-->PONE-D-25-52871-->-->Identifying and Validating ITGB2 and HNRNPAB as diagnostic biomarkers in chronic obstructive pulmonary disease Using Bioinformatics and Integrated Machine Learning Methods-->-->PLOS ONE

Dear Dr. Zhang,

Thank you for submitting your manuscript to PLOS One. Firstly, we would like to apologize for the delay in processing your manuscript. It has been exceptionally difficult to secure reviewers to evaluate your study. We have now received one completed review, which is available below. The reviewer has raised scientific concerns about the study that need to be addressed in a revision.

Please note that we have only been able to secure a single reviewer to assess your manuscript. We are issuing a decision on your manuscript at this point to prevent further delays in the evaluation of your manuscript. Please be aware that the editor who handles your revised manuscript might find it necessary to invite additional reviewers to assess this work once the revised manuscript is submitted. However, we will aim to proceed on the basis of this single review if possible.

Please submit your revised manuscript by Jan 09 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 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’.
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-->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,

Miquel Vall-llosera Camps

Senior Staff Editor

PLOS One

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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

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4. 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.

5. 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. 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

**********

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

Reviewer #1: 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

**********

-->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

**********

-->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: The manuscript by Zhang et al.” Identifying and Validating ITGB2 and HNRNPAB as diagnostic biomarkers in chronic obstructive pulmonary disease Using Bioinformatics and Integrated Machine Learning Methods" identifies COPD biomarkers from multiple gene expression datasets deposited in the GEO database, by combining several bioinformatics approaches including WGCNA, immune infiltration profiling, transcription factor and regulatory network analysis, enrichment analyses, MR, GSVA, PCR validation, and integrated machine-learning methods. Two of the identified biomarkers, ITGB2 and HNRNPAB, were further validated through PCR, IHC and IF experiments in a mouse model.

The experiments are well designed, appropriately analyzed, and the methodology is clearly described throughout the manuscript. The results are of interest to the field and, therefore, I recommend publication after minor revisions aimed at further improving the clarity and completeness of the work.

Minor comments:

1. Methods and Materials (line 100):

The sentence “…and GSE1650 consists of 18 COPD patient samples and 12 healthy control samples [17]…” is incomplete. Please clarify for what purpose this dataset was used in the study.

2. Identification of DEGs in COPD (lines 109–110):

The authors state that genes were defined as DEGs if they met the criteria of |log Fold Change| ≥ 0.585 and adjusted p-value < 0.05.

Why was the threshold set at 0.585 specifically, rather than 0.586, 0.584, or a more conventional 0.5? Please provide a justification for this choice.

3. Discussion section:

The results obtained from the validation of ITGB2 and HNRNPAB in the mouse model should be revisited and discussed in this section, clearly justifying their purpose and contribution to the interpretation and validation of the findings.

In addition, the sentence “The transcriptional factors FOXC1 and NFIC play an important role in the regulation of COPD” (lines 446–447) should be better integrated with the other proteins/pathways identified and discussed in the manuscript.

**********

-->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

**********

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

-->

<div>PONE-D-25-52871R1-->-->Identifying and Validating ITGB2 and HNRNPAB as diagnostic biomarkers in chronic obstructive pulmonary disease Using Bioinformatics and Integrated Machine Learning Methods-->-->PLOS One

Dear Dr. Zhang,

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 Mar 20 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,

Tomasz W. Kaminski

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.

Additional Editor Comments:

Dear Authors,

Based on the reviewer feedback, we will proceed with a major revision. The current reviews are constructive and provide a clear framework for strengthening the manuscript. Please address all comments carefully in your revision and response.

Kind regards,

Tomasz W Kaminski

Academic Editor

PLOS One

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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 #2: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

**********

-->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

Reviewer #3: No

Reviewer #4: Yes

**********

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

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: 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 #2: Yes

Reviewer #3: Yes

Reviewer #4: 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 #2: No

Reviewer #3: No

Reviewer #4: 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 #2: I understood the authors' purpose and motivation for this study. Unfortunately, however, methods of some analyses and experiments were totally unclear or insufficiently described, making the results and conclusion of this study totally unconvincing and not supported with valid evidence. In particular, I do not think that it is reasonable and well-supported to focus only on ITGB2 and HNRNPAB genes from the analysis results.

