BACKGROUND: PANoptosis has the features of pyroptosis, apoptosis, and necroptosis. Numerous studies have confirmed the diverse roles of various types of cell death in acute liver failure (ALF), but limited attention has been given to the crosstalk among them. In this study, we aimed to explore the role of PANoptosis in ALF and uncover new targets for its prevention or treatment. METHODS: Three ALF-related datasets (GSE14668, GSE62029, and GSE74000) were downloaded from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs). Hub genes were identified through intersecting DEGs, genes obtained from weighted gene co-expression network analysis (WGCNA), and genes related to PANoptosis. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein‍‒‍protein interaction (PPI) analyses and gene set enrichment analysis (GSEA) were performed to determine functional roles. Verification was performed using an ALF mouse model. RESULTS: Our results showed that expression of seven hub genes (B-cell lymphoma-2-modifying factor (BMF), B-cell lymphoma-2-interacting protein 3-like (BNIP3L), Caspase-1 (CASP1), receptor-interacting protein kinase 3 (RIPK3), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA), uncoordinated-5 homolog B receptor (UNC5B), and Z-DNA-binding protein 1 (ZBP1)) was up-regulated in liver samples of patients. However, in the ALF mouse model, the expression of BNIP3L, RIPK3, phosphorylated RIPK3 (P-RIPK3), UACA, and cleaved caspase-1 was up-regulated, while the expression of CASP1 and UNC5B was down-regulated. The expression of ZBP1 and BMF increased only during the development of ALF, and there was no significant change in the end stage. Immunofluorescence of mouse liver tissue showed that macrophages expressed all seven markers. Western blot results showed that pyroptosis, apoptosis, and necroptosis were always involved in lipopolysaccharide (LPS)/ d-galactosamine (d-gal)‍-induced ALF mice. The ALF cell model showed that bone marrow-derived macrophages (BMDMs) form PANoptosomes after LPS stimulation. CONCLUSIONS: Our results suggest that PANoptosis of macrophages promotes the development of ALF. The seven new ALF biomarkers identified and validated in this study may contribute to further investigation of diagnostic markers or novel therapeutic targets of ALF.
Animals ; Liver Failure, Acute/genetics* ; Computational Biology ; Mice ; Pyroptosis/genetics* ; Humans ; Protein Interaction Maps ; Apoptosis/genetics* ; Necroptosis/genetics* ; Gene Regulatory Networks ; Gene Ontology ; Gene Expression Profiling ; Disease Models, Animal

Recent data suggest that vascular endothelial growth factor receptor inhibitor (VEGFRi) can enhance the anti-tumor activity of the anti-programmed cell death-1 (anti-PD-1) antibody in colorectal cancer (CRC) with microsatellite stability (MSS). However, the comparison between this combination and standard third-line VEGFRi treatment is not performed, and reliable biomarkers are still lacking. We retrospectively enrolled MSS CRC patients receiving anti-PD-1 antibody plus VEGFRi (combination group, n=54) or VEGFRi alone (VEGFRi group, n=32), and their efficacy and safety were evaluated. We additionally examined the immune characteristics of the MSS CRC tumor microenvironment (TME) through single-cell and spatial transcriptomic data, and an MSS CRC immune cell-related signature (MCICRS) that can be used to predict the clinical outcomes of MSS CRC patients receiving immunotherapy was developed and validated in our in-house cohort. Compared with VEGFRi alone, the combination of anti-PD-1 antibody and VEGFRi exhibited a prolonged survival benefit (median progression-free survival: 4.4 vs. 2.0 months, P=0.0024; median overall survival: 10.2 vs. 5.2 months, P=0.0038) and a similar adverse event incidence. Through single-cell and spatial transcriptomic analysis, we determined ten MSS CRC-enriched immune cell types and their spatial distribution, including naive CD4⁺ T, regulatory CD4⁺ T, CD4⁺ Th17, exhausted CD8⁺ T, cytotoxic CD8⁺ T, proliferated CD8⁺ T, natural killer (NK) cells, plasma, and classical and intermediate monocytes. Based on a systemic meta-analysis and ten machine learning algorithms, we obtained MCICRS, an independent risk factor for the prognosis of MSS CRC patients. Further analyses demonstrated that the low-MCICRS group presented a higher immune cell infiltration and immune-related pathway activation, and hence a significant relation with the superior efficacy of pan-cancer immunotherapy. More importantly, the predictive value of MCICRS in MSS CRC patients receiving immunotherapy was also validated with an in-house cohort. Anti-PD-1 antibody combined with VEGFRi presented an improved clinical benefit in MSS CRC with manageable toxicity. MCICRS could serve as a robust and promising tool to predict clinical outcomes for individual MSS CRC patients receiving immunotherapy.
Humans ; Colorectal Neoplasms/drug therapy* ; Male ; Female ; Immunotherapy ; Middle Aged ; Aged ; Tumor Microenvironment/immunology* ; Retrospective Studies ; Microsatellite Instability ; Transcriptome ; Single-Cell Analysis ; Programmed Cell Death 1 Receptor/immunology* ; Gene Expression Profiling ; Immune Checkpoint Inhibitors/therapeutic use* ; Adult ; Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors*

