Search Results (417)

Marfan syndrome (MFS) is characterized by progressive aortic aneurysm formation resulting from mutations in the fibrillin-1 (Fbn1) gene. Although the thoracic aorta is the primary site of pathology, accumulating evidence indicates that vascular dysfunction in MFS extends beyond the aorta to involve multiple arterial beds. Statins have been shown to attenuate aneurysm progression in experimental models of MFS; however, their effects on systemic vascular remodeling and arterial stiffness outside the aorta remain poorly characterized. In this study, we investigated the impact of chronic atorvastatin therapy on vascular structure and hemodynamic function across multiple vascular beds in the Fbn1^C1041G/+^ mouse model of MFS. Male and female control and MFS mice received drinking water with or without atorvastatin (1 g/kg/day) from 4 weeks to 6 months of age, enabling the effects of atorvastatin to be assessed in both healthy and MFS arteries. High-frequency ultrasound imaging was used to assess vascular parameters in the aorta, left common carotid artery (LCCA), and posterior cerebral artery (PCA). Atorvastatin treatment significantly attenuated aortic root dilation in both male and female MFS mice and reduced aortic pulse wave velocity (PWV), indicating improved arterial compliance. In the carotid circulation, atorvastatin significantly reduced LCCA wall thickness and carotid PWV, although carotid wall strain did not improve. Atorvastatin raised both systolic and diastolic blood pressure in male and female MFS mice relative to untreated MFS animals, reaching levels not significantly different from untreated controls in both sexes, while having little effect in healthy controls apart from a rise in female diastolic pressure. In the posterior cerebral artery, peak systolic velocity, a hemodynamic index rather than a direct measure of perfusion, showed similarly sex-dependent changes, increasing in female MFS mice but decreasing further in males after atorvastatin. Collectively, these findings demonstrate that atorvastatin exerts systemic but heterogeneous vascular effects in MFS, improving arterial stiffness and structural remodeling across multiple arterial beds while producing sex-specific hemodynamic responses that warrant further investigation. Full article

Background: Gastric adenocarcinomas constitute a histologically and genomically heterogeneous group of cancers. The genomic classification of gastric cancers in four groups by The Cancer Genome Atlas (TCGA) has defined a framework for pathogenic discoveries. One of the groups is associated with infection by the gamma herpes virus Epstein–Barr virus (EBV) and represents a distinct subset of gastric cancers with potential therapeutic opportunities. Methods: The EBV-associated cancers from the TCGA gastric cancer cohort were analyzed to determine specific mutational and mRNA expression profiles that set these cancers apart from other gastric cancer subtypes. The cBioportal for Cancer Genomics site was used for downloading and analyzing the primary data. Results: EBV-associated cancers represented about 7% of the cohort. Mutations in the catalytic alpha subunit of PI3K kinase, PIK3CA, and the epigenetic modifiers ARID1A and BCOR were common. PIK3CA mutations were observed in 80% of EBV-associated cancers and frequently affected the hotspot codons E542 and E545. The few cases without PIK3CA mutations displayed frequent alterations in ERBB2 or in the regulatory unit of PI3K. EBV-associated cancers did not display excess cytidine to thymine (C>T) transitions compared with other gastric cancer genomic subtypes, as would be expected from the high genome methylation caused by the virus. In contrast, an increased rate of T to G (T>G) transversions was observed in EBV-associated cancers. Translesion polymerase eta (POLH), which produces a signature characterized by a preponderance of T>G, was up-regulated in EBV-associated gastric cancers and may be a contributing factor in this increase, up-regulated by wild-type p53 and over-expression of transcription factor IRF1. Conclusions: The data presented here suggest that mutagenesis in the EBV-associated gastric cancers is not a direct consequence of the virus-derived hypermethylation. Up-regulation of kinase PI3K and its pathway is a prerequisite for EBV transformation, and epigenetic alterations are frequently present, suggesting therapeutic avenues. Full article

