Internally quenched fluorescent (IQF) peptides offer a powerful, modular platform for studying the enzymatic dynamics of post-translational modifications (PTMs) on lysine and arginine. Here we report a versatile IQF system that enables monitoring of PTM installation and removal via proteolytic cleavage by trypsin. This platform is compatible with both native PTMs and PTM mimetics, including acetylation, various other acylations, mono-/di-/trimethylation and citrullination across both histone and non-histone derived peptide substrates. Using synthetically accessible thialysine and thiaarginine analogs, we developed cysteine conjugation chemistries to access a wide array of PTM mimics, including novel reagents for lysine lactylation, β-hydroxybutyrylation and methyl-acetylation. Application of the system revealed distinct substrate preferences and site-specific activities for enzymes such as SIRT3, HDAC2, HDAC6, KDM3A, KDM4A and PAD4. Notably, the system uncovered enzymatic selectivity for acyl chain type and methylation state and demonstrated resistance of the emerging PTM methyl-acetyllysine to erasers. The system was also used to study the recently reported reversibility of acylation modifications by HDAC2 and 6 and is capable of evaluating enzymatic crosstalk between neighboring post-translational modifications. Our platform's adaptability and readout simplicity offer a generalizable chemical biology toolkit for PTM profiling, enzyme characterization, and inhibitor discovery.

BackgroundAberrant DNA methylation plays a pivotal role in cancer progression by enhancing oncogene activation or silencing tumor suppressor genes, contributing to malignant phenotypes. Methylation driver genes (MDGs) are characterized by an inverse correlation between DNA methylation levels and mRNA expression, making them critical targets for cancer research.MethodsWe analyzed the liver hepatocellular carcinoma (LIHC) dataset from The Cancer Genome Atlas (TCGA) using the R package MethylMix to identify MDGs. Prognostic models were developed through univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and multivariate Cox regression to identify core genes. We further evaluated the associations of these genes with the tumor immune microenvironment, immune checkpoint inhibitors (ICIs), and chemotherapeutic sensitivity. Finally, liver cancer tissue organoid culture experiments combined with DNA methylation sequencing were conducted to validate predictions of drug sensitivity.ResultsA total of 21 MDGs were identified, among which GNA14, glutaminase (GLS), and GNG4 were selected to construct a prognostic risk score model. The model demonstrated robust predictive performance, with Receiver Operating Characteristic (ROC) values of 0.723, 0.764, and 0.716 for 1-, 3-, and 5-year survival, respectively. Among these, GLS emerged as a key gene, showing low methylation levels and high mRNA expression, which were associated with poor prognosis, significant alterations in the tumor immune microenvironment, and differential sensitivity to ICIs and chemotherapeutic agents.ConclusionThe three-gene MDG-based prognostic model effectively predicts survival outcomes in LIHC patients. Moreover, the methylation status of GLS serves as a biomarker for assessing immune microenvironment characteristics, responsiveness to immunotherapy, and chemotherapy sensitivity, highlighting its potential as a therapeutic target in liver cancer.

