Search Results (4,676)

RNA virus populations exist as quasispecies-complex, dynamic clouds of closely related but genetically diverse variants generated by high mutation rates during replication. Assessing quasispecies structure and diversity is crucial for understanding viral evolution, adaptation, and response to antiviral treatments. However, comparing single quasispecies observations from individual biosamples, especially at different infection or treatment time points, presents statistical challenges. Traditional inferential tests are inapplicable due to the lack of replicate observations, and resampling-based approaches such as the bootstrap and jackknife are limited by biases and non-independence, particularly for diversity indices sensitive to rare haplotypes. In this study, we address these limitations by applying the delta method to derive analytical variances for a set of quasispecies structure indicators specifically designed to assess the quasispecies maturation state. We demonstrate the utility of this approach using high-depth next-generation sequencing data from hepatitis C virus (HCV) quasispecies evolving in vitro under various conditions, including free evolution and exposure to antiviral or mutagenic treatments. Our results reveal that with highly fit HCV quasispecies, sofosbuvir inhibits quasispecies genetic diversity, while mutagenic treatments accelerate maturation, compared to untreated controls. We emphasize the interpretation of results through absolute differences, log-fold changes, and standardized effect sizes, moving beyond mere statistical significance. This framework enables robust, quantitative comparisons of quasispecies diversity from single observations, providing valuable insights into viral adaptation and treatment response. The R code and session info with required libraries and versions is provided in the supplementary material. Full article

Background/Objectives: RAS mutations are among the most common genetic alterations in thyroid cancer and are generally associated with less aggressive behavior. However, when co-occurring with TERT (telomerase reverse transcriptase) promoter mutations, known markers of poor prognosis, tumors exhibit markedly more aggressive features. The allele frequency (AF) of RAS may serve as a potential indicator of clonal dominance and the likelihood of additional high-risk mutations, such as TERT mutation. This study aims to assess whether a high RAS AF correlates with the presence of coexisting TERT promoter mutations and other molecular alterations. Methods: A retrospective chart review was performed on 111 patients with thyroid nodules harboring RAS mutations, either alone or in combination with TERT promoter mutations. All patients underwent molecular testing with ThyroSeq v3 and subsequent thyroidectomy at McGill University teaching hospitals. RAS AF was analyzed in relation to TERT mutation status, nodule size, and other molecular alterations including copy number alterations (CNA) and gene expression profiles (GEP). Results: The mean RAS AF was significantly higher in nodules with both RAS and TERT mutations (38.1%) compared to those with RAS mutations alone (22.1%) (p = 0.002). Nodules with coexisting TERT mutations were also significantly larger (mean size: 3.7 cm vs. 2.4 cm; p = 0.005). Malignant nodules, regardless of TERT status, showed a trend toward higher RAS AF than benign nodules (23.0% vs. 16.3%; p = 0.052). Higher RAS AF was also associated with the presence of CNA and/or GEP positivity. Notably, GEP was positive in 100% of nodules with both RAS and TERT mutations, compared to 37.5% in RAS-only nodules (p = 0.002). Conclusions: A high RAS AF increases the likelihood of a TERT promoter mutation and other genetic alterations, highlighting the importance of RAS AF in optimizing patient care and management. Full article

