Mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE) is a recently defined malformation of cortical development that is an important cause of childhood-onset drug-resistant epilepsy. Clinically, the epilepsies associated with MOGHE are heterogeneous, with infantile epileptic spasms syndrome (IESS) being the most common manifestation. Histopathologically, MOGHE demonstrates subtle cortical dyslamination, heterotopic neurons in the white matter, hypomyelination, and a distinctive increase in the density and clustering of oligodendroglial cells, features that distinguish it from other malformations of cortical development such as focal cortical dysplasia (FCD). Recent genetic analyses of epileptogenic tissue resected from individuals with MOGHE have identified somatic mosaic loss-of-function variants in SLC35A2. This gene encodes the Golgi transmembrane UDP-galactose transporter, suggesting disrupted N-glycosylation as a distinct pathogenic mechanism underlying epilepsy in this disorder. In this review, we present the current clinical, histopathological, and molecular understanding of MOGHE, with a particular focus on recent insights gained from experimental rodent and human cellular models of SLC35A2 deficiency. We contextualise these findings against established models of mTORopathies including FCD type 2, placing MOGHE within the broader malformation of cortical development spectrum. Synthesising this evidence, we observe that neuronal activity in models of both MOGHE and mTORopathies such as FCD type 2 converge on reduced action potential firing, despite their distinct genetic aetiologies. Finally, we discuss how these findings inform our understanding of epileptogenesis, especially the emergence of infantile epileptic spasms, and the development of future precision therapeutic strategies across malformations of cortical development.
Upper aerodigestive tract squamous cell carcinoma (UATSCC) is often easy to recognize in its conventional form. However, a subset of tumors loses histologic evidence of squamous differentiation and, instead, become sarcomatoid (spindled). These cases may be difficult or impossible to appropriately classify on biopsy material, even with immunohistochemistry. DNA methylation has emerged as a tool to help in classifying undifferentiated malignancies. We sought to explore whether conventional and sarcomatoid UATSCC harbor the same epigenetic signature. Cases of sarcomatoid UATSCC were collected and reviewed. The conventional and sarcomatoid components were documented. Only specimens with concomitant epithelial dysplasia and/or invasive SCC were included. A panel of keratins (CK AE1/3, CAM5.2 and CK5/6) was performed on the sarcomatoid component of each tumor and scored as follows: 0, < 1%; 1+, 1-25%; 2+, 26-50%, 3+, > 50%. DNA methylation profiling was performed and compared with profiles from publicly available UATSCC and a control cohort of pleomorphic malignancies including undifferentiated sarcoma, leiomyosarcoma, angiosarcoma, atypical fibroxanthoma/pleomorphic dermal sarcoma, and melanoma, as well as sarcomatoid carcinoma of cutaneous origin. 21 UATSCC samples from 15 patients were retrieved (9 M, 6 F; 47-88 years, median 71 years) from sites including tongue (7), larynx (5), pharynx (3), maxillary/mandibular alveolar ridge (3), "neck" (2), and nasal cavity (1). 12 samples were entirely sarcomatous, while six were conventional SCC and three contained both sarcomatoid and conventional components. Of the sarcomatous components, six were entirely negative (score = 0) for all keratins performed. Using dimensionality reduction analysis of DNA methylation data by Uniform Manifold Approximation and Projection (UMAP) and unsupervised hierarchical clustering, 11 of 12 sarcomatoid UATSCC samples, were positioned with conventional UATSCC. Methylomes of sarcomatoid carcinoma of upper aerodigestive tract and cutaneous origin overlapped. Copy number profile analysis did not show significant differences between conventional and sarcomatoid histologies or tumor time point. The epigenetic signatures of conventional and sarcomatoid UATSCC are similar, even in cases that lack expression of multiple keratins. DNA methylation profiling may be leveraged in biopsies and entirely sarcomatoid UATSCC to accurately diagnose these cases and differentiate them from pleomorphic sarcomas and melanoma.
