-->PONE-D-25-32798-->-->Multimodal Anti-Helicobacter pylori Effects of Lactobacillus casei HY001: Evidence from In Vitro and In Vivo Studies-->-->PLOS ONE

Dear Dr. Lv,

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #2: No

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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: This manuscript assesses anti–Helicobacter pylori properties of Lacticaseibacillus (Lactobacillus) casei HY001 using in-vitro assays and a mouse pretreatment model. The linkage between cellular readouts, histology/inflammation, and gastric microbiota is a strength. However, the central claim of “reduced colonization” is not supported by a direct quantitative gastric burden (CFU/qPCR). The prophylactic design is at times generalized to therapeutic benefit; the microbiota methods/terminology contain inconsistencies requires reinforcement; and nomenclature/English editing (e.g., Proteroidota) need correction. Data deposition for raw 16S reads is also recommended. In my view, the work is potentially publishable after major revision.

Major points

1. Quantification of gastric H. pylori burden is insufficient

Mouse infection status relies on rapid urease test, without CFU plating or qPCR of gastric tissue. RUT is semi-quantitative and not a gold-standard bacterial load measure. Please add a direct quantitative readout of gastric colonization—such as CFU counts and/or qPCR from gastric tissue.

Across the Discussion/Conclusion you state that HY001 effectively reduced colonization, yet no direct quantitative burden (gastric CFU or qPCR) is reported. Without such metrics, please soften the language (e.g., “associated with reduced markers of infection”) or add the missing quantification.

2. Animal study design demonstrates prophylaxis, not treatment

HY001 was given before and through infection, demonstrating pretreatment/prophylactic effects rather than therapy of established infection; claims drift toward “therapeutic” benefit. Please align Abstract/Discussion/Conclusion phrasing accordingly and avoid general therapeutic claims such as “foundation for an anti-H. pylori drug” unless a post-infection arm is added.

If you want to discuss a therapeutic effect, please include a post-infection treatment arm in which HY001 is initiated after HP colonization is established.

3. Microbiota analysis: terminology accuracy and method modernity

Across the text, figures, and legends, phylum-level names are inconsistently reported using a mixture of legacy and updated nomenclature. Because terminological consistency is essential for clarity and reproducibility, please apply it uniformly throughout. Concretely, we see both Bacteroidetes vs. Bacteroidota and Proteobacteria vs. Proteroidota (The manuscript uses “Proteroidota,” which is not a recognized phylum name in major taxonomic frameworks and appears to be a typographical error. It likely intends Pseudomonadota).

The manuscript states “Principal Component Analysis (PCA) based on the binary Jaccard algorithm,” which is methodologically inconsistent: Jaccard is a non-Euclidean distance, whereas PCA is defined in Euclidean (covariance) space. For β-diversity ordination you should use Principal Coordinates Analysis (PCoA) on the Jaccard distance (or NMDS), rather than PCA.

In addition, no statistical test of between-group differences is reported. Please provide a PERMANOVA with the pseudo-F, R², p-value, number of permutations, and apply multiple-testing correction (e.g., Benjamini–Hochberg) for pairwise contrasts when applicable.

4. 16S data transparency and availability

Methods list the 16S pipeline, but no repository accessions are provided; the Data Availability statement currently points to “within the manuscript,” which is insufficient for sequencing data. Please deposit raw reads and provide accession IDs.

Minor points

•Abstract scope drift: The latter half of Abstract lists multiple taxa (phylum/family/genus), which dilutes focus. In an abstract, avoid laundry lists; instead, state that the gastric microbiota composition shifted significantly. Leave taxon-level details to the Results.

•Figure legend scope: Some legends refer to “fecal microorganisms,” but the study focuses on gastric samples—please correct to “gastric.”

•Taxonomic spellings: Correct misspellings such as “Protebacteria → Proteobacteria,” “Muribaculacea → Muribaculaceae,” “Lachnosoiraceae → Lachnospiraceae.” “Protebacteria” also appears in Figure 7.

•Method citations: Ensure references in Methods directly support the techniques used (e.g., ELISA procedures, microbiome pipelines) and remove unrelated citations.

Reviewer #2: Materials and methods

Prior to screening, the selection criteria for the six Lactobacillus casei strains are not clearly mentioned beyond their isolation from sour milk. It would be better if authors give rationale for why these specific strains were chosen or their genomic/phenotypic differences.

Authors have used only one gastric cell line in their in vitro adhesion assays. What would be the justification for reliance on a single cell line? It would limit the generalizability of adhesion and displacement results.

Authors have used on female mice as animal models. The gender differences in studying immune response and microbiota composition could influence results. Therefore, it would be better if both sexes are included in the study.

The methodology for the formulation of L. casei is not explained.

Authors should describe explicitly the inclusion of some appropriate negative or vehicle controls.

