PONE-D-24-55722Identification of AURKB , BUB1 , CCL2 , and TOP2A  as biomarkers, and immune infiltration in kawasaki disease through bioinformatics analysis: a preclinical studyPLOS ONE

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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: Yes

Reviewer #3: Partly

Reviewer #4: Partly

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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: No

Reviewer #3: No

Reviewer #4: 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: Thank you for your invitation.

The manuscript is about the Identification of AURKB , BUB1 , CCL2 , and TOP2A  as biomarkers, and immune infiltration in kawasaki disease through bio informatics analysis. It is of certain value, the structure is clear and the charts are appropriate, but please consider some details as outlined below:

1.Please check through the manuscript for writing. There are differences in the use of statistical notation throughout the manuscript. "p" in line 74 and 78 should be italicized, and in line 99 presented as "p-values". In line 87, "xCell" should be capitalized as "XCell", etc.

2.When using GO and KEGG to identify the enriched functions and pathways, how do you handle the issue of functional redundancy to prevent over-interpretation caused by a single gene's involvement in multiple pathways?

Reviewer #2: This manuscript presents a bioinformatics study that identifies potential biomarkers (AURKB, BUB1, CCL2, and TOP2A) and investigates immune cell infiltration patterns in Kawasaki disease (KD). The study offers valuable insights into the molecular mechanisms underlying KD and provides evidence for the potential use of these biomarkers in diagnosis and targeted therapy. However, there are several areas where the manuscript could be improved in terms of structure, clarity, language, and scientific depth. (1)The manuscript lacks a clear, cohesive structure in some sections, particularly in the Results and Discussion. There is frequent overlap, repetition, and mixing of topics;(2) The manuscript contains frequent grammatical errors, awkward phrasing, and informal expressions, which reduce its readability and professionalism. (3)Many ideas, such as the roles of AURKB and BUB1 in mitosis or the enrichment of immune-related GO terms, are repeated across sections without adding new insights. This leads to redundancy. (4) While the manuscript provides detailed descriptions of hub gene functions and pathways, the relevance to KD is not consistently emphasized. Discussions about cancer-related roles of AURKB, BUB1, and TOP2A dominate, while their specific roles in KD are underexplored. Below are partial problems listed in the order they are stated in the paper

1.Use Greek letters consistently (e.g., TNF-α instead of TNF-a) to adhere to academic conventions.

2. 'However, patients do no show apparent clinical manifestations, thus delaying diagnosis and treatment.' better changes to 'However, KD patients often lack apparent clinical manifestations, leading to delayed diagnosis and treatment.'

3. Some expressions could be made more concise and formal to enhance the academic tone of your writing.

"The data were cleaned using Limma11packages in R software (4.3.0)." , it could be better revised as "Data normalization and preprocessing were performed using the limma package in R (version 4.3.0)."

4. "log fold change" should be clarified as "log2 fold change" to be consistent with standard terminology.

5. The current structure is mostly fine, but for better readability and organization, please start each subsection with a brief explanation of the purpose of the analysis. For example:In 2.2, briefly state the purpose of constructing the PPI network (e.g., to identify interactions between differentially expressed genes), etc.

6. Some expressions are too general or lack specificity. Consider the following refinements:

2.2: PPI Network Construction:

Specify the STRING database threshold (e.g., combined score cutoff for significance);

Explain how hub genes were identified (e.g., via centrality metrics like degree or betweenness).

Include the version of Cytoscape for reproducibility.

2.3: Immune Cell Infiltration Analysis:

Shorten and simplify the description of xCell without losing its technical essence.

State how input data were normalized or preprocessed before applying xCell.

Reorganize for better readability: explain the analysis, significance threshold, and visualization in sequence.

2.4: Correlation Analysis and PCA

Specify "hub genes" and "immune cells" explicitly in the correlation analysis description.

Use consistent package names (e.g., "ggplot2" in backticks).

For Spearman analysis: indicate how correlation coefficients were interpreted (e.g., thresholds for "strong" or "weak" correlations).

7. The logical flow of the results section is unclear, and the connections between subsections are weak and the results feel fragmented, and there is no clear summary of key findings in some sections. Please reorganize the results section to create a more coherent flow. Use a logical order such as:

--Identification of DEGs: Provide the results of DEG analysis and briefly explain its significance.

--GO and KEGG Enrichment Analysis: Highlight the biological relevance of the enriched pathways and terms.

--PPI Network Construction and Hub Gene Identification: Emphasize the key findings related to hub genes.

--Immune Cell Infiltration Analysis: Explain the immune landscape differences in KD.

--Correlation Analysis and PCA: Highlight the relationships between immune cells and hub genes, and show how PCA distinguishes KD and NC samples.

8. Replace vague phrases like "immune infiltration" with "immune cell infiltration scores."

9. Section 3.3 is better suited as a methods description rather than a presentation of results.

10. In Fig. 3E, the bottom-right point in the PCA plot may be treated as an outlier and removed before further analysis.

11. The discussion lacks a clear structure, making it difficult to follow the key findings and their implications. There is no clear progression from specific results to broader implications.Multiple ideas (e.g., hub gene functions, immune mechanisms, PI3K-Akt pathway) are mixed within the same paragraph without proper separation, which makes the discussion overwhelming and repetitive. Please restructure the discussion into clearly defined subsections for better readability.

