Search Results (574)

Genetic variation in four wild Prunus taxa (P. fruticosa, P. erythrocarpa, P. verrucosa and P. griffithii var. tianshanica) was investigated for the first time using six chloroplast DNA regions (matK, r rpl16, ycf1_1, ycf1_2, ndhF and trnH–psbA) analysed through CAPS-based SNP detection. The results revealed weak chloroplast differentiation among P. erythrocarpa, P. verrucosa and P. griffithii var. tianshanica. However, chloroplast variation exhibited a strong geographic signal across the studied populations. The observed chloroplast variation primarily reflected geographic structuring rather than clear differentiation among these closely related taxa. In contrast, P. fruticosa showed distinct chloroplast haplotypes not shared with the other taxa. These findings demonstrate that the developed chloroplast CAPS marker system is effective for detecting chloroplast haplotype variation but has limited discriminatory power among closely related wild Prunus taxa. Further studies using nuclear markers and genome-wide approaches will be required to better resolve their genetic relationships and evolutionary history. Full article

Methyl-dependent restriction endonucleases are promising tools for analyzing eukaryotic DNA methylation patterns. However, quantitative assessment of their substrate specificity requires the determination of the kinetic parameters of enzymatic reactions. Here, we present a method for determining initial reaction rates based on fluorescent probes and real-time monitoring of changes in fluorescence intensity. Initial rates of methyl-dependent GlaI and BlsI restriction endonucleases were determined as the slope of the linear part of the kinetic curves, after which the Michaelis–Menten constants (K_M) and reaction rates (V_max) were calculated using nonlinear regression. For both enzymes, K_M values were determined for the first time, indicating a high affinity of the methyl-dependent restriction endonucleases for methylated sites. K_M values for fully methylated duplexes were in the range of (4.4–7.0)·10² nM for GlaI and 2.4–55 nM for BlsI. K_M values were significantly lower for the hemimethylated duplexes: (1.9–8.0)·10² nM for GlaI and (0.7–15.0)·10² nM for BlsI; and even lower for unmethylated duplexes: (27–46)·10² nM for GlaI and (0.28–23)·10² nM for BlsI. Maximum reaction rates varied within relatively narrow limits: V_max values were in range (4.5–19.5)·10⁻⁴ nM/s for GlaI and (1.4–18)·10⁻⁴ nM/s for BlsI, respectively. V_max values were depended weakly on the degree of methylation compared to the K_M. The proposed fluorescence method was applied to determine the kinetic parameters of methyl-dependent restriction endonucleases for the first time. It may serve as a simpler and more environmentally friendly alternative to traditional electrophoretic approaches that use radioactive labels. Full article

Apoptosis is traditionally considered a definitive barrier against oncogenesis, as caspase activation typically eliminates damaged and genetically unstable cells. Here, we report that caspase-8, an initiator of extrinsic apoptosis, paradoxically promotes genetic instability and carcinogenesis following exposure to radiation. We observed that a substantial fraction of mammalian cells exposed to ionizing radiation can survive despite caspase-8 activation. This sublethal activation of caspase-8 facilitated persistent DNA damage, which was attenuated by the expression of a dominant-negative caspase-8 (C360A, Casp8DN) or short hairpin RNA (shRNA)-mediated knockdown of caspase-8 in mammalian cells. The facilitative role of caspase-8 in radiation-induced genomic instability was further validated in caspase-8 heterozygous mice. Moreover, inhibition of caspase-8 abolished iron-ion radiation-induced oncogenic transformation in both soft agar and nude mice. Mechanistically, sublethal caspase-8 activation promoted the nuclear translocation of mitochondrial endonuclease G and persistent DNA damage, accompanied by activation of non-canonical nuclear factor κB (NF-κB) signaling. Collectively, our findings demonstrate that caspase-8 can act as a causative driver of radiation-induced malignancy, challenging the dogma of caspases as universal anticancer barriers and providing important insights into the long-term health risks associated with space radiation and radiotherapy. Full article