Retinoblastoma is a rare but malignant pediatric retinal tumor, affecting 1 in 15,000–20,000 live births annually. It arises from biallelic mutations in the RB1 tumor suppressor gene (chromosome 13q14.2), leading to uncontrolled cell cycle progression. Clinically, it presents as unilateral (60%) or bilateral (40%) disease, with leukocoria and strabismus as hallmark signs. Untreated, retinoblastoma is fatal due to metastatic spread. The disease follows Knudson’s two-hit model: heritable forms (30–40% of cases) involve a germline RB1 mutation (M1) and a somatic second hit (M2), predisposing to bilateral/multifocal tumors and secondary cancers. Non-heritable cases (60–70%) result from somatic RB1 mutations or, rarely, MYCN amplification (2%). Genetic testing is critical to classify risk (H0, H1, and HX categories), guide surveillance, and inform family counseling. Bilateral cases almost always harbor germline mutations, while 15% of unilateral cases may carry germline/mosaic RB1 defects. Advanced techniques (Sanger/NGS sequencing for mutation detection, NGS for copy number alterations, and methylation assays) detect RB1 mutations, CNVs, and epigenetic silencing. Tumor DNA analysis resolves ambiguous cases. H1 patients require intensive ocular and brain MRI surveillance, while H0 cases need no follow-up. Prenatal/preimplantation genetic diagnosis (PGD) can prevent transmission in high-risk families. Emerging research explores additional genes (BCOR, CREBBP) and MYCN-amplified subtypes. Genetic counseling addresses recurrence risks, reproductive options, and long-term cancer monitoring. Integrating genetic insights into clinical practice enhances precision medicine, reducing morbidity and healthcare costs. Future directions include whole-genome sequencing and functional studies to refine therapeutic strategies. Full article

The overuse of carbapenems has driven the rise of carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Enterobacterales (CRE), against which ceftazidime–avibactam (CAZ-AVI) offers an alternative treatment. This study phenotypically determined resistance profiles of P. aeruginosa (PA), Escherichia coli (EC), and Klebsiella pneumoniae (KP) clinical isolates to CAZ-AVI and investigated molecular resistance mechanisms genotypically. A total of 394 PA, 90 EC, and 84 KP isolates were collected from the American University of Beirut Medical Center. Antimicrobial susceptibility testing (AST) and whole-genome sequencing (WGS) were performed on 30 isolates per species. Results showed that 100% of KP, 63% of EC, and 100% of PA isolates were carbapenem-resistant. Among these, 73% of KP, 79% of EC, and 60% of PA were CAZ-AVI-resistant. WGS revealed diverse sequence types, plasmids, and antimicrobial resistance genes. Additionally, 100% of KP, 93% of EC, and 89% of PA isolates produced metallo-β-lactamases (MBLs). Mutations in ampD, ampR, and mexR were identified in CAZ-AVI-resistant, non-MBL-producing PA, whereas mutations in ompC, marR, and ampC were detected in CAZ-AVI-resistant, non-MBL-producing EC. While CAZ-AVI remains effective against most CRE and CRPA lacking MBLs, resistance to CAZ-AVI is multifactorial, commonly involving overexpression of efflux pumps and AmpC β-lactamases, loss of porin channels, and the presence of oxacillinases. Full article

Purpose: Evaluation of predictors and outcomes in NSCLC patients treated with an immune checkpoint inhibitor (ICI) following a severe immune-related adverse event (irAE). Methods: We included all NSCLC patients receiving ≥1 ICI cycle and corticosteroids for CTCAE Grade ≥3 irAEs between 2017 and 2023 from three UK NHS teaching hospitals. Progression-free survival (PFS) and overall survival (OS) after the 1st irAE, best overall response (BOR) to ICI, and predictors of clinical benefit were evaluated. Kaplan–Meier, Cox and logistic regression models, and Wilcoxon tests were used. Results: We screened 1658 NSCLC patients and identified 80 eligible subjects. The majority of patients had metastatic (n = 50, 63%) vs. localized (n = 30, 37%) NSCLC. Most patients developed a single ≥Grade 3 irAE on 1st line ICI (n = 71, 89%). Overall, 14 (18%) patients developed 2nd irAEs, 7 after rechallenge with ICIs. In the complete cohort, median OS after 1st irAE was 15.84 months (95% CI, 12.45–26.91). Lower neutrophil-to-lymphocyte ratio (NLR), patients receiving >4 cycles of ICI or median duration of ICI of >2.76 months before 1st irAE were associated with improved OS (p < 0.05), the latter two with PFS (p < 0.05). Age, gender, stage, KRAS mutation, PD-L1 and ICI type were not associated with PFS or OS. Pneumonitis as 1st irAE had the worst PFS and OS (p < 0.05). Median starting corticosteroid dose of ≤60 mg for 1st irAE had an improved OS (p = 0.04). Post 1st irAE response associated with better PFS and OS (p < 0.05). Number and duration of irAEs and additional immunosuppressive agents (14% of patients) were not associated with PFS or OS. Conclusions: In ≥Grade 3 irAEs patients managed with corticosteroids, lower baseline NLR, longer ICI use, response to ICI after 1st irAE, and a ≤60 mg corticosteroid dose had promising outcomes. Full article

