Search Results (2,157)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a widespread enzymopathy affecting approximately 500 million individuals that represents a significant global health issue. Among the more than 230 identified mutations in the G6PD gene, six class A variants—G6PD Utrecht (Pro409Ser), G6PD Suwalki (Pro409Arg), G6PD Merlo (Pro409Gln), G6PD Kawasaki (Gly410Ala), G6PD Shinagawa (Gly410Asp), and G6PD Riverside (Gly410Cys)—are located in the beta-loop near the NADP+ binding site. These mutations are of particular interest due to their association with severe hematologic phenotypes, including chronic hemolytic anemia, as well as their proposed role in the allosteric regulation of G6PD multimerization. This study presents a comprehensive biochemical and functional characterization of these clinically relevant G6PD variants. The variant enzymes were cloned, expressed, and purified for characterization. Kinetic parameters and thermal stability assays, complemented by molecular dynamics simulations (MDS), were employed to elucidate the structural impacts of the mutations. Our results demonstrate that these mutations significantly impair protein function, characterized by reduced affinity for glucose-6-phosphate (G6P) and NADP⁺, as well as altered thermal stability compared with wild-type G6PD. MDS revealed that point mutations in the βN- and βM-sheets in the NADP⁺s region propagate subtle conformational changes, ultimately affecting the NADP⁺c region and the G6P binding cavity. Furthermore, secondary structure element analyses of the simulation data showed that Pro409 and Gly410 point mutations propagate several changes around residues 195–210 (G6P binding site) and 380–400 (NADP⁺s), explaining their effect on overall catalytic performance. These findings enhance our understanding of the molecular mechanisms underlying G6PD deficiency and its clinical implications, providing a foundation for future therapeutic strategies aimed at mitigating the effects of these variants. Full article

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

Lateral Spreading Tumors (LSTs) are a type of non-polypoid lesion known for their flat morphology, which often leads to them going undetected. However, especially nongranular (NG) LSTs have the potential for malignant transformation. Recent advances in endoscopic technologies have improved the detection of these lesions. Despite growing research interest in their role in colorectal cancer (CRC) development, a comprehensive molecular characterization of LSTs is still lacking. The aim of this review is to highlight the current knowledge of the molecular characteristics of LSTs, that may help in determining whether LSTs can be prognostic indicators and identifying cases where they may rapidly progress to CRC through characteristic molecular pathways. From a mutational point of view, LSTs seem to be more closely associated with inflammatory bowel diseases (IBDs) than with polypoid lesions. Nonetheless, they have peculiar epigenetic and genetic traits, which set them apart from other adenomas or bowel diseases. Elucidating their role in CRC development would provide benefits for their classification and management, by enhancing clinical surveillance strategies for patients diagnosed with these lesions in order to improve the efficient prevention of colorectal cancer. Full article

Human methyltransferase-like protein 16 (METTL16) installs N⁶-methyladenosine on U6 small nuclear RNA (snRNA) and other RNAs. Multiple X-ray crystal structures of METTL16 have been published; however, we do not yet fully understand the structure–function relationships of specific residues. We designed 38 mutants, including seven cancer-associated mutants, and used electrophoretic mobility shift assays and single-turnover kinetic assays to better understand the functional roles of specific domains and amino acid residues in binding to U6 snRNA, formation of the METTL16•U6 snRNA•S-adenosylmethionine (SAM) complex, and the rate of methylation. While point mutations in the methyltransferase domain mildly weaken the binding of METTL16 to U6 snRNA, the C-terminal vertebrate conserved regions (VCRs), particularly the arginine-rich region (R382 to R388), mediate cooperative binding and contribute more to RNA binding. All METTL16 K-loop mutants displayed tighter SAM binding, suggesting that the K-loop blocks SAM binding. In addition, residues E133 and F227 are critical for stabilizing SAM binding. Mutations in the ₁₈₄NPPF₁₈₇ catalytic core and R282A abolished methyltransferase activity. Two METTL16 somatic cancer-associated mutants (G110C and R241Dfs*2) displayed reduced methylation activity. This mutational analysis expands our understanding of how specific domains and residues contribute to substrate-binding activity and methylation of U6 snRNA catalyzed by METTL16. Full article