The aim of this study was to determine the association between matrix metalloproteinase (MMP1, MMP2, and MMP3) gene variations and susceptibility to Peyronie’s disease, and to investigate their effects on disease progression. This cross-sectional case–control study included a total of 182 individuals—91 patients with Peyronie’s disease and 91 healthy controls. Peripheral venous blood samples (5 mL) were collected, and genomic DNA was isolated using standard spin-column extraction kits. The promoter regions of MMP1, MMP2, and MMP3 were amplified via polymerase chain reaction (PCR), and genotyping was performed using restriction fragment length polymorphism (RFLP) assays with specific restriction endonucleases. All genotyping procedures were conducted in a blinded fashion. Hardy–Weinberg equilibrium analysis (HWE), binary logistic regression with rigorous multiplicity adjustments (Bonferroni-adjusted p-values), and non-parametric Kruskal–Wallis/Mann–Whitney tests with Dunn–Bonferroni post hoc comparisons were utilized. The frequency of the 2G allele for MMP1 (−1607 1G/2G) was significantly higher in the patient group (0.4830) compared to the control group (0.2360; p < 0.05). Similarly, significant differences in allele frequencies were observed for MMP2 (−735 C/T) and MMP3 (−1171 5A/6A) between the cohorts (p < 0.05). HWE analysis confirmed that all investigated variations were in equilibrium in both groups (all p > 0.05). Logistic regression analysis showed that the MMP1 1G/1G genotype was associated with lower odds of Peyronie’s disease (OR: 0.250, p < 0.001), while the 1G/2G (OR: 2.032, p = 0.023) and 2G/2G (OR: 3.509, p = 0.005) genotypes were significantly associated with increased odds. Temporally, carriers of the MMP1 2G/2G genotype presented predominantly in the acute phase, exhibiting a significantly shorter median symptom duration (6.00 months, IQR: 3.00–9.00) compared to 1G/1G carriers (12.00 months, IQR: 5.25–36.00; p = 0.016, adj p = 0.048). For MMP2 (−735 C/T), the CC genotype was associated with higher odds of Peyronie’s disease (OR: 3.049, p < 0.001), while the TT genotype was associated with lower odds (OR: 0.278, p = 0.002). Furthermore, penile plaques were detected in 90% of patients with the CC genotype (p < 0.001). Patients with the CC genotype presented with a median symptom duration of 6.00 months (IQR: 3.00–12.00), whereas TT carriers exhibited a significantly longer duration (24.00 months, IQR: 4.00–48.00; p = 0.002, adj p = 0.006). Regarding MMP3 (−1171 5A/6A), the 5A/5A (OR: 3.930, p = 0.002) and 5A/6A (OR: 2.050, p = 0.022) genotypes were significantly associated with increased odds of the disease, while the 6A/6A genotype showed an inverse association (OR: 0.226, p < 0.001). Patients carrying the 5A allele presented characteristically in the acute phase, with a median symptom duration of 6.00 months (5A/5A) or 4.00 months (5A/6A), which was significantly extended to a median of 24.00 months (IQR: 7.50–54.00) in the 6A/6A genotype (p < 0.001, adj p < 0.001). The findings indicate robust statistical associations between the MMP1 2G, MMP2 C, and MMP3 5A alleles and increased susceptibility to Peyronie’s disease, as well as distinct correlations with compressed symptom durations, acute-phase characteristics, and plaque presence. Because this study relied on peripheral blood DNA genotyping without direct tissue expression quantification in a cross-sectional design, these genetic variations reflect strong exploratory associations rather than direct causal evidence. These markers highlight significant prognostic potential that warrants validation in larger, prospective, longitudinal cohorts. Full article

Casimicrobium huifangae SJ-1 is one of the 28 core microbial groups in the activated sludge of municipal wastewater treatment plants. It exhibits strong environmental adaptability and interactions with other microbes. To develop a genetic manipulation system for this strain, we tested several broad-host-range plasmids, and pBBR1MCS-2 was successfully transformed into strain SJ-1. However, only the plasmid extracted from the transformant could be retransformed into strain SJ-1, suggesting strong host defense systems. We investigated the genome-wide DNA methylation and obtained a DNA methylation map and nine methylation-targeting motifs from C. huifangae. A modified pBBR1MCS-2 lacking all targeting motifs achieved transformation efficiency comparable to that of the host-derived plasmid, implicating the restriction modification (R-M) barrier of C. huifangae. REBASE annotation revealed nine R-M systems in the genome, including two of Type I, six of Type II, and one of Type III. Quantitative PCR showed that the restriction endonuclease component of the Type I RM-C system was transcriptionally upregulated in the transformant harboring pBBR1MCS-2. The subunits responsible for the methyltransferase and endonuclease functions of RM-C were expressed in Escherichia coli, purified, and characterized. The targeting motif of RM-C was implicated as 5′-GAGNNNNNNNTGCT-3′ based on in vitro cleavage assays and SMRT methylation calls. These findings reveal the DNA methylation characteristics of C. huifangae SJ-1 and outline the possible methylation-based defense architecture, providing a reference for future genetic manipulation. Full article

