PONE-D-19-23187

First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation

PLOS ONE

Dear Dr Jonathan D. Smith,

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 convincingly addresses the points raised by both the reviewers.

Since some of the points such as those involving unfolding and aggregation issues may require new experiments, we would appreciate receiving your revised manuscript by October 30th. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Maria Gasset, 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

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have a patent relating to material pertinent to this article. Please provide an amended statement of Competing Interests to declare this patent (with details including name and number), along with any other relevant declarations relating to employment, consultancy, patents, products in development or modified products etc. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” If 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.

This information should be included in your cover letter; we will change the online submission form on your behalf.

3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[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: 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: 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: Brubaker et al. present a characterization of apoA1 lipidation mechanism via unfolding of the N-terminal α-helices. Although the structural information on apoA1 is rather abundant including available structures of different conformations of the protein - particularly from the Davidson group - the authors provide convincing evidence that the very first N-terminal residues including Trp8 have a functional role solubilizing DMPC lipids or in cholesterol transport. Based on fluorescence measurements, the authors propose a mechanism by which unfolding of an N-terminal helix bundle is required for apoA1 lipidation. The experiments are performed with care and the data analyzed correctly. However, major concerns arise from the experiments shown.

1.- Figure 1 shows that deletion of the C-terminus (ΔC mutant) blocks apoA1 functionality, but the additional deletion of the N-terminus (ΔN/ΔC mutant) recovers lipid binding activity. This evidence goes in line with the model of monomeric, full-length apoA1 structure (Melchior et al NSMB 2017), in which the disordered C-terminus caps the N-terminal helix bundle. Lipid binding to the C-terminal domain of apoA1 induces a conformational switch on this region (Oda et al NSMB 2003) that favors the unfolding of the N-terminal helix bundle. The authors present compelling functional data supporting the hypothesis that unfolding of the N-terminal helical bundle is critical for functional lipidation (especially Figure 1B). Then the authors aim to follow the chemical and thermal unfolding of the different variants of apoA1 by monitoring the change in fluorescence maxima (Figure 1C, D). While full-length and ΔC apoA1 constructs contain 4 Trp residues, ΔN variants contain three Trp residues. More importantly, the structure (for instance, PDB code 3r2p) shows that, precisely, helix 1 clusters helices 2-5 in the bundle. Therefore, removal of helix 1 –assuming that the rest of the bundle remains folded- would expose the remaining Trp residues (Trp50, Trp72, Trp108) to the solvent in monomeric ΔN constructs. In this scenario, it is surprising that the authors observe a change in the fluorescence maxima upon chemical and thermal denaturation. One possible explanation would be that removal of helix 1 promotes oligomerization, in such way that the authors are actually following dissociation of oligomers in ΔN and ΔN/ΔC constructs in Figure 1C, D. To discard this possibility, the authors must show that ΔN, ΔC and ΔN/ΔC constructs are monomeric in the experimental conditions used. Full-length apoA1 is already shown to be monomeric in the SDS-PAGE presented in Figure 3B.

2.- Following protein denaturation by the change in fluorescence maxima is not a quantitative method to determine changes in the populations of folded and unfolded conformers. Different degrees of compaction in unfolded proteins will yield fluorescence maxima at different wavelengths. Authors should monitor the change in fluorescence in a single wavelength to follow protein unfolding.

3.- Proteins were purified in mild denaturing conditions. Proper refolding in the experimental conditions used should be assessed (by Circular Dichroism or comparable techniques). This is particularly important for the ΔN, ΔC and ΔN/ΔC deletion mutants. This point is additionally related to the previous point, since CD would not only be informative on the proper folding of the deletion mutants, but could also be very helpful to characterize protein unfolding upon thermal or chemical denaturation (by monitoring the change in ellipticity in a single wavelength, usually a minima characteristic of α-helical structures).