Cholangiocarcinoma (CCA) is a highly malignant tumor originating from the epithelium of the bile ducts. It has an insidious onset, is difficult to diagnose in its early stages, has a low rate of curative resection, and carries an extremely poor prognosis. Among these, intrahepatic cholangiocarcinoma (iCCA), as the most representative subtype, is a classic “immunologically cold tumor.” The response rate to single-agent immunotherapy is only 5–10%, and the mechanisms of immune resistance are complex and not yet fully elucidated. The tumor microenvironment, serving as the core site of immune resistance, forms a highly immunosuppressive network composed of cancer-associated fibroblasts, hypoxia, metabolic reprogramming, and epigenetic abnormalities; a population of immunosuppressive cells centered on tumor-associated macrophages further amplifies tolerance signals; and the gut–biliary microbiome exerts systemic immune regulation via the gut–liver axis. Based on mutant mouse models generated via tail vein injection and in-depth studies of mutations in key signaling pathways, our understanding of the mechanisms underlying iCCA’s immune resistance is deepening at both the molecular and systems levels. This article reviews the local and systemic regulatory mechanisms of immune resistance in primary iCCA, summarizes the research value of experimental and preclinical models, and reviews novel strategies such as tumor microenvironment remodeling, activation of immune cell networks, microbiome interventions, and multidimensional combination therapies. It analyzes current research bottlenecks and clinical challenges and outlines the future direction of precision immunotherapy, aiming to provide a theoretical basis and new insights for overcoming iCCA immunotherapy resistance and advancing clinical translation. Full article

Background: Antiretroviral resistance-associated mutations, within the broader context of HIV-1 genetic variability, represent a growing challenge for HIV-1 control, highlighting the need for continuous molecular surveillance and mechanistic understanding of drug resistance. This study aimed to characterize mutations in the pol gene associated with resistance to protease inhibitors and to explore their structural implications. Methods: Viral RNA was extracted from plasma samples of HIV-positive patients, and a 266 bp fragment of the HIV-1 pol gene was amplified by RT-PCR and sequenced using the Sanger method. Sequences showing ≥98% homology were aligned and analyzed using MEGA v11 and the Stanford HIV Drug Resistance Database to identify resistance-associated mutations, while viral subtypes were determined using COMET, jpHMM-HIV, and STAR tools. Amino acid sequences were used for structural modeling with AlphaFold, followed by molecular docking with Nelfinavir using the CB-Dock2 server. Results: Four samples exhibited resistance-associated profiles, including high-level, intermediate, and low-level resistance, with one isolate showing high-level resistance to multiple protease inhibitors. Structural analyses revealed that Nelfinavir preferentially binds to alternative hydrophobic cavities rather than the canonical catalytic site, lacking direct interactions with the Asp25/Asp25′ dyad. Conclusions: These findings suggest a structural mechanism of resistance based on non-canonical ligand binding that may impair effective protease inhibition. Full article

Background/Aims: While pathogenic germline variants play a critical role in pediatric cancer susceptibility, traditional clinical genetics primarily focuses on single-gene interpretations. Transitioning to a systems-level analysis of inherited variation can uncover shared biological vulnerabilities, informing genetic counseling, surveillance, and targeted therapeutics. This study aims to implement an unsupervised machine learning framework to identify and characterize Germline Mutation Signatures (GMS) across diverse pediatric malignancies, elucidating latent genomic patterns that reveal shared oncogenic mechanisms. Methods: We analyzed germline whole-exome and whole-genome sequencing (WES/WGS) data from a retrospective cohort of 420 pediatric cancer patients and matched non-cancer controls. Variants were deeply annotated to capture multi-dimensional features, including predicted pathogenicity, splice-site disruption, regulatory impact, population frequency, and sequence context. To enable robust modeling, we integrated an augmented feature set encompassing evolutionary constraint, loss-of-function intolerance, and compositionally normalized substitution spectra. These high-dimensional annotations were processed using a deep autoencoder for non-linear representation learning, followed by Gaussian Mixture Modeling (GMM) of the latent space. Results: The framework delineated 13 signatures (GMS1–GMS13), yielding an optimal Davies–Bouldin index of 1.051. These signatures map to fundamental biological processes, including DNA repair deficiencies, transcription-coupled damage, replication stress, and aberrant RNA regulation. Crucially, these GMSs transcend traditional tissue-of-origin classifications, manifesting across multiple distinct cancer types. This observation indicates convergent germline etiologies and suggests potential shared susceptibilities to pathway-directed therapies. Conclusions: The discovery of these cross-cancer signatures provides a scalable, biologically interpretable framework for decoding inherited pediatric cancer risk. While the therapeutic mapping networks identified are currently exploratory and serve as a hypothesis-generating foundation, this deep learning-driven paradigm establishes a robust basis for stratified precision medicine. Pending prospective clinical validation, this approach holds significant translational potential to move beyond single-gene paradigms toward unified, systems-level precision oncology strategies. Full article

