All reviews of published articles are made public. This includes manuscript files, peer review comments, author rebuttals and revised materials. Note: This was optional for articles submitted before 13 February 2023.
Peer reviewers are encouraged (but not required) to provide their names to the authors when submitting their peer review. If they agree to provide their name, then their personal profile page will reflect a public acknowledgment that they performed a review (even if the article is rejected). If the article is accepted, then reviewers who provided their name will be associated with the article itself.
All issues indicated by the reviewers were addressed and revised manuscript is acceptable now.
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
Please address the concerns of the reviewers and amend the manuscript accordingly.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
I read with interest this original work by Sun et al., which aimed to identify genes associated with MPTDN using a bioinformatics analysis. Through this analysis, the authors identified two genes, CASP1 and CASP4. The findings were also linked to an animal model to support the in vivo relevance of the data. Additionally, the data were analysed in relation to prior anti-TNF therapy.
The study design is valid.
1) In the discussion, I would significantly expand on the potential translational significance of this research.
Some suggestions:
1) In the introduction, I would include a brief mention of the pathogenesis of UC, particularly regarding the melanocortin system, which has been emerging in recent research (I recommend these two studies: (https://pubmed.ncbi.nlm.nih.gov/37508552/ , https://pubmed.ncbi.nlm.nih.gov/38577176/ ). These systems also impact mitochondrial function.
2) The term “total rectal colectomy” is quite cacophonic; I would correct it to “total proctocolectomy with ileal pouch-anal anastomosis (IPAA)” and spell out the acronym.
3) The acronym “ulcerative colitis (UC)” is repeated multiple times. It is sufficient to define it once at first mention.
**PeerJ Staff Note:** It is PeerJ policy that additional references suggested during the peer-review process should only be included if the authors are in agreement that they are relevant and useful.
The study is entitled "Biomarkers of mitochondrial permeability transition-driven
necrosis in the regulation of ulcerative colitis" is good designed study.
It is appropriate with proper experimental design
No Comment
No Comment
1. Language Clarity: The manuscript is well-written, but some sections, particularly in the Methods and Discussion, contain dense technical language. Consider simplifying or restructuring some sentences for better readability.
2. Introduction & Background: The introduction provides a comprehensive background on ulcerative colitis (UC) and MPT-driven necrosis, but the literature review could be expanded to include more recent studies on the role of mitochondria in UC beyond oxidative stress.
3.The Discussion could be more concise. Some parts, such as the role of inflammasomes and IFN signaling, are repeated multiple times.
1.The figures are well-labeled and relevant, but Figure 5 (Identification of Hub Genes) could be better explained, particularly the overlap between LASSO, SVM-RFE, and RF algorithms.
2. Research Question & Hypothesis: The study clearly defines a knowledge gap and presents a meaningful research question. However, additional justification is needed on why CASP1 and CASP4 were selected as key biomarkers over other MPTDN-related genes.
3.The DSS-induced UC model is well-established, but more information on how CASP1 and CASP4 expression was measured at the protein level (e.g., Western blot or immunohistochemistry) would be helpful.
4
1.The study presents statistically significant findings, but additional functional experiments (e.g., knockdown or overexpression of CASP1 and CASP4 in vitro) would further confirm their role in UC pathogenesis.
2. Immunoinfiltration Analysis: The correlation between CASP1/CASP4 and macrophages/neutrophils is well-described, but additional validation (e.g., flow cytometry analysis of immune cells in UC tissues) would strengthen these findings.
3. The study suggests that CASP1 could predict infliximab responsiveness. However, further clinical validation (e.g., in prospective UC patient cohorts) is needed before suggesting its use as a biomarker.
Nil
The manuscript is well-written, providing a clear background on ulcerative colitis (UC) and mitochondrial permeability transition-driven necrosis (MPTDN). It cites relevant literature, including recent studies on oxidative stress and inflammasome-mediated cell death, reinforcing the rationale. The structure follows the IMRAD format, but terminology should be consistent (e.g., “UC” vs. “Control”). Figures and tables are well-presented, though some, like Fig. 7, are information-dense, requiring clear labelling for readability.