Imprinted genes play a key role in regulating mammalian placental and embryonic development. Here, we generated glutaminyl-peptide cyclotransferase-knockout (Qpct^-/-) mice utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) platform and identified Qpct as a novel anti-angiogenic factor in regulating mouse placentation. Compared with Qpct^+/+ mice, placentae and embryos (Qpct^-/+ and Qpct^-/-) showed significant overgrowth at embryonic Day 12.5 (E12.5), E15.5, and E18.5. Using single-cell transcriptome analysis of 32 309 cells from Qpct^+/+ and Qpct^-/- mouse placentae, we identified 13 cell clusters via single-nucleus RNA sequencing (snRNA-seq) (8880 Qpct^+/+ and 13 577 Qpct^-/- cells) and 20 cell clusters via single-cell RNA sequencing (scRNA-seq) (6567 Qpct^+/+ and 3285 Qpct^-/- cells). Furthermore, we observed a global up-regulation of pro-angiogenic genes in the Qpct^-/- background. Immunohistochemistry assays revealed a notable increase in the number of blood vessels in the decidual and labyrinthine layers of E15.5 Qpct^-/+ and Qpct^-/- mice. Moreover, the elevation of multiple pairs of ligand-receptor interactions was observed in decidual cells, endothelial cells, and macrophages, promoting angiogenesis and inflammatory response. Our findings indicate that loss of maternal Qpct leads to altered phenotypic characteristics of placentae and embryos and promotes angiogenesis in murine placentae.
Animals ; Female ; Pregnancy ; Mice ; Placentation/genetics* ; Single-Cell Analysis ; Gene Expression Profiling ; Mice, Knockout ; Transcriptome ; Placenta/blood supply* ; Neovascularization, Pathologic/genetics* ; Genomic Imprinting ; Single-Cell Gene Expression Analysis ; Angiogenesis

Objective:Proteins are closely associated with the development, progression, and immunotherapy of head and neck squamous cell carcinoma（HNSCC）. However, few clinical models utilize proteomics to predict prognosis and immunotherapy efficacy. In this study, we developed a protein prognostic model（PPM） to stratify survival outcomes and differential immunotherapy responses in HNSCC patients. Methods:Based on proteomic profiling, we constructed a PPM comprising 11 protein markers. Patients were classified into high-and low-risk groups according to PPM scores. The prognostic value of risk scores was evaluated using Cox regression analysis, and predictive accuracy was assessed via time-dependent receiver operating characteristic（ROC） curves. Additionally, we analyzed treatment responses to PD1/CTLA4 immunotherapy in PD1-or CTLA4-positive patients across risk groups. Results:Cox regression confirmed the risk score as an independent prognostic factor（HR=1.161, 95%CI 1.112-1.213, P<0.001）, with high-risk patients exhibiting significantly poorer survival than low-risk counterparts. The model demonstrated robust predictive accuracy, with 1-year and 3-year time-dependent ROC areas under the curve（AUC） of 0.713 and 0.707, respectively. In PD1/CTLA4-positive subgroups, low-risk patients showed superior immunotherapy responses compared to high-risk patients. Conclusion:The PPM can provide reliable prognostic stratification and preliminary guidance for immunotherapy in HNSCC. However, further clinical studies and basic experiments are needed for further verification.
Humans ; Immunotherapy ; Squamous Cell Carcinoma of Head and Neck/therapy* ; Prognosis ; Head and Neck Neoplasms/genetics* ; Proteome ; Proteomics ; Gene Expression Profiling ; Transcriptome ; CTLA-4 Antigen ; Programmed Cell Death 1 Receptor ; Male ; Female ; Middle Aged