The infectious bronchitis virus (IBV) causes a severe infectious disease in poultry, leading to significant financial losses. The prevention and treatment of this disease are extremely challenging due to the virus’s rapid mutation. Therefore, quick diagnosis of IBV infections is crucial for controlling the disease. This study aimed to develop a real-time reverse transcription recombinase-aided amplification (RT-RAA) method for IBV. The most effective primer combination was selected for further validation. To determine the assay’s analytical sensitivity, a serial dilution from 10⁵ to 10⁰ EID₅₀/mL was used, and the limit of detection was calculated. The assay could detect down to 10² EID₅₀/mL. The limit of detection (95% Confidence Interval) was 67 EID₅₀ per reaction. There was no cross-reaction with common poultry diseases. When analyzing 39 clinical samples, RT-RAA and RT-PCR showed 100% diagnostic sensitivity and specificity. In conclusion, the IBV RT-RAA detection method is rapid, sensitive, and specific. This approach can be used to improve IBV diagnosis at the point of need. Full article

Ecological drought in terrestrial systems is a vegetation-functional degradation phenomenon triggered by the long-term imbalance between ecosystem water supply and demand. This process involves nonlinear coupling of multiple climatic factors, ultimately forming a compound ecological stress mechanism characterized by spatiotemporal heterogeneity. Based on meteorological and remote sensing datasets from 1982 to 2022, this study identified the spatial distribution and temporal variability of ecological drought in China, elucidated the dynamic evolution and return periods of typical drought events, unveiled the scale-dependent effects of climatic factors under both univariate dominance and multivariate coupling, as well as deciphered the response mechanisms of ecological drought to meteorological drought. The results demonstrated that (1) terrestrial ecological drought in China exhibited a pronounced intensification trend during the study period, with the standardized ecological water deficit index (SEWDI) reaching its minimum value of −1.21 in February 2020. Notably, the Alpine Vegetation Region (AVR) displayed the most significant deterioration in ecological drought severity (−0.032/10a). (2) A seasonal abrupt change in SEWDI was detected in January 2003 (probability: 99.42%), while the trend component revealed two mutation points in January 2003 (probability: 96.35%) and November 2017 (probability: 43.67%). (3) The drought event with the maximum severity (6.28) occurred from September 2019 to April 2020, exhibiting a return period exceeding the 10-year return level. (4) The mean values of gridded trend eigenvalues ranged from −1.06 in winter to 0.19 in summer; 87.01% of the area exhibited aggravated ecological drought in winter, with the peak period (88.51%) occurring in January. (5) Evapotranspiration (ET) was identified as the dominant univariate driver, contributing a percentage of significant power (POSP) of 18.75%. Under multivariate driving factors, the synergistic effects of ET, soil moisture (SM), and air humidity (AH) exhibited the strongest explanatory power (POSP = 19.21%). (6) The response of ecological drought to meteorological drought exhibited regional asynchrony, with the maximum correlation coefficient averaging 0.48 and lag times spanning 1–6 months. Through systematic analysis of ecological drought dynamics and driving mechanisms, a dynamic assessment framework was constructed. These outcomes strengthen the scientific basis for regional drought risk early-warning systems and spatially tailored adaptive management strategies. Full article

