Queuine, a bacterial-derived hypermodified nucleobase, shows protection in in vitro models of neurodegeneration

Patricia Richard; Lucie Kozlowski; Hélène Guillorit; Patrice Garnier; Nicole C. McKnight; Antoine Danchin; Xavier Manière

doi:10.1371/journal.pone.0253216

Peer Review History

Original SubmissionFebruary 3, 2021
19 Apr 2021 Decision Letter - Stephan N. Witt, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-21-03739 Queuine, a bacterial derived hypermodified nucleobase, shows protection in in vitro models of neurodegeneration PLOS ONE Dear Dr. McKnight, 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. 1) Reviewer #2 said that not all data in your study has been made available. Please address this concern. 2) Both reviewers are very positive about your manuscript. 3) Reviewer #2 has three major concerns relating to the synthesis of queuine, controls, and the experiments with MPP+. Please address these issues. 4) Reviewer #2 has six minor issues that should be addressed 5) Please address my editorial points Overall, the reviews are very positive. Addressing these various issues will improve your study. 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'. 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Witt, Ph.D. 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. To comply with PLOS ONE submissions requirements, please ensure you have provide methods of sacrifice in the Methods section of your manuscript for all experiments. 3. Thank you for stating the following in the Financial Disclosure section: 'The author(s) received no specific funding for this work.' We note that one or more of the authors are employed by a commercial company: Stellate Therapeutics a. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. c. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests 4. Please include a caption for figure 3. Additional Editor Comments: Both reviewers liked your manuscript. Reviewer #1 said, "It represents an important contribution to the field and will have a noticeable impact." Reviewer #2 said, "This manuscript has the potential to attract wide-spread readership." I have served as a academic editor for PlOS One for many years, and this is the first time I have seen both reviewers so positive about a manuscript and its potential impact. I strongly urge you to improve the manuscript by addressing the reviewer concerns. I have some comments that I also urge you to address: 1) Reviewer #2 said "...add a cartoon to describe what you are referring to when you state that queuine replaces guanine at the wobble position 34 of tRNAs with GUN anticodons." The Reader should see what the structure of queuine is and how it "replaces guanine at the wobble position..." 2) Synthetic queuine. (a) You should show a chromatogram of the compound. (b) What is its percent purity? (c) You must have performed mass spec on the synthesized queuine. What was the synthetic compound's mass as determined by mass spec? What is its theoretical mass? [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: This is an interesting and important study reporting a set of intriguing data on the effect of a bacterial derived hypermodified nucleobase queuine on aggregation potential of proteins associated with neurodegenerative diseases. The authors utilized two in vitro model of synucleinopathy (primary mouse cortical neurons exposed to the pre-formed alpha-synuclein (α-syn) fibrils (huPFFs) and rat DA neurons treated with MPP+ (1-methyl-4-phenylpyridinium)) and an acute Aβ1-42 oligomer injury model of AD in primary cultures of rat cortical neurons and microglia. The authors also showed that in healthy humans, queuine levels are not age-dependent but higher in women. The manuscript is well-written and concise. It represents an important contribution to the field and will have a noticeable impact. Reviewer #2: This is an interesting manuscript describing a chemical that represents a bacterial product found in the gut of humans responsible for modifying a nucleobase. Here, the authors use a chemically synthesized form of this product to show that varying levels of this chemical are associated with diseases of protein misfolding. Major comments: 1. The manuscript has very little information about the synthesis of STL-101 (results, methods or figure legends). Please describe this somewhere. For example, the referenced paper for synthesis states that additional analogues of queuine are possible. Is this an analog or the original? Was this compound dissolved in water or a solvent? If a solvent is used, which one and at what final concentration in the experiments? 