-->PONE-D-25-63496-->-->Monitoring radiation exposure through skin swab multi-omic profiling-->-->PLOS One

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Animal procedures realized at Lawrence Berkeley National Laboratory were supported by the IARPA TEI-REX contract number 20008-D2021-2107310008. The views and conclusions contained should not be interpreted as necessarily representing the official policies, either expressed or implied, of ODNI, IARPA, ARO, or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright annotation therein. The project described above was also supported by award no. P30 CA051008 (P.I. Louis Weiner) from the National Cancer Institute (NCI). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NCI or the NIH. Raw 16 S rRNA sequencing data have been deposited in the NCBI Sequence Read Archive under accession number [PRJNA1308618] and are available at < https://www.ncbi.nlm.nih.gov/bioproject/1308618>. 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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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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:  Summary

This manuscript presents a multi-omic study proposing skin swab–based metabolomic, lipidomic, and microbiome profiling as a non-invasive biodosimetry platform for ionizing radiation exposure. Using colonized human skin equivalents and mouse models exposed to 0, 1, or 4 Gy of x-rays, the authors apply DIABLO-based multiblock sPLS-DA to identify correlated, dose-responsive molecular and microbial signatures. While the experimental execution is extensive and the datasets are large, the study ultimately prioritizes multivariate discrimination over biological rigor. Many conclusions rely heavily on supervised classification, post-hoc feature selection, and pathway over-interpretation, without sufficient control for overfitting, temporal dependence, or biological confounding. As a result, the manuscript reads more as a methodological showcase than a validated biodosimetry framework, and its translational claims are substantially overstated.

Strengths

The study addresses a relevant and timely problem in radiation biology: the need for rapid, non-invasive biodosimetry. The use of skin swabs is innovative and practically attractive, and the parallel analysis of human skin equivalents and mouse skin is conceptually sound. The experimental workflows for metabolomics, lipidomics, and 16S profiling are technically competent and well described. The authors make effective use of modern multi-omic integration tools and present a large amount of data in a transparent manner. The inclusion of ROC analyses and dose-resolved comparisons demonstrates an effort to evaluate diagnostic performance rather than relying solely on descriptive trends.

Recommendations

The central weakness of the manuscript lies in its analytical strategy. The study relies almost exclusively on supervised sPLS-DA (DIABLO) to define “radiation-responsive” features, yet provides insufficient safeguards against overfitting.

Sample sizes per group are small relative to the number of features, time points are pooled rather than modeled explicitly, and the same datasets are repeatedly used for feature selection and performance evaluation.

High AUC values reported for refined metabolite panels, particularly those approaching 0.99, are therefore not convincing without independent validation, permutation testing, or nested cross-validation. At minimum, the authors should demonstrate model robustness through repeated resampling and clearly separate training and evaluation steps.

Temporal structure is inadequately handled. Samples collected at 1, 3, and 7 days post-irradiation are pooled for many analyses, despite the well-known time dependence of radiation responses.

Pooling obscures whether identified signatures represent true dose effects or simply reflect differences in recovery kinetics. A proper longitudinal analysis, or at least stratified presentation by time point, is necessary to support claims of dose-specific signatures.

Biological interpretation consistently overreaches the data. Pathway enrichment results are derived from putatively identified metabolites selected via supervised models, yet are discussed as evidence of coordinated biological programs (e.g., barrier disruption, mitochondrial dysfunction, systemic cardiovascular risk).

These interpretations are speculative in the absence of orthogonal validation such as histology, barrier function assays, inflammatory readouts, or targeted biochemical measurements. The frequent extension of skin molecular changes to systemic health implications is particularly problematic given that no systemic endpoints were measured.

Microbiome analyses raise similar concerns. Changes in taxa such as Lachnospiraceae and Lactobacillales are repeatedly framed as radioprotective or reparative, but the study design cannot distinguish direct radiation effects from secondary ecological shifts or housing effects. Moreover, 16S-based taxonomic resolution is insufficient to support functional claims without complementary metagenomic or metabolomic corroboration.