4.- Figure 1A shows that ΔN and ΔN/ΔC mutants partially recover activity. The authors should state that the mutants “only partially” recover the activity (in Figure legend 1A page#9). Do the authors have any explanation for the observed difference between WT and ΔN and ΔN/ΔC mutants in solubilizing DMPC (only about 50% recovery)?

Minor concerns:

1.- Figure 1A should contain error bars.

2.- Authors state that “the C-terminus stabilized the folded state” based on fluorescence data. However, the ΔC mutant shows much lower change in fluorescence maxima upon thermal denaturation, probably because of the high entropic contribution of the disordered C-terminal region present in WT. Thus, it seems that the C-terminus indeed destabilized the folded state, since in its absence the change in unfolded populations is minor compared to the WT.

3.- Text should be carefully edited: last line in the Introduction reads “who’s” and it should be “whose”. 3 lines before the last in page#5: Remove “of” in “counting radioactivity to measure of cholesterol”… Line 8, page 10, should read “completely” rescued instead of “completed”

4.- Abbreviations should be consistent. The protein is called apoA1 and even apoAI. The C-terminal deletion mutant is termed ΔC and also in page#11 it is called Δ183-243 apoA1.

5.- Labeling of the helices in Figures 6C-D would be appreciated.

Reviewer #2: This study aims to characterize determinants of helical bundle unfolding necessary for lipidation of apolipoprotein A-1 that is mediated by the first eight residues that interact with C-terminus of the protein. The approach uses a Cholesterol Efflux and liposome clearance assay that are now standard to estimate the functionally of the apoA1. In addition to both assays, the authors perform denaturation assay in presence of Guanidium Chloride and Temperature influence. The results suggest that the first eight residues, and specially Trp8, are essential to control the unfolding of the helical bundle necessary for further ApoA1 lipidation. Several published work, spanning the last 20 years, have presented evidence consistent with the current manuscript, but nonetheless, this paper adds a little bit more details and tend to clarify some tenets of apoA1 lipidation and its mechanism.

Taken together the results of the paper are suitable for publication in Plos One. However, the are some points that should be amended or clarified before the manuscript may be accepted. These are detailed in the following:

Major revisions:

- Authors used WMF for unfolding experiments as a red shift is produced when the ApoA1 Trp present in the structure go from a hydrophobic to an aqueous environment during the unfolding process. When Trp are exposed to the hydrophilic environment, an increase of the intensity of the fluorescence is generally observed. The manuscript would gain reproducibility if the authors could provide at least one Trp fluorescence spectrum where the red shift and intensity changes are observed, at least for the Wt and �C and �N isoforms

- In the results corresponding to figure 2, authors mutated Trp8 to Phe, Leu and Ala to discuss the role of the aromatic residue as in the N-terminus unfolding. Trp is an aromatic/amphipathic residue, and it is changed to Phe (aromatic/ hydrophobic) and Leu/Ala (hydrophobic). More disruptive Trp8 changes are needed to clarify the key role of Trp8. Mutations as Trp8 to some polar residue (as Asp and/or Lys and/or Ser) and less bulky residue (Gly) look essential to establish the proper function of the Trp8 into the native structure. Finally, more discussion is needed for mutation Trp8 to Tyr or at least, hypothesize what happens in their model when substitution is performed.

- It is a clear effect that deletion of the first eight residues into �C isoform affects into the unfolding process of the helical bundle. Manuscript would improve if the �1-7 and �1-8 could be performed into the Wt protein and observe the possible effect and comparison wit the isoform �1-8 �C.

Minor revisions:

- Authors proposed in the abstract part “we demonstrate that apoA1 lipidation can occur when the barrier to this bundle unfolding is lowered”. Authors should clarify the term “barrier”, if is from a “thermodynamic or energetic” barrier or another type.

- Introduction lane 2-3: in vivo should be in italics.

- In the introduction, it is stated “the C-terminal truncation of residues 183-243 (called hereafter the �C

- isoform), but authors used a C-terminal truncation of residues of 185-243. Authors should explain why residues 183-184 were not deleted and its possible influence.