Abstract What? We present a new suite of bioinformatic tools (PHYFUM, PHYFUMflow, and PHYFUMr) to track the evolution of human tissues organized in clonal stem-cell populations using methylation data from multiple samples per patient. We demonstrate the validity of our methods, characterize their accuracy under a broad simulation parameter space, and use them to analyze a total of 40 colon, 28 small intestine, and 31 endometrium samples extracted from 23 patients. We found statistically significant differences (p.adj << 0.001, Dunn’s test with Holm correction) between the shapes of the endometrial and intestinal trees (both types), highlighting the power of our method to develop evolutionary biomarkers for cancer progression. Why? The development of cancer from healthy somatic cells is fundamentally an evolutionary process. Understanding this process is crucial for enhancing its clinical management and developing more effective treatment strategies. We cannot use human cell-labeling techniques in vivo; however, tracing the fates of somatic cells would advance somatic evolutionary theory and our understanding of normal tissue function and its changes throughout development, aging, and cancer progression. Population genetic and phylogenetic models can leverage the information recorded in heritable alterations to reconstruct the history of the unobserved somatic cells. Most such methods are limited by the few parameters they can estimate and restricted to neoplastic samples due to the lack of adequate evolutionary signal in healthy somatic cells. How? Gabbutt et al. recently introduced fluctuating methylation clocks (FMCs)—tissue-specific CpG sites that randomly change their methylation state—and a model that uses them to reconstruct the evolution of a clonal stem-cell population that can be sampled together, like crypts (e.g., intestine, Barrett’s esophagus) and glands (e.g., endometrium). We have developed a multi-sample extension of this model that simultaneously reconstructs the evolutionary dynamics of stem cells within a niche and the niches within the tissue. This phylodynamic model generates additional estimates, including ancestral relationships between sampled clonal populations, their calendar divergence times, the number of effective evolutionary niches, and how these parameters change over time. So what? The stochastic nature of neoplastic progression makes it unlikely for any particular somatic alteration to be highly predictive of its outcome. This can explain why developing reliable traditional biomarkers has been so difficult. Alternatively, evolutionary biomarkers measure the characteristics of the process itself and thus should apply to all neoplasms. Parametric phylogenetic reconstruction models like the one introduced here will enable us to develop such universal biomarkers. Our ultimate goal is for evolutionary biomarkers to join evolutionary therapies in the revolution of cancer clinical management. Citation Format: Diego Mallo, Pablo Bousquets-Muñoz, Calum Gabbutt, Heather E. Grant, Darryl Shibata, Trevor A. Graham, Carlo C. Maley. PHYFUM: Reconstructing the evolutionary dynamics of human tissues using fluctuating methylation clocks [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Cancer Evolution: The Dynamics of Progression and Persistence; 2025 Dec 4-6; Albuquerque, NM. Philadelphia (PA): AACR; Cancer Res 2025;85(23_Suppl):Abstract nr B034.

Inflammatory bowel disease (IBD) represents a multifaceted, chronic inflammatory disorder of the gastrointestinal tract, with its pathogenesis modulated by a confluence of genetic, environmental, and immunological factors. Recent advancements have underscored the significance of the interaction network between gut microbiota and host epigenetic regulation, particularly via DNA methylation, as a pivotal area of investigation in elucidating the mechanisms underlying IBD. The gut microbiota exerts influence on host gene expression through its metabolic byproducts, thereby modulating immune responses and intestinal barrier integrity, while changes in host DNA methylation status both reflect and mediate this complex interaction. This review delineates the molecular mechanisms that regulate the interplay between gut microbiota and host DNA methylation, examines the impact of environmental factors on the onset and progression of IBD through microbiota-epigenetic pathways, and highlights recent multi-omics research findings and their potential applications in precision medicine. The objective is to furnish a theoretical framework and research trajectory for deciphering the molecular underpinnings of IBD and fostering the development of innovative therapeutic strategies.

Based on information from the international database The Cancer Genome Atlas, the methylation status of the RASSF1 gene promoter was studied in 57 non-small cell lung cancer tissue samples (adenocarcinoma and squamous cell lung carcinoma) and 27 normal lung tissue samples. Promoter regions characterized by specific methylation profiles in non-sm all cell lung cancer, including various histological types, were identified. The obtained data was then used to design primers for methylation-specific PCR, thereby confirming the feasibility of using the RASSF1 marker for tumor DNA detection. A methodology for detecting ctDNA in the blood plasma of patients with non-small cell lung cancer was developed using droplet digital PCR.

DNA methylation plays a crucial role in the progression and metastasis of colorectal cancer (CRC), yet the mechanisms underlying its effect at specific loci remain poorly understood. We previously identified cg24067911 hypomethylation as a potential biomarker associated with poor CRC prognosis. To investigate the role of cg24067911 methylation in CRC metastasis and elucidate its underlying molecular mechanisms, we utilized the CRISPR-dCas9-DNMT3a system to specifically modify the methylation status of cg24067911. Then, we performed high-throughput transposase-accessible chromatin sequencing, RNA sequencing, and chromatin immunoprecipitation analysis. We demonstrated that cg24067911 was located within an enhancer region of the ATXN1 gene, where it was bound by BCL6. Hypomethylation of cg24067911 improved the binding of BCL6 to this enhancer, leading to upregulated transcription of ATXN1. Furthermore, ATXN1 was found to act as a transcription factor that upregulates CDH2, promoting epithelial-mesenchymal transition and facilitating CRC metastasis. These findings not only reveal that cg24067911 methylation modulates CRC metastasis through a novel epigenetic mechanism involving ATXN1 and CDH2, but also highlight cg24067911 as a potential prognostic marker for CRC and suggest that targeted epigenetic therapies could be an effective strategy for treating CRC metastasis in the future.