The data of some cytokine is showing nonsignificant trends but authors have discussed them as biologically relevant without sufficient statistics.

Results

It would be better if authors give clinical relevance of their findings of in vitro and animal models.

Authors have studied microbiota changes but did not establish functional causality between specific microbial shifts and observation of anti-inflammatory or anti-H. pylori effects.

Authors should critically discuss the contradiction of conflicting microbiota results for their increased abundance of genera such as Helicobacter in the treatment group.

Authors have not addressed the potential for probiotics causing adverse effects, especially with long-term administration.

Discussion

Authors have merely discussed the recent development and contradictory findings in probiotic therapy against H. pylori.

Authors should add comparative discussion on why HY001 is superior or mechanistically different.

Writing and Presentation

The manuscript has grammatical errors, awkward phrasing, and redundancy.

Need corrections for some terms such as anaplasmosis portal and thick-walled portal which are related to microbiota analysis. These are unclear or not correctly used.

The abbreviations are inconsistently used in different sections, which are confusing.

Authors should strengthen experimental design by including additional gastric cell lines and/or both sexes in animal studies or provide strong justification for their choices.

Authors should provide more robust statistical reporting and improve figure legends for independent comprehension.

**********

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Reviewer #1: No

Reviewer #2: Yes: Rawaba Arif

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-->PONE-D-25-32798R1-->-->Multimodal Anti-Helicobacter pylori Effects of Lactobacillus casei HY001: Evidence from In Vitro and In Vivo Studies-->-->PLOS One

Dear Dr. Lv,

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 Jan 29 2026 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 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'.
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• 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,

Ghulam Mustafa, PhD

Academic Editor

PLOS One

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If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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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: All comments have been addressed

**********

-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: 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

**********

-->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

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-->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 substantially improved the manuscript in response to the previous review, particularly regarding the prophylactic framing of the animal model, data deposition for 16S rRNA sequencing, and additional analyses linking gastric microbiota to inflammatory markers. However, there remain several important issues that, in my view, still warrant a further round of revision before the manuscript can be considered for acceptance.

Major Comments

1. Quantification and interpretation of gastric H. pylori burden

The authors still do not provide a direct quantitative measure of gastric H. pylori burden (e.g., CFU counts or qPCR from gastric tissue), and rely instead on RUT results and relative abundance of Helicobacter from 16S rRNA amplicon sequencing as indirect indicators.

Given this limitation, the Discussion and Conclusion occasionally still use wording that implies a reduction in “load” or “colonization” in a way that suggests direct quantitative evidence. This overstates what the available data can support.

Request:

• Throughout the Discussion and Conclusion, I ask the authors to: Further soften and standardize the language so that claims are clearly limited to:

• reductions in Helicobacter relative abundance (from 16S data), and

• improvements in infection-related biomarkers (RUT, urease, PGI/PGII, histology, cytokines),

rather than “reducing H. pylori colonization” or “reducing bacterial load” per se.

• For example, expressions such as “reduced the load/colonization of H. pylori” should be systematically revised to formulations like “reduced Helicobacter relative abundance and infection-related markers”.

• In the Limitations section (or the final part of the Discussion), the authors should explicitly acknowledge that:

• Gastric H. pylori burden was not directly quantified using CFU counts or qPCR.

• The conclusions regarding anti–H. pylori effects therefore rely on RUT and relative abundance from 16S rRNA gene sequencing, rather than absolute bacterial load measurements.

This clarification is important to align the strength of the claims with the actual data and to transparently communicate methodological limitations to readers.

2. Microbiota analysis – β-diversity methodology and PERMANOVA

The authors state in their response that they have re-analysed β-diversity using PCoA on the Jaccard distance and performed PERMANOVA. However, the current manuscript text remains internally inconsistent and does not fully reflect these methodological changes.

(a) PCA vs PCoA inconsistency

In Methods (Section 2.5.3), β-diversity is still described as being analysed by PCA based on Jaccard (e.g., “PCA was employed to further analyze beta diversity”). In contrast, in Results (Section 3.4.3) and the relevant figure legend, the analysis is described as Principal Coordinate Analysis (PCoA) based on the binary Jaccard algorithm.

This discrepancy leaves it unclear what analysis was actually performed.

Request:

• Please ensure that the description of β-diversity analysis is fully consistent throughout the manuscript.

• If PCoA on the binary Jaccard distance was indeed used (which is methodologically appropriate), then Methods 2.5.3 must be revised to say PCoA (not PCA), and the language in all sections (Methods, Results, figure legends) should be harmonized accordingly.

(b) PERMANOVA – description and reporting

In the response letter, the authors state that they performed PERMANOVA with pairwise comparisons and FDR correction.

However, in Methods (Statistics, Section 2.6), there is no mention of PERMANOVA or any distance-matrix–based multivariate test. Only one-way ANOVA, Tukey’s post hoc test, and correlation heatmaps are described. In the Results and figure legends, there is no reporting of PERMANOVA outputs (no pseudo-F statistic, R², p-value, or number of permutations).

