Search Results (945)

Background/Objectives: Tea, the world’s second most consumed beverage after water, contains diverse phytochemicals that have garnered growing interest for their potential ability to modulate inflammasome activation. This study examined the antioxidant and anti-inflammatory properties of oolong tea (OLT) extracts, with a specific focus on their regulatory effects on NLRP3 inflammasome assembly—a critical mediator in chronic inflammatory diseases. Methods: OLT extracts were prepared from the Jin-Xuan cultivar with quantification for bioactive components (total phenolics, flavonoids, condensed tannins, and proanthocyanidins). J774A.1 murine macrophages were primed with LPS and stimulated with ATP to induce inflammasome activation. Therapeutic potentials of OLT extracts were assessed by measuring cytokine secretion, expression of NLRP3 inflammasome-related proteins (NLRP3, ASC, Caspase-1, and IL-1β), inflammasome complex formation, and ROS generation via biochemical assays, immunoblotting, and fluorescence microscopy. Results: OLT extracts, particularly at 100 µg/mL, markedly suppressed both the priming and activation phases of NLRP3 inflammasome formation. OLT treatment reduced IL-1β secretion by more than 50%, attenuated ASC oligomerization and speck formation, inhibited caspase-1 cleavage, and lowered intracellular ROS levels by approximately 50%. Conclusions: These findings suggest that OLT extracts exert potent anti-NLRP3 inflammasome activity and offer immunomodulation potential in preventing inflammation-related diseases such as infections, cancer, and neurodegenerative disorders. Further in vivo investigations, followed by clinical applications and epidemiological studies, are warranted to validate these preventive effects in human populations. Full article

Mitochondria are crucial for a wide range of cellular processes. One of the most important is innate immunity regulation. Apart from functioning as a signaling hub in immune reactions, mitochondrial nucleic acids can themselves act as damage-associated molecular patterns (DAMPs) to participate in immune processes directly. This review synthesizes the current understanding of mitochondrial RNA (mtRNA) biology and its link to immune activation through aberrant accumulation. We focus on its origin through bidirectional mitochondrial transcription and metabolism, encompassing maturation (cleavage, polyadenylation, modification) and degradation. Dysregulation of mtRNA metabolism leads to mt-dsRNA (mitochondrial double-stranded RNA) accumulation, which escapes mitochondria via specific channels into the cytosol and serves as DAMPs to trigger an immune response. We discuss the critical roles of key regulatory factors, including PNPT1 (PNPase, Polyribonucleotide Nucleotidyltrans ferase 1), in controlling mt-dsRNA levels and preventing inappropriate immune activation. Finally, we review the implications of mt-dsRNA-driven inflammation in human diseases, including autoimmune disorders, cellular senescence, and viral infection pathologies, highlighting unresolved questions regarding mt-dsRNA release mechanisms. Full article

Background/Objectives: Global warming and concomitant extreme weather events have markedly increased the incidence of heat stroke. Heat stroke (HS) poses a substantial threat to cerebral health by triggering neuroinflammation and accelerating neurodegenerative processes. The activation of the Nod-like receptor protein 3 (NLRP3) inflammasome for interleukin-1β (IL-1β) secretion has been implicated as a critical mechanism underlying HS-related fatalities. However, the potential role of specific dietary factors to counteract heat stroke-induced neurotoxicity remains largely underexplored. We previously reported that eicosapentaenoic acid (EPA) and urolithin A (UroA), a gut metabolite of ellagic acid, effectively suppress NLRP3 inflammasome activation against metabolic or pathogenic insults. This study aimed to assess the impact of eicosapentaenoic acid (EPA), urolithin A (UroA), and their combination on mitigating heatstroke-mediated NLRP3 inflammasome activation in microglial cells. Methods: In vitro heatstroke conditions were replicated by subjecting murine BV2 microglial cells to a high temperature (41 °C) under hypoxic conditions. To achieve nutrient loading, BV2 cells were preincubated with either EPA (50 µM) or UroA (10 µM). NLRP3 inflammasome activation was evaluated by proinflammatory gene expression, caspase-1 cleavage in cells, and IL-1β secretion to the medium. The caspase-1 activation was determined using a luciferase-based inflammasome and protease activity reporter (iGLuc) assay. Results: Exposure to high temperatures under hypoxia successfully mimicked HS conditions and promoted NLRP3 inflammasome activation in BV2 cells. Both EPA and UroA substantially attenuated the heat stroke-induced priming of proinflammatory genes. More importantly, EPA and UroA demonstrated a synergistic effect in mitigating HS-induced active caspase-1 production, leading to a dramatic decrease in IL-1β secretion. This synergistic effect between EPA and UroA was further confirmed by the iGLuc reporter assay. Conclusions: Dietary enrichment with EPA and UroA precursors may constitute an efficacious strategy for mitigating heat stroke-mediated neuroinflammation and neurodegenerative diseases. Full article

