Search Results (307)

Glioblastoma and diffuse gliomas pose major therapeutic challenges due to marked intratumoral heterogeneity, limited tissue accessibility, and the blood–brain barrier. Tissue-based next-generation sequencing (NGS) remains essential for WHO CNS5 molecular classification, yet it is invasive and poorly suited to serial monitoring. Two complementary non- or minimally invasive approaches have advanced rapidly: radiogenomics, which correlates multiparametric MRI features with genomic alterations, and cerebrospinal fluid (CSF) liquid biopsy sequencing, which detects circulating tumor DNA with high tissue concordance. This review examines the independent progress and synergistic integration of radiogenomics and CSF-NGS. Imaging signatures can non-invasively predict key drivers (IDH1/2, EGFR, TERT, PTEN, TP53) and molecular subtypes, while CSF-ctDNA sequencing enables real-time assessment of clonal evolution, therapy resistance (including post-temozolomide hypermutation), and residual disease. We discuss technical considerations, performance metrics, multimodal artificial-intelligence fusion, and emerging clinical applications for diagnosis, prognosis, treatment selection, and longitudinal surveillance. Critical challenges, standardization, prospective validation, and workflow integration are highlighted. By combining the spatial phenotypic information of radiogenomics with the temporal genomic resolution of CSF sequencing, this multimodal strategy offers a promising path toward precision neuro-oncology and reduced reliance on repeated invasive sampling. Full article

Directed evolution has revolutionized protein engineering by applying the principles of natural selection to the laboratory. However, traditional in vitro methods are quite labor-intensive, while common in vivo methods suffer from low mutation rates and high rates of off-target mutations. To address these issues, researchers have developed targeted mutagenesis tools for rapid in vivo evolution of biomolecules. In this review, we discuss recent in vivo hypermutation tools that enable rapid sampling of the vast evolutionary landscape, all while supporting simultaneous selection of the best proteins within living organisms. We focus on three main mechanisms of hypermutation: (i) orthogonal replication, which uses error-prone replication machinery to replicate the target gene with low fidelity; (ii) CRISPR-Cas-guided mutators, where mutagenic proteins are localized to virtually any user-defined loci; and (iii) transcription-coupled mutagenesis, a simple, yet elegant tool that exploits the innate processivity of orthogonal ribonucleic acid (RNA) polymerases to guide mutagenic proteins along the target gene during transcription. We highlight key advantages of these systems, as well as some clinically- and biotechnology-relevant applications. We discuss important limitations and how they could be addressed in the future to make hypermutation tools with broad mutational spectra and windows that span entire genes with minimal off-target effects. Full article

Historically, mpox was thought to be a geographically constrained ‘disease of poverty,’ leading to decades of neglect by global health actors. Waning population immunity from the cessation of smallpox vaccination and prolonged scientific neglect created conditions that enabled the monkeypox virus (MPXV) to adapt cryptically. This ultimately contributed to the emergence of unprecedented global public health crises. This review aims to systematically trace the history, epidemiology, genomic evolution, and reservoir ecology of mpox in West Africa from 1970 to 2025. Following PRISMA guidelines, 110 articles met the inclusion criteria and were synthesized to map the virus’s trajectory. For nearly four decades, an “Era of Silence” (1970–2016) masked the silent enzootic circulation of MPXV within West African wildlife, primarily rodents and small mammals. This epidemiological quiescence ended with the 2017 re-emergence in Nigeria, which signaled a fundamental paradigm shift. The disease profile transitioned from sporadic, rural paediatric infections to sustained, urban and secondary transmission among young adult males. This shift was often associated with sexual networks, especially among men who have sex with men, and was characterized by novel clinical presentations, including genital and perianal lesions. Genomic analyses revealed that clade II diverged from clade I approximately 3500 years ago and is uniquely defined by the deletion of virulence factors, such as the complement-binding protein. Importantly, the clade IIb lineage, which triggered the 2022 global outbreak, exhibits accelerated microevolution consistent with APOBEC3-mediated hypermutation. This host-driven mutational signature provides genomic evidence supporting the hypothesis that clade IIb circulated cryptically within human-to-human transmission chains in West Africa as early as 2014. Ecologically, while no definitive reservoir has yet been identified, evidence suggests diverse rodents and an expanding host range. The transformation of mpox from a rare zoonosis to a global threat underscores the severe consequences of delayed intervention, demanding robust, integrated “One Health” surveillance. Full article