Background: Viral attachment mediated by host cell surface receptors is the first step in viral infection. As a key cell surface receptor, heparan sulfate (HS) mediates the attachment and entry of numerous non-enveloped viruses in livestock, thereby serving as a crucial molecular target for studying virus–host interactions. Methods: Based on the structural scaffold of a nanobody (Nb; PDB: 7TJC), we rationally designed and constructed a mutant Nb targeting HS, designated HS-Mut-Nb1, using molecular docking, site-directed mutagenesis, molecular dynamics (MD) simulations, and experimental characterization. Results: Molecular docking indicated that the active site of wild-type Nb for HS binding was located within the cavity jointly formed by the complementarity-determining region 3 (CDR3) and the framework regions (FRs) of the wild-type Nb. A comprehensive analysis integrating virtual alanine scanning, site-directed mutagenesis, and MD simulations revealed that the combination of three point mutations (Phe47Arg, Asp99Tyr, and Tyr108Pro) significantly enhanced the binding affinity of Mut-Nb1 for HS, with a calculated binding free energy (ΔG) of −83.26 ± 3.06 kcal/mol. Enzyme-linked immunosorbent assay (ELISA) results further confirmed that Mut-Nb1 exhibited high affinity for HS (K_D = 65.87 nM) and specificity (positive/negative ratio, P/N = 3.84; cross-reactivity, CR < 6.60%). Conclusions: This study not only provides novel candidate molecules for elucidating the mechanism of HS–virus interactions and developing related inhibitors but also offers a reference for the rapid construction of mutant Nbs. Full article

Background/Objectives: Mosquitoes, including Culex quinquefasciatus and Aedes aegypti, are important vectors of dengue fever, Zika virus, West Nile virus, Japanese encephalitis virus, Eastern equine encephalitis virus, etc. Biological control has always been urgent in mosquito prevention due to resistance developing to synthetic insecticides and environmental toxicity by insecticides. Methods: The leaf essential oil of Mosla. chinensis was isolated, and major components were identified via GC-MS, followed by olfactory behavior assays to evaluate its repellent activity against C. quinquefasciatus. Additionally, the odorant-binding protein 1 and odorant-binding protein 2 (CquiOBP1-2) genes were prokaryotically expressed, and their fluorescence competitive binding activities with the active components of essential oils were examined. Results: The bioassays indicated this essential oil greatly repels C. quinquefasciatus, which will significantly protect people against vector-borne diseases. In the fluorescence competitive binding experiments, the CquiOBP1-2 proteins exhibit great binding capacities to volatile components, including Citronellal, Citronellol, Geraniol, Limonene and Isopulegol. Furthermore, the behavioral experimental results also indicate that the mixture of these five ligand compounds has an obvious repellent effect on mosquitoes, highlighting that they may be applied as potential mosquito repellent agents. Moreover, molecular docking and site-directed mutation analysis further confirm Phe123 and Gln77 are both key amino acid residues of CquiOBP1-2 proteins involved in the olfactory recognition of repellent ligand compounds from M. chinensis essential oil. Conclusions: The behavioral experimental verification and the exploration of olfactory molecular mechanisms are helpful to promote the biological control of plant essential oils in mosquito pests. Full article