At least the authors should have concretely explained (1) the source of SNPs affecting protein expression levels; (2) rationale of “TF-gene regulatory network" (based on TF binding sites? or co-expression?); (3) excuse for arbitrarily selecting the IVW method in the MR analysis while some other methods returned insignificant results; (4) how to establish the “COPD mouse model” and its validity; and (5) whether antigen retrieval in immunohistochemistry on the model tissue was surely succeeded. In addition, there are many typos and mis-description in this manuscript which probably cause misunderstanding and misleading: always spell the two "key" genes at least correctly; legends of Fig. 3D and Fig. 6 do not correspond to the contents of those figures.

Reviewer #3: The study by Zhang et al. combines two publicly available COPD microarray datasets with multiple computational approaches, including WGCNA, immune cell analysis, Mendelian randomization, and machine-learning models, to identify diagnostic biomarkers for COPD. The authors highlight ITGB2 and HNRNPAB as key candidate genes and attempt to validate them using qPCR, IHC, and IF in cell line and mouse COPD models. This work is relevant because there is still a clear need for reliable biomarkers in COPD, and integrative computational approaches are increasingly used to study disease complexity. A major strength of the study is the broad computational pipeline that brings together multiple analytical methods and external validation datasets. However, the manuscript has major weaknesses that limit its scientific rigor and clarity, specially the mRNA/protein level discrepancy. Also, the writing style is unscientific and reflects conceptual misunderstandings, which makes the conclusions difficult to evaluate. In its current form, I cannot recommend this manuscript for publication unless these issues are carefully addressed. Please see my comments below.

1. A major weakness of the manuscript is the use of unscientific and conceptually incorrect language, along with a writing style that points readers to figures instead of explaining the results. For example, sentences such as “a gradient volcano plot (Fig. 2A) visualizes…” and “a heatmap (Fig. 2B) illustrates…” describe figures rather than findings and belong in figure legends, not the results section. Also, the GSVA section uses incorrect terminology (e.g., “ITGB2 exhibited expression changes across pathways” or “KEGG pathway enrichment of ITGB2”). ITGB2 is a gene, and genes are not “expressed across pathways”, pathways do not have expression. These sound like conceptual errors rather than wording issues, and should be corrected throughout the manuscript.

2. I am not convinced by the way the mRNA/protein level mismatch is addressed in the manuscript. In their MLE‑12 cell model, ITGB2 and HNRNPAB transcripts go up, but in the mouse COPD model tissue, these proteins go down. Opposite mRNA–protein changes are very rare and should be addressed by additional experiments and validation. Without cell‑matched protein quantification by western blot and some mechanistic follow‑up, this is a red flag rather than supportive evidence, and the ‘key protein biomarker’ claims should be toned down.

3. The manuscript reports “positive t-values” for GSVA results but never explains what these t-values represent, how they were calculated, or what comparison they correspond to. The t-statistics is not a p-value, and reporting “t-values” without p-values or FDR makes the results hard to interpret.

4. The reported AUC of 0.989 for a 12-gene COPD classifier trained on relatively small microarray datasets is unusually high for this kind of problem and raises concerns about overfitting. The cross-validation strategy and how the training and test sets were separated need to be explained much more clearly and evaluated more rigorously.

5. The authors did not compare the 112 genes or the final 12-gene diagnostic panel with previously reported COPD transcriptomic signatures or genes linked with COPD (e.g., PMID-25265030 linking ITGB2 expression to COPD), making it difficult to assess what is truly novel versus a rediscovery of known signals.

6. The FOXC1/NFIC–HNRNPAB axis is highlighted as a key regulatory mechanism, but the TF–gene network is built only from JASPAR motif predictions. There is no integration of expression correlations, no analysis of public ChIP‑seq data or promoter/enhancer motifs, and no functional assays (e.g., NFIC knockdown) to actually support these regulatory claims.