Thus, the manuscript does not currently support the claim that PERMANOVA was performed.

Request:

• If PERMANOVA has indeed been carried out:

• Add a clear description in Methods (Statistics) of:

• the distance matrix used (e.g., Jaccard),

• the PERMANOVA procedure (e.g., number of permutations),

• and how multiple testing was handled for pairwise comparisons (e.g., Benjamini–Hochberg).

• In the Results (and/or relevant figure legend), report at minimum the overall PERMANOVA result with pseudo-F, R², p-value, and number of permutations.

———

Minor Comments

1. Taxonomic spelling and nomenclature

Despite substantial improvement, several taxonomic spelling errors remain and should be corrected.

• In the Discussion: “Proteobactria” → “Proteobacteria”

• In the Abstract and Results: “unclassified-Muribaculaceaeae” → “unclassified-Muribaculaceae”

• In the Results: “Oscillospiracea”(Line 406) → “Oscillospiraceae”

2. Other wording and typographical issues

A few additional typographical and wording errors should be corrected for clarity and precision.

• Introduction (around line 83): “alleviating tail inflammation” should be corrected to “alleviating gastric inflammation”

• Methods (around lines 232–233): “Interleuki-8, Interleuki-1β, Interleuki-6” → “Interleukin-8, Interleukin-1β, Interleukin-6”

• Results (around line 371): “binary jaccard algorith”→ “binary Jaccard algorithm”

• Results (around line 364, IL-6 description)

The text currently states that the mean IL-6 concentration in the HY001 group is “higher than that of the HP group”, which contradicts the protective direction of effect described elsewhere and in the Discussion.

This is almost certainly a typo and should be corrected to “lower than that of the HP group”,

provided that the underlying data support this (if not, the interpretation in the Discussion should be adjusted accordingly).

• Figure 4 legend

The definitions of the HP and HY001 groups are currently confusing and partly duplicated, e.g.:

• “HP: treated by H. pylori SS1 group vs. pretreated by L. casei HY001 and treated by H. pylori SS1.”

• “HY001: treatment using L. case HY001 and treatment using H. pylori SS1; HP: treatment using H. pylori SS1.”

Please simplify and unify the legend.

• Figure 6 legend (gastric vs fecal)

In one place, the legend refers to the “composition of gastric microorganisms”, whereas in another location (around line 869) it refers to “composition of fecal microorganisms”. Since this study is based on gastric samples, all such references in the figure legends and text should be unified as “gastric microbiota” or “gastric microorganisms”, and any inadvertent “fecal” wording should be removed.

Reviewer #2: All comments have been addressed and authors have revised their article. Some references should be checked and updated if possible. Authors should carefully proofread their article as their are still some typos and grammatical mistakes that should be corrected.

**********

-->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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-->PONE-D-25-32798R2-->-->Multimodal Anti-Helicobacter pylori Effects of Lactobacillus casei HY001: Evidence from In Vitro and In Vivo Studies-->-->PLOS One

Dear Dr. Lv,

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 Apr 02 2026 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 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,

Ghulam Mustafa, PhD

Academic Editor

PLOS One

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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: (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

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

Reviewer #1: Yes

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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 #1: 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

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-->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: Overall recommendation

The manuscript has improved substantially, and the key methodological/statistical issues (PCoA vs PCA; PERMANOVA reporting; explicit limitations regarding lack of absolute H. pylori quantification) are largely addressed. I recommend Minor revision prior to acceptance, but several important consistency/wording issues remain.

β-diversity methodology and PERMANOVA: largely fixed, but internal inconsistencies remain

R2 requested consistent PCoA terminology and full PERMANOVA description/reporting.

The Methods/Statistics now correctly describe PCoA (binary Jaccard) and PERMANOVA (adonis2, 9999 permutations, BH-FDR, pairwise).

The Results now report pseudo-F, R², p, and permutations.

Critical issue(@ Results 3.4.3): the text states HP vs HY001 is “not significant” but shows p-adj = 0.012; this contradicts the usual p<0.05 threshold and likely reflects a typo (e.g., 0.12) or a misstatement. Please correct based on the actual output.

Minor comments

(Line 826)

Figure legends/group definitions remain confusing (Figure 4 legend mixes definitions and comparisons; “L. case” should be “L. casei”; group naming should be consistent across all figures/text).

(Line 832)

Figure 6 legend still contains “fecal microorganisms,” which must be “gastric microorganisms.”

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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

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-->

Multimodal Anti-Helicobacter pylori Effects of Lactobacillus casei HY001: Evidence from In Vitro and In Vivo Studies

PONE-D-25-32798R3

Dear Dr. Lv,

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.

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Kind regards,

Ghulam Mustafa, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

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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

**********

-->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)

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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

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