Fungal iterative non-reducing polyketide synthases (NR-PKS) contain a unique product template (PT) domain for aromatic cyclization. Among them, some NR-PKSs, such as the sorbicillin NR-PKS (SorB), have an apparently inactive PT. It is unknown what role such PT plays in NR-PKS programming. In this study, the PT domain of SorB was first dissected and engineered. Removal of the PT domain from SorB did not change the product profile, but the yield decreased. Meanwhile, a significantly lower transcriptional level of the ketoacyl synthase (KS) domain was observed in the knockout mutant (UvSorB∆PT). Phylogenetic tree analysis and multiple sequence alignments revealed this PT belongs to group I (C2–C7, monocyclic ring), and mutations were found at catalytic dyad sites when compared with functional fungal PTs. However, mutating these residues back to the conserved ones did not give rise to products corresponding to a functional PT, but rendered the NR-PKS unproductive. Likewise, removal of the C-methyltransferase (CMT) domain from SorB destroyed the polyketide production. Furthermore, in an attempt to alter the methylation pattern, mutations of the key substrate-binding sites of the CMT domain were made. Site-directed mutations of the C-MT led to cessation of the polyketide production. This reveals CMT is vulnerable to engineering in a collaborating NR-PKS (SorB). These results provide additional insights for catalytic reprogramming in fungal NR-PKS. Full article

Background: Doramectin (CHC-B1) is an excellent antiparasitic drug produced by feeding cyclohexanecarboxylic acid (CHC) to Streptomycesavermitilis bkd⁻ mutants. AveC, a bifunctional enzyme encoded by aveC (sav_0940), catalyzes the stereospecific spiroketalization and selective dehydration of dihydroxy ketone polyketide intermediates and modulates both the yield and the proportion of avermectin/doramectin in Streptomyces avermitilis. In our previous work, we constructed a strain harboring a synthetic aveC* gene encoding ten amino acid mutations, which produced nearly pure doramectin. However, the doramectin yield achieved only approximately 60% of the total doramectin and CHC-B2 output observed in the parental strain. Methods: To investigate the roles of Ser138 and Ala139 of AveC in the biosynthesis of doramectin and avermectin, site-directed mutagenesis was performed at both sites. The production and proportion of avermectin and doramectin were determined using high-performance liquid chromatography (HPLC). AlphaFold2-based molecular docking simulations were used to interpret the results. Results: Among the tested mutants, S138G, S138T, and A139H exhibited the highest doramectin production, achieving 143.87%, 151.22%, and 153.36% of the control level, respectively. Unfortunately, almost none of the tested mutants showed a positive effect on avermectin production. Molecular docking simulations revealed distinct affinities of these mutants for the dihydroxy ketone polyketide intermediate, both with and without a cyclohexyl group. Notably, all three mutants displayed larger substrate-binding cavity volumes compared with the wild-type enzyme, which likely facilitates doramectin synthesis by effectively accommodating the cyclohexyl moiety. Docking results further indicated that Ser138 and Ala139 are positioned within the binding cavity but probably do not directly participate in the dehydration activity. Conclusions: These findings suggest that optimizing cavity size through residue substitutions can enhance substrate specificity for doramectin production while preserving catalytic functionality. Full article

Andes virus (ANDV), a highly pathogenic orthohantavirus, enters host cells through low pH–triggered membrane fusion mediated by the Gc glycoprotein, a class II fusion protein containing a single C-terminal transmembrane domain (TMD). While the ectodomain has been extensively characterized, the role of the TMD in late-stage fusion remains unclear. Here, we investigated the minimal functional length and sequence requirements of the ANDV Gc TMD using site-directed mutagenesis. C-terminal deletion mutants and serine-to-alanine substitutions were evaluated for protein expression, virus-like particle production, cell–cell fusion, pseudotyped vector entry, and hemifusion activity. Deletion of the Gc cytoplasmic tail (CT) or a single C-terminal TMD residue was tolerated, whereas deletion of two or more residues impaired particle production and fusion, indicating that at least 21 of the 22 TMD residues are required for efficient membrane fusion and viral entry. Hemifusion assays showed that deletion of two or three residues, or substitution of the strictly conserved S1121, allowed lipid mixing but blocked progression to full fusion, while deletion of four residues also abolished hemifusion. In contrast, mutation of the less conserved S1126 had minimal effect. These results identify a precise TMD length and a conserved polar TMD residue as critical determinants of fusion pore formation in ANDV. Full article