Libraries for next-generation sequencing were prepared using primers directed to framework 2, framework 3 and D and the 6 J regions of the rearranged immunoglobulin gene from 50 diagnosis myeloma bone marrow samples. After sequencing and bioinformatic analysis, a marker sequence suitable for minimal residual disease analysis by HAT-PCR was obtained for 90% of the 50 samples studied. In 88% of samples a complete VDJ sequence was obtained. Failure to detect a marker sequence was principally due to a low percentage of plasma cells in the marrow sample. Evaluation of 24 pairs of primers showed that they were highly specific and, when used in HAT-PCR, they provided accurate quantification. The high frequency of detection of a suitable marker sequence indicates that minimal residual disease can be monitored by HAT-PCR in the great majority of instances. Full article

Anti-interferon-γ (IFN-γ) autoantibody (AAb)-associated adult-onset immunodeficiency (AOID) is an emerging disorder in East Asian adults characterized by severe opportunistic infections despite negative HIV status. The mechanisms driving anti-IFN-γ AAb production remain unclear. This study investigated B-cell gene expression, serum cytokine profiles, associations with AAb levels, and IFN-γ genetic variations in AOID patients. A cross-sectional study was conducted in 63 AOID patients and 30 healthy controls at Chiang Mai University Hospital, Thailand. Patients were classified as active or inactive according to infection status. B-cell gene expression, serum cytokines, anti-IFN-γ Aabs levels, and IFN-γ polymorphisms were analyzed by quantitative PCR, multiplex assays, ELISA, and nucleotide sequencing, respectively. AOID patients exhibited increased PAX5 and BLIMP1 expression and reduced XBP1, BCL6, KRAS, and BAFF expression. Active patients had higher levels of TNF-α, IL-6, IL-17A, IL-10, IL-21, and more chemokines than inactive patients. AAb levels positively correlated with TNF-α, IL-6, and IL-10. Correlation analysis showed positive associations among PAX5, XBP1, and KRAS, and a negative association between BLIMP1 and BCL6. No significant IFN-γ sequence differences were identified within the analyzed transcript region. These findings suggest altered B-cell gene expression and inflammatory cytokine profiles in AOID, which may be associated with the immunological alterations observed in these patients and may provide insights into potential mechanisms underlying anti-IFN-γ AAb production. Full article

Objective: The study was conducted to investigate the frequency of MED12 gene (rs5030619) mutations and to evaluate DNA methylation patterns in uterine leiomyomas among women at a tertiary care hospital in Karachi, Pakistan. Methods: In this cross-sectional study, 200 women with uterine fibroids and 50 controls were recruited from tertiary care hospitals after ethical approval. Baseline data and history were collected. Serum levels of estradiol, progesterone, and FSH were measured. Genomic DNA was extracted from fibroid and myometrial tissues and analyzed using allele-specific PCR for MED12 genotyping. DNA methylation profiling was conducted to evaluate epigenetic modifications. Statistical analyses were performed using the Statistical Package for social sciences (SPSS) version 27.0. Results: The majority of cases were aged 31–50 years, with 41.5% overweight and 24.5% obese. An increased WHR was observed in 65% of cases, compared to only 36% of controls. Serum estradiol, progesterone, and FSH levels were significantly higher in cases across all menstrual phases (p < 0.01). The MED12 A/C genotype was significantly associated with fibroid risk (OR = 11.72, 95% CI: 5.59–24.57, p < 0.001), and the C/C genotype conferred the highest risk (OR = 18.17, 95% CI: 4.00–82.54, p = 0.001). C allele was associated with increased disease susceptibility (OR = 4.65, 95% CI: 2.69–8.03, p < 0.001). Hyper-methylation was the most prevalent finding, observed in 125 cases of leiomyomas (69.4%). Multivariable logistic regression identified age (OR = 1.04, p = 0.012), BMI (OR = 1.09, p = 0.004), multiple fibroids (OR = 2.31, p = 0.005), intramural fibroid location (OR = 1.88, p = 0.041), and positive family history (OR = 2.74, p = 0.003) as significant independent predictors. Conclusions: Uterine leiomyomas may be influenced by a combination of hormonal, metabolic, genetic, and epigenetic factors. Overall findings suggest that MED12 C allele and A/C genotype variation was significantly associated with increased DNA methylation in uterine leiomyomas. Full article