Background: Multiple Myeloma (MM) is a malignant plasma cell dyscrasia that progresses through the consecutive asymptomatic, often undiagnosed, precancerous stages of Monoclonal Gammopathy of Undetermined Significance (MGUS) and Asymptomatic Multiple Myeloma (SMM). MM is characterized by low survival rates, severe complications and drug resistance; therefore, understanding the molecular mechanisms of progression is crucial. This study aims to detect genetic mutations, both germline and somatic, that contribute to disease progression and drive tumorigenesis at the final stage of MM, using samples from patients presenting MGUS or SMM, and newly diagnosed MM patients. Methods: Mutations were identified through a fully computational pipeline, implemented in a Linux and RStudio environment, applied to each patient sequence, obtained through single-cell RNA-sequencing (scRNA-seq), separately. Structural and functional mutation types were identified by stage, along with the affected genes. The analysis included quality control, removal of the Unique Molecular Identifiers (UMIs), trimming, genome mapping and result visualization. Results: The findings revealed frequent germline and somatic mutations, with distinct structural and functional patterns across disease stages. Mutations in key genes were identified, pointing to molecules that may play a central role in carcinogenesis and disease progression. Notable examples include the HLA-A, HLA-B and HLA-C genes, as well as the KIF, EP400 and KDM gene families, with the first four already confirmed. Comparative analysis between the stages highlighted molecular transition events from one stage to another. Emphasis was given to novel genes discovered in newly diagnosed MM patients, that might contribute to the tumorigenesis that takes place. Conclusions: This study contributes to the understanding of the genetic basis of plasma cell dyscrasias and the transition events between the stages, offering insights that could aid in early detection and diagnosis, guide the development of personalized therapeutic strategies, and improve the understanding of mechanisms responsible for resistance to existing therapies. Full article

Kobuvirus is a new genus of viruses in the Picornaviridae family causing diarrhea in animals. Porcine kobuvirus (PKV) is an important pathogen with a high rate of infection in pig herds. In this study, a total of 10,990 fecal swabs and tissue samples were collected from different areas of Guangxi province in southern China during 2021–2025 and then tested for PKV using RT-qPCR. The results showed a 19.19% (2109/10,990) PKV positivity rate. Sixty-two PKV-positive samples, which were selected according to sampling regions, sampling seasons, and detection Ct values, were used for PCR amplification and gene sequencing. A sequence comparison showed that the nucleotide and amino acid identities of VP1, 2B, and 3D genes were 78.6–99.5% and 83.5–100%, 77.7–99.8% and 80.9–100%, and 90.9–99.8% and 94.9–99.9%, respectively, indicating that the 3D gene was more conserved than the VP1 and 2B genes. The phylogenetic trees based on these three genes revealed that the PKV VP1 gene sequences from different countries could be classified into two groups (Groups I and II), and the PKV VP1 gene sequences obtained from Guangxi province were distributed in Groups I and II and formed independent clades. The 2B and 3D gene sequences could also be classified into two groups (Groups I and II). Bayesian analysis indicated a state of population growth for PKV strains from the time of their discovery until 2009, at which point it began to decline. Amino acid sequence analysis of the VP1 gene identified mutations and insertions in the obtained PKV strains. Recombinant analysis showed that no recombinant event was found in the VP1, 2B, and 3D genes of the obtained strains. The results indicated the geographically specific inheritance and variation in PKV, provided more information on the prevalence and genetic evolution of PKV in Guangxi province, Southern China, and emphasized the importance of regularly monitoring genetic variation in PKV for better comprehension of PKV. Full article

Background and Objectives: Non-small-cell lung cancer (NSCLC) often has epidermal growth factor receptor (EGFR) mutations, which are key targets for therapy. EGFR mutation subtypes, especially exon 19 deletions and exon 21 L858R mutations, influence responses to EGFR tyrosine kinase inhibitors (TKIs) and patient survival. Despite progress in TKI treatments, resistance and different responses remain challenges. This study explores the relationship between EGFR mutation subtypes, PD-L1 expression, and patient outcomes after anti-EGFR therapy. Materials and Methods: We studied 176 cases of EGFR mutation-positive NSCLC. Next-generation sequencing was used to analyze EGFR and other mutations, while PD-L1 expression was evaluated through immunohistochemistry. We analyzed EGFR mutation subtypes, PD-L1 status, treatments, and survival outcomes. Results: Among 176 cases, 88.6% were adenocarcinomas. Within the EGFR mutation spectrum, exon 19 deletions were the most common subtype, accounting for 40.9% of cases, followed by the point mutation in exon 21, which occurred in 35.8% of cases. Less frequent alterations, making up 23.3% of all detected mutations, included mutations in exon 18, insertions, and point mutations such as S768I and T790M in exon 20, as well as changes in exon 2, exon 7, and other less frequently affected regions. Exon 19 mutations were associated with older age, female sex, adenocarcinoma, and bone metastasis (p < 0.05). TP53 was the most common concurrent mutation (44.3%). PD-L1 positivity (TPS ≥ 1%) was observed in 48.3%, with high expression (TPS ≥ 50%) in 25.9%. Exon 21 mutations were significantly linked to PD-L1 negativity (p = 0.008). The median overall survival was longest with TKI therapy (51 months), and this was also observed in PD-L1-positive patients, although the difference was not statistically significant. The median progression-free survival for patients treated with TKIs and those with EGFR mutations was 14 months. PD-L1-positive patients receiving TKIs had significantly longer survival than those who did not (51 vs. 17 months, p = 0.003). Conclusions: EGFR mutation subtypes and PD-L1 expression seem to affect treatment outcomes and survival in NSCLC. The observed links emphasize the potential value of combining molecular and immunological markers to guide therapy choices. Full article