Glioblastoma multiforme (GBM) remains one of the most aggressive primary brain tumors with limited therapeutic options. The efficacy of standard temozolomide chemotherapy is critically influenced by O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status. While monoclonal antibody (mAb)-based biologics represent a promising therapeutic approach, current clinical trials have failed to demonstrate significant improvement in overall patient survival. In this study, systematic screening identified histamine H1 receptor (HRH1) as a potential therapeutic target. Genetic silencing of HRH1 significantly inhibited GBM cell proliferation, migration, and invasion both in vitro and in vivo. Pharmacological validation using the HRH1-specific antagonist terfenadine confirmed the anti-tumor effects of HRH1 targeting. Based on these findings, we developed an HRH1-targeting mAb that demonstrated potent anti-tumor activity in GBM xenograft models. Mechanistic investigations revealed that the therapeutic effect was mediated through protein kinase C (PKC)-dependent regulation of the RAF/MEK/ERK signaling pathway. In conclusion, we provided a new target, HRH1, for treating GBM and constructed an effective therapeutic mAb.

Sepsis is a major cause of acute kidney injury (AKI). Dysregulation of N7-methyladenosine (m7G) methylation is a pathogenic mechanism of sepsis. However, the role of m7G methylation in renal damage remains poorly understood. In this study, we investigated the regulation of METTL1, an m7G writer, on pyroptosis in sepsis-induced AKI. HK-2 cells were treated with lipopolysaccharide (LPS), and pyroptosis was assessed using enzyme-linked immunosorbent assays and western blotting. The m7G methylation status of NLRP3 was analyzed through methylated-RNA immunoprecipitation (Me-RIP), RNA immunoprecipitation (RIP), and dual-luciferase reporter assays. Renal injury in mice subjected to cecal ligation and puncture (CLP) was evaluated using hematoxylin and eosin (H&E) staining. Our results demonstrated that METTL1 expression was significantly upregulated in both LPS-treated HK-2 cells and the CLP-induced mouse model. Interfering with METTL1 suppressed LPS-induced pyroptosis in vitro and attenuated kidney damage and pyroptosis in vivo. Furthermore, METTL1 knockdown inhibited m7G methylation of NLRP3, thereby reducing its stability. Overexpression of NLRP3 abrogated the inhibition of pyroptosis caused by METTL1 knockdown. In conclusion, silencing of METTL1 alleviates sepsis-induced AKI by inhibiting m7G methylated NLRP3-mediated pyroptosis in renal tubular epithelial cells. These findings suggest that targeting METTL1 may represent a promising therapeutic strategy for managing sepsis-associated AKI.

Cereblon (CRBN) serves as the substrate recognition subunit of the CRL4-CRBN E3 ubiquitin ligase complex and isthe primary target of immunomodulatory drugs (IMiDs) in cancer therapy. While CRBN mutations occur infrequently in cancer, reduced CRBN expression correlates with poor IMiD response and treatment resistance, suggesting nongeneticregulatory mechanisms may govern CRBN expression. We investigated CRBN expression patterns across multiple cancer types and examined the role of promotermethylation in regulating CRBN expression and its clinical significance. We analyzed CRBN expression patterns using GENT2 database analysis and examined CRBN methylation status indiverse human cancer cell lines using methylation-specific PCR (MSP) and bisulfite genomic sequencing. We analyzedthe clinical significance of CRBN methylation and expression using TCGA and GENT2 databases with Kaplan-Meiersurvival analyses. CRBN expression was significantly downregulated in breast, cervical, head and neck, lung, and skin cancers comparedto normal tissues. We identified a CpG island 1kb upstream of the CRBN transcriptional start site, with cancer celllines exhibiting partial methylation patterns. Bisulfite sequencing confirmed specific CpG methylation sites withinCRBN promoter regions. CRBN promoter hypermethylation significantly correlated with increased mortality risk inbreast, lung, acute myeloid leukemia, and bladder cancers, while low CRBN expression was associated with poorsurvival in breast, lung, brain, and ovarian cancers. Combined low expression and high methylation stronglypredicted poor outcomes in breast and lung cancers. CRBN expression is partially regulated by promoter methylation in specific cancer types, with epigenetic silencingcontributing to treatment resistance and poor prognosis. CRBN methylation and expression serve as important pancancerprognostic biomarkers, highlighting the potential for epigenetic therapies to restore CRBN function andovercome therapeutic resistance.