The authors provide extensive raw data, including gene lists, expression matrices, R code, and qPCR results, ensuring reproducibility. However, several refinements are suggested:
1. Clarify Statistical Thresholds: Indicate whether p-values for differential expression are adjusted (FDR) and explicitly state cutoffs.
2. Correct Notation Issues: Use proper statistical symbols (e.g., “r = 0.54” instead of “P = 0.54”) and resolve character encoding errors (e.g., “TNF-α” instead of “TNF³”).
3. Define WGCNA Trait Coding: Explain how UC vs. normal was coded for correlation analysis to resolve inconsistencies.
4. Data Availability Statement: Clearly state that raw data and code are accessible, specifying GEO accessions and supplemental files.
Justify Use of Multiple Feature Selection Methods: In the Methods, briefly explain why three different ML methods were used to identify feature genes. For example: We employed LASSO, SVM-RFE, and Random Forest in parallel to increase confidence in the selected biomarkers.
Consider Combining Feature Genes in a Model: As a suggestion (not required, but for completeness), you might evaluate if a simple two-gene combined model (CASP1+CASP4) outperforms each alone. For instance, a logistic regression using both CASP1 and CASP4 might yield an AUC even higher than 0.91 on validation. If it does, you could report that as a supplementary note. If not, it reinforces that CASP4 alone is sufficient. Given CASP4’s dominance, it may not improve much, but it’s worth checking.
Address Batch Effect Handling: In the Methods, note that analyses of different datasets were kept separate to avoid batch effects. If any batch correction was needed within a dataset (sometimes GEO series have multiple batches), mention if applicable.
MPT Gene List Source: Provide a bit more detail on the source of the 39 MPTDN genes (Appendix 1). You cite Liu et al. 2023, was that a supplemental table of genes in a cancer context?
Animal Model needs a justification of Acute vs Chronic: Acknowledge that the DSS model is acute and predominantly innate-driven, which matches the processes of interest (MPT, inflammasomes are innate). A good argument could be: “We used an acute DSS colitis model, which replicates the innate inflammatory response of UC flares and is well-suited to observe changes in CASP1/4 expression. While it does not capture chronic relapsing pathology, it serves to validate the involvement of these genes in colitic inflammation.”
Murine Caspase Nomenclature: As noted, clarify the mouse CASP4 issue. The mouse functional homolog of human CASP4 is caspase-11.
Multiple Comparisons in Immune Analysis: You might mention if you adjusted p-values or if not, which differences remained significant after correction. Possibly you did not adjust given the exploratory nature. It’s acceptable, but you could note that the concordant results in two independent datasets argue the differences (like neutrophils, M1, M2) are real and not false positives.
Discuss CASP1/4 in Context of UC vs Crohn’s: It might strengthen the discussion to mention whether these biomarkers are UC-specific or pan-IBD. The title and text focus on UC. If you have data or literature on Crohn’s disease (CD), you could compare.
Mechanistic Insights and Further Validation: In Discussion, suggest mechanistic follow-ups.
Anti-TNF Response Discussion: Refine the interpretation of CASP1 as a predictor of therapy response. Make it clear it’s an on-treatment marker. Clinically, if mucosal CASP1 remains elevated after induction therapy, it might predict a lack of remission, signaling the need for alternative treatments.
Wider Context, PANoptosis: To frame the findings in the latest conceptual context, you might mention PANoptosis (programmed cell death crosstalk). A recent concept in immunology is that pyroptosis, apoptosis, and necroptosis can co-occur in IBD (Lu J, Li F, Ye M. PANoptosis and Autophagy-Related Molecular Signature and Immune Landscape in Ulcerative Colitis: Integrated Analysis and Experimental Validation. J Inflamm Res. 2024 May 20;17:3225-3245. doi: 10.2147/JIR.S455862. PMID: 38800594; PMCID: PMC11122227.).
Figures for CASP1/4 validation: If possible, include a small panel in Fig 9 showing the actual qPCR results for CASP1 and CASP4.