- Assays with �C and �N are well-designed and provided information but more discussion is needed. Specifically, authors should discuss why �C is not “more resistant” to unfolding as Wt isoform.

- In Figure 1, it is confusing what black box is. It should be explained, at least, in Figure Legend 1

- Authors mutated Leu 38 and Met 112 to create the 38C/112C helix bundle. Authors should explain better why these residues are chosen as in a quick look of the structure there are more possible candidates.

- In figure 3E, black box should be explained at least in Figure Legend 3

- In figure 4B, red line is not explained or mentioned.

- Page 12, authors mentioned “Thus, like Trp18, D18 and �N/C isoforms….”. It is not clear what D18 is as there are not results refereed to it. Authors should clarify the role of D18 in this context.

- Page 13, authors enlisted a large number of cross links that are not relevant in the discussion part or at least in the main text. It would be more illustrative as supplementary or in a table.

**********

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: Manuel Bano-Polo

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-19-23187R1

First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation

PLOS ONE

Dear Dr Jonathan D. Smith,

Thank you for submitting your revised manuscript to PLOS ONE. Both reviewers agree in the manuscript improvement, but to defend the final mechanistic model displayed in Figure 6 the oligomerization control is essential. This control was requested in the first review round and could easily provide it. The study deserves publication in PLoS One but only once this control is provided. Therefore, we invite you to submit a revised version of the manuscript that addresses specifically this point.

We would appreciate receiving your revised manuscript by december 12. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Maria Gasset, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

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: Partly

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 have thoroughly reviewed the manuscript and improved it. However, major concerns still remain regarding data interpretation and the reply of the authors is not fully convincing.

This reviewer is concerned about the conformational state and energy of the deletion mutants that the authors used to build their hypothesis. I understand that the goal of the study is not to describe the effect of the deletion mutants on the distribution of populations of ApoA1 conformations. But since the authors finally build a mechanistic model on monomeric ApoA1 (Figure 6), they must show that the effects on stability and unfolding presented in the study account on the same protein species as the WT. In this reviewer’s opinion, they still fail to do so.

All the protein constructs are shown to be dimers. Different values of fluorescence maxima at 0 GdmHCl may reflect that deletion mutants are partially unfolded or they adopt a molten globule state. This reviewer asked the authors to follow unfolding at a single wavelength as it is the only method to quantitatively monitor protein denaturation and determine the fraction of folded and unfolded protein at each GdmHCl concentration. This reviewer is unable to find the solicited analysis in the author’s reply. The dependence on the concentration argued by the authors is true, but the authors claim they are using the same protein concentrations in all the fluorescence experiments.

Regarding CD measurements, it is true that quantification of secondary structure based on changes in ellipticity may not be very robust. Authors claim that CD follows secondary structure and the study aims to describe changes in tertiary structure. This reviewer is concerned about the effect that quaternary structure may induce in the fluorescence data since it has been completely neglected from the interpretation. CD would be ideal to show if the deletion mutants adopt a molten globule conformation at 0 M GdmHCl. Thermal denaturing (particularly DSC) would inform on the possible sequential dimer dissociation-unfolding. The authors added a new line showing the values of secondary structure elements. They must show the CD spectra at increasing concentrations of GdmHCl.

All in all, it is still not clear whether the destabilizing effects on the helix bundle upon lipidation of the C-terminal tail happens intramolecularly or intermolecularly. The data presented by the authors is insufficient to answer this question. The authors still should reply or propose an explanation why mutants only partially recover the ability to solubilize DMPC (New Figure 1B).

This reviewer suggests further editing of the text; some minor mistakes are still present.

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: Yes: Manuel Bano-Polo

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation

PONE-D-19-23187R2

Dear Dr. Jonathan D. Smith,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Maria Gasset, 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 #1: 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

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: 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

**********

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

**********

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)

**********

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