2. As a related comment, there are no solvent controls mentioned in the manuscript that I can find. Please include in Figure 1D and in Figure 2B-D, and Figure S1 A-D. This will serve as a negative control and as a comparison to the activity observed vs. the neuronal incitor in each of these experiments. 3. The concentration of STL-101 required for significant protection of MPP+ protection of DA neurons was much higher than that for huPFFs (1 uM vs. 0.1 uM) or the Abeta acute model. The abstract describes the utility of queuine for the facilitation of protein misfolding and prevention of protein aggregation. This concept should be further described within your dataset and compared with the MPP+ model which is certainly not considered a traditional protein misfolding model (and required much higher amounts of STL-101 for efficacy). Minor comments: 1. This manuscript has the potential to attract wide-spread readership. However, many readers will not be familiar with GUN anticodons and wobble position replacement. Therefore, as part of the introduction, could you please add a cartoon to describe what you are referring to when you state that queuine replaces guanine at the wobble position 34 of tRNAs with GUN anticodons? 2. Similarly, I muddled through reading the terms queuine vs. queosine. Please add something of emphasis to the writing (or in an introductory figure) to help the time-crunched reader remember the difference the first time they read it. 3. What program was used to calculate statistics? This should be included in the Stats section. 4. In figure 1C, these data are described in micromolar. However, in figure 1B (legend and results section), the concentration here is described in nanomolar. While the conversion is easy enough, it is inconsistent. Additionally, in Figure 1C, there is a 0 micromolar data point. What was the solvent? 5. Similarly, in Figure 2, there is a discrepancy between nanomolar and micromolar terminology. 6. In the first sentence of the discussion, the word “remarkable” seems a bit overboard and could probably be eliminated without changing the sentence or meaning of your nice work, at all. Moreover, stating in this same sentence that queuine is exclusively produced by bacteria and causes neurodegeneration seems misleading, since, in this study, the STL-101 was made in a lab. Perhaps you could break these thoughts into two different sentences to remind the reader that naturally, queuine is exclusively made by bacteria, but in this study it was chemically synthesized. ****** 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:** Vladimir N. Uversky 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.0253216.r001
Revision 1
23 May 2021 Author Response Response to the academic editor and reviewers The academic editor and reviewers’ comments highlighted several important points that needed further clarification which we have provided. We found these comments valuable and below we describe our response point by point. Academic editor and reviewers’ comments are in blue. Academic editor: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. As recommended, we followed PLOS ONE’s style requirements. 2. To comply with PLOS ONE submissions requirements, please ensure you have provided methods of sacrifice in the Methods section of your manuscript for all experiments. Methods of sacrifice have been added in the Materials and Methods section for each experiment. 3. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. Updated Funding and Competing Interests Statements have been included in the cover letter: “The authors received no specific funding for this work. Authors [PR, LK, HG, PG, NCM, XM] are affiliated to Stellate Therapeutics. The funder provided support in the form of salaries for authors [PR, LK, HG, NCM, XM], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. This affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.” 4. Please include a caption for figure 3 A caption for Figure 3 (now Figure 4) already existed and can be found on page 12 of the manuscript. Additional Editor Comments: 1. Reviewer #2 said "...add a cartoon to describe what you are referring to when you state that queuine replaces guanine at the wobble position 34 of tRNAs with GUN anticodons." The Reader should see what the structure of queuine is and how it "replaces guanine at the wobble position..." We added a figure (Figure 1) that is now part of the introduction explaining the exchange of guanine by queuine into tRNAs and showing the structure of queuine. 