Lignin, the most abundant aromatic biopolymer, represents a key renewable feedstock for sustainable biorefineries, yet its structural complexity poses a formidable challenge for enzymatic degradation. While ligninolytic enzymes such as laccases (LACs), lignin peroxidases (LiPs), and manganese peroxidases (MnPs) exhibit remarkable catalytic versatility, the molecular mechanisms underlying their ability to balance substrate specificity and structural flexibility remain unresolved. Here, we employed all-atom molecular dynamics (MD) simulations and virtual mutagenesis to dissect the dynamic interactions between these enzymes and lignin model compound (β-O-4-linked H-type dimers). Our simulations revealed a dual recognition mechanism in which polar residues (such as Asp, Glu, Arg and His) formed hydrogen bonds with hydroxyl and keto groups near catalytic cleavage sites, ensuring precise alignment for bond scission, while aromatic residues stabilized diverse lignin conformations via hydrophobic interactions with conserved aromatic rings. Conformational dynamics of active-site residues enabled adaptive adjustments to substrate heterogeneity, reconciling enzymatic specificity with structural promiscuity. Virtual mutation experiments further demonstrated that aromatic residues were indispensable for binding stability, whereas polar residues dictated cleavage-site selectivity. These findings provide atomic-scale insights into the catalytic mechanism of ligninolytic enzymes, with implications in the rational design of superior biocatalyst for lignin biorefineries. Full article

To express and purify staphylococcal enterotoxin M (SEM) using immobilized metal affinity chromatography (IMAC), a signal peptide-truncated (ΔNsp) wild-type SEM (SEM_WT) was N-terminally fused in pET-28a(+) to a polyhistidine tag (His_6×-) and thrombin cleavage site (TCS; LVPR↓GS), generating His_6×-TCS-ΔNspSEM_WT. Unexpectedly, 4 °C desalting reduced the fusion protein’s molecular weight by ~2.0 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequencing and mass spectrometry identified cleavage specifically at the arginine (R) and glycine (G) peptide bond (R–G bond) within the TCS motif. AlphaFold 3 revealed an exposed serine protease catalytic triad: histidine 172, serine 178, and aspartic acid 212 (H₁₇₂/S₁₇₈/D₂₁₂) in the β-grasp domain, suggesting intrinsic thrombin-like activity (TLA). Sequential IMAC and size-exclusion high-performance liquid chromatography (SE-HPLC) purification eliminated contaminant concerns, while chromogenic substrate S-2238 (S-2238) assays demonstrated increasing specific activity and purification fold, supporting intrinsic TLA. Critically, the mutation of serine at position 178 to alanine (His_6×-TCS-ΔNspSEM_S178A) abolished TLA but preserved the secondary/tertiary structure, confirming the activity’s origin within the wild-type construct. Molecular dynamics (MD) simulations probed the atomistic mechanism for specific R–G bond cleavage. This work establishes a foundation for understanding ΔNspSEM_WT’s TLA. Full article

Recent progress in assisted reproductive medicine has introduced novel therapeutic possibilities for couples experiencing various reproductive challenges or subfertility. A critical concern in this field is the diminished ovarian response to hormonal treatments preceding ovum pickup, necessitating personalised and optimised protocols to enhance ovarian response across different age groups. Furthermore, a common male factor in IVF couples, oligozoospermia, characterised by a low sperm count, significantly impacts the success rates of assisted reproductive technologies, posing an increasing challenge for in vitro fertilisation clinics. Lifestyle choices, dietary habits, and overall health behaviours have also demonstrably affected fertility outcomes in the 21st century. This original article aims to highlight the synergistic importance of both partners’ health, specifically addressing poor ovarian response and oligozoospermia, in achieving successful conception. Our study analysed intracytoplasmic sperm injection outcomes in couples affected by both aforementioned conditions and proposed an optimal management strategy. This study shows that oligozoospermia significantly reduced ICSI fertilisation and cleavage rates. Poor ovarian responders experienced more cancelled cycles due to fewer embryos. While blastocyst rates relative to zygotes were comparable, overall success was lower in groups with male factor infertility and poor ovarian response, necessitating personalised treatment approaches. Full article