BackgroundGlioblastoma (GBM) is the most common and aggressive primary brain tumor, with limited survival despite multimodal treatment strategies. O6-Methylguanine-DNA Methyltransferase (MGMT) promoter methylation is a well-established predictive biomarker for response to temozolomide (TMZ) therapy. However, determining an optimal quantitative methylation-specific PCR (qMSP) cut-off value remains a challenge in clinical practice.ObjectiveThis study aimed to establish an optimal qMSP cut-off value for MGMT promoter methylation and validate its prognostic significance in GBM patients. The impact of MGMT methylation status on survival outcomes was analyzed concerning surgical extent, tumor localization, and white matter tract involvement.MethodsA retrospective analysis of 101 GBM patients (IDH-wildtype) diagnosed between 2008 and 2022 was performed. All patients underwent surgical resection (total/partial excision or stereotactic biopsy) followed by standard chemoradiotherapy. MGMT promoter methylation status was assessed using real-time qMSP. The optimal cut-off value was determined via receiver operating characteristic curve analysis. Kaplan-Meier survival analysis and Cox regression models evaluated the association between MGMT methylation levels, clinical characteristics, and overall survival (OS).ResultsAmong 101 patients with IDH-wildtype glioblastoma, a qMSP cut-off value of 0.242% demonstrated strong diagnostic performance for MGMT methylation status (AUC = 0.875), with 78% sensitivity and 86% specificity. Patients with high methylation levels (≥ 0.242%) showed significantly longer median overall survival compared to those with low methylation (24 vs. 12 months; p = 0.006). This prognostic relevance persisted across surgical and anatomical subgroups. Multivariable Cox regression identified high qMSP methylation (HR ≈ 0.45, p < 0.001) and extent of resection ≥ 90% (HR ≈ 0.30, p = 0.002) as independent predictors of improved survival, whereas TERT promoter mutation (HR ≈ 1.9, p = 0.017) was associated with worse survival. Stratified analysis revealed that TERTp-mutant tumors with low methylation had the worst outcomes. Additionally, excisional surgery and neocortical tumor involvement were associated with significantly better survival (p = 0.0010 and p = 0.0218, respectively). These findings validate within our institutional setting the clinical utility of the 0.242% qMSP threshold for prognostic stratification in glioblastoma, although external multicenter validation is warranted before generalization to routine clinical practice.ConclusionThe identified qMSP cut-off value (0.242) based on the procedure described in this study provides a robust prognostic stratification tool for GBM patients. High MGMT methylation correlates with improved survival, supporting its integration into clinical decision-making. Further multi-center validation studies are warranted to establish standardized MGMT assessment methodologies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-025-15225-2.

