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PONE-D-21-38603Comprehensive analysis of different tumor cell-line produced soluble mediators on the differentiation and functional properties of monocyte-derived dendritic cellsPLOS ONE

Dear Dr. Türk-Mázló,

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 submit your revised manuscript by May 09 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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PLOS ONE

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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

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: No

Reviewer #2: Yes

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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

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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: Referee comments on PONE-D-21-38603

Burai, S, et al “Comprehensive analysis of different tumor cell-line produced soluble mediators on the differentiation and functional properties of monocyte-derived dendritic cells”

Türk-Mázló and colleagues investigate the impact of tumor environment on dendritic cell differentiation. To do so, they analyze and compare the effect of the tumor cell line-derived condition media using eight different adenocarcinoma and melanoma cell lines on in vitro differentiation of dendritic cells according to several variables. Their results highlight the different effects on DC differentiation between melanoma and adenocarcinoma cell lines.

Overall, they provide an interesting approach for the in vitro study of different tumor-promoting DCs, however, revisions seem to be needed.

Major revisions:

1) There are several inconsistencies between the text and the results presented in the figures. In particular:

“HT29-, HeLa-CM-moDCs and in accordance with the literature, DexDCs also showed significantly higher CD14 expression [23] compared to the control moDCs (Fig 1A).” (lines 270-271): Fig 1 does not show significant result for HeLa cell line.

“HT29, WM278, WM1617, and WM983B-conditioned moDCs expressed significantly more CD14 and CD209 molecules than their MDA- or WM983A-educated counterparts.” (lines 280-282): This should be re-worded to refer to higher frequency of co-expression of CD14 and CD209 instead of more expression. Fig 1 also does not show that the difference between WM1617 and MDA or WM983A cell lines is significant.

“… significant difference was observed between the WM983A and DexDC groups).” (line 293): this difference is between DexDC and WM278 and not between DexDC and WM983A.

“… the phagocytic capacity was increased slightly but not significantly compared to control moDCs. These results indicate that only certain tumors can alter the phagocytic capacity of the DCs.” (line 374-376) and “… indicating that tumor microenvironment influences the phagocytic capacity of DCs” (line 522): The authors state that the results indicate that only certain tumors may impair or influence the phagocytic capacity of DCs, stating that no significant results are shown. I think the authors should be less categorical in the Discussion.

Fig 5 needs more thorough description of gating. Panel A would be better visualized as a 2D plot for CD4 and CD8 instead of independent density/histogram plots. For the quantification on the right it is not entirely clear what the denominators are. CD8+ IFNg+ should probably be reported as a percentage of total CD8+ cells and similarly CD4+CD25+FOXP3+ should be percentage of total CD4+. It is not clear whether CD4+CD25+IL10+ should be reported as percentage of CD4+ or CD4+CD25+.

“Based on our correlation analysis, between HLA-ABC, HLA-DR and PD-L1, their expression was similarly regulated in the case of melanoma cell lines-educated DCs, whereas strong negative correlation was observed in the group of moDCs conditioned by adenocarcinomas with the respect of HLA-ABC, HLA-DR and PD-L1 expressions.” (lines 539-543): Regarding adenocarcinoma cell lines, only HLA-ABC and PD-L1 are strongly anti-correlated, the other pairs (HLA-ABC with HLA-DR and HLA-DR with PD-L1) are just not correlated. This should be described more accurately.

2) There are some errors in the annotation of the figures and the accompanying descriptions. I encourage the authors to review the annotation of their figures and their descriptions. In particular:

Lines 291 and 301 refer to Fig 1C, however, Figure 1 contains only two parts (Fig 1A and Fig 1B).

Presenting Fig 3B as a heatmap makes it very difficult to compare with Fig 3A. Can another visualization be found (e.g. perhaps the same as Fig 3A) that makes these results easier to interpret?

The references to Fig 5 do not target the correct part of the figure. For example, the reference to Fig 5C on line 408 should probably be in the previous sentence and replaced here by Fig 5D.

There is an annotation problem between Fig 6 and its description (the description mentions Correlogram B and C while the correlograms make up Fig 6C and Fig6D).

3) The authors propose an interesting approach using bioinformatics to enrich the interpretation of their results. Although the authors have the good idea to use the correlation circle as a complement to the PCA, they did not apply it on the variables (as described in the figure description) but on the cell lines. Correcting this error and interpreting the results obtained could bring possibly interesting information and help to enrich the bioinformatic results.

“In summary, PCA analysis revealed differences in the strategy of melanomas and adenocarcinoma cell lines in the manipulation of moDC differentiation, thus in their ability to orchestrate T cell responses.” (lines 449-451): PCA shows clear differences between the cell lines and the control (RPMI and DexDC) but it doesn’t show differences between the different cell lines. Can the authors perhaps provide a PCA after removing the RMPI and DexDC controls to see if it validates this conclusion?

Minor revisions:

1) A better description of the method of the bioinformatics part would be helpful (package version, function parameters, ...).

