Search Results (138)

Background: Neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), are genetically complex and often linked to structural genomic variations such as copy number variants (CNVs). Current diagnostic strategies face challenges in interpreting the clinical significance of such variants. Methods: We developed a customized, gene-oriented chromosomal microarray (CMA) targeting 6026 genes relevant to neurodevelopment, aiming to improve diagnostic yield and candidate gene prioritization. A total of 39 patients with unexplained developmental delay, intellectual disability, and/or ASD were analyzed using this custom platform. Systems biology approaches were employed for downstream interpretation, including protein–protein interaction networks, centrality measures, and tissue-specific functional module analysis. Results: Pathogenic or likely pathogenic CNVs were identified in 31% of cases (9/29). Network analyses revealed candidate genes with key topological properties, including central “hubs” (e.g., NPEPPS, PSMG1, DOCK8) and regulatory “bottlenecks” (e.g., SLC15A4, GLT1D1, TMEM132C). Tissue- and cell-type-specific network modeling demonstrated widespread gene involvement in both prenatal and postnatal developmental modules, with glial and astrocytic networks showing notable enrichment. Several novel CNV regions with high pathogenic potential were identified and linked to neurodevelopmental phenotypes in individual patient cases. Conclusions: Customized CMA offers enhanced detection of clinically relevant CNVs and provides a framework for prioritizing novel candidate genes based on biological network integration. This approach improves diagnostic accuracy in NDDs and identifies new targets for future functional and translational studies, highlighting the importance of glial involvement and immune-related pathways in neurodevelopmental pathology. Full article

Microfluidic modulation spectroscopy-infrared (MMS) offers a label-free, high-sensitivity approach for quantifying changes in protein secondary structures under native solution conditions. MMS subtracts the solvent backgrounds from sample signals by alternately flowing proteins and matched buffers through a microfluidic chamber, yielding clear amide I spectra from microliter volumes. In this study, we validated MMS on canonical globular proteins, bovine serum albumin, mCherry, and lysozyme, demonstrating accurate detection and resolution of α-helix, β-sheet, and mixed-fold structures. Applying MMS to the intrinsically disordered protein Tau, we detected environment-driven shifts in transient conformers: both the acidic (pH 2.5) and alkaline (pH 10) conditions increased the turn/unordered structures and decreased the α-helix content relative to the neutral pH, highlighting the charge-mediated destabilization of the labile motifs. Hyperphosphorylation of Tau yielded a modest decrease in the α-helical fraction and an increase in the turn/unordered structures. Comparison of monomeric and aggregated hyperphosphorylated Tau revealed a dramatic gain in β-sheet and a loss in turn/unordered structures upon amyloid fibril formation, confirming MMS’s ability to distinguish disordered monomers from amyloids. These findings establish MMS as a robust platform for detecting protein secondary structures and monitoring aggregation pathways in both folded and disordered systems. The sensitive detection of structural transitions offers opportunities for probing misfolding mechanisms and advancing our understanding of aggregation-related diseases. Full article

Objective: Epilepsy, a common neurological disorder marked by recurrent seizures often starting in childhood, has a complex etiology. Advances in high-throughput sequencing now confirm that 70–80% of cases have a genetic basis. Accordingly, this study aims to evaluate the clinical relevance of genetic variations detected through epilepsy panels and whole exome sequencing (WES) in pediatric-onset epilepsy patients. Methods: For this study, we enrolled a cohort of pediatric patients involving 205 subjects with a preliminary diagnosis of epilepsy. Targeted next-generation sequencing panels for epilepsy and whole exome sequencing was performed using the NextSeq 500 platform. The results were analyzed with the QIAGEN Clinical Insight bioinformatic platform and were further confirmed and approved by the Human Genome Mutation Database and ClinVar databases. Results: In this study, an epilepsy panel was conducted in 138 patients, and whole exome sequencing was performed in 67 patients. No clinically relevant variants were identified in 29 (21.0%) patients who underwent the epilepsy panel and 27 (40.3%) patients who underwent WES. Variants were detected in 128 different genes in the epilepsy panel group and in 54 different genes in the WES group, with the frequency of these variants limited to one or two patients. Significance: In both the epilepsy panel and WES groups, variants in sodium channel proteins, specifically in the SCN1A, SCN8A, and SCN9A genes, were found to have a high frequency. Collectively, these findings suggest that sodium channel proteins may play an important role in epilepsy. Full article