7. The paper repeatedly labels ITGB2 and HNRNPAB as “key biomarkers” and frames them as a basis for personalized treatment, but all data are from mouse cells/mouse lungs and retrospective gene‑expression datasets. With no prospective human validation or demonstration that these markers improve on standard clinical measures, those clinical claims feel overstated and need to be toned down.

8. The discussion section is excessively long and repetitive, with too much speculation that goes beyond the data presented. It should be shortened and focused on the main findings that are directly supported by the results and key limitations, with speculative sections reduced or removed to improve clarity and impact.

Reviewer #4: In this manuscript, Zhang et al. integrate multiple bioinformatics and machine-learning approaches to identify potential COPD biomarkers from gene expression data derived from multiple datasets. The study combines network-based analyses (co-expression and regulatory networks), pathway-level analyses, Mendelian randomization, and machine-learning methods to prioritize candidate biomarkers, followed by in vivo validation. Among the candidates, ITGB2 and HNRNPAB were validated by PCR, IHC, and IF experiments in a mouse model.

The manuscript is generally well written, and the analyses are appropriately conducted. I believe that minor revisions would further improve the clarity, consistency, and completeness of the work prior to publication.

1. Lines 123–124:

The sentence ending at line 124 appears to be incomplete, as it ends with a comma. Please revise the sentence to ensure it is grammatically complete.

2. Figure 3D:

Figure 3D is not explicitly described or referenced in the main text.

Please add a brief explanation in the Results section to clarify the purpose and interpretation of this panel.

Also, the figure legend for Fig. 3D appears to be identical to that of Fig. 3B. Please revise the legend to accurately describe the content of Fig. 3D.

4. Figure 4 legends:

The figure legends for Fig. 4B and Fig. 4C appear to be reversed.

Please confirm and correct the legends to match the corresponding panels.

5. Lines 486–488:

Two consecutive sentences begin with “On the other hand.”

I recommend revising this part to improve readability and sentence flow.

6. Reproducibility and code availability:

For clarity and reproducibility, it may be helpful for the authors to indicate whether the analysis code used in this study is available, for example in a public repository or upon reasonable request.

**********

-->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

Reviewer #3: No

Reviewer #4: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

<div>PONE-D-25-52871R2-->-->Identifying and Validating ITGB2 and HNRNPAB as diagnostic biomarkers in chronic obstructive pulmonary disease Using Bioinformatics and Integrated Machine Learning Methods-->-->PLOS One

Dear Dr. Zhang,

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 24 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.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only the individual author can complete the verification step; PLOS staff cannot verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Tomasz W. Kaminski

Academic Editor

PLOS One

Journal Requirements:

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.

Additional Editor Comments:

Dear Authors,

Thank you for submitting the revised version of your manuscript.

We appreciate the effort you have made in addressing the previous comments. The manuscript has improved in several areas, particularly with respect to the expansion of methodological descriptions and overall clarity.

However, after careful evaluation of the revised version and the additional reviewer feedback, several important concerns remain.

Importantly, one of the reviewers continues to recommend rejection, noting that key methodological and interpretational issues are still insufficiently addressed. We agree that these concerns are substantial and require careful revision.

Key points to address:

a) Methodological justification

Please provide a clearer rationale for key analytical choices, particularly:

-the use of the IVW method as the primary Mendelian randomization approach,

-the selection and source of SNPs used in the analysis.

b) TF-gene regulatory network

Please clarify the basis of this network (e.g., binding site prediction vs co-expression vs functional inference) and discuss its limitations.

c) Gene prioritization

The focus on ITGB2 and HNRNPAB requires stronger justification. Please clearly explain why these genes were selected over other candidates.

d) Coonsistency of interpretation

The reference to “treatment response” in the WGCNA analysis is inconsistent with the study design (COPD vs control) and should be revised.

e) Methods presentation

Sections describing HE staining, immunohistochemistry, and immunofluorescence should be rewritten in a concise scientific format rather than step-by-step protocol style.

f) Technical and formatting issues

Please correct formatting errors in Section 2.13.

