some survival statistics so it is clear to the reader the current outcome for these patients. Furthermore, since the study describes putative new therapies for these patients, please can the authors include details of the current treatment strategies, why these are inadequate and therefore why new treatments are necessary. The authors report previous work revealing the importance of a variety of signalling pathways, including the JAK-STAT pathway in MF/SS, and importantly suggest the use of inhibitors targeting this pathway as a possible therapeutic strategy. However, it is unclear as to why the authors have chosen to examine the combined effect of two compounds targeting the same pathway and proteins. It would be useful to have this explained more clearly in the introduction. Since it is unlikely these compounds would be employed as a monotherapy, it would be important to establish if any synergy exists with current therapies. This should be addressed in the discussion. Although the authors described decreased “proliferation” of the cells in response to combination treatment, in this study the MTT assay has been employed, which does not measure the proliferation rate of cells. The MTT assay is a colorimetric assay which merely reflects the number of cells present based on the activity of the mitochondria. It is a major error to refer to this data as measuring proliferation. Although in line with previous data from the literature, the authors describe data from 1 cell line from a MF and a SS lymphoma. They report the data as an “in vitro model of CTCL”, which is misleading. It should be acknowledged that these findings need to be validated in a wider panel of cells, preferably primary patient derived cells, to confirm the efficacy of this therapeutic strategy in vitro. i.e. expand comment on line 338. It is unclear as to the vehicle solution in which the drugs have been dissolved. Furthermore, it is not clear as to whether vehicle control treated cells have been used as the “control” cells (line 175) or are these really completely “untreated” cells (line 174). If a vehicle treated cell control has not been employed, this seriously limits the impact of the findings described throughout the study as it is wholly incorrect. The statistics in this manuscript have been performed correctly, however, data from multiple independent repeats must be presented with standard error of the mean as opposed to standard deviation (line 163). Furthermore, some conclusions have been formed from only 2 independent repeats of experiments (line 162 “at least twice”). It is most appropriate to run 3 independent repeats of all experiments. The authors describe a decrease in the number of cells and increased apoptosis following treatment with both resminostat and ruxolitinib, showing this drug combination represents an interesting strategy for investigation. However, in places throughout the manuscript, including the title, the authors refer to synergy between the two compounds (line 245, 339). Synergy between the inhibitors has not been shown or investigated in this manuscript. To examine synergistic or additive effects between 2 compounds, a range of concentrations of both drugs must be investigated alone and in combination using the CalcuSyn analyser of combined drug effects. It is wrong to suggest synergy has been observed in this study. The authors refer to testing the efficacy of resminostat and ruxolitinib across a range of concentrations. Please can this data be included to explain why the concentrations of 1um and 15um, respectively, were chosen for future experiments. What was the reason for this choice of concentration? The manuscript is written in standard English. There are some small errors for correction: Line 197: “same wise” – please find an alternative. Line 307: typo “\\” Line 265: extra space Line 267: extra comma Throughout: et al. must be in italics ******* 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:** Dr. Arindam Pramanik 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.0248298.r001
Revision 1
6 Feb 2021 Author Response Dear Editor-in-Chief, We would like to thank you and the reviewers for your thoughtful evaluation of our manuscript and for your most welcome comments/suggestions. Accordingly, we have now revised our manuscript to reflect these comments. In the revised Text all changes/additions/modifications made in response to the Reviewers’ points are marked with track changes. Please find below a point-by-point response to the issues raised by the Reviewers: Academic Editor: Please revise the use of MTT assay as a cell proliferation assay. As correctly stated by the reviewer this assay is a cytotoxicity assay rather than a proliferative assay. To determine the effect on cell proliferation the authors are encouraged to perform cell cycle analyses with PI and BrdU staining and analyses of cyclin expression by immunoblots WB analyses. RESPONSE: We would like to thank the academic editor for this comment. We have revised the use of MTT as a proliferation assay to cytotoxicity assay and we have also performed cell cycle analyses with 7AAD and BrdU staining. Data are shown in Figure 1. Due to time restriction, we have not performed cyclin expression, but we believe that 7AAD/BrdU analysis clearly demonstrated the effect of the two agents in cell proliferation. Reviewer #1: The author here shows an interesting finding of synergistic effect of ruxolitinib and resminostat to inhibit proliferation and induce apoptosis on Cutaneous T-cell lymphomas. Although there are certain concerns, I would like the author to address: 1. Figure 1B shows cell survival of SeAx cells around 60% at 24h whereas 80% at 48h for combination treatment with 1uM resm/15 uM rux. This is surprising as mostly in case for any anticancer drug, cell death should be increasing with increase in time point, or we may say cell survival should be decreasing. Also, its contradicting to the result obtained from Apoptosis study in Figure 3, where percentage of apoptotic cells (early + late) are more in 48h (almost 60%) compared to 24 h (35%). The author should explain this discrepancy. Response: We have re-evaluated the effect of ruxolitinib and resminostat in cell proliferation using the 7AAD/BrdU staining and provide the new data in the revised manuscript. Based on these experiments, cell viability, cell proliferation and apoptotic data are in agreement (Figures 1 and 2). 