Nipah virus (NiV), a highly lethal zoonotic pathogen causing encephalitis and respiratory diseases with mortality rates up to 40–70%, faces research limitations due to its strict biosafety level 4 (BSL-4) containment requirements, hindering antiviral development. To address this, we generated two NiV minigenome replicons (Fluc- and EGFP-based) expressed via helper plasmids encoding viral N, P, and L proteins, enabling replication studies under BSL-2 conditions. The minigenome replicon recapitulated the cytoplasmic inclusion body (IB) formation observed in live NiV infections. We further demonstrated that IB assembly is driven by liquid–liquid phase separation (LLPS), with biochemical analyses identifying the C-terminal N core domain of the N protein, as well as N₀ and XD domains and the intrinsically disordered region (IDR) of the P protein, as essential structural determinants for LLPS-mediated IB biogenesis. The targeted siRNA silencing of the 5′ and 3′ untranslated regions (UTRs) significantly reduced replicon-derived mRNA levels, validating the regulatory roles of these regions. Importantly, the minigenome replicon demonstrated sensitivity to type I/II/III interferons and antivirals (remdesivir, azvudine, molnupiravir), establishing its utility for drug screening. This study provides a safe and efficient platform for investigating NiV replication mechanisms and accelerating therapeutic development, circumventing the constraints of BSL-4 facilities while preserving key virological features. Full article

IQSEC2 is a guanine nucleotide exchange factor that modulates synaptic transmission, the excitatory/inhibitor balance and memory consolidation. Pathogenic mutations in the IQSEC2 gene result in epilepsy, cognitive dysfunction and autism spectrum disorder. The most common de novo IQSEC2 mutation in the IQSEC2 gene, associated with a particularly severe phenotype in males as compared to other IQSEC2 mutations, is due to a frameshift mutation near the C terminus, resulting in an extension of the open reading frame [IQSEC2 S1474Qfs*133]. The objective of this study was to understand the pathophysiology of this specific IQSEC2 mutation using molecular modeling protein–protein interaction assays and a conditional transgenic mouse model of the mutation. Molecular modeling studies showed that the mutation results in the generation of a new domain that may bind ATP. The mutant IQSEC2 protein failed to interact with proteins that normally interact with IQSEC2, most notably with PSD-95. Finally, mice expressing the human mutation displayed marked developmental delays and abnormal social behavior. We conclude that diseases associated with the IQSEC2 S1474Qfs*133 may be due not only to the loss of function of IQSEC2 but also to the appearance of new detrimental activity. The conditional mouse model will allow for the identification of brain regions that are critical for IQSEC2 expression and will serve as a platform for the development of personalized therapies for this disease. Full article

Rare diseases typically evade the application of the standard drug discovery and development pipelines due to their understudied molecular etiology and the small market size. Herein, we report a rare disease-directed workflow that rapidly studies the molecular features of the disorder, establishes a high-throughput screening (HTS) platform, and conducts an HTS of thousands of approved drugs to identify and validate repositioning drug candidates. This study examines the pediatric neurological disorder caused by de novo mutations in YWHAG, the gene encoding the scaffolding protein 14-3-3γ, and the workflow discovers nuclear relocalization and a severe drop in 14-3-3γ binding to its phosphorylated protein partners as the key molecular features of the pathogenic hotspot YWHAG mutations. We further established a robust in vitro HTS platform and screened ca. 3000 approved drugs to identify the repositioning drug candidates that restore the deficient 14-3-3γ-phosphotarget interactions. Our workflow can be applied to other 14-3-3-related disorders and upscaled for many other rare diseases. Full article