Gene expression is regulated by transcription factors (TFs), which recognize specific DNA sequence motifs. Several hundred putative human TFs, identified mainly by an apparent DNA-binding domain, lack known binding motifs1. Furthermore, even for well-characterized TFs, it remains controversial the degree to which motifs accurately reflect binding sites in living cells2. Here we describe a systematic effort ('Codebook') to determine the sequence specificity of 332 putative and poorly characterized human TFs. More than 4,000 independent experiments, encompassing multiple in vitro and in vivo assays, produced motifs for just over half (177; 53%) of the TFs, of which most are associated with only a single protein. These results extend the vocabulary of sequence recognition encoded by human TFs by around 130 distinct motifs. Moreover, binding motifs identified in vitro are strongly enriched in cellular binding sites. Collectively, the data reveal tens of thousands of previously unknown, conserved and direct TF-binding sites across the human genome. These sites are concentrated in promoter regions and are predictive of gene expression. In summary, this new codebook provides an important step forward in decoding the human genome.
This study aims to investigate the expression and function of DNA methyltransferases (DNMTs) and histone methyltransferase EZH2 in regulating the expression of thyroid-stimulating hormone receptor (TSHR) and fibrotic markers by orbital fibroblasts from Graves' ophthalmopathy (GO) patients. DNMTs mRNA levels were measured in orbital fibroblasts from healthy controls (control orbital fibroblasts), active GO fibroblasts, and inactive GO fibroblasts (iGOFs). TSHR and other fibrotic markers were measured in iGOFs treated with platelet-derived growth factor (PDGF)-BB and decitabine (a DNMT inhibitor) or DZNep (an EZH2 inhibitor) or small interfering RNA by RT-qPCR or Western blotting. DNMT1 and DNMT3A mRNA expressions were significantly higher in active GOFs compared with iGOFs and control orbital fibroblasts. Among all DNMTs, DNMT1 expression was at the highest levels. Upon PDGF-BB stimulation, DNMT1 and DNMT3A mRNA levels in iGOFs were significantly induced. Decitabine significantly inhibited PDGF-BB-induced cell viability and TSHR mRNA levels in iGOFs, whereas DNMT1 silencing partly reduced TSHR protein. Because a positive correlation between DNMT1 and EZH2 expression in iGOF was observed, the regulatory role of EZH2 on TSHR expression was also investigated. DZNep slightly decreased TSHR mRNA expression in iGOFs and iGO orbital tissue, whereas EZH2 silencing partly affects TSHR expression. This study demonstrates that DNMT1 is the major DNMT during the active stage of GO. Moreover, DNMT1 is inducible upon PDGF-BB stimulation in iGOFs, suggesting an important role of PDGF-BB and DNMT1 during an active stage of GO. Our findings also highlighted that the inhibition of DNMT or EZH2 potentially suppressed TSHR mRNA expression, suggesting potential therapies targeting DNMT/EZH2 in controlling GO progression.
The chances of oral squamous cell carcinoma (OSCC) being diagnosed at an early stage are very low, and this leads to a poor prognosis. The presence of circulating miRNAs (miRNAs) in blood or saliva is now a potential non-invasive diagnostic biomarker, although the diagnostic accuracy of the studies reported remains vastly different due to differences in biofluids, miRNA panels, disease spectrum, and platforms. We did a meta-analysis of diagnostic accuracy and a systematic review in accordance with PRISMA-DTA 2020 guidelines (PROSPERO: CRD420251274288). Articles that evaluated circulating miRNAs in any biofluid in diagnosing OSCC were taken into consideration. A Bayesian bivariate random-effects model was used to pool together the sensitivity, specificity, likelihood ratios, diagnostic odds ratios and the summary receiver operating characteristic (sROC) curves. Heterogeneity sources were analyzed using pre-constructed subgroup analysis and meta-regression. The number of studies that were evaluated in totality was seventeen and seven were incorporated in the meta-analysis. The pooled sensitivity of the circulating miRNAs towards the detection of OSCC was 0.89 (95% CI: 0.77-0.95) and the pooled specificity was 0.88 (95% CI: 0.75-0.95). It had a pooled positive likelihood ratio (PLR) of 7.25 (95% CI: 3.42-16.08), a negative likelihood ratio (NLR) of 0.13(95% CI: 0.05-0.27), and a diagnostic odds ratio (DOR) of 59.69(95%CI:14.33-209.95). Good performance in discriminating was also noted in the sROC curve with an area under the curve value of approximately 0.90. However, substantial between-study heterogeneity was observed (τ ≈ 0.93 for sensitivity; τ ≈ 0.83 for specificity), and the 95% credible intervals around the DOR were wide (14.33-209.95). Individual studies of multi-miRNA panels reported AUCs up to 0.98, though pooled subgroup estimates were limited by small study numbers and wide uncertainty. Circulating miRNAs and in particular multi-miRNA saliva-based profiles provide promising but heterogenous diagnostic accuracy for OSCC. While individual panel studies have reported high AUCs, the current pooled evidence-derived from only seven small, mostly case-control studies of advanced-stage disease-does not yet establish utility for true early detection or population screening.
