PONE-D-23-03945Negative regulation of angiogenesis and the MAPK pathway may be a shared biological pathway between IS and epilepsyPLOS ONE

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: N/A

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: i accept the manuscript titled "Negative regulation of angiogenesis and the MAPK pathway may be a shared

biological pathway between IS and epilepsy " as the study concluded that the first comorbidity common between

epilepsy and ischemic stroke.

Reviewer #2: In this study, Longhui Fu et al. reported the combined RNA expression analysis of ischemia stroke and epilepsy by using published bulk and single cell sequencing datasets. They found that negative regulation of angiogenesis and the MAPK signaling pathway may functions in a conserved and mechanistic way in the pathogenesis of these two diseases. Further DEG analysis revealed the hub genes, in addition to the potential drugs revealed by prediction of compounds binding. Findings from this study provide novel insights into the common pathogenic mechanisms and drugs underlying ischemia stroke and epilepsy. The expriments are well designed and performed in a high standard. I have some concerns regarding the methods, text format and data analysis.

1. Move the figure legends from the main text to somewhere indicated by the publish policy of Plos One.

2. Authors should consider to change the name of method 2.1 “microarray datasets”, since this part includes bulk and single cell data as well.

3. Detailed parameters used in the software should be disclosed in the method part for reproducibility. For example, in method 2.3, the specific commands used for Metascape and ClueGO, Cytoscape and GSEA should be fully listed. Similar requirements are applied to other packages.

4. The session “Data Availability Statement” should be presented somewhere before “Reference”. Authors may have to rearrange some sessions according to the regulation of Plos One.

5. For scRNA-seq analysis in Figure 4, authors should present the cell markers of each cluster annotated in the main figure, in the format of either dotplots or heatmap. This will make the analysis transparent and readable to the readers.

6. For scRNA-seq analysis in Figure 4, authors should explain the phenotypes that some cell clusters consist of subpopulations characterized by different sample origin (Sham vs Mcao), especially in the clusters of microglia and ¬endothelial cells. The segregation by conditions indicate that these subsets of cells are condition-specific, and may represent a different cell type/status. Authors should address these issues, and consider to reannotate the clusters accordingly. In addition, the resolution of UMAPs is too low to clearly identify cell labels. High resolution images are required all through the figures.

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Reviewer #1: No

Reviewer #2: Yes: Le Xu

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Negative regulation of angiogenesis and the MAPK pathway may be a shared biological pathway between IS and epilepsy.

PONE-D-23-03945R1

Dear Dr. Gong,

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,

Yuzhen Xu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #2: All comments have been addressed

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

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

Reviewer #2: Yes

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

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 #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Authors have addressed all my concerns. I have no further comments and suggest the next step towards publication.

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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 #2: No

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