2. Synthetic queuine. (a) You should show a chromatogram of the compound. (b) What is its percent purity? (c) You must have performed mass spec on the synthesized queuine. What was the synthetic compound's mass as determined by mass spec? What is its theoretical mass? Additional details about STL-101 have been added to the STL-101 synthesis paragraph in the Materials and Methods section on page 17 (see response to the major comment #1 of reviewer #1 below). HPLC, IR, NMR and LC-MS spectra can be found in supporting information as the “queuine dihydrochloride analyses” file. Reviewer #1: Major comments: 1. The manuscript has very little information about the synthesis of STL-101 (results, methods or figure legends). Please describe this somewhere. For example, the referenced paper for synthesis states that additional analogues of queuine are possible. Is this an analog or the original? Was this compound dissolved in water or a solvent? If a solvent is used, which one and at what final concentration in the experiments? Spectra of STL-101 analyses can now be found as supporting information and the paragraph “STL-101 synthesis” in the Materials and Methods page 17 now indicates the required information about synthesized queuine (STL-101): “STL-101 is queuine dihydrochloride (C12H15N5O3 (2HCl)) with a molecular weight (MW) of 350.20 g.mol-1 (natural queuine has a MW of 277.28 g.mol-1). STL-101 purity was determined at 99% by HPLC and was solubilized in water, PBS or DMSO at 10-30 mM as stock solution. Spectra related to STL-101 analysis can be found in the supporting information file entitled “queuine dihydrochloride analyses”. The final concentration of STL-101 used is indicated on each graph. 2. As a related comment, there are no solvent controls mentioned in the manuscript that I can find. Please include in Figure 1D and in Figure 2B-D, and Figure S1 A-D. This will serve as a negative control and as a comparison to the activity observed vs. the neuronal incitor in each of these experiments. The vehicle used in each experiment is now indicated in Materials and Methods and in Figure captions. For Figure 1A-C (now Fig 2A-C), the vehicle used is PBS (now indicated on page 17/18 of the “Primary culture of cortical neurons and huPFFs challenge” section and Fig 2C caption on page 9). For Figure 1D (now Fig 2D), culture medium was used as indicated on page 19 in the “Culture of mesencephalic dopaminergic neurons, STL-101 pretreatment and MPP+ injury at 10μM and immunostaining” section and in the figure caption of Fig 2D on page 9. For Figure 2B-D (now Fig 3B-D) and S1A-D (now Fig S2A-D), STL-101 has been solubilized in DMSO and diluted in culture medium (final concentration in DMSO = 0.1%). The vehicle used for control (Ctrl) = culture medium with 0.1% DMSO as indicated in Figure 3D (page 11) and S2D (page 33) captions. This information can now also be found on page 22 of the Materials and Methods section. For Figure S1, vehicle = culture medium as indicated in Fig S1B caption (page 33) and on page 19 of the “Culture of mesencephalic dopaminergic neurons, STL-101 and MPP+ (4μM) co-treatment, immunostaining and analysis” section of the Materials and Methods. 3. The concentration of STL-101 required for significant protection of MPP+ protection of DA neurons was much higher than that for huPFFs (1 uM vs. 0.1 uM) or the Abeta acute model. The abstract describes the utility of queuine for the facilitation of protein misfolding and prevention of protein aggregation. This concept should be further described within your dataset and compared with the MPP+ model which is certainly not considered a traditional protein misfolding model (and required much higher amounts of STL-101 for efficacy). We performed another in vitro experiment using an MPP+ injury at 4μM instead of 10μM and a co-treatment with STL-101 instead of a pre-treatment of 1 day. The data can now be found in Figure S1. We obtained a similar neuroprotective effect of STL-101 (although a significant effect is seen at 3μM only probably due to the lack of pretreatment) in this model than the one previously observed (Fig 2D). In the same model, we also looked at the α-syn signal by IF since it has previously been reported that MPP+ also triggers α-syn aggregation. Indeed Callizot et al., 2019 showed that increased signal of α-syn in DA neurons corresponds to hyper-phosphorylated/aggregated α-syn. Our result (Fig S1B) shows a significant decrease in the α-syn signal in DA neurons when the cells are treated with STL-101 at 1μM and above. In order to fully address comment #3, we added the following text in the discussion on page 13 of the manuscript: “We note that STL-101 concentration required for significant protection is higher with the MPP+ model than the other models (1µM vs. 0.1-0.3µM respectively). STL-101 was added 4 days prior huPFFs exposure, which might contribute to a better protection than no pretreatment or 1 day pretreatment before injury (MPP+ or Aβ1-42). It is also possible that the mitochondrial stress induced by MPP+ alters STL-101 function (e.g., oxidation