Background/Aim: Fibroblast growth factor receptor 4 (FGFR4) is a tyrosine kinase involved in cell growth, proliferation, and angiogenesis. While FGFR1–3 are well studied in cancer, FGFR4 remains relatively understudied, and the distribution of its mutations across cancers and patient populations is not well defined. Materials and Methods: A retrospective pan-cancer analysis was performed using the AACR Project GENIE v12 database via cBioPortal. Tumors with somatic FGFR4 mutations were included, excluding copy number alterations and structural variants. Mutations were grouped by hotspot (amino acid 401) and major protein domains. Comparative analyses assessed cancer type distribution, demographics, mutation burden, and co-occurring genomic alterations using chi-square testing with multiple comparison correction. Results: A total of 4565 tumor samples (4283 patients) were analyzed. FGFR4 alterations were observed across diverse malignancies, most commonly non-small cell lung cancer, colorectal cancer, and melanoma. Mutations clustered primarily in the tyrosine kinase and immunoglobulin I-set domains, with no significant variation in distribution across cancer types. Sex was not associated with the mutation group, while race and ethnicity showed significant differences. The FGFR4 hotspot 401 group demonstrated a higher mutation burden, driven by a subset of hypermutated tumors, and showed enrichment for co-occurring alterations in chromatin remodeling, DNA repair, tumor suppressor, and receptor tyrosine kinase genes; however, sensitivity analyses indicated this association was largely attributable to mutation burden rather than a mutation-specific effect. Domain-based mutation groups had lower mutation burdens and fewer co-alterations. Conclusions: FGFR4 alterations occur across a broad range of cancers with consistent domain-level patterns. The hotspot 401 mutation shows a higher mutation burden and co-alteration frequency driven largely by a subset of hypermutated tumors, rather than acting as an isolated driver. Full article

Temozolomide (TMZ) remains the cornerstone of chemotherapeutic agent for glioblastoma (GBM), yet intrinsic and acquired resistance severely limits its clinical benefit. While O⁶-methylguanine-DNA methyltransferase (MGMT)–mediated repair of TMZ-induced O6-methylguanine (O⁶-meG) lesions has been extensively studied, the DNA mismatch repair (MMR) pathway is increasingly recognized as a key determinant of TMZ cytotoxicity. Canonical MMR, mediated by MutSα (MSH2–MSH6) and MutLα (MLH1–PMS2) complexes, recognizes O⁶-meG: thymine mispairs generated during replication and initiates futile repair cycles that culminate in replication stress, replication fork collapse, and apoptotic signaling; intact canonical MMR is, therefore, required for TMZ-induced cell death. Disruption of canonical MMR, frequently via acquired MSH6 mutations, confers TMZ tolerance and drives hypermutated recurrent GBM. Beyond mismatch correction, MMR proteins perform non-canonical functions in DNA damage signaling, replication stress responses, transcriptional regulation, chromatin dynamics, and immune modulation. These activities may shift the outcome from cytotoxic futile repair toward replication stress adaptation, Translesion synthesis (TLS)-mediated lesion tolerance, immune remodeling, and therapeutic resistance. Notably, partial attenuation or functional diversion of MMR may decouple lesion recognition from cytotoxic signaling, enabling TLS-mediated lesion tolerance without complete loss of MMR activity. This review integrates current insights into canonical and non-canonical MMR functions in GBM, defines their distinct contributions to TMZ sensitivity and resistance, and highlights therapeutic opportunities to exploit MMR-associated dependencies, including synthetic lethal strategies and immunotherapeutic vulnerabilities linked to MMR deficiency-driven hypermutation. Full article

Background: Endometrial carcinoma (EC) is one of the most common gynaecological malignancies, with a steadily increasing incidence worldwide. In 2013, a novel molecular classification of ECs enabled patient stratification into four groups with important clinical management implications: (1) DNA Polymerase Epsilon (POLE)-mutated, (2) microsatellite instability-high (MSI-H/dMMR) hypermutated, (3) tumours with low somatic copy number alteration (low-SCNA), and (4) tumours with high SCNA and p53 deficiency. This molecular classification should also be used as a tool to identify hereditary cancer syndromes, as MSI-ECs can be associated with Lynch Syndrome (LS, ORPHA:144) while somatic POLE mutations may reflect polymerase proofreading-associated polyposis (PPAP syndrome, ORPHA:447877). Methods: From 2022 to 2024, molecular classification of ECs was routinely performed on 188 consecutive cases. The results were correlated with family history and constitutional genetic testing. Results: Somatic testing revealed that 45 ECs were dMMR and 23 carried POLE variants. Five POLE-mutated ECs also displayed concomitant MSI. For all 68 patients, cancer genetic counselling was proposed, but only 41 accepted, and of these, 26 patients were eligible for the genetic test. None of the POLE somatic variants were proven to be constitutive, whereas LS was diagnosed in seven patients with dMMR-EC; interestingly, two of these patients displayed a POLE-mutated EC. Conclusions: Our data strongly suggest that molecular classification of ECs is important to improve the identification of LS and highlight the relevance of investigating all molecular markers at once in order to identify overlaps between POLE mutations and MMR defects. Full article