Enhancer of zeste homolog 2 (EZH2) is a key epigenetic regulator implicated in tumor progression; however, its expression pattern and subcellular localization across different stages of endometrial carcinogenesis remain incompletely characterized. This study evaluated EZH2 expression in two biologically distinct endometrial carcinoma cell lines (Ishikawa and MFE-319) and in archived human endometrial tissues representing proliferative and secretory endometrium, hyperplasia, and Type I and Type II endometrial carcinomas. Histopathological evaluation was performed using hematoxylin and eosin staining. EZH2 expression was assessed by immunocytochemistry and Western blot analysis in the cell lines and by immunohistochemistry in tissue specimens, while apoptosis was evaluated by TUNEL assay in tissue samples. MFE-319 cells demonstrated significantly higher EZH2 expression than Ishikawa cells (H-score: 352.9 ± 78.9 vs. 137.6 ± 31.5, p < 0.0001), and Western blot analysis confirmed the same direction of change. Among tissue specimens, the highest EZH2 immunoreactivity was observed in Type II endometrial carcinoma (366.0 ± 63.7), with significantly higher expression than proliferative endometrium (231.5 ± 59.3), secretory endometrium (190.5 ± 52.4), and hyperplasia without atypia (261.0 ± 56.9), whereas no significant differences were detected among several intermediate histopathological groups. Nuclear localization of EZH2 became more prominent in atypical hyperplasia and carcinoma tissues. Apoptotic indices were significantly higher in both Type I and Type II carcinomas than in normal endometrium and hyperplasia groups, representing an association with increased EZH2 expression rather than evidence of a direct mechanistic relationship. These findings demonstrate that EZH2 expression differs across histopathological categories of endometrial lesions, with the highest expression observed in Type II endometrial carcinoma. The observed predominance of nuclear EZH2 in atypical hyperplasia and carcinoma further supports its association with aggressive tumor biology, although additional functional and clinicopathological studies are required to establish its clinical and biological significance.
Eggerthella lenta (E. lenta) is an opportunistic anaerobic pathogen associated with severe systemic infections, yet rapid and accurate diagnostic tools remain limited. To address this challenge, we developed a highly sensitive and specific dual-readout diagnostic platform integrating recombinase-aided amplification (RAA) with the CRISPR/Cas13a system, targeting the highly conserved rsmG gene of E. lenta. The assay offers two detection modalities: a real-time fluorescence readout and a visually interpretable lateral flow strip. Analytical evaluation demonstrated that the fluorescence-based assay achieved a limit of detection (LOD) of 4.4 copies per reaction (95% CI: 3.7-5.6 copies/reaction), while the instrument-free lateral flow assay yielded an LOD of 10⁴ copies per reaction. The platform exhibited exceptional specificity, showing no cross-reactivity with 10 common non-target bacterial species. Clinical validation was performed using 24 synovial fluid samples, all confirmed positive for E. lenta by Sanger sequencing. The fluorescence assay successfully detected all 24 samples, achieving a detection rate of 100% (24/24). In parallel, the lateral flow assay detected 21 of the 24 positive samples, yielding a detection rate of 87.5% (21/24). The three samples undetected by the lateral flow strip were verified as true positives by sequencing, indicating that the discrepancy was due to the lower analytical sensitivity of the strip format rather than a lack of specificity. In conclusion, this dual-mode RAA-CRISPR/Cas13a platform serves as a robust and practical tool for rapid clinical diagnosis and point-of-care (POC) triaging of E. lenta infections. The fluorescence format is optimal for high-sensitivity laboratory testing, whereas the lateral flow variant provides a deployable alternative for rapid, point-of-care screening in resource-limited environments.