Enterovirus D68 (EV-D68) is a non-polio enterovirus that can cause a polio-like paralysis condition, acute flaccid myelitis (AFM). EV-D68-associated AFM cases waned in the US after 2018, and the reasons for this are unknown. It has recently been demonstrated that EV-D68 containing point mutations in viral structural proteins VP1 and VP3 resulted in decreased paralysis in different neonatal mouse models. However, phenotypes of these mutations in a human multicellular central nervous system (CNS) model are unknown. We hypothesize that mutations in VP1 and VP3 will similarly direct neurotropism in human spinal cord organoids (hSCOs). To investigate this, we recreated viruses with mutations in VP3 (I88V) or VP1 (L1I/N2D/T98A/E283K or L1P/V148A/K282R) and infected hSCOs. We found that VP3 I88V and VP1 L1I/N2D/T98A/E283K resulted in decreased titer and viral protein staining, consistent with attenuated neurovirulence in previously published murine models. We also found through immunofluorescence that VP1 L1P/V148/K282R mutations altered cellular tropism, primarily infecting glial cells rather than neuronal cells. When these mutations were combined, their effects on neurotropism were not additive. Sequence analysis of recently circulating EV-D68 strains reveals that VP3 I88 and VP1 E283 have remained the dominant amino acid residues since 2014, whereas VP1 sites 1, 2, and 98 have higher population diversity, indicating that these residues may be contributing to newly reduced neurovirulence after 2018. Full article

Background: The molecular concordance between primary lung adenocarcinoma and metastatic lesions remains incompletely characterized despite its direct implications for precision oncology and biopsy-driven therapeutic decision-making. This prospective monocentric paired-sample study evaluated genomic concordance between primary lung adenocarcinoma and synchronous thoracic metastatic lesions using targeted next-generation sequencing (NGS). Methods: We identified 27 treatment-naïve patients with histologically confirmed lung adenocarcinoma who underwent paired molecular profiling of the primary tumor and a synchronous thoracic metastatic site (pleural or intrapulmonary). DNA and RNA were analyzed using validated institutional NGS platforms. Genomic alterations, including clinically actionable oncogenic drivers consistently covered by the sequencing panel used in each pair, were compared across matched samples. Concordance was assessed using exact binomial confidence intervals, Cohen’s κ statistics, McNemar tests, and paired Wilcoxon signed-rank tests. Results: Actionable driver alterations were identified in 17 of 27 patients (63.0%; 95% CI 42.4–80.6), including EGFR mutations (40.7%), KRAS alterations (18.5%), and one ALK gene rearrangement (3.7%). TP53 concurrent mutations were detected in 14 cases (51.9%). Across all 27 paired samples, driver-level concordance was 100% (95% CI 87.2–100), with perfect agreement for EGFR, KRAS, and ALK alterations (κ = 1.00). TP53 mutations showed high concordance (92.9%; κ = 0.85), while CNVs were concordant in 88.0% of evaluable pairs. Variant allele frequency (VAF) comparisons, adjusted for tumor cellularity, further supported the apparent clonal stability of driver alterations across paired samples. Conclusions: This study demonstrates very high molecular concordance between primary lung adenocarcinomas and their synchronous pleural or intrapulmonary metastases. The observed 100% concordance of actionable driver alterations across paired specimens supports the clinical reliability of thoracic metastatic biopsies for baseline molecular profiling in treatment-naïve disease. Although limited by sample size, these findings support the biological stability of actionable driver alterations during early thoracic metastatic dissemination. Full article