2. Although the author have written a detailed discussion of their finding, but it would be better for the manuscript to have a short (conclusion section to summarize the manuscript for ease of an reader) separately following the discussion and before author contribution. Not just as a part of abstract section. Response: We have included a conclusion section following the discussion. 3. I would prefer the author to mention in full before using abbreviations like WGS for Whole genome sequencing or NGS for Next-Generation Sequencing if they mean so. (Introduction section, page 11 line 68). Response: The full descriptions of abbreviations are provided (line 76-77). 4. The figures representations could be improved (eg. In fig 3B and fig 3D the asterisk marks does not have consistent font size within or in comparison as well). Response: The figure representations were improved and the asterisks in Figure 3 (B and D) were corrected with consistent font size. 5. Each figure caption could start with a title in bold (to explain what this data/figure is about in short) then followed by the legend. This helps the reader to quick glance through. (eg., Fig 1. Cell viability assay of resminostat and/or ruxolitinib on CTCL. Effect of resminostat and/or ruxolitinib in cell proliferation in Myla (A) and SeAx (B) cell lines at 24h and 48h. Values are the mean � standard deviation of three experiments. p <0.05, p<0.001 statistically significant differences versus untreated and single drugs or combination. Response: All figure captures have been corrected as proposed by the reviewer (figure legend file). Reviewer #2: This manuscript describes an interesting study, reporting the induction of apoptosis in 2 lymphoma cell lines following treatment with specific concentrations of resminostat and ruxolitinib alone and in combination. 1. However, the manuscript is lacking 3 independent repeats of all data and despite a clear increase in efficacy when resminostat and ruxolitinib are employed in combination as opposed to their use as individual compounds, to describe the result as synergistic is incorrect as this has not been investigated in the study. Response: All experiments performed in triplicates and three independent repeats were performed. The authors corrected this statement at line 198-199. We have also determined the synergistic activity of resminostat and ruxolitinib by calculating the combination index (CI). The synergistic activity of the two drugs (CI<1 for all combinations) is described in the revised manuscript [see sections Materials and Methods (line 174-183), Results (line 270-272), Discussion (line 353-355) and Figure 3. Dr Maria Dalamaga has performed the synergistic experiments and she has been added as a co-author to this manuscript. 2. Describing the MTT as a proliferation assay is also incorrect. However, the assessment of cell cycle using annexin v/PI staining and the status of proteins in a variety of cell signalling pathways, using the MILLIPLEX MAP Multi-Pathway Magnetic Bead is sound. Response: ΜΤΤ is a colorimetric assay for assessing cell metabolic activity. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. However, the authors agreed to assess the proliferation (rapidly dividing cells) by performing cell cycle analysis with 7AAD and BrdU. The corresponding method is described in Material and Methods section (line 153-160), the data are described in Results Section (line 237-249) and in Discussion section (line 340-341). These experiments were performed by Aris Spathis and Periklis G. Foukas, and have been added as co-authors in the revised manuscript. 3. The authors refer to a poor survival rate (line 57) for those with lymphoma (MF). Please can the authors include some survival statistics, so it is clear to the reader the current outcome for these patients. Response: On line 64-65, statistics of the poor survival for the advanced stage disease have been added. 4. Furthermore, since the study describes putative new therapies for these patients, please can the authors include details of the current treatment strategies, why these are inadequate and therefore why new treatments are necessary. The authors report previous work revealing the importance of a variety of signalling pathways, including the JAK-STAT pathway in MF/SS, and importantly suggest the use of inhibitors targeting this pathway as a possible therapeutic strategy. However, it is unclear as to why the authors have chosen to examine the combined effect of two compounds targeting the same pathway and proteins. It would be useful to have this explained more clearly in the introduction. Since it is unlikely these compounds would be employed as a monotherapy, it would be important to establish if any synergy exists with current therapies. This should be addressed in the discussion. Response: The authors have added a more descriptive paragraph at the beginning of the Discussion (line 303-315) and at the end of Introduction (line 125-126). 