From the corona virus pandemic in Japan that started with the “Diamond Princess” accident, it became clear that rapid detection, a high sensitivity, multiple diagnostic items, one-step one-base point mutation detection, a fast speed of system development, portability (small size and light weight), full automation, random access, and other conditions are required for future point-of-care testing systems. The Eprimer-SmartAmp technology that was developed possesses characteristics fully aligned with these requirements. Building upon this platform, the “GenPad” system was subsequently established. The GenPad system is widely applicable not only to emerging foreign infectious diseases, but also to cancer, lifestyle-related diseases, and other areas of healthcare through telemedicine and intraoperative nucleic acid diagnoses. In collaboration with telecommunication systems, GenPad is expected to contribute to the establishment of a smart medical city with a countermeasure against emerging foreign infectious diseases, where individuals can check their own health conditions in all healthcare areas. Full article

In the modern era of HIV treatment, people co-infected with HIV and HBV still face poor liver outcomes, including liver fibrosis, liver cirrhosis, and hepatocellular carcinoma. We investigated baseline characteristics and long-term liver function outcomes in 435 people living with HIV and HBV co-infection, focusing on HCC-associated point mutations (PMs) and PreS region deletion mutations. PMs were present in 72.9% of participants and were associated with male predominance, lower HBV genotype C prevalence, reduced HBV DNA and HBeAg levels, and higher HBsAg and HBeAb positivity. However, PMs did not significantly impact liver function or fibrosis progression over six years of ART follow-up. In contrast, PreS deletions were found in 21.8% of cases and stratified into PreS1, PreS2, and PreS1+2 deletions. PreS2 and PreS1+2 deletions were linked to older age, higher HBsAg and AFP levels, elevated liver enzymes, and lower platelet counts. These groups also exhibited significantly worse liver fibrosis markers (APRI and FIB-4), with PreS2 deletions consistently showing the highest values throughout the follow-up. Despite the initial improvement with ART, patients with PreS2 and PreS1+2 deletions maintained higher fibrosis and cirrhosis risks over six years. In summary, while PMs were not predictive of liver disease progression, PreS deletion mutations (especially in the PreS2 region) were associated with poorer liver outcomes, indicating their potential as biomarkers for fibrosis risk in co-infected individuals with long-term ART. Full article

Acute lymphoblastic leukemia (ALL) is a heterogeneous hematologic malignancy defined by the uncontrolled proliferation of lymphoid precursors. Accurate diagnosis and effective therapeutic strategies hinge on a comprehensive understanding of the genetic and molecular landscape of ALL. This review synthesizes the latest updates in cytogenetic and molecular classifications, emphasizing the 2022 World Health Organization (WHO) and International Consensus Classification (ICC) revisions. Key chromosomal alterations such as BCR::ABL1 and ETV6::RUNX1 and emerging subtypes including Ph-like ALL, DUX4, and MEF2D rearrangements are examined for their prognostic significance. Furthermore, we assess novel diagnostic tools, notably next-generation sequencing (NGS) and optical genome mapping (OGM). While NGS excels at identifying point mutations and small indels, OGM offers high-resolution structural variant detection with 100% sensitivity in multiple validation studies. These advancements enhance our grasp of leukemogenesis and pave the way for precision medicine in both B- and T-cell ALL. Ultimately, integrating these innovations into routine diagnostics is crucial for personalized patient management and improving clinical outcomes. Full article