OBJECTIVES: To identify the key genes and immunological pathways shared by type 2 diabetes mellitus (T2DM) and chronic obstructive pulmonary disease (COPD) and explore the potential therapeutic targets of T2DM complicated by COPD. METHODS: GEO database was used for analyzing the gene expression profiles in T2DM and COPD to identify the common differentially expressed genes (DEGs) in the two diseases. A protein-protein interaction network was constructed to identify the candidate hub genes, which were validated in datasets and disease sets to obtain the target genes. The diagnostic accuracy of these target genes was assessed with ROC analysis, and their expression levels and association with pulmonary functions were investigated using clinical data and blood samples of patients with T2DM and COPD. The abundance of 22 immune cells was analyzed with CIBERSORT algorithm, and their relationship with the target genes was examined using correlation analysis. DGIdb database was used for analyzing the drug-gene interactions and the druggable genes followed by gene set enrichment analysis. RESULTS: We identified a total of 175 common DEGs in T2DM and COPD, mainly enriched in immune- and inflammation-related pathways. Among these genes, CXCL12 was identified as the final target gene, whose expression was elevated in both T2DM and COPD (P<0.05) and showed good diagnostic efficacy. Immune cell infiltration correlation analysis showed significant correlations of CXCL12 with various immune cells (P<0.01). GESA analysis showed that high CXCL12 expression was significantly correlated with "cytokine-cytokine receptor interaction". Drug-gene analysis showed that most of CXCL12-related drugs were not targeted drugs with significant cytotoxicity. CONCLUSIONS CXCL12 is a potential common key pathogenic gene of COPD and T2DM, and small-molecule targeted drugs against CXCL12 can provide a new strategy for treatment T2DM complicated by COPD.
Humans ; Pulmonary Disease, Chronic Obstructive/complications* ; Diabetes Mellitus, Type 2/genetics* ; Chemokine CXCL12/metabolism* ; Protein Interaction Maps ; Gene Expression Profiling

OBJECTIVES: To explore the role of oxidative stress and immune infiltration in rheumatoid arthritis (RA). METHODS: RA datasets GSE55235 (10 RA vs 10 normal samples) and GSE55457 (13 RA vs 10 normal samples) from the GEO database were merged as the test set to identify the differentially expressed genes (DEGs) in RA using R. The DEGs were intersected with oxidative stress-related genes to obtain oxidative stress-associated DEGs. KEGG and GO enrichment analyses of the DEGs were performed, and the RA-related pathways and biological processes were analyzed using GSEA. A protein-protein interaction (PPI) network was constructed using STRING and Cytoscape, and the top 10 key genes were obtained using the Degree algorithm. The validation dataset GSE1919 from GEO database was used for ROC analysis of the key genes to obtain the core genes, and their correlations with infiltrating immune cells were analyzed using CIBERSORT. The results were verified by RT-qPCR for detecting expression levels of the core genes in RA and normal joint samples. RESULTS: We identified 89 oxidative stress-associated DEGs. Enrichment analysis suggested that these DEGs were involved in the biological processes including oxidative stress, chemical stress response, reactive oxygen species response, and lipopolysaccharide response. ROC analysis showed that the 5 core genes (STAT1, MMP9, MYC, CCL5, and JUN) all had AUC values >0.7, indicating their high diagnostic sensitivity and specificity for RA. These genes were closely correlated with immune cells, particularly T cells. RT-qPCR confirmed significant differential expressions of the core genes between RA and normal samples. CONCLUSIONS Oxidative stress and diverse immune responses are features of RA, and the immune responses contribute to activation of oxidative stress. The identified core genes can potential serve as new diagnostic markers for RA.
Arthritis, Rheumatoid/genetics* ; Oxidative Stress/genetics* ; Humans ; Computational Biology ; Protein Interaction Maps ; Gene Expression Profiling ; Gene Regulatory Networks

OBJECTIVES: To explore the value of ferroptose-related genes in the diagnosis and prediction of ulcerative colitis (UC). METHODS: We used UC dataset from the GEO database to screen for differentially expressed genes (DEGs) in UC. The DEGs related to ferroptositis were screened from the FerrDb database and their functions were analyzed. The hub genes were identified by constructing the protein-protein interaction network (PPI), the differences in immune infiltration levels between UC and the control group were evaluated using CIBERSORT, and the diagnostic values of the hub genes for UC were verified by using the training set. In a mouse model of UC, we examined the expression levels of the hub genes in the colon tissues of the mice using real-time fluorescence quantitative PCR (qPCR). RESULTS: We identified a total of 76 DEGs related to ferroptosis. Functional enrichment analysis showed that these genes were significantly enriched in ferroptosis and hypoxia pathways. The PPI network identified 10 hub genes, and 9 of them were highly expressed in UC. Analysis of immune cell infiltration showed that 27 cell types were significantly increased in UC (P<0.05), and the immune checkpoints-related genes had the strongest correlation with the hub gene PPARG (P<0.05). Verification analysis using the training set showed that P4HB, PPARG and STAT3 had the best predictive value for UC (P<0.05). In the UC mouse model, the expression of PPARG was significantly decreased and the expressions of P4HB and STAT3 were significantly increased in the colon tissues of the mice as compared with the normal mice. CONCLUSIONS Ferroptose-related genes have significant value for diagnosis and prediction of UC.
Colitis, Ulcerative/genetics* ; Animals ; Mice ; Ferroptosis/genetics* ; Humans ; Protein Interaction Maps ; Disease Models, Animal ; Gene Expression Profiling ; STAT3 Transcription Factor/genetics*