TRPM4, a nonselective cation channel, has been implicated in a variety of tumorigenic processes and is closely linked to insulin secretion. However, its functions and underlying mechanisms in pancreatic ductal adenocarcinoma (PAAD) progress remain elusive, and the potential as PAAD biomarker and therapeutic target remains unexplored. This study was meticulously crafted to experimentally validate the expression of TRPM4 in PAAD and to undertake a thorough examination of its effects on expression patterns, prognostic significance, mutational landscapes, methylation statuses, protein-protein interaction networks, as well as the correlation between TRPM4 expression and immune infiltration in PAAD. Our analytical approach included KEGG pathway analysis and GO enrichment analysis to identify signaling pathways associated with TRPM4. Our findings reveal a significant overexpression of TRPM4 in PAAD tissues, with heightened expression levels tightly correlated with unfavorable clinical outcomes. A comprehensive analysis utilizing the cBioPortal database unveiled a high mutation rate of TRPM4 in pancreatic cancer, predominantly attributed to gene amplification events. Examination of PPI networks and co-expression gene profiles highlighted notable interactions between TRPM4 and crucial ion channel proteins, including voltage-gated sodium channels and calcium release-activated calcium channels, shedding light on its potential role in PAAD progression. Functional enrichment analysis implicated TRPM4 in a range of biological processes, encompassing immune responses, neural signaling, and cellular electrophysiology, with particular emphasis on endocrine system regulation, especially pancreatic hormone secretion. Furthermore, TRPM4 expression exhibited striking associations with infiltration patterns of diverse immune cell populations in PAAD, particularly demonstrating strong correlations with Th17 cells and iTregs, hinting at its potential regulatory role in the tumor immune microenvironment. TRPM4 demonstrates heightened expression in pancreatic cancer tissues relative to normal tissues. It is also linked to the prognosis of pancreatic cancer patients, the intratumoral immune microenvironment, and the dissemination of the disease. These findings imply a potential oncogenic role for TRPM4. Moreover, TRPM4 potentially interacts with other ion channels, including sodium and calcium channels, to regulate the behavior of pancreatic cancer cells.

Mild cognitive impairment (MCI) is a prodromal stage of Alzheimer's disease characterized by subjective cognitive decline and objective evidence of cognitive impairment revealed through neuropsychological examinations. Dopamine receptors play a pivotal role in the modulation of memory and cognitive functions. DRD4 is a dopamine receptor gene enriched in memory-related brain regions, modulates synaptic plasticity, and its promoter methylation status. While DRD4 promoter methylation is implicated in neurodevelopmental disorders, its role in MCI pathogenesis remains uninvestigated across ethnically diverse populations. Our study focused on the association between DRD4 methylation and MCI in Uygur and Han populations. 192 age-sex-matched participants (48 Uygur MCI, 48 Uygur controls, 48 Han MCI, 48 Han controls) were recruited from Xinjiang and Zhejiang, China. MCI diagnosis followed DSM-IV criteria with cognitive assessment (MMSE/MoCA). Peripheral blood DNA underwent bisulfite conversion, followed by pyrosequencing of DRD4 promoter CpG sites 1-4 (PyroMark Q24). Group comparisons used independent t-tests with two-way ANOVA for covariate adjustment (age/sex/ethnicity); Pearson/Spearman tests assessed methylation-biomarker correlations. Methylation was measured using bisulfite pyrosequencing. Our results indicated that MCI-related hypermethylation was detected in the Uygur (CpG1) and Han (CpG1-3) populations, and subgroup analyses by sex in the Uygur population displayed consistent results, while in the Han subgroup, DRD4 hypermethylation was only observed in the male group. Ethnic differences in CpG4 (CpG: cytosine phosphate guanine) in male cases, CpG1 in overall control, and CpG3 in male controls. Regional distinctions were shown between CpG1and CpG4 in the male subgroup. Diverse correlations were observed. CpG1 methylation in the Uygur male control groups, Uygur female case groups, and Han female control groups was significantly correlated with age, Glu, and HDL-C, respectively. CpG2 in Uygur male controls and Uygur female patients showed correlations with Glu and TG levels. CpG3 in Uygur male and Han male controls was significantly correlated with TC and HDL-C levels. CpG4 in Uygur female controls and Han female patients correlated with Glu and age. Our findings provide novel insights into DRD4 methylation in Uygur and Han populations.