While the integration of multi-omic skin swab data is technically impressive, the study does not yet establish a deployable biodosimetry tool. Claims regarding real-time monitoring, emergency response utility, or spaceflight applications are premature and should be clearly labeled as future directions rather than inferred outcomes.

Conclusion

This is a data-rich and technically sophisticated study, but it is analytically and interpretively overextended. The reliance on supervised multivariate methods without adequate validation, the pooling of temporally distinct samples, and the speculative biological extrapolations substantially weaken the manuscript’s conclusions. As currently presented, the work does not convincingly demonstrate a robust, generalizable skin-based biodosimetry platform. Major restructuring of the analysis, substantial tempering of claims, and additional validation would be required to support publication.

Reviewer #2: This study systematically investigates the dose-dependent response of skin to ionizing radiation by integrating multi-omics (microbiome, metabolome, lipidome) and cross-model (human skin equivalent coHSE, mouse) approaches. The research design is novel, and the data volume is substantial. It is the first to propose a "host-microbe co-metabolism" framework for radiation response at the skin level and to screen biomarker combinations with high diagnostic potential, which holds significant value for the development of radiation biodosimetry. However, the paper has deficiencies in the rigor of experimental details, the depth of data interpretation, and logical coherence, requiring substantial revisions to support its important conclusions.

1.P150: Briefly explain why the control group has a larger sample size.

2.Briefly state the rationale for selecting doses of 1 Gy and 4 Gy.

3.Shaving itself is a strong physical and mildly traumatic stimulus that can significantly disrupt the skin barrier, alter the local microenvironment (e.g., temperature, humidity), and trigger skin inflammation and repair responses. This greatly perturbs the structure of the skin microbial community before sampling, making it unrepresentative of the "steady-state" skin flora, instead reflecting a "post-perturbation recovery" state. The animal experiment design of "shaving 24 hours before sampling" severely interferes with skin microbial homeostasis. This constitutes a fundamental flaw that must be modified or provide strong reasons.

4.The temporal design alignment between the human skin equivalent (coHSE) and mouse experiments is excellent, and the use of the same irradiation device is a major advantage. However, the inconsistency in key irradiation physical parameters (energy, filter, dose rate) means the radiation received in the two experiments differs in physical characteristics. The rationale for selecting these parameters should be briefly explained.

5.The current statistical description is the weakest part of the methods section. It fails to match the complex time-dose multi-factorial design of your study. It is recommended to rewrite the statistics section with a more rigorous and detailed description.

6.P445-447: It is suggested to change to: "1 Gy radiation exposure was primarily associated with the activation of carbohydrate metabolism pathways, while concurrent changes in the abundance of microbial taxa such as Lachnospiraceae were observed."

7.P474: Confirm the accuracy of the lipid nomenclature for "TAG(52:7)-FA(16:0), and (-2)Cl66:3-FA(16:1)".

8.P500: Change "1Gy" to "4Gy".

9.Much of the Discussion section reiterates specific observations already detailed in the Results, without sufficient interpretation, synthesis, or attribution of new meaning. The Discussion should avoid being a secondary narration of the results. It is recommended to significantly condense the restatement of specific results and instead emphasize the new concepts, models, or insights distilled from these results.

Reviewer #3: Overall comment:

This study demonstrated that radiation caused dose-dependent shifts in the skin microbiome in both human skin equivalents and mice. Several taxa - especially Lachnospiraceae/Oscillospiraceae/Bacillaceae - emerged as candidate biomarkers for non-invasive radiation biodosimetry. Although the study demonstrates the potential of skin swab multi-omic profiling for biodosimetry, the translational application of these findings to human settings remains uncertain. Additional research is needed to validate the findings in human subjects and develop practical tools for real-time monitoring. The study uses relatively small sample sizes for both coHSEs and mouse models, which may limit the statistical power and generalizability of the results. Additionally, the study pools microbial samples from human donors to inoculate coHSEs, which may not fully replicate the diversity and complexity of individual human skin microbiomes.