Plague, caused by Yersinia pestis, remains a significant public health concern, yet the determinants of many toxin-associated proteins encoded by this pathogen remain poorly understood. Here, we characterized two previously unexplored Rearrangement hotspot (Rhs) proteins, YPO3609 and YPO3615, using a combination of bioinformatic analyses, bacterial toxicity assays, DNA degradation assays, SOS reporter assays, and RT-qPCR analysis. The C-terminal domains of YPO3609 and YPO3615 confer nuclease toxicity, with YPO3609 containing a WHH motif of the HNH nuclease superfamily and YPO3615 harboring a restriction endonuclease-like aspartate dyad. Heterologous expression of these C-terminal toxin domains in Escherichia coli inhibited growth, caused DNA degradation, elicited SOS-related responses, and induced cellular filamentation. Co-expression experiments identified YPO3610 and YPO3616 as the cognate immunity proteins of YPO3609 and YPO3615, respectively, while site-directed mutagenesis further showed that H411 and D1442/D1447 contribute to the nuclease-associated activities of the two toxins. Homologs of both toxin domains were broadly distributed in Pseudomonadota, with the C-terminal domain of YPO3609 showing a wider distribution. These findings characterize two functional Rhs toxin–immunity modules encoded by Y. pestis, and provide new insights into Rhs-associated nuclease toxins and their related homologs. Full article

In this study, we used our own prediction models to assess the antiviral potential of pyrimidin-4(3H)-one derivatives against the A/H1N1 influenza virus strain. This assessment allows us to identify promising structures. The models are based on machine learning algorithms and molecular modeling results. In general, the prediction results are consistent with experimental data. The most promising compound, namely 12 (6-amino-2-(dimethylamino)pyrimidin-4(3H)-one), inhibits the reproduction of the A/Puerto Rico/8/34 (H1N1) influenza virus strain in vitro, likely by affecting the function of the endonuclease domain of the viral polymerase complex. Compound 12 can be used to create new PAN inhibitors by modifying its structure. Full article

CRISPR-associated protein 9 (Cas9)-mediated editing generates numerous mutations. Existing detection methods, such as ACT-PCR, T7EI endonuclease cleavage, HRM analysis, and high-throughput sequencing, often require stringent conditions, expensive reagents, or specialized instruments. Here, we introduce Mismatch-Enhanced Specific PCR (MES-PCR), a method that offers sensitivity for detecting mutations under non-stringent experimental conditions. Combined with quantitative PCR (MES-qPCR), it facilitates the calculation of sgRNA efficiency and enables screening for heterozygous mutations. We validated this method in soybean (Glycine max (L.) Merr.) and Arabidopsis thaliana, confirming its practicality. This approach significantly enhances the efficiency and reduces the cost of mutation screening, presenting a powerful tool to accelerate precision breeding and functional genomics research in crops. Full article

Over a decade of advances in Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-based technologies have culminated in the first-ever FDA-approved CRISPR/Cas-based therapy. Aside from this approved therapy for sickle cell anemia, several CRISPR/Cas-based therapies are currently under development or testing for a range of chronic diseases, including viral diseases like human immunodeficiency virus type 1 (HIV-1) infection, genetic diseases like familial hypercholesterolemia, and cancer. The success of these therapies hinges on the effective delivery of CRISPR/Cas9 components to target regions, efficient Cas endonuclease editing, repair profiles generated, and their resulting outcomes. Here, we discuss the factors that influence the generation of CRISPR/Cas9-generated repair edits, the overall profiles, and outcome prediction(s), as well as the analytical tools that have been developed to date. Finally, how this technology has been used towards a functional HIV-1 cure is discussed. Full article