Di (2-ethylhexyl) phthalate (DEHP) is an endocrine disruptor commonly present in environment, with metabolic disturbances and reproductive toxicity. DEHP remains one of the most widely utilized plasticizers, is still consumed in large quantities with limited prospects for complete replacement in short term. Consequently, human population is inevitably at risk of DEHP exposure. Previous studies have revealed the adverse effects of maternal perinatal DEHP exposure on offspring glucose homeostasis, however, whether paternal pre-pregnancy DEHP exposure disrupts offspring glucose metabolism through epigenetic inheritance remains unclear. In this study, we aimed to investigate the transgenerational epigenetic mechanism of abnormal glycometabolism in paternal DEHP exposed offspring, and to identify potential intervention targets. Here, male rats are administrated DEHP (0.1, 0.5 and 1.0 mg/kg body weight/d) by gavage for 15 weeks and then mated with females to produce offspring. This study finds that paternal DEHP exposure impaired glycometabolism in adult male offspring across generations. Mechanistically, paternal DEHP exposure down-regulated miR-10a-5p expression in the F0 generation sperm and offspring pancreatic islets, then led to Klf11-dependent β cell dysfunction and insulin synthesis reduction. Further evidence confirmed that miR-10a-5p bound Klf11 mRNA 3'-untranslated region (3'- UTR) and repressed its expression. Therefore, DEHP induced miR-10a-5p reduction resulted in Klf11 disinhibition, which functions as a transcriptional suppressor of Kit and Ins2 genes expression, eventually exacerbating glycometabolism disorders. Ultimately, our findings reveal that long-term DEHP exposure induced glycometabolism disorders exhibits transgenerational genetic characteristics and identify miR-10a-5p/Klf11 axis as a potential molecular target to mitigate offspring metabolic risk associated with paternal environment exposures.
A lateral flow analysis (LFA) strip for detecting dual miRNAs (miR155 and miR21) was constructed based on the "AND" logic gate and DNA self-assembly. The ternary synergistic strategy of "logic gating-signal amplification-visual output" enables highly sensitive and visual detection of miR21 and miR155 for breast cancer diagnosis. Only if both miR21 and miR155 are present can the trigger DNA (TDNA) in the sensor specifically dissociate and activate the "AND" logic gate (output signal = "1"); in the absence of either miRNA, the system remains in an inactive state (output signal = "0"). TDNA initiates catalytic hairpin assembly (CHA), and the product of the CHA reaction cascaded triggers the hybridization chain reaction (HCR) to achieve dual signal amplification. Gold nanoparticles (AuNPs) were used to label DNA for the construction of the LFA strip, allowing naked-eye observation of the detection results of miR21 and miR155. Detection results showed that the signal intensity of the test line (T line) had a positive correlation with the concentration of miRNAs (with a miR155:miR21 ratio of 1:1). Within the concentration range of 0.1-10 nM, the average grayscale value of the T line exhibited a good correlation with the logarithm of miRNA concentration (correlation coefficient r = 0.9983), and the limit of detection (LOD) was 30.98 pM. This LFA strip has been preliminarily validated to the detection of human serum samples, and the results indicated that the levels of miR155 and miR21 in the serum of breast cancer patients were significantly higher than those in normal individuals. Free from the limitation of large-scale instruments, this strip holds great commercialization potential.
This study describes the development and analytical validation of a DNA-based quantitative PCR (qPCR) assay for detecting the NPM1 Type A mutation (NPM1-A mut) in acute myeloid leukemia (AML). The assay employs a modified wild-type blocker (WTB) to preferentially suppress amplification of wild-type (WT) alleles, thereby enriching mutant targets. The modified WTB was synthesized as a 3'-ddC-terminated LNA/DNA chimera and used together with a 6-FAM-MGB-Eclipse hydrolysis probe. The analytical performance of the WTB-qPCR assay was compared with that of an otherwise identical qPCR assay lacking WTB. Key parameters such as linearity, precision, limit of detection (LoD), and limit of quantification (LoQ) were evaluated following the guidelines outlined in CLSI EP17-A2. Concurrently, modified WTB was implemented in Sanger sequencing to improve the sensitivity of mutation detection. Incorporation of WTB yielded an LoD of 0.00302% and an LoQ of 0.00528%, corresponding to approximately 3.02 and 5.28 NPM1-Amut copies, respectively, per 100,000 WT copies. This represented a clear improvement over the non-WTB assay. The WTB-qPCR showed excellent linearity (R2 > 0.999) and precision (CV < 5%), with 100% analytical specificity and no detectable cross-reactivity. Application of modified WTB in Sanger sequencing increased analytical sensitivity to ~ 0.1% mutant allele frequency. The modified WTB-qPCR assay offers a rapid, highly sensitive, and cost-effective DNA-based method for MRD monitoring in NPM1-A-mutated AML. Its strong analytical performance and straightforward workflow support its implementation as a routine diagnostic tool in clinical laboratories.