Numerous studies on the formation and consolidation of memory have shown that memory processes are characterized by phase-dependent and dynamic regulation. Memory retrieval, as the only representation of memory content and an active form of memory processing that induces memory reconsolidation, has attracted increasing attention in recent years. Although the molecular mechanisms specific to memory retrieval-induced reconsolidation have been gradually revealed, an understanding of the time-dependent regulatory mechanisms of this process is still lacking. In this study, we applied a transcriptome analysis of memory retrieval at different time points in the recent memory stage. Differential expression analysis and Short Time-series Expression Miner (STEM) depicting temporal gene expression patterns indicated that most differential gene expression occurred at 48 h, and the STEM cluster showing the greatest transcriptional upregulation at 48 h demonstrated the most significant difference. We then screened the differentially-expressed genes associated with that met the expression patterns of those cluster-identified genes that have been reported to be involved in learning and memory processes in addition to dipeptidyl peptidase 9 (DPP9). Further quantitative polymerase chain reaction verification and pharmacological intervention suggested that DPP9 is involved in 48-h fear memory retrieval and viral vector-mediated overexpression of DPP9 countered the 48-h retrieval-induced attenuation of fear memory. Taken together, our findings suggest that temporal gene expression patterns are induced by recent memory retrieval and provide hitherto undocumented evidence of the role of DPP9 in the retrieval-induced reconsolidation of fear memory.
Animals ; Fear/physiology* ; Male ; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics* ; Memory Consolidation/physiology* ; Time Factors ; Mental Recall/drug effects* ; Mice ; Gene Expression Profiling

The regulatory processes in developmental biology research are significantly influenced by long non-coding RNAs (lncRNAs). However, the dynamics of lncRNA expression during human tooth development remain poorly understood. In this research, we examined the lncRNAs present in the dental epithelium (DE) and dental mesenchyme (DM) at the late bud, cap, and early bell stages of human fetal tooth development through bulk RNA sequencing. Developmental regulators co-expressed with neighboring lncRNAs were significantly enriched in odontogenesis. Specific lncRNAs expressed in the DE and DM, such as PANCR, MIR205HG, DLX6-AS1, and DNM3OS, were identified through a combination of bulk RNA sequencing and single-cell analysis. Further subcluster analysis revealed lncRNAs specifically expressed in important regions of the tooth germ, such as the inner enamel epithelium and coronal dental papilla (CDP). Functionally, we demonstrated that CDP-specific DLX6-AS1 enhanced odontoblastic differentiation in human tooth germ mesenchymal cells and dental pulp stem cells. These findings suggest that lncRNAs could serve as valuable cell markers for tooth development and potential therapeutic targets for tooth regeneration.
Humans ; RNA, Long Noncoding/metabolism* ; Odontogenesis/genetics* ; Tooth Germ/embryology* ; Cell Differentiation ; Gene Expression Regulation, Developmental ; Mesoderm/metabolism* ; Tooth/embryology* ; Gene Expression Profiling ; Sequence Analysis, RNA ; Dental Pulp/cytology*

Ancient traditional Chinese medicine (TCM) doctrine says "The superior doctor prevents illnesses," pointing out preventative medicine as the ultimate goal for medical care. TCM recognizes that genetic predisposition and environmental and lifestyle influences contribute to diseases. It divides people into eight constitutions in addition to one normal/healthy kind. People with one of the eight subhealth constitutions are prone to develop different kinds of corresponding illnesses. The goal for this type of categorization is to help people take preemptive measures to prevent or delay disease onset. As the peripheral immune system through surveying the body, it can capture information from essentially all organs and reflect anomalies occurring in each organ. Thus, the detailed profiling of the peripheral immune-system function can generally reflect a person's overall heath state. In this study, we performed the single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) from individuals with Tanshi (phlegm dampness) constitution. They were prone to develop metabolic disorders including diabetes. scRNA-seq revealed greatly reduced mucosal-associated invariable T cell content and heightened TNFα-NFκB, JAK-STAT, and interferon signaling. These findings indicated heightened chronic inflammation, as well as increased hypoxia/apoptosis responses, likely resulting from frequent sleep apnea that Tanshi individuals experienced. Altogether, this pilot study demonstrated effectiveness in using scRNA-seq to reveal molecular-immunological bases for constitution categorization, thereby substantiating that preventative medicine originated from TCM.
Humans ; Leukocytes, Mononuclear/metabolism* ; Male ; Female ; Gene Expression Profiling ; Single-Cell Analysis ; Middle Aged ; Adult ; Medicine, Chinese Traditional ; Transcriptome ; Single-Cell Gene Expression Analysis