Background: GM3 Synthase Deficiency (GM3SD) is a rare autosomal recessive neurodevelopmental disease characterized by recurrent seizures and neurological deficits. The disorder stems from mutations in the ST3GAL5 gene, encoding GM3 synthase (GM3S), a key enzyme in ganglioside biosynthesis. While enzyme deficiencies affecting ganglioside catabolism are well-documented, the consequences of impaired ganglioside biosynthesis remain less explored. Methods: To investigate GM3SD, we used a Human Embryonic Kidney 293-T (HEK293-T) knockout (KO) cell model generated via CRISPR/Cas9 technology. Lipid composition was assessed via high-performance thin-layer chromatography (HPTLC); glycohydrolase activity in lysosomal and plasma membrane (PM) fractions was enzymatically analyzed. Lysosomal homeostasis was evaluated through protein content analysis and immunofluorescence, and cellular bioenergetics was measured using a luminescence-based assay. Results: Lipidome profiling revealed a significant accumulation of lactosylceramide (LacCer), the substrate of GM3S, along with increased levels of monosialyl-globoside Gb5 (MSGb5), indicating a metabolic shift in glycosphingolipid biosynthesis. Lipid raft analysis revealed elevated cholesterol levels, which may impair microdomain fluidity and signal transduction. Furthermore, altered activity of lysosomal and plasma membrane (PM)-associated glycohydrolases suggests secondary deregulation of glycosphingolipid metabolism, potentially contributing to abnormal lipid patterns. In addition, we observed increased lysosomal mass, indicating potential lysosomal homeostasis dysregulation. Finally, decreased adenosine triphosphate (ATP) levels point to impaired cellular bioenergetics, emphasizing the metabolic consequences of GM3SD. Conclusions: Together, these findings provide novel insights into the molecular alterations associated with GM3SD and establish the HEK293-T KO model as a promising platform for evaluating potential therapeutic strategies. Full article

The development of targeted drugs for diseases of the central nervous system (CNS) is a significant challenge due to the structural complexity and functional specificities of these systems. Recently, exosomes have emerged as a promising therapeutic platform, given their unique capacity to traverse the blood-brain barrier and deliver bioactive molecules to target cells. This review examines recent advances in exosome research with a particular focus on CNS diseases, emphasizing their role as carriers of therapeutic cargo, including proteins, RNAs, and lipids. Nevertheless, significant challenges remain before exosome-based therapies can be translated from preclinical research to clinical applications. These include the need for scalable production and standardized isolation methods. Despite these hurdles, ongoing studies continue to shed light on the mechanisms of exosome-mediated neuroprotection and neurodegeneration. This paves the way for innovative therapeutic strategies to address CNS disorders. Full article

Impaired function of Polymerase-γ (Pol-γ) results in impaired replication of the mitochondrial genome (mtDNA). Pathogenic mutations in the POLG gene cause dysfunctional Pol-γ and dysfunctional mitochondria and are associated with a spectrum of neurogenetic disorders referred to as POLG spectrum disorders (POLG-SDs), which are characterized by neurologic dysfunction and premature death. Pathomechanistic studies and human cell models of these diseases are scarce. SH-SY5Y cells (SHC) are an easy-to-handle and low-cost human-derived neuronal cell model commonly used in neuroscientific research. Here, we aimed to study the effect of reduced Pol-γ function using stable lentivirus-based shRNA-mediated knockdown of POLG in SHC, in both the proliferating cells and SHC-derived neurons. POLG knockdown resulted in approximately 50% reductions in POLG mRNA and protein levels in naïve SHC, mimicking the residual Pol-γ activity observed in patients with common pathogenic POLG mutations. Knockdown cells exhibited decreased mtDNA content, reduced levels of mitochondrial-encoded proteins, and altered mitochondrial morphology and distribution. Notably, while chemical induction of mtDNA depletion via ddC could be rescued by the mitochondrial biosynthesis stimulators AICAR, cilostazol and resveratrol (but not MitoQ and formoterol) in control cells, POLG-knockdown cells were resistant to mitochondrial biosynthesis-mediated induction of mtDNA increase, highlighting the specificity of the model, and pathomechanistically hinting towards inefficiency of mitochondrial stimulation without sufficient Pol-γ activity. In differentiated SHC-derived human neurons, POLG-knockdown cells showed impaired neuronal differentiation capacity, disrupted cytoskeletal organization, and abnormal perinuclear clustering of mitochondria. In sum, our model not only recapitulates key features of POLG-SDs such as impaired mtDNA content, which cannot be rescued by mitochondrial biosynthesis stimulation, but also reduced ATP production, perinuclear clustering of mitochondria and impaired neuronal differentiation. It also offers a simple, cost-effective and human (and, as such, disease-relevant) platform for investigating disease mechanisms, one with screening potential for therapeutic approaches for POLG-related mitochondrial dysfunction in human neurons. Full article

