PONE-D-24-34139Bioinformatics analysis of genes associated with disulfidptosis in spinal cord injuryPLOS ONE

Dear Dr. Tian,

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PLOS ONE

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Reviewers' comments:

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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: No

Reviewer #2: 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: 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: Comments:

The paper performs bioinformatics analyses of transcriptomic data to assess differences in gene expression following spinal cord injury, with a particular focus on disulfidptosis-related genes. The study employed datasets from multiple species and used human patient-derived SCI and control samples to support their findings. The authors utilised online databases for deeper investigations of their genes of interest. Overall, the authors frequently reach conclusions that are not supported by the paper's findings and tend to overinterpret their analyses. In particular, the origin of the list of disulfidptosis-related genes that is relied upon very heavily in the paper is not described. More detailed information on the settings of bioinformatic tools used in the work are also required in order to accurately assess the appropriateness of much of the analysis. I recommend that this article is not accepted for publication in its current state. Very major revisions should be made or the article should be rejected.

Methods:

The methods should contain more information on how exactly batch effects were accounted for, and how the differential expression was performed. What settings were used in each of the programs, which versions, and what design formula was used for the differential expression analysis. More details are needed for how almost all of the bioinformatics tools were used.

Results:

1.For the differential expression analysis, it would be good to see a histogram of p-values to know that the test used is appropriate for the data. This could be put in the supplementary data.

2.It would also be informative to see a principal component analysis plot for the different samples shown in Figure 1. This could also go in supplementary data.

3.How were the genes chosen in Figure 1A? Is it the top 20 up- and downregulated genes? If so, this should be stated.

4.The bar chart in figure 1C should not have an arrow on the X axis, as this axis is showing discrete categories, not a continuous variable. This information is also provided in the Venn diagram, so may not be necessary to include. It would be better if the sizes of the circles in the Venn diagram were plotted relative to the size of each group. Furthermore, a statistical test like Fisher’s exact test should be performed to determine if this overlap is more than expected by chance.

5.Where was the list of DRGs obtained? I couldn’t find anything in the text regarding this. Was it from previous literature? If so, that needs to be accurately cited. This is an essential part that is missing from this paper.

6.Figure 1D is very hard to understand. The connected lines convey that there is a connection between individual samples, which as I understand, there is not. It would be better to plot this as a heatmap.

7.The figures are in too low resolution to clearly read gene names and gene ontologies.

8.In Figure 2, it would be better to plot an enrichment score rather than the number of genes. Given that we aren’t given information on the number of genes that make up each GO term, the number of genes that overlap here could be not very relevant if the GO term lists are of very different sizes.

9.In figure 3, it is best practice to show the control first on the plot, and the injury sample after.

10.In line 282, it should state that STAT1 was predicted as a transcription factor targeting CAPZB, not “the transcription factor for CAPZB”. The evidence in the paper is not sufficient to claim that STAT1 is a direct transcriptional regulator of CAPZB. Same goes for the rest of the transcription factors mentioned here. The authors should also be careful to double-check for italicizing of gene names.

11.Most of these are quite general transcription factors, with many global effects so the authors should write cautiously about using them as drug targets. Futhermore, more empirical/experimental evidence is required to demonstrate that the identified SCI-DRGs are actually involved in the pathology of SCI before they could be considered as drug targets.

12.For the ceRNA analysis, the authors should perform co-expression analysis of the protein-coding mRNAs, lncRNAs and miRNAs and integrate these results with the database search they have performed.

Discussion:

13.In line 374-376 of the discussion, the authors make conclusions that are not supported by the data of the paper.

14.Line 444-447, they are also making claims that are not supported by the data of the paper. The authors should not use the word “confirm” based only on bioinformatic analysis of transcriptomic data.

15.Line 453-454, the data of this paper does not provide sufficient evidence for this claim.

Reviewer #2: I believe this manuscript meets the publication requirements of PLOS ONE. Therefore, I recommend its acceptance pending minor revisions. Below are my comments for the authors:

1) The figure legends should be reworded. I suggest assigning clear titles to each figure and describing each panel separately.

2) Once a term is abbreviated, use only the abbreviation in subsequent mentions (examples: lines 47–56, 66–100, 38–79).

3) I recommend adding a sentence at the end of the last paragraph of the introduction to summarize the major takeaway of the study.

4) The section 2.4 is too dense and wordy. Consider renaming it “RNA extraction and qRT-PCR,” and move the latter part into a new subsection (e.g., 2.5).

5) Provide details about the technical and biological replicates used in the qRT-PCR experiments.

6) Sections 3.1, 3.2, 3.3 are overly descriptive. At the beginning of each paragraph, include a concise rationale explaining why the analysis was performed. Conclude each paragraph with a brief summary of the findings.

7) What does the literature say about the roles of CAPZB and SLC3A2? (lines 250–254).

8) Double-check punctuation (e.g., line 278).

9) When repeating your findings in the discussion, cite the corresponding figures whenever possible (examples: lines 336–341, 353–354).

10) Lines 376–379: Is this observation a finding of the current study or derived from the literature? In either case, cite the appropriate figures or references.

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Reviewer #1: No

Reviewer #2: No

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Bioinformatics analysis of genes associated with disulfidptosis in spinal cord injury

PONE-D-24-34139R1

Dear Dr. Tian,

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,

Zhiwen Luo

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

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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 #2: The authors have addressed my concerns, therefore, I recommend the publication of this manuscript in PLOS ONE, provided that Reviewer 1's concerns are also addressed satisfactorily.

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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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