5. Although the authors described decreased “proliferation” of the cells in response to combination treatment, in this study the MTT assay has been employed, which does not measure the proliferation rate of cells. The MTT assay is a colorimetric assay which merely reflects the number of cells present based on the activity of the mitochondria. It is a major error to refer to this data as measuring proliferation. Response: As it has been mentioned above, the authors agreed and assessed the proliferation assay by performing cell cycle analysis with 7AAD and BrdU. 6. Although in line with previous data from the literature, the authors describe data from 1 cell line from a MF and a SS lymphoma. They report the data as an “in vitro model of CTCL”, which is misleading. It should be acknowledged that these findings need to be validated in a wider panel of cells, preferably primary patient derived cells, to confirm the efficacy of this therapeutic strategy in vitro. i.e. expand comment on line 338. Response: In current line 432-434 (previous line 338) the phrase ‘…. in primary patient derived cells (cultured isolated T lymphocytes from peripheral blood and skin biopsies)…. validating the efficacy of this therapeutic strategy in vitro….’ was added. 7. It is unclear as to the vehicle solution in which the drugs have been dissolved. Furthermore, it is not clear as to whether vehicle control treated cells have been used as the “control” cells (line 175) or are these really completely “untreated” cells (line 174). If a vehicle treated cell control has not been employed, this seriously limits the impact of the findings described throughout the study as it is wholly incorrect. Response: On line 139-142, a description was added where it is stated where the drugs had been dissolved. All cells that were characterized as ‘untreated’, where cells that were treated with 0.1% DMSO (vehicle control). 8. The statistics in this manuscript have been performed correctly, however, data from multiple independent repeats must be presented with standard error of the mean as opposed to standard deviation (line 163). Furthermore, some conclusions have been formed from only 2 independent repeats of experiments (line 162 “at least twice”). It is most appropriate to run 3 independent repeats of all experiments. Response: On line 199, the authors changed the phrase ‘at least twice’ by the phrase ‘in 3 independent repeats’. 9. The authors describe a decrease in the number of cells and increased apoptosis following treatment with both resminostat and ruxolitinib, showing this drug combination represents an interesting strategy for investigation. However, in places throughout the manuscript, including the title, the authors refer to synergy between the two compounds (line 245, 339). Synergy between the inhibitors has not been shown or investigated in this manuscript. To examine synergistic or additive effects between 2 compounds, a range of concentrations of both drugs must be investigated alone and in combination using the CalcuSyn analyser of combined drug effects. It is wrong to suggest synergy has been observed in this study. Response: The authors added the following sentence: ‘The synergistic effect between resminostat and ruxolitinib was determined by the combination index (CI) based on the Chou-Talalay method [31], as previously described [32]. The CI value was determined by the following equation: CI=sum of apoptosis of single agent treatment/apoptosis upon combined treatment. The combination effect was defined as “synergistic”, “additive” or “antagonistic” when CI was <1, 1 and >1, respectively’. A combination index (CI) of > 1 indicates antagonism, a CI of 1 denotes additivity, and a CI of < 1 indicates synergism. More specifically, CI values ranging from 0.1–0.3 are considered to indicate strong synergism, 0.3–0.7 synergism, and 0.7–0.85 moderate synergism. (line 174-183). 10. The authors refer to testing the efficacy of resminostat and ruxolitinib across a range of concentrations. Please can this data be included to explain why the concentrations of 1um and 15um, respectively, were chosen for future experiments. What was the reason for this choice of concentration? Response: The authors have added the MTT assay with the various concentrations of both drugs to choose the concentrations for the combined experiments. Therefore, supplementary figure 1 has been added. 11. The manuscript is written in standard English. There are some small errors for correction: Line 197: “same wise” – please find an alternative. Line 307: typo “\\” Line 265: extra space Line 267: extra comma Throughout: et al. must be in italics Response: We have performed a grammar and spell check throughout the manuscript. In more detail: Previous line 197: line 262 changed to ‘similarly’ Previous line 307: line 399, the typo ‘/’ was deleted Previous line 265: extra space on line 256 was deleted Previous line 267: extra comma on line 359 was deleted Et al. was changed to italics throughout the manuscript. Trusting that we have adequately addressed the Reviewers’ concerns, we would like to thank you for your help in significantly improving our work. With kind regards, Fani Karagianni Corresponding author Attachments Attachment Submitted filename: PONE-D-20-21841R1 -ANSWERS TO REVIEWERS COMMENTS.doc* https://doi.org/10.1371/journal.pone.0248298.r002
24 Feb 2021 Decision Letter - Salvatore Papa, Editor Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma PONE-D-20-21841R1 Dear Dr. Karagianni, 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, Salvatore Papa, 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: (No Response) 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: (No Response) Reviewer #2: (No Response) ****** 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.0248298.r003
Formally Accepted
2 Mar 2021 Acceptance Letter - Salvatore Papa, Editor PONE-D-20-21841R1 Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma Dear Dr. Karagianni: 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 Salvatore Papa Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0248298.r004

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