Activation of TP53 signaling during ribosome biogenesis is an essential part of erythroid development, whereas the pathologic activation of TP53 in ribosomopathies such as Diamond-Blackfan anemia (DBA) and del (5q) myelodysplastic syndrome (MDS) prevents the normal expansion of erythroid precursors. TP53 can also be linked to the pathogenesis of DBA and MDS via ferroptosis, a form of iron-mediated cell death propagated by excess polyunsaturated fatty acid-containing oxidizable phospholipids and loss of lipid peroxide repair capacity. The primary objective of this work was to establish how overexpression and mutation of the TP53 gene influences lipid composition, erythroid differentiation, and ferroptosis sensitivity in K-562 cells, an in vitro model for studying erythropoiesis. Employing a reverse genetics approach, we generated four isogenic cell lines that either lacked functional TP53 expression, expressed wild-type (WT) TP53, or expressed one of the two most common TP53 mutation types, R175H or R282W. We then utilized non-targeted lipidomics to quantify and identify changes in specific lipid species that occur with induction of WT and mutant TP53 expression. We also analyzed differences in gene expression, ferroptosis sensitivity, and hemoglobinization by qPCR, CCK-8 cytotoxicity assay, and o-dianisidine staining, respectively. The abundance of 337 distinct lipid species was impacted by induction of WT TP53 expression compared to K-562 cells expressing a nonfunctional P53 protein. Yet only 17 lipid compounds were differentially impacted between cells expressing WT TP53 and either of the mutant TP53 genes tested. Similarly, while the TP53 null K-562 cells displayed modest sensitivity to ferroptosis, cells expressing both WT and mutant TP53 genes were remarkably resistant to ferroptosis. However, terminal differentiation and hemoglobinization were significantly impacted in R175H mutant TP53-expressing K-562 cells. Findings from this work provide novel insights into the role of TP53 in lipid metabolism and terminal erythropoiesis. Full article

Background: Tuberculosis (TB) remains a primary global health concern, despite the widespread availability of effective chemotherapeutic interventions. The emergence and dissemination of drug-resistant strains of Mycobacterium tuberculosis, particularly those exhibiting resistance to rifampicin, present significant obstacles to the success of TB control programs. Consequently, there is an urgent need for rapid, sensitive, and specific molecular diagnostic tools to inform timely clinical decision-making and reduce the transmission of disease. Loop-mediated isothermal amplification (LAMP) has gained attention as a promising alternative to conventional polymerase chain reaction (PCR) techniques. This method, which facilitates DNA amplification under constant temperature conditions, offers advantages including high specificity, rapid turnaround time, and operational simplicity—features that render it especially suitable for implementation in resource-limited settings. Methods: In this study, a LAMP assay targeting the rpoB gene was developed, with particular focus on detecting the codon 531 C→T mutation associated with rifampicin resistance. A set of four to six primers was designed to recognize six distinct regions of the target sequence. Allele-specific amplification was achieved by incorporating a deliberate single nucleotide mismatch at the 3′ terminus of the B2 primer to enable precise discrimination between wild-type and mutant alleles. The assay was conducted at an optimized temperature of 61 °C for 60 min, followed by visual detection using a lateral flow dipstick (LFD) within five minutes. Results: The LAMP-LFD assay demonstrated 100% concordance with drug susceptibility testing (DST) and DNA sequencing. No cross-reactivity with wild-type strains was observed, underscoring the assay’s high specificity. Conclusions: This platform offers a robust, field-deployable solution for detecting the codon 531 C→T mutation associated with rifampicin resistance in low-resource settings. Full article