Sudden Unexplained Death in Youth (SUDY) requires thorough investigation to identify underlying causes and guide prevention strategies. In the Netherlands, cases are investigated using the standardized Postmortem Evaluation of Sudden Unexplained Death in Infants and Children (PESUDIC), in which autopsy is offered as a standard component. However, its invasive nature and associated time burden may limit parental acceptance. This study evaluated to what extent the cause of death can be established using a limited set of diagnostic tests compared to the standard procedure including autopsy. In this observational study, children > 2 years of age who died suddenly and unexpectedly and underwent PESUDIC, including imaging and autopsy, were included. Two expert panels, consisting of a forensic and a pediatric specialist, independently assessed early diagnostic tests available before autopsy. Cases were classified by level of diagnostic certainty and need for autopsy. Panel conclusions were compared with the reference standard: a multidisciplinary audit incorporating all available information, including autopsy findings. Sixty-six cases were included (median age 12 years (IQR 4-14.7), 63% male). Panels identified indicative information for a cause of death in 60 patients (91%). Nevertheless, autopsy was required in most cases (n = 59) to confirm the diagnosis. In 7 cases (10.6%), panels were sufficiently confident to establish the cause of death without autopsy with complete agreement with the reference standard. Causes included obstructive gastrointestinal pathology (n = 5) and diabetic ketoacidosis with dehydration (n = 2).  Only a small proportion of children > 2 years of age with sudden unexpected death have a cause of death that can be established with sufficient certainty at an early stage. In the vast majority of cases, the cause of death remains uncertain, supporting the recommendation to perform an autopsy. • Autopsy in sudden unexplained death in youth is worldwide recognized as the gold standard for postmortem examination. However, its invasive nature and associated time burden may limit parental acceptance. • In 7 of 60 sudden and unexplained deceased minors (10%), sufficient certainty regarding the cause of death can be obtained by other minimal invasive diagnostic test, so without the need of an autopsy. Causes without the need of an autopsy included obstructive gastrointestinal pathology and diabetic ketoacidosis with dehydration.
Transcription factors (TFs) are key players in eukaryotic gene regulation, but the DNA binding specificity of many TFs remains unknown. Here, we assay 284 mostly uncharacterized putative human TFs using selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq), revealing 74 new DNA binding motifs. To investigate whether TFs lacking detectable motifs preferably bind epigenetically modified DNA, we develop methylation-sensitive SMiLE-seq (meSMiLE-seq), a microfluidic assay that simultaneously probes binding to methylated and unmethylated DNA. Using meSMiLE-seq, we assay 114 TFs and identify DNA-binding models for 48 proteins, including known methylation-sensitive binding modes for POU5F1 and RFX5. 11 TFs prefer methylated DNA or display alternative methylation-dependent motifs (e.g. PRDM13), while 13 show aversion to methylated sequences (e.g. USF3). Finally, we identify ZHX2 as a putative Z-DNA binder. Altogether, our study significantly expands the human TF codebook, while providing a versatile platform to quantitatively assay the impact of DNA modifications on TF binding.