Lactic acid bacteria (LAB) are central to food, feed, and health biotechnology, yet their genomes have long resisted rapid, precise manipulation. This review charts the evolution of LAB genome-editing strategies from labor-intensive RecA-dependent double-crossovers to state-of-the-art CRISPR and CRISPR-associated transposase systems. Native homologous recombination, transposon mutagenesis, and phage-derived recombineering opened the door to targeted gene disruption, but low efficiencies and marker footprints limited throughput. Recent phage RecT/RecE-mediated recombineering and CRISPR/Cas counter-selection now enable scar-less point edits, seamless deletions, and multi-kilobase insertions at efficiencies approaching model organisms. Endogenous Cas9 systems, dCas-based CRISPR interference, and CRISPR-guided transposases further extend the toolbox, allowing multiplex knockouts, precise single-base mutations, conditional knockdowns, and payloads up to 10 kb. The remaining hurdles include strain-specific barriers, reliance on selection markers for large edits, and the limited host-range of recombinases. Nevertheless, convergence of phage enzymes, CRISPR counter-selection and high-throughput oligo recombineering is rapidly transforming LAB into versatile chassis for cell-factory and therapeutic applications. Full article

Background: DEAD/H box 5 (DDX5) serves as a transcriptional coactivator for several transcription factors including E2F1, the primary target of the tumor suppressor pRB. E2F1 physiologically activated by growth stimulation activates growth-related genes and promotes cell proliferation. In contrast, upon loss of pRB function due to oncogenic changes, E2F1 is activated out of restraint by pRB (deregulated E2F1) and stimulates tumor suppressor genes such as ARF, which activates the tumor suppressor p53, to suppress tumorigenesis. We have recently reported that DDX5 augments deregulated E2F1 activity to induce tumor suppressor gene expression and apoptosis. During the analyses, we noted that over-expression of E2F1 increased DDX5 expression, suggesting a feed forward loop in E2F1 activation through DDX5. Objective: We thus examined whether the DDX5 gene is a target of deregulated E2F1. Method: For this purpose, we performed promoter analysis and ChIP assay. Result: The DDX5 promoter did not possess typical E2F binding consensus but contained several GC repeats observed in deregulated E2F1 targets. Insertion of point mutations in these GC repeats decreased responsiveness to deregulated E2F1 induced by over-expression of E2F1, but scarcely affected responsiveness to growth stimulation. ChIP assays showed that deregulated E2F1 induced by over-expression of E2F1 or expression of E1a, which binds pRB and releases E2F1, bound to the DDX5 gene, while physiological E2F1 induced by growth stimulation did not. Conclusions: These results suggest that the DDX5 gene is a target of deregulated E2F1, generating a feed forward loop mediating tumor suppressive E2F1 activity. Full article

Accurate dynamic flow statistics for trackless vehicles are critical for efficiently scheduling trackless transportation systems in underground mining. However, traditional discrete time-point methods suffer from “time membership discontinuity” due to RFID timestamp sparsity. This study proposes a fuzzy five-region membership (FZFM) model to address this issue by subdividing time intervals into five characteristic regions and constructing a composite Gaussian–quadratic membership function. The model dynamically assigns weights to adjacent segments based on temporal distances, ensuring smooth transitions between time intervals while preserving flow conservation. When validated on a 29-day RFID dataset from a large coal mine, FZFM eliminated conservation bias, reduced the boundary mutation index by 11.1% compared with traditional absolute segmentation, and maintained high computational efficiency, proving suitable for real-time systems. The method effectively mitigates abrupt flow jumps at segment boundaries, providing continuous and robust flow distributions for intelligent scheduling algorithms in complex underground logistics systems. Full article