Background/Objectives: The Germ Cell Theory of cancer posits that human primordial germ cells (hPGCs) are the cells of origin for malignancies. While this theory is well established for germ cell cancers, a germ cell origin for somatic cancers has been largely overlooked despite clinical observations of malignant somatic transformation (MST), wherein germ cell cancers give rise to diverse somatic cancer phenotypes, often without additional mutations. Methods: To test the Germ Cell Theory experimentally in somatic cancer, we established a virus-driven MST model linking hPGC-like cells (hPGCLCs) to Merkel cell polyomavirus (MCPyV)-positive Merkel cell carcinoma (MCC), a highly aggressive somatic cancer with a germ cell cancer-like, low-mutation epigenetic profile. The MCPyV genome was transduced into human induced pluripotent stem cells (hiPSCs) or hPGC-like cells by lentiviral transfection, followed by xenotransplantation. Results: Virus-positive MCC (VP-MCC)-like tumors were consistently induced without additional oncogenic mutations. These tumors recapitulated VP-MCC’s high-grade neuroendocrine carcinoma histology and molecular profiles. DNA methylation analysis revealed near-complete global hypomethylation in VP-MCC-like tumors, matching the unique epigenetic state of late-stage hPGCs. Notably, pluripotent intermediates were neither necessary nor sufficient for MST; transformation required acquisition of a late-hPGC-like epigenetic state. Conclusions: This is the first MST model of a somatic cancer arising through an aberrant germline-to-soma transition. Our findings unify VP-MCC and germ cell cancer biology, challenge mutation- and soma-centric paradigms, and provide a tractable platform to investigate developmental and epigenetic mechanisms of oncogenesis. This MST model supports a unifying germ cell origin for both germ cell and non-germ cell somatic malignancies. Full article

Regulatory T cells (Tregs) have anti-inflammatory immunomodulatory activity and hold therapeutic potential for chronic neuroinflammatory neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). We are developing engineered human Tregs with enhanced disease-modifying activity for treating ALS. A combination of a disease-specific chimeric antigen receptor (CAR) recognizing misfolded human superoxide dismutase-1 (hSOD1) and constitutive expression of brain-derived neurotrophic factor (BDNF) was tested. The scFv region of CAR demonstrated binding to anterior horn tissues of ALS patients with and without familial ALS mutations in SOD1. Tregs transduced to express BDNF showed the ability to secrete BDNF and protect co-cultured neuronal cells from peroxidase toxicity. Co-expression of BDNF did not inhibit CAR Treg expansion, Treg markers, or CAR-mediated anti-inflammatory cytokine production. Human Tregs co-expressing CAR and BDNF were tested for activity in G93A hSOD1-NSG transgenic mice, which develop an early-onset and aggressive ALS-like disease and do not reject human cells. Human Tregs expressing CAR and BDNF delayed the onset of disease development, extended survival, and decreased spinal cord neuroinflammation. The engineered Tregs showed enhanced disease-modifying activity and hold promise as a therapy for ALS. Full article