The rapid surge of Electric Two-Wheelers (E2Ws) in high-density urban villages imposes severe strain on low-voltage residential distribution networks. Unlike formal Electric Vehicles, E2W charging is decentralized and highly constrained by short pedestrian walking thresholds, frequently forcing users to adopt non-compliant “fly-wire charging” when public facilities are scarce. Traditional top-down load models fail to capture these localized, micro-behavioral single-phase grid impacts. To address this deficit, this study proposes a GIS-integrated “Digital Sandbox” simulation framework that projects individual behavioral mutations directly onto feeder networks via a hyper-granular “particle tracking” mechanism, treating each E2W as an autonomous agent. As a case study focused on a representative urban-village area, we validate the model using field data from the site. A 30-day simulation reveals that unmanaged fly-wire charging generates a peak load of 17.64 kW (nearly double the public station peak) and accounts for 38.9% of aggregate energy consumption—concentrated within the top 10 buildings and coinciding with evening peaks, inducing severe phase imbalance. While the numerical results are case-specific, the framework itself is transferable to other service areas through re-calibration against local data. This foundational digital twin blueprint shifts E2W planning from guesswork to particle-level risk prediction. Full article

Developing high-quality and reliable solver code for the job shop scheduling problem (JSSP) remains a challenging and expertise-intensive task because generated code must stay executable, produce feasible schedules, and achieve strong scheduling results. This paper proposes a large language model (LLM)-driven multi-agent evolution framework for scheduling solver code generation, where LLMs act as hyper-heuristics for program-space search under external evaluation. The framework forms a closed-loop process with three collaborating agents. A seed heuristic generation agent uses a structured constraint template and a shared solver skeleton to synthesize, screen, and diversify seed programs to construct a competitive initial code pool. An evolutionary operator agent updates the pool through program-space crossover and best-so-far mutation. A code reflection agent analyzes solver code and maintains trajectory-aware reflective memory to generate structured guidance for later revision. Experiments on standard JSSP benchmarks show that the framework outperforms representative metaheuristics across heterogeneous instance families and scales while reaching best-known reference quality on a subset of instances. Ablation results further confirm the contributions of the initialization design and the reflection-guided revision mechanism. More broadly, the proposed framework helps reduce manual heuristic design effort and offers a practical approach to production scheduling optimization in intelligent manufacturing environments. Full article

Classic Hodgkin lymphoma (cHL) is a distinctive B-cell malignancy defined by the presence of scarce but pathobiologically dominant Hodgkin Reed-Sternberg (HRS) cells within an inflammatory tumor microenvironment (TME). Although representing less than 10% of total tumor cellularity, HRS cells shape the TME by recruiting and functionally polarizing immune and stromal elements through cytokine- and chemokine-mediated signaling. Morphologically, HRS cells are large, atypical, often binucleated or multinucleated cells with prominent eosinophilic nucleoli and abundant cytoplasm, giving rise to the classic “owl’s eye” appearance. Distinct morphological variants—including lacunar, mummified, mononuclear, and anaplastic forms—contribute to the histopathologic diversity across cHL subtypes such as nodular sclerosis, mixed cellularity, lymphocyte-rich, and lymphocyte-depleted disease. The immunophenotype of HRS cells is equally characteristic, with strong and uniform CD30 expression, frequent CD15 positivity, reduced expression of B-cell markers (CD20, CD79A/B), and partial retention of PAX5, reflecting profound lineage dysregulation. Aberrant expression of activation markers and immune-evasion molecules, including PD-L1 driven by recurrent 9p24.1 amplification, underscores their capacity for immune escape. Genetically, HRS cells display alterations affecting NF-κB, JAK/STAT, and PI3K/AKT pathways, facilitated by somatic mutations, chromosomal gains, and epigenetic remodeling that silence B-cell-defining genes. Despite reprogramming, clonality and somatic hypermutation patterns confirm their origin from germinal center B-cells, even in EBV-associated cases. Collectively, the morphology, phenotype, and genotype of HRS cells reveal a complex pathogenic network in which intrinsic oncogenic pathways and extrinsic TME interactions co-operate to sustain malignant transformation. Understanding these integrated mechanisms provides a biological foundation for current therapeutic strategies. Full article