Objective: To compare the prognostic impact of different extents of resection among patients with molecularly defined glioma subtypes. Methods: This retrospective cohort study included 1191 glioma patients who underwent surgical treatment at Beijing Tiantan Hospital, Capital Medical University, between January 2011 and January 2021. The cohort comprised 692 males and 499 females, with an age of (44.5±11.9) years (range: 18 to 75 years). Tumors were classified according to the 2021 WHO Classification of Tumors of the Central Nervous System (5th edition), and the extent of resection was assessed using postoperative MRI. Kaplan-Meier survival analyses and log-rank tests were used to evaluate the effects of resection extent on progression-free survival (PFS) and overall survival (OS) within each molecular subtype. Cox proportional hazards models were applied to identify independent prognostic factors for PFS and OS. Follow-up was completed in January 2024. Results: Among the 1 191 patients, 291 (24.43%) had isocitrate dehydrogenase(IDH)-mutant, 1p/19q-codeleted oligodendroglioma (OG), 338 (28.37%) had IDH-mutant astrocytoma, and 562 (47.19%) had IDH-wild-type glioblastoma (GBM). Patients with IDH-mutant, 1p/19q-codeleted OG had the best prognosis, with median PFS and OS not reached. In IDH-wild type GBM, the median PFS and OS were 12.0 and 24.0 months, respectively; in IDH-mutant astrocytoma, the median PFS and OS were 61.0 and 102.0 months. Differences in PFS and OS among the three groups were statistically significant (P<0.01). In patients with IDH-wild type GBM, supratotal resection yielded better PFS and OS than gross total resection (P<0.05). In IDH-mutant astrocytoma, PFS and OS did not differ between supratotal and gross total resection (P>0.05), while gross total resection was superior to subtotal resection (P<0.05). In IDH-mutant, 1p/19q-codeleted OG, PFS and OS did not differ significantly across resection categories (P>0.05). Multivariate analyses identified age, Karnofsky Performance Status, extent of resection, tumor grade, and O6-methylguanine-DNA methyltransferase promoter methylation status as independent predictors of both PFS and OS (P<0.05). Conclusions: For IDH-wild type GBM, maximal efforts should be made to achieve supratotal resection. For IDH-mutant astrocytoma, maximal safe resection is recommended with preservation of neurological function. For IDH-mutant, 1p/19q-codeleted oligodendroglioma, a relatively conservative approach may be appropriate to protect neurological function.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy. This study aimed to construct a competing endogenous RNA (ceRNA) network to identify potential molecular targets and validate their biological functions. Gene expression profiles from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases were integrated to construct a miRNA-mediated mRNA-mRNA network within a ceRNA framework. Pathway enrichment analysis was applied to identify candidate genes. Differential expression in clinical specimens was validated by Western blot and qRT-PCR. Functional assays were performed after target gene silencing in PTC cell lines. Methylation specific PCR (MSP) was used to assess the relationship between promoter methylation and gene expression. A total of 160 potential ceRNA pairs were identified, of which 51 were associated with methylation status. Functional enrichment analysis further narrowed the candidates to eight tumorigenesis-related genes. Among these, integrin alpha 2 (ITGA2) was significantly overexpressed in PTC tissues. ITGA2 knockdown in PTC cells markedly suppressed proliferation, invasion, and metastatic capacity. MSP analysis demonstrated reduced promoter methylation of ITGA2 in PTC cells relative to controls, indicating that its upregulation is linked to promoter hypomethylation. This study established a ceRNA regulatory network in PTC and identified ITGA2 as a potential therapeutic target. Its dysregulated expression is closely associated with epigenetic alterations, offering new insights into the molecular mechanisms of PTC progression.

DNA methylation of peripheral blood (PB) B cells of warm autoimmune hemolytic anemia (wAIHA) patient was investigated. B cell DNA methylation level and the methylation-regulatory genes mRNA from 34 wAIHA patients and 16 healthy controls (HCs) were detected. The correlation between B cell DNA methylation and clinical indicators was analyzed. The effects of IL-6 on the B cell DNA methylation were examined. B cell DNA methylation level and DNMT1 mRNA expression of hemolytic episode patients were lower than that in remission patients and HCs. B cell DNA methylation in glucocorticoid-resistant patients was low. The hemolytic degree, response and relapse rate were correlated with B cell DNA methylation. Serum IL-6 concentration in hemolytic episode patients was higher than that in HCs and IL-6 inhibited DNMT1 expression in B cell of patients. IL-6 may inhibit the B cell DNA methylation levels and methylation regulatory genes of wAIHA patients.