Post-COVID syndrome (PCS) is an ongoing legacy of the SARS-CoV-2 pandemic. Defined as symptoms persisting beyond 12 weeks following SARS-CoV-2 infection unexplained by an alternative diagnosis, many individuals continue to experience symptoms for months post-infection. Fatigue, breathlessness, cognitive difficulties and sleep disturbances are all commonly reported, frequently impairing daily functioning and quality of life. Acceptance and commitment therapy (ACT), which enhances psychological flexibility and supports values-based behaviour change, has shown effectiveness across long-term health conditions. This protocol describes the evaluation of Balance-ACT as a novel, adapted ACT-based intervention to address the complex physical and psychological needs of people with PCS. A randomised controlled trial (RCT), co-developed with people with PCS, will evaluate the efficacy and cost-effectiveness of Balance-ACT for PCS. A total of 196 adults with PCS will be recruited from specialist long COVID services within secondary care across NHS organisations in England and randomised 1:1 to Balance-ACT or treatment as usual (TAU). Balance-ACT consists of 10 therapist-delivered sessions delivered over 13 weeks. TAU comprises brief educational videos and a self-help leaflet for COVID-19 recovery. Outcomes will be assessed at 7, 14 and 20 weeks post-randomisation. The primary outcome is health-related quality of life, measured using the SF-36 Physical Component Score at 14 weeks post randomisation. Secondary outcomes include physical health-related quality of life at 20 weeks, perceived fatigue, clinical global improvement, sleep disturbance, cognitive functioning, depressive symptoms, anxiety symptoms, post-exertional malaise, breathlessness, muscle strength, muscle fatigue and heart-rate variability. Mediation analyses will examine whether the intervention leads to changes in psychological flexibility, fear-avoidance beliefs, damage beliefs, embarrassment-avoidance, symptom focusing, muscle strength and physical activity and whether these changes are associated with physical health outcomes. We will also conduct a nested qualitative study to explore participants' views of the intervention. Cost-effectiveness will be assessed by comparing health and social care service costs and quality of life outcomes between Balance-ACT and TAU at 20 weeks. Ethical approval has been granted by the NHS Camberwell St Giles Research Ethics Committee (reference 23/LO/0941). The study results will be disseminated in peer-reviewed journals and presented at international conferences. NCT06231238.
Due to shared ß-hexosamindase A deficiencies and significant clinical overlap, the predominating subtypes of late-onset GM2 gangliosidosis, late-onset Tay-Sachs (LOTS) and late-onset Sandhoff disease (LOSD) have been considered essentially indistinguishable. However, growing evidence supports several distinctions between the two entities. We highlight these distinctions through the cross-sectional evaluation of 27 (21 LOTS and 6 LOSD) late-onset GM2 gangliosidosis participants. Study protocol included physical examinations, assessments of gait, balance, muscle strength, ataxia, and nerve conduction velocities, and brain magnetic resonance imaging. Lower limb weakness and later development of upper limb weakness was highly prevalent in both diseases. Accompanying gait disturbances, balance issues, and dysmetria were also prevalent in both cohorts. Strength evaluations showed weakness in both the LOTS and LOSD cohorts compared to controls primarily impacting extensor muscles. In contrast, BARS scores for dysarthria and oculomotor dysfunction were present and heterogenous in LOTS participants and absent in LOSD participants. Twenty-four percent of LOTS participants and none of the LOSD participants had a history of neuropsychiatric symptoms. Cerebellar volume including lobules V and VI were lower in LOTS compared to LOSD and normative data. However, severe length-dependent sensory neuropathy was present in all LOSD participants but not in LOTS participants. The finding of lower cerebellar volume in LOTS suggests the distinctive features of the LOTS phenotype are related to cerebellar dysfunction. However, the cause of the phenotypic differences between LOTS and LOSD remains a mystery, and the molecular and biochemical basis for the dichotomy requires further investigation.
Gain-of-function (GoF) missense variants in the two-pore domain (K2P) K+ channel TASK-1 (KCNK3) result in developmental delay with sleep apnea (DDSA), a neurodevelopmental channelopathy, while loss-of-function (LoF) variants cause pulmonary arterial hypertension. However, for the related TASK-3 channel (KCNK9), both LoF and GoF variants underlie a distinct neurodevelopmental disorder, KCNK9 imprinting syndrome (KIS). The relationship between genotype and phenotype in these disorders is further complicated because TASK-1 and TASK-3 can co-assemble into heteromeric channels with distinct functional properties. Here, we report additional patients with missense variants in KCNK3 and KCNK9 and investigate the effect of four novel genetic variants on the functional properties of homomeric and heteromeric TASK channels. Interestingly, two of these new pathogenic GoF variants (R131H and L122V) are found in both TASK-1 and TASK-3 and have equivalent functional effects on heteromeric TASK-1/TASK-3 channel activity, yet result in different clinical phenotypes. We have also determined a cryo-EM structure for the pathogenic L122V mutant TASK-3 channel, which suggests that subtle changes in gating and permeation within the inner cavity are responsible for its activatory effect. Overall, these results highlight the dominant role that homomeric TASK channels likely play in defining their associated channelopathies as well as the complexity of interpreting K+ channel dysfunction in pathophysiology.