Immune dysregulation is being increasingly recognized as a prominent feature of a wide range inborn errors of immunity (IEIs) with different molecular backgrounds. Among the manifestations of immune dysregulation, inflammatory arthritis has emerged as an important yet underrecognized complication that may occur across multiple IEI categories, including humoral immunodeficiencies (such as X-linked agammaglobulinemia, hyper-IgM syndrome, common variable immunodeficiency, and others), complement deficiencies, disorders of immune dysregulation (STAT3 gain of function mutation, CTLA4 and LRBA haploinsufficiency), and combined immunodeficiencies. In some patients, arthritis may represent the first or predominant clinical manifestation, resulting in a diagnostic challenge in the rheumatologic setting. The pathogenesis of arthritis in IEIs reflects different immunological mechanisms, including the defective clearance of immune complexes, dysregulated B- and T-cell responses, impaired regulatory T-cell function, and aberrant cytokine signaling. Clinically, IEI-associated arthritis may mimic classical rheumatologic conditions such as juvenile idiopathic arthritis, rheumatoid arthritis, or other connective tissue diseases, although distinctive immunological and histopathological features are often present. Recognizing arthritis as a potential manifestation of IEIs has important clinical implications. The presence of specific “red flags”, including treatment refractoriness, recurrent infections, or additional signs of immune dysregulation (other autoimmune diseases, atopy, lymphoproliferation, enteropathy), should prompt targeted immunological evaluation. While management often relies on conventional immunosuppressive therapies, advances in the molecular characterization of IEIs are increasingly enabling the use of targeted treatments directed at the underlying pathogenic mechanisms. This paper provides an overview of the current knowledge of arthritis associated with IEIs, highlighting diagnostic challenges, underlying immunopathogenic mechanisms, and emerging therapeutic perspectives. Full article

Achromobacter spp. are opportunistic pathogens in people with cystic fibrosis (PwCF), yet the role of the upper airways in their persistence and adaptation remains poorly understood. We investigated whether the sinonasal compartment may act as reservoir and evolutionary niche for Achromobacter spp. during airway infection. Twenty-two isolates obtained from paired nasal lavage and sputum samples of seven PwCF were analysed by whole-genome sequencing. Within each PwCF, identical clone types were detected in both airway compartments, supporting bacterial exchange between upper and lower airways. Despite clonal relatedness, substantial genomic diversification was observed between paired isolates. Genomic signatures indicative of elevated mutation rates were detected in a high number of isolates (73%) and in both airway compartments, highlighting widespread genomic diversification across the respiratory tract. Mobilome analysis revealed compartment-specific variations in insertion sequences, prophages, and integrative elements, suggesting genome plasticity. Additionally, mutation in an aspartate kinase gene was consistently associated with loss of biofilm formation in vitro, highlighting a potential link between this pathway and biofilm phenotype. Overall, our findings indicate that upper and lower airways represent interconnected but partially independent ecological niches where Achromobacter populations can diverge during colonization, supporting the view that both compartments contribute to their persistence and evolution in CF airways. Full article

Chronic myeloid leukaemia (CML) is driven by the t(9;22) forming the BCR::ABL1 fusion gene, leading to the development of hyper-myeloid proliferation. This led to development of tyrosine kinase inhibitors (TKIs) such as Imatinib, Nilotinib, and Ponatinib. However, resistance or intolerance to ATP-competitive TKIs remains a challenge for some patients. asciminib (ABL001), a novel TKI, targets the myristoyl pocket of ABL1 instead of the ATP-binding site, reducing resistance to mutations. As asciminib is linked to thrombocytopenia, its effects on platelet activation, endothelial function, and inflammation must be studied to assess its potential to promote thrombosis. The main objective of this study is to determine the potential of asciminib as a monotherapy in inducing pathological responses to platelets and endothelium over time within the vasculature. This study assessed the effects of TKIs including asciminib on platelets and thrombotic biomarkers. Washed platelets were used to measure granule secretion, thrombus formation, surface expression of glycoproteins, apoptosis, and viability. Plasma from chronically Asciminib-treated CML patients was analysed using sandwich ELISA for inflammatory and platelet–endothelial biomarkers, and thrombin generation assays were performed to study coagulation. This approach combined in vitro and ex vivo methods to explore the impact of asciminib on platelet function and thrombotic potential. The study shows that acute treatment with asciminib does not promote platelet activation or thrombus formation. Instead, it exhibits an inhibitory effect on thrombus formation in vitro and is associated with reduced thrombo-inflammatory biomarkers ex vivo in chronically treated CML patients. Asciminib was associated with increased thrombin generation over time, suggesting an effect on secondary haemostasis. Asciminib does not appear to induce a prothrombotic or proinflammatory state under the conditions studied, which may be advantageous for CML patients. However, the observed increase in thrombin generation over time suggests a potential effect on secondary haemostasis that warrants further investigation in controlled studies. Full article