2) Adding titles to the figures would help the reader to follow (e.g.: add adenocarcinoma above the corresponding correlogram, ...)

3) Improve the title of figure 6 "PCA" which does not take into account the half of the figure.

4) Figure2: results observe using MFI histograms and the barplots are sometimes different, perhaps more representative MFI histograms should be used instead (e.g.: HLA-ABC of WM983B cell line shows clear differences by MFI plot at top of Fig 2A but much more moderate typical effect in barplot below).

5) “Our result suggests that tumor cell lines and dexamethasone alter the epigenetic regulation of the CD14 and DC-SIGN expression in moDCs. All tumor cell type-derived factors induced the co-expression of CD14 and DC-SIGN.” (lines 495-498): Authors say cell lines and dexamethasone alter the epigenetic regulation of CD14 and DC-SIGN expression in moDCs, however, only CD14 expression is altered as described after, maybe this sentence could be reformulated to explain better this observation.

6) “We have found that the expression pattern of MHCII and PD-L1 was also correlated with the ability of TU-CM-conditioned moDCs to stimulate CD4+CD25+FoxP3+ T cells. (line 545-547) : Authors could be precise these observation is in the melanoma cell lines.

7) “Overall, TU-CM-educated DCs induced Treg differentiation, especially the subtype of IL-10 producing Tregs.” (lines 554-555): Add the figure reference.

8) “… Interestingly, this correlation was the opposite in the case of melanoma cell-line-CM-conditioned moDCs” (lines 576-577): Authors precise the TNFα and CD4+CD25+FoxP3+ T cells positively correlate in melanoma cell lines, however, only WM1617 cell line expresses TNFα well. Authors should describe this observation more carefully.

9) “IL-8 production by adenocarcinomas negatively correlated with the expression of MHC and costimulatory molecule CD86 as well as IL-10 producing helper T cell polarizing capacity of moDCs” (lines 585-587): It is only the case with MHCII, not MHCI.

Suggestions:

1) Unless I have not seen the file, it would be useful to provide an excel or csv file containing all the variables used for the bioinformatics analyses.

2) For the correlation analyses, adding in additional figures the plots of correlations of the most interesting pairs of variables could help to better understand the data. Moreover, it could allow to see if some melanoma or adenocarcinoma cell lines behave differently from others.

3) To complete PCA, authors could be produce a a clustering/heatmap on the same data.

Reviewer #2: In this manuscript, the authors aim to decipher the impact of tumor cell-derived soluble factors on monocyte differentiation into monocyte-derived DC (mo-DC). They use a well described in vitro model (GM-CSF + IL-4). The authors claim that monocyte exposure to factors derived from tumor cell modify the phenotype of mo-DC and their expression of co-stimulatory molecules. The authors next focus on typical functions of mo-DC: phagocytosis and polarization of T cells. They conclude that tumor-derived soluble factors alter phagocytosis and polarization of T cells. In the last part, the authors show correlations between different properties or functions of mo-DC: phenotype, cytokine secretion, ability to polarize T cells and phagocytosis.

Major comments:

1. In figure 1, the authors define mo-DC as CD14+ or CD209+. However, CD14 and CD209 are not specific markers of mo-DC and can be expressed by macrophages too. Moreover, it has been shown by mass cytometry that the mo-DC population (differentiated from monocytes with GMCSF and IL-4) is homogeneous, with some cells expressing CD163 and MERTK suggesting the presence of mo-mac in this model (Sander, Immunity 2017). Are the authors sure that tumor cell derived factors modify only surface markers and not the identity of the cells? In line with this comment, the authors show that CD1a (typical mo-DC marker) expression is decreased by factors secreted by tumor cells.

2. Figure 2 : it is not clear how the authors define the positive population for the different markers. In comparison with the unlabeled control, it seems that almost all the cells are positive. They should rather analyze the MFI.

3. Figure 3 : the authors show that some tumor cell lines secrete IL8 and IL6. When measuring the secretion of cytokines by mo-DC, did the authors use a control to exclude cytokines coming from the conditioned medium?

4. In figures 4 and 5, the authors should increase the n. It is hard to reconcile how they obtain statistically significant results with n=3.

5. In figure 6, the staining for Foxp3 should be improved. A better staining allowing the identification of T regs could modify the conclusion.

Minor comments:

1. In the introdcution, the authors should mention the DC1 population. It has been shown that this subset of DC is involved in the anti-tumor responses.

2. In the section « DC-T cell co-cultures » of the material and methods, more details about the procedure are needed. How were T cells isolated? Were CD8 and CD4 T cells cultured together with mo-DC (3 cell types in the same well)? Did the authors use frozen PBMCs to isolate T cells (as they fo autologous co-cultures)?

3. The authors should specify which statistical tests were performed.

4. In figure 1 and 2, the authors should include an FMO for each condition and not only the control one (RPMI).

5. The authors should explain why they choose to investigate the phagocytosis of bacteria in the context of anti-tumor immunity.