Carefully proofread the manuscript for typographical errors, gene name consistency, and figure legend accuracy.

g) Positioning of conclusions

The proposed gene signature should be presented more cautiously as exploratory, given the nature of the data and analytical pipeline.

Overall, while the manuscript has improved, the remaining issues are substantial and must be fully addressed before the manuscript can be considered further.

We therefore invite you to submit a revised version that carefully responds to all points above.

With best regards,

Tomasz W Kaminski

Academic Editor

[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 #3: All comments have been addressed

Reviewer #4: 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 #3: Partly

Reviewer #4: Partly

**********

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

Reviewer #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: No

**********

-->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 #3: The revised manuscript has improved significantly. I am still somewhat unconvinced by the mRNA–protein discrepancy, but this limitation is now acknowledged and possibilities are discussed. Overall the language is acceptable, key claims have been toned down, limitations are recognized, and the conceptual errors have been corrected. I therefore recommend the manuscript for acceptance.

Reviewer #4: I appreciate that the authors have addressed my previous comments in the revised manuscript. On reviewing the revised version, however, I have identified several additional issues that should be addressed to further improve the clarity and consistency of the manuscript.

1. Methods: Section 2.13 Lung Function Test:

There appears to be a formatting issue in this section, where the content is inserted between the text "Methods for Lung Function Testing". I recommend carefully proofreading the manuscript prior to submission to avoid such issues.

2. HE staining, Immunohistochemistry and Immunofluorescence methods:

The description of methods is currently presented in a highly step-by-step manner, for example repeated use of imperative expressions such as "Place the sections ...". While detailed protocols are useful, this style reads more like a protocol than a methods section appropriate for a research article.

3. Results: Section 3.2 (WGCNA analysis):

The interpretation of the WGCNA results may require clarification. The statement "confirming its critical role in the treatment response" appears inconsistent with the study design, which is based on COPD versus control samples rather than treatment conditions. It is unclear whether any treatment-related variables were included in the analysis. This may be due to the labeling (e.g., "Treat" vs "Ct") used in the dataset, which could potentially lead to confusion in interpretation. I recommend revising these labels and the associated text to ensure that the interpretation is consistent with the analyzed dataset conditions.

Overall, the manuscript has improved, and addressing the points above would further strengthen it.

**********

-->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 #3: No

Reviewer #4: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

-->PONE-D-25-52871R3-->-->Identifying and Validating ITGB2 and HNRNPAB as diagnostic biomarkers in chronic obstructive pulmonary disease Using Bioinformatics and Integrated Machine Learning Methods-->-->PLOS One

Dear Dr. Zhang,

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 Jun 06 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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We look forward to receiving your revised manuscript.

Kind regards,

Tomasz W. Kaminski

Academic Editor

PLOS One

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Additional Editor Comments:

Dear Authors,

Thank you for your revised manuscript. The study has improved and is now close to being suitable for publication. Before final acceptance, I would ask you to address a few minor points to improve clarity and interpretability:

a) In the Results section (Sections 3.9-3.10), you report increased mRNA but decreased protein levels for ITGB2 and HNRNPAB. Please acknowledge this discrepancy already in the Results (not only in the Discussion) to guide the reader.

b) Your validation includes both MLE-12 cells and whole lung tissue. Please briefly clarify which cell types are likely contributing to the observed signals in tissue-based analyses and note potential differences between epithelial cells and mixed tissue samples.

c) The reported AUC values (up to 0.989) are very high. Please add a brief comment in the Discussion acknowledging the potential for overfitting or dataset-specific bias, even though external validation cohorts were used.

These are relatively minor clarifications and should be straightforward to address.

Best regards,

Tomasz W Kaminski

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Identifying and Validating ITGB2 and HNRNPAB as diagnostic biomarkers in chronic obstructive pulmonary disease Using Bioinformatics and Integrated Machine Learning Methods

PONE-D-25-52871R4

Dear Dr. Zhang,

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,

Tomasz W. Kaminski

Academic Editor

PLOS One