12.The discussion sometimes strays from Kawasaki disease (KD) and focuses too much on general roles of genes (e.g., their roles in tumor progression or mitosis). While these are important, the relevance to KD is not always clearly established. For example, the discussion on IL-4 mentions IVIG studies in mice but does not sufficiently connect these findings to KD in humans.

13. "It can be conclused that the four-gene above mentioned take part in the course of immune cell-mediated development in KD patients." (grammatical and awkward phrasing)

14.The conclusion is too brief and does not adequately summarize the key findings or their broader implications. Please provide a stronger, more detailed conclusion that highlights the study’s key contributions and potential impact:

--Recap the four hub genes and their potential as diagnostic biomarkers.

--Emphasize the unique immune cell infiltration patterns identified in KD.

--Discuss how the findings provide a foundation for future research into KD mechanisms and therapeutic strategies.

Reviewer #3: In this study, the authors looked at up- and downregulated genes in Kawasaki disease (KD) patients compared to healthy patients. They clustered the genes by pathways revealing immune system-related processes, as expected in KD, and PI3K-Akt signaling as potentially important for KD. Further protein-protein interaction analysis led to the identification of AURKB, BUB1, CCL2, IL4 and TOP2A as potential protein biomarkers for KD. After looking at the genes, they looked at the immune cell distribution suggesting that innate immune cells are more present in patients with KD, but adaptive immune cells are less present. They ended the study by using PCA to look at relationship between immune cell levels and gene expression levels for the previously identified ones potentially involved in KD.

While identifying biomarkers for KD is of great need due to the lack of KD-specific biomarkers at the moment, the reviewer believes that this study lacks discussion and critical analysis of the results.

Here are some major comments:

1) The rationale for correlating immune cells and gene expression, and how the link between both supports the role of the gene-associated proteins in KD remains unclear.

2) The study limitations such as the size of the dataset, the usage of a single KD dataset, the specificity/sensitivity for KD of these potential biomarkers or the lack of experimental validation needs to be discussed.

3) The language needs to be more conservative as there is no proof that the identified hub genes are valid biomarkers, while the study definitely is supporting their role in KD. For example, line 148 “In this study, five hub genes involved in KD (AURKB, BUB1, CCL2, IL-4 and TOP2A) 148 were identified.” could be “In this study, five candidate hub genes for KD (AURKB, BUB1, CCL2, IL-4 and TOP2A) were identified.”.

4) Similarly, the title needs to be changed as this is far from qualifying as a preclinical study without any experimental data to validate the findings.

5) The discussion needs to rely more on previous literature and link the role of identified hub genes with KD as the link remains unclear.

6) In general, the study is not fluid to read, with regular typos/language mistakes making it difficult to read. I would suggest taking advantage of professional language editing.

Here are some other comments:

7) Line 10: There is a missing noun word for the adjective “immune”

8) Line 12: Please clarify what “immune infiltration situation” refers to.

9) Line 14: Please add more details about GSE73461 such as specifying how many KD and control patients were included, where are they from, when was this set obtained.

10) Line 62: There are two “provide(s)”, please remove one.

11) Lines 99-101: The result part would benefit from more description of gene up- and downregulation: in the volcano plot some look significantly more upregulated and downregulated, can you comment on that? |log FC| > 8 or 10.

12) Fig 1B: The heatmap figure does not help the reader to understand the study, the results can be visualized in Fig 1A. I would suggest removing Fig 1B or moving it to the supplementary information.

13) Lines 103-108: I would suggest commenting on the results, as some of them were to be expected for KD.

14) Fig. 2: The images are too small, please ensure the text is readable.

15) Fig. 2A-2F: I would suggest moving some of the figures to a supplementary information document as some of the information is redundant why not uninteresting. This would allow for larger figures and facilitate readability of the manuscript.

16) Fig 2G: It is impossible to read it due to the size and the identified hubs are then unclear. Please update figure.

17) Fig. 3: I would suggest moving some of the figures (A, B, C, E) to the SI and maybe generating a summary figure to make the results easy to interpret for the reader supporting the statements from line 119 to line 139. Also noting that the immune cell name formatting needs to be corrected.

18) Fig. 3D: The color legend is missing, please add it to the figure.

Reviewer #4: 1) The paper lacks novelty. There are already published papers related to the same topic of the paper. For example, there is a peer reviewed published paper here https://pubmed.ncbi.nlm.nih.gov/37837870/ named Identification of hub biomarkers and immune-related pathways participating in the progression of Kawasaki disease by integrated bioinformatics analysis. The paper conducted more comprehensive and used more datasets to identify biomarkers for Kawasaki disease.

2) The methodology is too simple, just differential expression analysis, resulting in more than one thousand genes. Additionally, selecting hub genes from the protein-protein interaction (PPI) network is insufficient for establishing reliable biomarkers.

3) The paper only used a single microarray dataset, which is outdated and may not reflect current advancements in technology or data availability.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Identification of Potential Biomarkers associated with Immune Cell Infiltration Patterns in Kawasaki Disease via Bioinformatics

PONE-D-24-55722R1

Dear Dr. Wang,

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.

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Kind regards,

Xiaosheng Tan

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

Reviewer #2: All comments have been addressed

Reviewer #3: 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: (No Response)

Reviewer #2: Yes

Reviewer #3: (No Response)

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

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: (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 #1: (No Response)

Reviewer #2: Yes

Reviewer #3: (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 #1: (No Response)

Reviewer #2: Yes

Reviewer #3: (No Response)

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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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (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

Reviewer #2: No

Reviewer #3: No

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