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disorder characterized by serious physical and cognitive impairments. Recent research underscores the role of immune dysfunction, including the role of autoantibodies, in ME/CFS pathophysiology. Expanding on previous studies, we analyzed 7542 antibody–antigen interactions in ME/CFS patients using two advanced platforms: a 1134 autoantibody Luminex panel from Oncimmune and Augmenta Bioworks, along with Rapid Extracellular Antigen Profiling (REAP), a validated high-throughput method that measures autoantibody reactivity against 6183 extracellular human proteins and 225 human viral pathogen proteins. Unlike earlier reports, our analysis of 172 participants revealed no significant differences in autoantibody reactivities between ME/CFS patients and controls, including against GPCRs such as β-adrenergic receptors. However, subtle trends in autoantibody ratios between male and female ME/CFS subgroups, along with patterns of herpesvirus reactivation, suggest the need for broader and more detailed exploration. Full article

As a traditional Chinese medicine, the adverse hepatotoxicity effects of Pleuropterus multiflorus (Thunb.) Nakai (PM) have been documented. However, nephrotoxicity has been neglected as studies related to kidney toxicity mechanisms are limited. Our previous research reported that extract D [95% ethanol (EtOH) elution, PM-D] in a 70% EtOH PM extract showed more significant hepatotoxicity than other extracts. In the current study, PM-D was continuously administered to mice for 7 days at a dose of 2 g/kg (equivalent to a human dose of 219.8 mg/kg), which increased renal biochemical indexes and caused pathological kidney injury, suggesting renal toxicity. Therefore, network pharmacology and spatially resolved metabolomics were conducted to explore nephrotoxicity mechanisms underpinning PM-D. Network pharmacology indicated that BCL2, HSP90, ESR1, and CTNNB1 genes were core targets, while the phosphoinositide 3-kinase (PI3K)/protein kinase B(AKT)/signaling pathway was significantly enriched. Spatially resolved metabolomics indicated heterogeneous metabolite distribution in the kidney, further indicating that PM-D nephrotoxic metabolic pathways were enriched for α-linolenic acid and linoleic acid metabolism, pyrimidine metabolism, carnitine synthesis, and branched-chain fatty acid oxidation. Our comprehensive analyses highlighted that nephrotoxicity mechanisms were related to oxidative stress and apoptosis induced by disordered energy metabolism, lipid metabolism issues, and imbalanced nucleotide metabolism, which provide a platform for further research into PM nephrotoxicity mechanisms. Full article

Exosome-like nanovesicles (ELNs) derived from natural products are gaining attention as innovative therapeutic agents due to their biocompatibility, low immunogenicity, and capability to transport bioactive molecules such as proteins, lipids, and nucleic acids. These plant-derived ELNs exhibit structural similarities with mammalian exosomes, making them suitable for drug delivery, microbiome-targeted therapies, and regenerative medicine. Recent studies highlight their potential in treating cancer, inflammation, and metabolic disorders. Additionally, ELNs have applications in cosmetics, agriculture, and the food industry. This review combines the latest advancements in research on plant-derived ELNs, focusing on isolation techniques, pharmacological effects, and therapeutic applications. Although plant-derived ELNs offer promising opportunities, several challenges must be addressed, including standardization, large-scale production, and in vivo efficacy. By summarizing cutting-edge studies and suggesting future directions, we aim to inspire further development of plant-derived ELNs as next-generation therapeutic platforms. Full article