Klebsiella pneumoniae, due to its capacity to produce numerous virulence factors and form biofilms, is one of the most significant etiological agents of nosocomial infections. The extensive and often unwarranted use of antibiotic therapy has driven the emergence of various mutations, adaptive mechanisms, and horizontal gene transfer among K. pneumoniae strains, resulting in resistance to most beta-lactam antibiotics, carbapenems, and the last-resort drug—colistin. A promising alternative or adjunctive treatment is the application of rotating magnetic fields (RMFs). The present study aimed to evaluate changes in colistin susceptibility among 20 extended-spectrum beta-lactamases (ESBLs) and 20 K. pneumoniae carbapenemase (KPC)-positive K. pneumoniae strains isolated from hospital infections following exposure to RMF at frequencies of 5 and 50 Hz. Exposure to RMF at 5 Hz resulted in decreased colistin minimum inhibitory concentration (MIC) values in over half of the tested (ESBLs) and (KPC)-positive strains. Additionally, RMF at 50 Hz reduced colistin MIC values in 30% of (ESBL)-positive and 40% of (KPC)-positive strains. Therefore, in the future, RMF may be developed as a supportive therapeutic strategy to improve the efficacy of antibiotics in the treatment of infections caused by multidrug-resistant (MDR) pathogens, including colistin-resistant K. pneumoniae. Full article

Introduction: Elevated succinylacetone (SA) is the hallmark of tyrosinemia type 1, which requires immediate treatment. Mild SA elevation has also been recently reported in maleylacetoacetate isomerase deficiency (MAAID). Methods: We report on two cases of MAAID, review clinical features of MAAID and discuss its management. Results: Both cases displayed elevated SA and normal Tyrosine levels at newborn screening. Case 1 showed intermittent SA elevation; Nitisinone and dietary treatment were started, then discontinued after the identification of two variants in the GSTZ1 gene and the definitive diagnosis of MAAID. Case 2, showing no SA elevation at the confirmatory tests and two variants in the GSTZ1 gene, did not start treatment. mRNA analysis confirmed the pathogenicity of the c.68-12 G>A variant, found in both patients. Discussion: MAAID should be considered in newborns showing elevated SA and no variants in the FAH gene. Our study reports for the first time the course of SA in a patient affected by MAAID. Furthermore, it expands the molecular epidemiology of this rare disease, also investigating the pathogenicity of a novel splicing mutation. Although our data argue against medical treatment in MAAID, longer follow-up data are warranted. Full article

Focusing on joint-space time-optimal trajectory planning for industrial robots, this study integrates 3-5-3 piecewise polynomial parameterization with an improved Fire Hawk Optimization algorithm (TFHO). Subject to joint position, velocity, and acceleration limits, segment durations are optimized as decision variables. TFHO employs Tent-chaotic initialization to improve the uniformity of initial solutions and a two-phase adaptive Lévy–Gaussian–Cauchy hybrid mutation to balance early global exploration with late local exploitation, mitigating premature convergence and enhancing stability. On benchmark functions, TFHO attains the lowest mean area under the convergence curve (AUC; lower is better). Wilcoxon signed-rank tests show statistically significant improvements over FHO, PSO, GWO, and WOA $(p\le 0.05)$. Ablation studies indicate a pronounced reduction in run-to-run variability: the standard deviation decreases from 0.3157 (FHO) to 0.0023 with TFHO, a 99.27% drop. In an ABB IRB-2600 simulation case, the execution time is shortened from 12.00 s to 9.88 s (−17.66%) while preserving smooth and continuous kinematic profiles (position, velocity, and acceleration), demonstrating practical engineering applicability. Full article

Human mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase, mHS) synthase is a key enzyme in ketogenesis and is located mainly in the liver, but also in the colon, skeletal muscle, heart, pancreas, and testes. It is an inner mitochondrial membrane-associated protein. Mutations in the HMGCS2 gene, which encodes this enzyme, lead to “mHS deficiency,” a rare, autosomal recessive, inherited metabolic disorder. To date, about 100 patients with this disorder have been described. The disorder usually appears during the first year of life, often after a period of starvation or an intercurrent illness. A total of 77 different DNA mutations has been described that are considered responsible for mHS deficiency, although the mechanisms leading to loss of function are not fully understood. To study how the different missense variants affect the enzymatic activity of the protein on an atomic scale, we used molecular dynamics computational simulation techniques for variants whose activity could be measured “in vitro.” The study included a total of 46 molecular dynamics trajectories of enzyme–substrate/product interaction simulations, each 500 ns long (23 microseconds total). Currently, the atomic and biophysical effects of the mHS variants on their catalyzed reactions have not been studied in detail experimentally. To our knowledge, molecular dynamics simulations are one of the most promising tools for understanding the molecular basis of the phenotypic consequences of these variants. In the present work, molecular dynamics simulations reliably reproduce most experimental enzyme activity measurements, supporting their future application to the study of new mHS mutations. Full article