(1) Background: Integrated bioarchaeological approaches combining osteological and ancient DNA analyses provide powerful insights into health, disease, and population history in past societies. However, the relationship between rare skeletal variations, genetic disorders, and ancestry remains insufficiently explored within single individuals. This study aimed to investigate the combined osteological, paleopathological, and genetic characteristics of a Roman-period individual from southwestern Anatolia. (2) Methods: A multidisciplinary approach was applied to the skeletal remains of an adult male recovered from the Sekköy excavation site. Osteological analysis was conducted to assess cranial morphology, pathological lesions, and dental status. Ancient DNA was extracted from petrous bone under strict contamination control. The hemoglobin beta (HBB) gene was analyzed using Next Generation Sequencing and validated by Sanger sequencing. Y-chromosomal STR analysis was performed to determine paternal lineage. (3) Results: Osteological examination revealed a rare craniovertebral anomaly consistent with a third occipital condyle, along with porotic hyperostosis and extensive antemortem dental pathology, indicating prolonged physiological stress. Genetic analysis identified a heterozygous hemoglobin S mutation (HbAS; rs334), confirmed by both next-generation sequencing and Sanger sequencing, providing direct molecular evidence of hereditary hemoglobinopathy. Y-STR profiling assigned the individual to haplogroup R1b (predicted based on Y-STR data), indicating affiliation with Western Eurasian paternal lineages. (4) Conclusions: Despite the presence of comparable skeletal stress indicators, the integration of osteological and genetic data revealed a complex interaction between anatomical variation, chronic physiological stress, and inherited disease. The co-occurrence of a rare cranial anomaly, HbS mutation, and a defined paternal lineage represents a unique bioarchaeological case. These findings highlight the value of integrating skeletal and molecular approaches to reconstruct individual health profiles in archaeological contexts and demonstrate the methodological potential of interdisciplinary bioarcheological analysis. Full article

The orphan insulin receptor-related receptor (IRR), in contrast to its homologs from the insulin receptor family, is activated by a mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by two synergistic sites located in the dimeric extracellular domain. Here, we describe artificial mutations in the IRR transmembrane domain that promote receptor activation. First, using molecular modeling based on the NMR-derived structure, we proposed amino acid substitutions that could enhance non-covalent interactions between the transmembrane segments of the IRR dimer. These mutations were subsequently tested for effects on pH sensing by IRR. We showed that double-mutant A938E-A939R was highly phosphorylated at neutral pH and still sensitive to alkaline pH. Remarkably, the double substitution of V929E-G930R resulted in strong basal phosphorylation of the receptor over the pH titration range. Through site-directed mutagenesis, we demonstrated that the transmembrane domain plays a critical role in IRR activation, allowing for targeted control of functioning of the receptor, including its pH sensitivity. Full article

Endophytic fungi are promising sources of novel antiviral compounds, and the crude extract from Alternaria alternata PO4PR2 has previously shown anti-HIV-1 activity. This study evaluated its efficacy against integrase strand-transfer inhibitor (INSTI)-resistant HIV-1 and its mechanism of action. Key resistance mutations (Y143H, G118R, N155H, and R263K) were introduced into the HIV-1 pNL4.3 clone via site-directed mutagenesis and confirmed through Sanger sequencing. Viral infectivity was assessed in TZM-bl cells, while cytotoxicity was measured using an MTT assay. Antiviral activity was determined through a luciferase-based assay, and integration inhibition was evaluated using integrase activity assays and Alu-gag nested PCR. The extract demonstrated potent inhibition of resistant mutants, with low IC₅₀ values (0.02971–0.1652 μg/mL), and showed minimal cytotoxicity (CC₅₀ = 300 μg/mL), maintaining over 80% cell viability. It inhibited integrase activity by 67%, specifically targeting the strand-transfer step, and significantly reduced integrated viral DNA. Molecular docking of 14 compounds identified coumarin derivatives as key bioactive metabolites, exhibiting mutation-tolerant binding within the integrase catalytic pocket. Overall, these findings highlight PO4PR2 as a promising source of compounds for developing new therapies targeting drug-resistant HIV-1 integrase. Full article