ObjectivesO‐6‐methylguanine DNA methyltransferase (MGMT) promoter methylation status is a critical prognostic factor in glioblastoma. The aim of this study is to evaluate the feasibility of diagnosing MGMT status in a rapid, non‐invasive manner using multiparametric magnetic resonance imaging (mpMRI). The proposed method seeks to reduce reliance on stakeholders, thereby facilitating potential clinical applications in the future.Materials and methodsThis study employed a Siamese neural network (SNN) as the backbone of the model to effectively leverage information from various mpMRI modalities. Off‐the‐shelf deep learning features extracted from pre‐trained networks was used to represent the information from mpMRI and adopted as the inputs of SNN. Delta deep features from T1 modality were integrated as additional branch of SNN to enhance model's performance. Finally, external validation was performed to increase the robustness of study. The proposed method was applied to one of the largest publicly available mpMRI datasets, comprising 585 participants, with an additional 81 samples used for external validation.ResultsThe proposed method achieved an average area under the curve (AUC) of 0.666 with a standard error of the mean (SEM) of 0.031, average precision of 0.591 (SEM 0.021), and average recall of 0.630 (SEM 0.064). In external validation, the method yielded an average AUC of 0.624 (SEM 0.022), precision of 0.674 (SEM 0.050), and recall of 0.810 (SEM 0.101).ConclusionThe results demonstrate that our method outperforms existing approaches on a single‐CPU platform. Ablation studies confirmed the effectiveness of incorporating delta T1 deep features, while external validation confirmed the method's reliability across different datasets.

Silver-Russell syndrome (SRS) is aclinically and genetically heterogeneous imprinting disorder. The most common molecular defects are loss of methylation of the H19/IGF2:IG-DMR on chromosome 11p15.5, followed by maternal uniparental disomy of chromosome 7. Further molecular lesions are genetic variants in the PLAG1 oncogene, as well as in the transcription factor HMGA2 and the fetal growth factor IGF2. A phenotypic overlap exists between SRS and Temple syndrome (TS14) that is also characterized by growth restriction but associated with abnormalities in the imprinted chromosome 14q32 gene cluster. In TS14 patients, the germline MEG3/DLK1: IG-DMR is hypomethylated and the MEG8:Int2-DMR gains methylation probably as consequence of transcriptional readthrough from the MEG3 promoter on the paternal chromosome. However, the functional role of the MEG8 DMR remains unknown. We analysed the DNA methylation of 11-12 imprinted regions in 17 cases with clinical SRS features and heterozygous for a PLAG1 variant. We observed a specific loss of methylation of the MEG8:Int2-DMR in the ten cases carrying pathogenic PLAG1 variants that result in stable aberrant proteins. Normal MEG8 methylation was observed in the cases carrying variants of uncertain pathogenicity or gene deletions. Most of the PLAG1 cases are familial and both epigenetic and genetic defects co-segregated within the families. Additionally, we assessed the methylation status of the MEG8:Int2-DMR in several SRS patients with HMGA2 or IGF2 variants, H19/IGF2:IG-DMR-LoM and upd(7)mat and all of them showed normal methylation. Our results indicate that pathogenic PLAG1 variants leading to stable aberrant PLAG1 proteins and possibly acting in a dominant-negative manner influence methylation of the MEG8 locus. This study suggests a new pathogenetic mechanism of the PLAG1 gene in SRS, involving imprinted genes in the chr14q32 cluster through deregulation of the MEG8:Int2-DMR and provides an epigenetic signature that may be used to assess the damaging potential of the PLAG1 variants.

Epidermal growth factor receptor (EGFR) is a transmembrane protein belonging to the receptor tyrosine kinase (RTK) superfamily, reported as a promising anticancer target in treating diverse malignancies. Previous studies on microarray gene expression and methylation status in cell and animal models revealed the differential expression of EGFR at the early stages of cellular transformation. Additionally, an unpublished study of methylation analysis of EGFR gene promoters conducted in human cancer-related samples showed a several-fold increase in EGFR gene expression, suggesting epigenetic upregulation in tumors. Considering these findings, in the present study, we selected inactive (DFGout) (D: aspartic acid, F: phenylalanine, G: glycine) and active (DFGin) confirmations of EGFR to identify novel lead molecules against aberrant EGFR activity in cancer. Extra precision (XP) docking, molecular mechanics/generalized born surface area (MM/GBSA), molecular dynamics (MD) simulations, and ADME/T were performed, and the results showed that the lead 1 molecule of each target exhibited a better binding affinity and favorable stability than the existing ligands.