Background: Genetic factors pertinent to Parkinson’s disease (PD) might predispose an individual to post-traumatic stress disorder (PTSD). Humans who are heterozygous for the glucocerebrosidase 1 (GBA) L444P Gaucher mutation have an increased PD risk and elevated levels of alpha synuclein (aSyn). Mice that are heterozygous for the GBA mutation and express aSyn with the A53T mutation show elevated anxiety levels at 20 months of age compared to those expressing only A53T. Objective: This study aims to assess whether A53T and A53T-L444P affect the risk of developing PTSD phenotypes and whether sex and age modulate this risk. Methods: Young (5.1 ± 0.2 months) and older (11.3 ± 0.2 months) A53T and GBA L444P female and male mice were tested for fear learning and memory extinction in the contextual fear conditioning and passive avoidance paradigms. Subsequently, the mice were tested for measures of activity and anxiety in the open field and for depressive-like behavior in the forced swim test. Results: In the contextual fear memory extinction paradigm, only young A53T female mice showed contextual fear memory extinction, while older A53T female mice showed increased activity levels over subsequent days. In the passive avoidance memory paradigm, no mice showed extinction of passive avoidance memory. When the frequency of entering the more anxiety-provoking center of the open field was analyzed, a test history x sex x age interaction was observed. In the forced swim test, test history affected the depressive-like behavior in mice trained; there was more depressive-like behavior in mice trained in the contextual fear memory extinction paradigm than in mice trained in the passive avoidance memory extinction paradigm. Moreover, there was an effect of age with more depressive-like behavior in older than in younger mice, and an effect of genotype with more depressive-like behavior in A53T-L444P compared to A53T mice. When cortical phosphorylated tau (pS 199) levels were analyzed, there was an effects of genotype, a sex x age interaction, and ant age x test history interaction. Conclusions: A53T and A53T-L444P affect the risk of developing PTSD phenotypes. Fear extinction test history, genotype, and age affect depressive-like behavior and tau pathology. Full article

Isocitrate dehydrogenase (IDH) catalyzes the conversion of NAD(P)⁺ and isocitrate to NAD(P)H and α-ketoglutarate (αKG). The cytoplasmic enzyme IDH1 is important for producing NADPH for biosynthesis and for protecting against oxidative stress. IDH1 mutants, such as R132H found in glioblastomas and other types of human cancers, have a neomorphic activity that uses NADPH to reduce αKG to 2-hydroxyglutarate (2HG). 2HG interferes with the activity of important enzymes such as histone demethylases and TET demethylases. We hypothesized that Caenorhabditis elegans could be a good model system for studying oncogenic properties of mutant IDH1. To test this, we purified C. elegans cytoplasmic IDH-1 and two mutants, G98N and R133H, which correspond to human IDH1 mutants G97N and R132H, respectively. We found that the wild-type IDH-1 had similar kinetic properties to human IDH1, and it could produce small amounts of 2HG. We also found that the R133H mutant had a lower K_M for αKG than human R132H in steady-state enzyme kinetic experiments, and it produced almost exclusively 2HG in the presence of NADPH and αKG. The G98N mutant had a higher k_cat in the forward direction than the comparable human G97N mutant, and the G98N mutant produced a smaller amount of 2HG compared to the R133H mutant. These results suggest that C. elegans strains with IDH-1 mutations could be a good model system for studying the effects of 2HG in eukaryotic organisms. Full article