Especially, the coxsackievirus B group of enteroviruses has been linked to the development of islet autoimmunity and type 1 diabetes in genetically susceptible individuals. Our aim was to study the possible associations of 10 different microbial infections with islet autoimmunity in a large international prospective study. In a nested case-control study within the TRIGR study, follow-up serum samples from 240 islet autoantibody-positive case children and 436 age- and country-matched control children were analysed for IgG class antibodies against 10 different respiratory and gastrointestinal microbes using an enzyme immunoassay, and for neutralising antibodies against all six coxsackievirus B types. The samples were obtained at several time points prior to and at the time of seroconversion for multiple islet autoantibodies. All children had a first-degree relative with type 1 diabetes, and they carried risk-associated HLA-DQ alleles. Coxsackievirus B5 was associated with an increased risk of islet autoimmunity (OR 2.22, 95% CI 1.29-3.80, p = 0.004, corrected p = 0.024), which remained after adjustment for HLA, sex, and maternal type 1 diabetes. This association was particularly seen for infections occurring more than 12 months (OR 2.02, 95% CI 1.06-3.84, p = 0.032) and 0-6 months (OR 2.66, 95% CI 1.11-6.35, p = 0.028) before the first detection of multiple islet autoantibodies. After correction for multiple comparisons, none of the other viruses showed an association with islet autoimmunity. This study supports previous evidence of the risk association of coxsackievirus B infections. These results support the role of certain virus infections as possible modulating factors in the pathogenesis of type 1 diabetes.
Accurate identification of actionable somatic variants is essential for therapeutic stratification in colorectal cancer (CRC). While next-generation sequencing (NGS) enables comprehensive genomic profiling, targeted approaches may provide faster and more practical alternatives for routine diagnostics. This study evaluated the analytical performance of the Agena Bioscience iPLEX® High Sensitivity (HS) Colon Panel using MassARRAY MALDI-TOF technology for detection of hotspot variants in KRAS, NRAS, BRAF, and PIK3CA from formalin-fixed paraffin-embedded (FFPE) specimens. A total of 60 unique clinical and reference samples were analyzed, targeting 26 single-nucleotide variants and compared with an orthogonal targeted NGS assay. Limit of detection (LOD) was assessed using serial dilutions of reference materials, and intra- and inter-run reproducibility was evaluated across multiple runs. Analytical performance metrics including positive and negative percent agreement, predictive values, and error rates were calculated. The assay demonstrated complete concordance with NGS across all evaluated variants, requiring only 20 ng of DNA input, compared to 80-120 ng for a successful NGS run. LOD studies showed reliable detection of multiple variants at approximately 5% variant allele frequency, with BRAF p.V600E detectable near 1%. Intra- and inter-run analyses achieved 100% concordance, confirming assay reproducibility. Aggregated performance metrics demonstrated high sensitivity and specificity across a heterogeneous sample set. These findings establish the iPLEX® HS Colon Panel as a reliable platform for rapid detection of clinically actionable hotspot mutations. This study represents analytical validation using mixed FFPE tumor specimens; evaluation in larger colorectal cancer cohort, is warranted.