6. In figure 5, when gating T cells, the authors should not exclude the cells with higher FSC and SSC. After proliferation, T cells become bigger and more granular.

7. In figure 5D, it is not clear why the authors gate on two different populations (black and red).

8. I would recommend the authors to improve the language to facilitate the readability of the text.

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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.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-38603R1Comprehensive analysis of different tumor cell-line produced soluble mediators on the differentiation and functional properties of monocyte-derived dendritic cellsPLOS ONE

Dear Dr. Mázló,

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 submit your revised manuscript by Aug 08 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Jean Kanellopoulos, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear Dr Anett Mázló,

You will find the comments of reviewers N°1 and N°2. They contain some requests and criticisms that need to be addressed carefully.

I would like to stress that the flow cytometry analyses must be clarified as requested by reviewer N°2. For figure N°4, a negative control must be introduced in typical experiments.

For figure N°2, you must precise how you define the positive populations and modify your initial conclusions if needed.

Yours Sincerely,

Dr Jean Kanellopoulos

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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: (No Response)

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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: Partly

**********

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

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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: Referee comments on PONE-D-21-38603

Burai, S, et al “Comprehensive analysis of different tumor cell-line produced soluble mediators on the differentiation and functional properties of monocyte-derived dendritic cells”

The revised version of the manuscript by Mázló and colleagues is significantly improved and addresses most of the original comments. I now recommend accepting the manuscript for publication although there are some essential, minor revisions that must be addressed (below). I trust the editor to evaluate these revisions, I do not need to see the manuscript again.

Minor revisions:

1) References should be added for statements on lines 100-107 and lines 627-628.

2) The description of the methods concerning the bioinformatics part is really much better. However, it is still necessary to add the parameter values used for these commands, even if just the default values were used. Similarly, the parameter values for pheatmap in figure 6 and S6 should be specified.

3) The text describing the results in Figure 3 is unclear.

The statement quoted in line 371-372 should reference Figure 3B instead of 3A. Conversely, the statement on line 381-382 should refer to Figure 3A.

Similarly, the statement in line 378-381 concerning WM1617 should refer to Figure 3A instead of 3B.

4) Figure 4B legend should be revised to refer to boxplots instead of histograms.

5) Figures 5 and 6 appear to have been swapped or mislabeled. Eg lines 422- 451 appear to refer to the figure labeled 6 instead of 5 and lines 453-530 refer to the figure labeled 5 instead of 6.

6) The two sentences on line 460-463 should be joined and reworded for clarity.

7) Several errors are present in the description of the correlation analyses of adenocarcinomas: CD1a and HLA-ABC are not positively correlated (line 475), CD1a is not negatively correlated with IL10-producing CD4+CD25+ but is negatively correlated with IFNγ-producing CD8+ cells (line 489) and HLA-DR is not negatively correlated with IFNγ-producing CD8+ but with IL8-production (line 493-494). In addition, t might be better to specify the correlations grouped in the term “T cell stimulatory potential” (line 513), such as: “…T cell stimulatory potential (IFNγ-producing and T cell activator)”.

8) There are some small errors in the discussion:

There is an error in line 622, WM278 should be replaced by WM1617.

There is an error in the sentence of line 638-639: the negative correlation between TNFα secretion and IL-10 producing CD4+CD25+ T cells is observed in melanoma and not in adenocarcinoma.

9) Several typos are present in the manuscript such as: "DC-SIGN/CD20916" (line 553) instead of "DC-SIGN/CD209", "DCsand" (line 589) instead of "DCs and", etc. Extensive proof reading is required.

Reviewer #2: The flow cytometry data still needs to be improved.

In figure 2 : it is still not clear how the authors define the positive population for the different phenopytpic markers. If the positive population is determined according to the unlabelled cells, the mean should be higher than 80% (and not 50% as shown in the graphs).

In figure s4 : a representative facs plot should be shown for CD163 and CD206.

In figure 4 : a negative control (monocyte-derived cells + Bacteria at 4°C) is lacking. The fluorescence measured should correspond to binding of the cell surface instead of phagocytosis.

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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

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[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.

Comprehensive analysis of different tumor cell-line produced soluble mediators on the differentiation and functional properties of monocyte-derived dendritic cells

PONE-D-21-38603R2

Dear Dr. Mázló,

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.

Your manuscript is very much improved. However, there is still numerous mistakes (typos etc...) such as :

Line 599 The following sentence is unintelligible : “These observations corroborate the importance the simultaneous consideration of variables”

Line 610 : “but it is quite differ from the other melanomas” instead of

“but it is quite different from the other melanomas”

line 618 : “In oppose to this in the group of moDCs…” instead of

In contrast to this  

Name misspelled line 614 : Johson instead of Johnson (49).

I recommand you to have your manuscript edited if possible by a native english speaker.

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,

Jean Kanellopoulos, M.D., 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

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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)

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

Reviewer #2: (No Response)

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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)

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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

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