Specific comments

Abstract

- Please provide more details on sample sizes of human skin equivalents and murine models and how the findings can be applied to human biodosimetry.

Introduction

- Please provide more detailed comparisons of existing biodosimetry methods and their shortcomings.

- Please 3xplain why skin swabs are particularly suitable for biodosimetry compared to other non-invasive methods.

- Please highlight the need for validation in human subjects and the challenges of translating findings from animal models and coHSEs.

Materials and Methods

- Please provide more details on the diversity of microbial samples used to inoculate coHSEs and how representative they are of human skin microbiomes.

- Please explain why 0 Gy, 1 Gy, and 4 Gy were selected and how these doses relate to real-world radiation exposure scenarios.

- Please elaborate on the rationale for using specific statistical approaches (e.g., DIABLO, sPLS-DA) and their limitations.

- Please address how variability in microbial and metabolomic profiles was controlled or accounted for in the study.

Results

- Please provide more detailed explanations of the biological significance of dose-specific microbial and metabolomic changes.

- Please provide more clarity on the implications of lipid remodeling and its connection to skin barrier function and radiation injury.

- Please address how variability in responses at higher doses (4 Gy) might affect the reliability of biodosimetry.

Discussion

- Please provide more details on how the findings can be applied to human biodosimetry and the steps needed to develop practical tools for real-time monitoring.

- Please further explain how microbial shifts contribute to skin recovery and systemic health, particularly in the context of radiation exposure.

- Please discuss the need for future studies to investigate a wider range of radiation doses to improve generalizability.

- Please provide more evidence or references to support the hypothesis that skin responses may reflect systemic health, including cardiovascular injury.

- Please clarify the functional significance of lipid remodeling and its potential as a biomarker for radiation exposure.

- Please discuss the functional roles of the identified microbial taxa and their potential contributions to skin recovery and systemic health.

Conclusion

Highlight limitations: Summarize the key limitations of the study and suggest specific areas for future research.

Expand on practical applications: Provide more details on how the findings can be translated into real-world biodosimetry tools, including the use of emerging technologies like Raman or infrared spectroscopy.

Acknowledgements

Clarify contributions: Provide more details on the specific roles of each author in the study.

Expand on funding sources: Clarify the role of funders in the study design, data collection, analysis, and publication decisions.

References

Ensure completeness: Verify that all references are properly cited and formatted.

Highlight key references: Identify and emphasize the most relevant references that support the study’s findings and conclusions.

Supporting Information

Provide more details: Expand on the supplementary figures and tables to ensure clarity and accessibility for readers.

Explain the significance: Clarify the importance of the supplementary data in supporting the main findings of the study.

By addressing these points, the study can improve its clarity, robustness, and potential for real-world application.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Monitoring radiation exposure through skin swab multi-omic profiling

PONE-D-25-63496R1

Dear Dr. Laiakis,

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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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?<br/><br/>The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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-->4. Have the authors made all data underlying the findings in their manuscript fully available?<br/><br/>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?<br/><br/>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<br/><br/>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 revision addresses the major concerns raised during review. The authors have clarified the exploratory role of the DIABLO analysis, added independent validation of the metabolite panel, included cross-validation and permutation testing, and toned down the interpretation of the AUC results. They also provide a clearer rationale for pooling early time points, dose selection, shaving procedures, irradiation parameters, donor microbiome diversity, and statistical methods. Importantly, the biological and translational claims are now more balanced, with microbiome shifts, pathway enrichment, systemic implications, and real-time biodosimetry applications presented as hypotheses or future directions rather than established conclusions. Overall, the manuscript is much improved, the remaining limitations are clearly acknowledged, and I have no further major concerns. I consider the manuscript ready for publication.

Reviewer #2: The author has already answered all the questions I raised.I have no further comments. I recommend publishing forPLOS One.

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Reviewer #1: No

Reviewer #2: No

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