of the molecule, reduction of cellular intake) which would require higher amount of the molecule to reach protection. Interestingly, significant decrease of α-syn aggregation in the MPP+ model is seen when STL-101 is added without pretreatment at 1µM and above while significant neuronal survival is seen with STL-101 at 3µM. This indicates that some MPP+-induced phenotypes can be rescued by STL-101 near physiological concentrations (α-syn aggregation) while others require STL-101 at pharmacological concentrations (cell survival). This could also indicate that STL-101 treatment reduces/prevents α-syn aggregation before affecting cell survival. It is possible that by optimizing mRNA translation and preventing misfolding of newly synthesized protein, STL-101 delays and/or prevents protein aggregation (in a seeding model such as huPFFs as well as in a model where aggregation might be triggered by mitochondrial dysfunction (e.g., MPP+ intoxication)) that needs to reach a certain level before it can prevent neuronal loss.” Minor comments: 1. This manuscript has the potential to attract wide-spread readership. However, many readers will not be familiar with GUN anticodons and wobble position replacement. Therefore, as part of the introduction, could you please add a cartoon to describe what you are referring to when you state that queuine replaces guanine at the wobble position 34 of tRNAs with GUN anticodons? We have now added a Figure (Figure 1) in the introduction describing queuine integration into tRNAs. 2. Similarly, I muddled through reading the terms queuine vs. queosine. Please add something of emphasis to the writing (or in an introductory figure) to help the time-crunched reader remember the difference the first time they read it. Figure 1 also states what is queuine and what is queuosine. 3. What program was used to calculate statistics? This should be included in the Stats section. GraphPad Prism was used to perform all the stats and this information is now in the “Statistical analysis” section on page 26. 4. In figure 1C, these data are described in micromolar. However, in figure 1B (legend and results section), the concentration here is described in nanomolar. While the conversion is easy enough, it is inconsistent. Additionally, in Figure 1C, there is a 0 micromolar data point. What was the solvent? The concentration has been changed to µM in the caption of Figure 2B (former Figure 1B). As now indicated in the caption of Figure 2C, the solvent is PBS. 5. Similarly, in Figure 2, there is a discrepancy between nanomolar and micromolar terminology. µM is now used in the caption of Figure 3 (former Figure 2). 6. In the first sentence of the discussion, the word “remarkable” seems a bit overboard and could probably be eliminated without changing the sentence or meaning of your nice work, at all. Moreover, stating in this same sentence that queuine is exclusively produced by bacteria and causes neurodegeneration seems misleading, since, in this study, the STL-101 was made in a lab. Perhaps you could break these thoughts into two different sentences to remind the reader that naturally, queuine is exclusively made by bacteria, but in this study it was chemically synthesized As suggested, the first sentence of the discussion is now: “Here we have shown protective properties of chemically synthesized queuine, a molecule naturally exclusively produced by bacteria, in several in vitro models of proteinopathies and neurodegeneration.” Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0253216.r002
31 May 2021 Decision Letter - Stephan N. Witt, Editor Queuine, a bacterial-derived hypermodified nucleobase, shows protection in in vitro models of neurodegeneration PONE-D-21-03739R1 Dear Dr. Richard, 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, Stephan N. Witt, Ph.D. 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 #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 #2: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: (No Response) ****** 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 #2: (No Response) ****** 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 #2: (No Response) ****** 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 #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 #2: No https://doi.org/10.1371/journal.pone.0253216.r003
Formally Accepted
29 Jul 2021 Acceptance Letter - Stephan N. Witt, Editor PONE-D-21-03739R1 Queuine, a bacterial-derived hypermodified nucleobase, shows protection in in vitro models of neurodegeneration Dear Dr. Richard: 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. Stephan N. Witt Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0253216.r004

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