Non-alcoholic fatty liver disease (NAFLD), now referred to as metabolic dysfunction-associated steatotic liver disease (MASLD), is the most prevalent liver disorder globally, linked to obesity, type 2 diabetes, and cardiovascular risk. Understanding its potential progression from simple steatosis to cirrhosis and hepatocellular carcinoma (HCC) is crucial for patient management and treatment strategies. The disease’s complexity requires innovative approaches for early detection and personalized care. Omics technologies—such as genomics, transcriptomics, proteomics, metabolomics, and exposomics—are revolutionizing the study of MASLD. These high-throughput techniques allow for a deeper exploration of the molecular mechanisms driving disease progression. Genomics can identify genetic predispositions, whilst transcriptomics and proteomics reveal changes in gene expression and protein profiles during disease evolution. Metabolomics offers insights into the metabolic alterations associated with MASLD, while exposomics links environmental exposures to MASLD progression and pathology. By integrating data from various omics platforms, researchers can map out the intricate biochemical pathways involved in liver disease progression. This review discusses the roles of omics technologies in enhancing the understanding of disease progression and highlights potential diagnostic and therapeutic targets within the MASLD spectrum, emphasizing the need for non-invasive tools in disease staging and treatment development. Full article

Alzheimer’s disease (AD) is a prevalent neurodegenerative disorder and a significant cause of dementia in elderly individuals, with a growing prevalence in our aging population. Extracellular amyloid-β peptides (Aβ), intracellular tau proteins, and their phosphorylated forms have gained prominence as critical biomarkers for early and precise diagnosis of AD, correlating with disease progression and response to therapy. The high costs and invasiveness of conventional diagnostic methods, such as positron emission tomography (PET) and magnetic resonance imaging (MRI), limit their suitability for large-scale or routine screening. However, electrochemical (EC) analysis methods have made significant progress in disease detection due to their high sensitivity, excellent specificity, portability, and cost-effectiveness. This article reviews the progress in EC biosensing technologies, focusing on the detection of tau protein biomarkers in the blood (a low-invasive, accessible diagnostic medium). The article then discusses various EC sensing platforms, including their fabrication processes, limit of detection (LOD), sensitivity, and clinical potential to show the role of these sensors as transformers changing the face of AD diagnostics. Full article

Tissue engineering has emerged as a promising approach for improved regeneration of native tissue and could increase the quality of life of many patients. However, the treatment of injured tissue transitions is still in its early stages, relying primarily on a purely physical approach in medical surgery. A biodegradable implant with a modified surface that is capable of biological active protein delivery via a nanoparticulate release system could advance the field of musculoskeletal disorder treatments enormously. In this study, interconnected 3D macroporous scaffolds based on Polycaprolactone (PCL) were fabricated in a successive process of blending, annealing and leaching. Blending with varying parts of Polyethylene oxide (PEO), NaCl and (powdered) sucrose and altering processing conditions yielded scaffolds with a huge variety of morphologies. The resulting unmodified hydrophobic scaffolds were modified using two graft polymers (CS-g-PCL_x) with x = 29 and 56 (x = PCL units per chitosan unit). Due to the chitosan backbone hydrophilicity was increased and a platform for a versatile nanoparticulate release system was introduced. The graft polymers were synthesized via ring opening polymerization (ROP) of ε-Caprolactone using hydroxy groups of the chitosan backbone as initiators (grafting from). The suspected impact on biocompatibility of the modification was investigated by in vitro cell testing. In addition, the CS-g-PCL modification opened up the possibility of Layer by Layer (LbL) coating with alginate (ALG) and TGF-β₃-loaded chitosan tripolyphosphate (CS-TGF-β₃-TPP) nanoparticles. The subsequent release study showed promising amounts of growth factor released regarding successful in vitro cell differentiation and therefore could have a possible therapeutic impact. Full article