Chromosomal rearrangements that lead to the formation of oncogenic gene fusions, such as EML4-ALK, are thought to arise from incorrect repair of double-strand breaks in DNA. However, the mechanisms and factors driving rearrangement formation remain poorly understood, and analysis of these processes is limited by detection methods that are labor-intensive, low-throughput, and not readily quantitative at single-cell resolution. Here, we developed a genetically encoded ALK reporter based on A549 lung adenocarcinoma cells, created by inserting an ALK-P2A-mCherry cassette into the endogenous ALK locus, so that induced EML4-ALK fusion activated mCherry fluorescence. Reporter activation yielded a readily quantifiable mCherry-positive subpopulation that could be measured and enriched by flow cytometry and correlated with EML4-ALK levels. Using this platform, we combined CRISPR-mediated rearrangement induction with knockdown of DNA repair factors using RNA interference. Of the factors involved in base excision repair, homologous recombination-related pathways and canonical non-homologous end joining, knockdown of the APEX1 gene encoding apurinic endonuclease 1 (APE1) selectively increased EML4-ALK levels both in the reporter cell line and in parental A549 cells. Together, this work provides a sensitive, single-cell A549-based ALK reporter platform and a framework for future studies aimed at identifying cellular and environmental factors that modulate oncogenic EML4-ALK rearrangement formation. Full article

Machupo virus (MACV) is the causal agent of Bolivian Hemorrhagic fever. It is highly pathogenic, has a high mortality rate, and currently lacks specific treatments or vaccines. MACV belongs to the Arenaviridae family, which uses a cap-snatching mechanism during the transcription process. Its viral polymerase, the L protein, harbors the endonuclease activity required for cap snatching, making it a suitable target for the development of antiviral therapeutics. We combined experimental and computational methods to characterize MACV endonuclease activity and evaluate inhibitors. A fluorescence resonance energy transfer (FRET) assay was used to measure the enzymatic activity of endonuclease and identify potent inhibitors via high-throughput screening. FRET assays identified BW-148, an inhibitor with a 48.4 µM (95% CI: 37.3–59.3 µM; R² = 0.98) IC₅₀, and a K_D of 13.7 µM (95% CI: 8.2–19.2 µM, n = 3). Docking studies reveal that BW-148 may bind near the MACV endonuclease catalytic site, inhibiting enzymatic activities by metal chelating. BW-148 is a useful lead compound for further optimization of Machupo endonuclease inhibitors. Full article

DNA oxidation damage and its repair are essential for maintaining genomic integrity in the human limbal epithelium, which harbors corneal epithelial stem cells. This study investigated the distribution of the DNA base oxidation 8-oxoguanine (8-oxoG) and the base excision repair (BER) enzymes 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease 1 (APE1) in non-cultured and eye-bank organ-cultured human limbal epithelia. Immunohistochemistry was used to assess the localization and staining intensity of 8-oxoG, OGG1, and APE1, evaluated semi-quantitatively and by image analysis. In situ hybridization was performed to detect the distribution of OGG1 and APE1 gene expression in organ-cultured tissue. In non-cultured limbal epithelia, nuclear 8-oxoG staining was more frequently observed in superficial epithelial layers, whereas nuclear OGG1 and APE1 staining predominated in basal layers. In organ-cultured epithelia, a higher proportion of superficial nuclei exhibited 8-oxoG staining, while the basal predominance of OGG1 was reduced and that of APE1 was preserved. Transcripts of OGG1 and APE1 were detected in basal- as well as in suprabasal layers of organ-cultured epithelia. These findings demonstrate the presence of DNA base oxidation and BER-related enzymes in basal and suprabasal human limbal epithelial cells during storage of corneal tissue under commonly used eye-bank organ-cultured conditions prior to transplantation. Full article