Potential Diagnostic Use: If CASP1 and CASP4 are to be biomarkers, consider how they might be measured clinically. Assess this paper (Demon D, Kuchmiy A, Fossoul A, Zhu Q, Kanneganti TD, Lamkanfi M. Caspase-11 is expressed in the colonic mucosa and protects against dextran sodium sulfate-induced colitis. Mucosal Immunol. 2014 Nov;7(6):1480-91. doi: 10.1038/mi.2014.36. Epub 2014 May 21. Erratum in: Mucosal Immunol. 2014 Nov;7(6):1504. PMID: 24850431; PMCID: PMC420521)
Supplemental Code Improvement: Since you provided code, ensure any personal file paths or unnecessary lines are removed to avoid confusion. Perhaps provide a brief README in the supplemental code to guide others on how to run it.
Primer Efficiency (if data available): In Supplemental Info 4, if you have primer efficiencies or validation, note if the PCR was quantitative in a linear range and normalized properly (ΔΔCt method presumably).
In conclusion, the findings of the paper are valid and reliable.
做得很好
This manuscript investigates the mitochondrial permeability transition pore (MPTP)-mediated necroptotic pathway in ulcerative colitis (UC). The study validates MPTP-related biomarkers (e.g., AIF, CypD) using animal models and clinical samples, correlating them with disease progression, and proposes novel therapeutic strategies targeting mitochondrial necrosis. The key contribution is the establishment of a dynamic monitoring system for MPTP activity and the identification of diagnostic marker combinations. However, the experimental design exhibits limitations, including insufficient time gradients, a single animal model, questionable raw data integrity (e.g., incomplete Western blot membrane images), inadequate analysis of upstream regulatory mechanisms (e.g., lack of ROS-Ca²⁺ axis), and incomplete ethical documentation (e.g., undisclosed clinical informed consent). Recommendations include supplementing the data with multi-time point dynamic monitoring, incorporating organoid model validation, and enhancing the mechanistic correlation analysis to improve the study's depth.
The paper explores the role of the mitochondrial permeability transition pore (MPTP)-driven necrosis in ulcerative colitis (UC), addressing a gap in the research of UC mechanisms and therapeutic targets, with potential clinical application. The study innovatively combines MPTP-related biomarkers (e.g., CASP1/CASP4) with the efficacy prediction of anti-TNF-α drugs (IFX), offering a novel approach for individualized treatment. However, the mechanistic investigation lacks depth, and direct functional validation of MPTP's role in UC through experiments (e.g., gene knockout or inhibitor intervention) is absent. Furthermore, the comparison of innovation is insufficient, with an incomplete discussion of the differences and advantages compared to existing UC markers (e.g., NLRP3 inflammasome-related molecules). Animal pain minimization measures (e.g., pain score monitoring) are not specified. It is recommended to incorporate animal welfare measures during the experiment (e.g., the use of analgesics).
① The pathological mechanism section fails to cite the latest review of the mitochondrial necrosis pathway published in Nature Reviews Gastroenterology in 2023.
② The rationale for selecting the GSE87466 and GSE75214 datasets lacks clarity, specifically the justification for their selection over alternative public datasets. It is recommended to augment the methods section with dataset screening criteria, including sample size and matching of clinical characteristics.
③ In the DSS-induced ulcerative colitis (UC) mouse model, the methodology for anesthesia and euthanasia, and compliance with ARRIVE guidelines, are not detailed. It is recommended to include an ethical statement, such as the IACUC approval number, and specific operational procedures, including the rationale for selecting a 2.5% DSS concentration.
④ Figure 9 is labeled "n=5," but the statistical basis for the sample size calculation is not provided. It is recommended to include a Power Analysis or cite the sample size standard from similar studies.
⑤ Figures 7 and 8 only indicate p-value ranges (e.g., p < 0.05), without specifying whether multiple comparison corrections (e.g., Bonferroni correction) were performed. It is recommended to provide details of the statistical methods and indicate the correction methods in the figure legends.
⑥ The relationship between CASP1/CASP4 and IFX treatment is presented solely through differences in expression levels, without exploring molecular mechanisms (such as the association with the NLRP3 inflammasome). It is recommended to incorporate mechanistic hypotheses in the discussion section or cite relevant literature to support it.
All text and materials provided via this peer-review history page are made available under a Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.