DNA methylation plays a pivotal role in the pathogenesis of Acute Lymphocytic Leukemia (ALL), a hematological malignancy marked by abnormal cellular behavior and immune dysregulation. This study aimed to investigate how alterations in DNA methylation affect lysosmal function in pediatric ALL. A total of 50 blood samples were collected from children diagnosed with ALL and analyzed for biochemical markers associated with the disease. Expression levels of key DNA methylation regulators, including DNA methyltransferase 1 (DNMT1) and DNMT3, were evaluated and compared with those from healthy controls. In addition, pro-inflammatory cytokines, interleukin-6 (IL-6), interleukin-27 (IL-27), and tumor necrosis factor-alpha (TNF-α), were monitored over a six-day period prior to treatment initiation. The study also assessed the expression of lysosome-associated membrane proteins, LAMP1 and LAMP2, which are essential for lysosomal function and the degradation of autophagosomes. To determine the DNA methylation status of the promoter regions of these genes, genomic DNA underwent sodium bisulfite treatment and digestion with methylation-sensitive and methylation-dependent restriction enzymes, followed by amplification with gene-specific primers. Our results revealed a significant upregulation of DNMT1 and DNMT3 in ALL samples, along with a marked downregulation of TET1 gene expression, which is responsible for DNA demethylation. This suggests that disrupted DNA methylation dynamics may contribute to the pathogenesis of the disease. Furthermore, methylation levels within the CpG islands of the LAMP1 and LAMP2 promoter regions were substantially elevated, showing more than a seven-fold increase in ALL samples compared to healthy control blood samples. In ALL samples, the expression levels of LAMP1 and LAMP2 were significantly reduced, may due to promoter region hypermethylation, which contributes to lysosomal dysfunction. In parallel, the expression of autophagy-related genes such as ATG5 and LC3B, markers of autophagy initiation and maturation, respectively, was markedly increased, suggesting an accumulation of autophagosomes that depend on functional lysosomes for complete degradation. Additionally, elevated levels of pro-inflammatory cytokines IL-6, IL-27, and TNF-α were consistently observed in ALL patients, indicating heightened immune activation that may drive disease progression. Collectively, these findings underscore the pivotal role of DNA methylation in disrupting lysosomal function, leading to autophagosome accumulation and impaired recycling of cytoplasmic components.

Various long non-coding RNAs (lncRNAs) have indicated their role in different regulatory processes and therapeutics in cervical cancer (CC). This study aims to assess the gene expression and methylation status of LINC00518 & MAFG-AS1 in CC patients. Methylation-specific PCR (MS-PCR) and quantitative real-time PCR (qRT-PCR) were performed on 81 patients. The association of the promoter methylation status of cancer tissues was studied with HPV infection and clinicopathological factors. The Kaplan-Meier curves were used from the GEPIA and TANRIC databases to analyze the overall survival of CC patients. The bioinformatics analysis of relative gene expression was carried out using the GEPIA database. The RNAinter database was also explored to find out the potential interacting partners. This is the first-ever research revealing that hypomethylation of the LINC00518 gene promoter may be relevant to its oncogenic behavior in CC (p < 0.05). However, no significant difference was observed between the MAFG-AS1 methylation status of cancerous and normal tissues. A notable association between the methylation status of LINC00518 promoter and clinicopathological factors, including age (p < 0.001), histological subtypes (p < 0.00001), and differentiation degree (p < 0.00001), has been observed, indicating its possible role in predicting the severity and prognosis of this disease. Overall survival analysis showed a significant value for LINC00518 using GEPIA (p < 0.05). Our findings about the gene expression of LINC00518 and its hypomethylated status in cancerous tissues suggest a potential mechanism that might contribute to its dysregulation in CC and could serve as a potential clinical biomarker.