Skeletal muscle excitation-contraction coupling (ECC) is a highly specialized process that converts membrane depolarization into contraction through tightly regulated intracellular Ca²⁺ dynamics. Recent advances in molecular genetics have expanded the spectrum of skeletal muscle disorders associated with ECC-related proteins, including congenital myopathies, malignant hyperthermia susceptibility, exertional rhabdomyolysis, exertional heat illness, and related episodic disorders. Although these disorders have traditionally been classified according to clinical manifestations, pathological findings, and causative genes, accumulating evidence suggests that genetically and clinically heterogeneous disorders converge on overlapping abnormalities in intracellular Ca²⁺ handling and ECC function. This review provides an integrative overview of ECC-related skeletal muscle disorders from the perspective of shared abnormalities in intracellular Ca²⁺ handling and the molecular mechanisms that underlie them. We discuss how pathogenic variants affecting the voltage-sensing skeletal muscle L-type calcium channel (CaV1.1), the ryanodine receptor type 1 (RyR1), and other triad-associated proteins disrupt Ca²⁺ release from the sarcoplasmic reticulum (SR), luminal Ca²⁺ regulation, and SR Ca²⁺ reuptake, leading to diverse but partially convergent phenotypes. We also discuss secondary pathological changes associated with chronic intracellular Ca²⁺ dysregulation, including mitochondrial dysfunction, oxidative stress, and skeletal muscle remodeling. Finally, we present a functional perspective centered on intracellular Ca²⁺ handling that complements conventional genetic and clinicopathological classifications of ECC-related skeletal muscle disorders.
Pseudohypoparathyroidism (PHP) is a rare disorder with clinical and genetic heterogeneity, resulting from inactivating variants or epigenetic alterations of the GNAS locus. To characterize the natural course of PHP, focusing on rarely defined endocrine features. Nineteen children (11 girls) from 17 families from a single center, diagnosed with PHP from 1992 to 2025, were enrolled. Genetic/epigenetic analyses, age at onset, and evidence of hormone resistance, radiologic findings, and long-term follow-up of anthropometric measurements were retrospectively reviewed, and cases were classified according to clinical and genetic features as PHP1A, PHP1B, or PPHP. The study group consisted of children with PHP1A (n=10), PHP1B (n=8), and PPHP (n=1). PHP1A cases harbored six different GNAS point mutations, one of which was novel. PHP1Bs exhibited GNAS methylation abnormalities. Except for one PHP1A case, all PHP1A/1Bs displayed PTH resistance, which developed earlier in PHP1A (6 years vs 11.2 years). All PHP1A and 5 PHP1B cases had clinical or subclinical hypothyroidism. One PHP1A and two PHP1B cases had low free T4 with inappropriately normal TSH, suggesting central hypothyroidism due to presumed TRH resistance. Clinical signs of gonadal dysfunction were observed in four of six girls with PHP1A. Skeletal features of AHO were present in 7 PHP1A and 3 PHP1B patients. Intellectual disability was identified in 50% of PHP1A. Phenotypic variability was noted among subjects with the same mutation. Overlapping PHP1A and PHP1B features may complicate differential diagnosis, necessitating molecular genetic analysis. The pattern and severity of endocrine involvement, including possible central hypothyroidism, vary widely among affected children.
Cervical cancer is the second most prevalent cancer among women in developing nations, including India. This study aims to assess the knowledge, attitude, and practice (KAP)  regarding cervical cancer screening, risk factors, and prevention strategies, along with the  barriers to participation in screening activities, among married tribal women in  Sirohi district, Rajasthan, to identify areas for improvement in this essential aspect of women's health. A cross-sectional, community-based study was conducted among 170 married tribal women recruited from the Tribal Sub-Plan (TSP) zone in Sirohi district, Rajasthan. A  standardised, fixed-response questionnaire was used to obtain data on participants' KAP regarding cervical cancer screening. The mean age of the study population was 32.81 ± 10.5 years. Out of 170, 28 (16.5%) women had heard of cervical cancer, and 20 (11.8%) women were aware of cervical cancer screening. Only 19.4% of women agreed to undergo screening for cervical cancer. A significant proportion, 86.5% of participants, had poor knowledge, 84.1% had a negative attitude, and only 0.6% demonstrated good practices regarding cervical cancer screening. Knowledge scores were significantly associated with age group, education, socioeconomic class, and number of children (p < 0.05). Attitude scores were significantly associated with age, contraceptive method used, and tobacco use. Practice scores were significantly associated with education and menstrual cycle characteristics (p < 0.05). Identified barriers to screening included lack of awareness, financial constraints, anxiety about vaginal examinations, feelings of embarrassment, and communication difficulties. The study findings indicate a critical deficiency in knowledge, attitude, and practice as well as low willingness to participate in cervical cancer screening among tribal women. Educational interventions  through culturally sensitive awareness programs should be conducted in the local tribal language to enhance preventive healthcare in the tribal population.