Background: Eosinophilic chronic rhinosinusitis (ECRS) is characterized by refractory nasal polyps and severely impaired mucociliary clearance (MCC). The molecular mechanisms underlying the modulation of mucociliogenesis following IL-4/13 blockade with dupilumab remain poorly understood, notwithstanding its proven clinical efficacy. Methods: Bulk RNA Barcoding and sequencing (BRB-seq) was performed on nasal polyp tissues collected from healthy controls (n = 6), patients with non-ECRS (n = 8), and patients with ECRS both before and four weeks after dupilumab treatment (n = 9) to identify the early molecular drivers underlying ciliary regeneration. Comprehensive gene-set scoring systems were developed to evaluate multiciliogenesis master regulators, master regulators of core/ciliary planar cell polarity (PCP) and PCP components. Interaction scores for epithelial-derived cytokines—thymic stromal lymphopoietin (TSLP), IL-25, and IL-33—were calculated based on ligand and cognate receptor subunit expression. Results: The ciliary master regulatory hierarchy (e.g., FOXJ1, RFX2/3), PCP components (CELSR1 and the ciliogenesis and planar polarity effector (CPLANE) module: FUZ, INTU, WDPCP), and structural ciliogenesis pathways were robustly restored following IL-4/13 blockade. The TSLP interaction score correlated with global mucosal damage, serving as a trigger for compensatory multiciliogenesis. The pre-treatment IL-33 interaction score emerged as a significant predictor of transcriptomic ciliary recovery (p < 0.05). DNASE1L3—the primary endonuclease for degrading eosinophilic extracellular traps (EETs)—remained persistently downregulated post-treatment. Conclusions: IL-4/13 blockade successfully restores the structural and directional “hardware” of the respiratory epithelium but fails to rectify the enzymatic “software” required for mucus degradation. This “residual molecular scar” may explain the persistent mucus hyperviscosity observed in some ECRS patients even after clinical polyp resolution. Full article

Background/Objectives: Pixavir marboxil is a novel oral cap-dependent endonuclease inhibitor that is rapidly converted to its active metabolite pixavir. This study aimed to characterize the population pharmacokinetics (PopPK) of pixavir, identify clinical covariates affecting its pharmacokinetics, and evaluate the exposure–response relationship to inform optimal dosing in the treatment of acute influenza. Methods: Data were pooled from three clinical studies spanning Phase I to Phase III trials and included adults and adolescents aged ≥12 years. PopPK modeling was conducted using NONMEM software. Individual exposure metrics, including the 24-h post-dose concentration, maximum concentration, and area under the concentration-time curve from time zero to infinity, were derived using Bayesian estimation based on the final PopPK model. Efficacy endpoints included time to alleviation of influenza-related symptoms and changes in viral titer and viral RNA load from baseline to Day 2. Safety was evaluated through the incidence of treatment-related adverse events. Results: Pixavir pharmacokinetics were best described by a two-compartment model. Body weight significantly influenced clearance and central volume of distribution, while dose amount had a notable effect on bioavailability. Food, sex and age had no clinically meaningful impact on pharmacokinetics. Exposure–response analysis showed no clear relationship between pixavir exposure and both efficacy and safety endpoints within the studied dosing range. Conclusions: The weight-based dosing regimen of pixavir marboxil (40 mg for <80 kg and 80 mg for ≥80 kg) is supported, as it reduces body-weight-driven exposure variability and maintains exposure within the clinically characterized range in adults and adolescents with acute influenza. Full article

DNA damage requires repair via the endonuclease IV-mediated base excision repair (BER) pathway, which corrects apurinic/apyrimidinic (AP) sites. Chlamydophila pneumoniae AP endonuclease IV (CpEndoIV), the sole AP endonuclease in this pathogen, is crucial for genomic integrity. As humans lack a homologous protein, it represents a potential therapeutic target. In this study, we report the first crystal structure of CpEndoIV at 1.97 Å resolution. The structure reveals two Zn²⁺, one Mg²⁺, and a malonate molecule bound in the active site, marking the first observation of Mg²⁺ coordination in the EndoIV family. Compared to the three-Zn²⁺ model with a narrow, deep pocket for precise AP-site cleavage, the Zn²⁺/Mg²⁺-bound state has a wider, shallower pocket that might promote diverse catalytic activities. Combined with enzymatic assays, we suggest that the mixed Zn²⁺/Mg²⁺ model is better adapted for CpEndoIV to operate under host oxidative stress. Malonate binds to the metal ions, occupying the positions normally coordinated by water molecules. This binding mode may mimic the coordination of the substrate to the metal ions, and the protein conformation resembles that of the enzyme upon substrate binding at the active site. This study provides a structural basis for the functional characterization of CpEndoIV and offers a reference for the development of targeted inhibitors against diseases caused by Chlamydophila pneumoniae. Full article