Background and Objectives: Cutaneous melanoma (CM) is widely regarded as the most aggressive form of skin cancer worldwide, showing a rising global incidence. It develops from the uncontrolled transformation of pigment-producing melanocytes. The aim of this study is to characterize the cytotoxic and anti-proliferative properties of two 1-(2-aryl-adamantyl)piperazine derivatives, 6 and 7, with a specific emphasis on their impact on melanoma cells. Both compounds are synthesized based on the adamantane core structure which increases drug-like properties of the lead compound phencyclidine I, without increasing toxicity. Materials and Methods: This study describes concentration-dependent effects on cell viability and clonogenicity. Results: SRB assays, clonogenic (long-term) assays, and scratch assays reveal a significant anticancer activity of these two agents at low μΜ levels with a selective activity against melanoma cells. Furthermore, Western blot experiments indicate that both 6 and 7 induce LC3 accumulation, procaspase 3 decrease, and PARP cleavage, suggesting the implication of multiple death pathways in their anticancer mechanism of action. Conclusions: This study sheds light on the in vitro anticancer potential of two novel 1-(2-aryl-2-adamantyl)piperazine derivatives. It highlights their differential activity against melanoma and emphasizes their potential as lead candidates for further therapeutic exploration. Full article

In November 2024, an outbreak of highly pathogenic avian influenza (HPAI) H5N1 was confirmed in backyard poultry in Timiș County, Western Romania. The index cases involved chickens and domestic geese found dead with lesions characteristic of HPAI. Laboratory confirmation was achieved by real-time RT-qPCR targeting the matrix, H5, and N1 genes, followed by virus isolation in embryonated specific-pathogen-free eggs. Sequencing of the hemagglutinin cleavage site revealed the multi-basic motif PLREKRRKR/GLFG, consistent with a highly pathogenic phenotype. To investigate potential viral persistence, tracheal and cloacal swabs were collected from apparently healthy selected backyard poultry (chickens, geese, ducks and pheasants). RNA extraction and RT-qPCR, performed using protocols validated by the European Union Reference Laboratory for Avian Influenza, yielded negative results for all samples. Internal controls confirmed assay reliability, excluding the possibility of PCR inhibition. The investigation confirmed the occurrence of HPAI H5N1 in backyard poultry and demonstrated the absence of detectable viral persistence in surrounding flocks under the tested conditions. These findings highlight the importance of rapid molecular diagnostics, active surveillance, and strict biosecurity in limiting virus spread. Continued monitoring under the One Health framework is essential to mitigate the risk of avian influenza at the human–animal–environment interface. Full article

Aortic valve (AV) disease affects about 5% of the aging population, with AV replacement as the only treatment option. Histopathology indicates that accumulation of extracellular matrix (ECM) proteoglycans correlates with dysfunctional AVs. Proteoglycan content is controlled by ECM proteolytic cleavage, with the cleaved and intact forms of the proteoglycan Versican (VCAN) occupying different cell lineage-specific regions throughout AV development. To test the hypothesis that VCAN cleavage is required for lineage specific cell behaviors and ECM stratification, the cardiac neural crest (CNC) lineage was traced in mice with global inactivation of the proteoglycan protease Adamts5. By mid-gestation, Adamts5^−/− mice exhibited disorganized CNC patterning with excess VCAN and enlarged semilunar valve (SLV) morphology. Use of the Adamts5 floxed mice indicated that Adamts5 was required in the endothelial cells and their mesenchymal derivatives (EndoMT lineage) to prevent VCAN accumulation, initiate ECM stratification, and promote normal SLV morphology. These data suggest that the ECM remodeling event of VCAN cleavage may orchestrate cell lineage distinct behaviors and interactions to control proteoglycan levels throughout AV development and to prevent disease. Understanding mechanisms that regulate VCAN content may lead to the discovery of effective pharmacological targets for the treatment of AV disease. Full article

This study systematically compares the crystal properties of fluorite (CaF₂) and dolomite [CaMg(CO₃)₂] through first-principle calculations. Density functional theory (DFT) simulations revealed fundamental differences in structural and electronic characteristics: fluorite exhibits purely ionic Ca-F bonds (Mulliken population: 0.08) with a wide bandgap; whereas dolomite demonstrates a hybrid bonding nature featuring ionic Ca-O/Mg-O bonds (populations: 0.09/0.18) and covalent C-O bonds (0.86), which are accompanied by a narrower bandgap. The charge density and density of states (DOS) analyses demonstrated fluorine’s dominant electronic reactivity in fluorite (F 2p states near Fermi level) versus the oxygen/calcium activity in dolomite. Cleavage studies identify preferential fracture planes, with fluorite’s {111} plane exhibiting higher unsaturated bond density (14.76 nm⁻¹) than dolomite’s {104} plane (10.55 nm⁻¹), which correlates with their distinct mechanical processing behaviors. This work establishes a theoretical foundation for developing selective separation strategies by exploiting crystal-specific surface properties. Full article

Topoisomerase 1 (Top1) removes transcription-related helical torsions and thus plays an important role in preventing genome instability instigated by the formation of non-canonical DNA secondary structures. The genetically tractable Saccharomyces cerevisiae model proved effective in defining the critical function of Top1 to prevent recombination and chromosomal rearrangement at G4-forming genomic loci and studying the human cancer-associated Top1 mutants through the expression of analogous yeast mutants. We previously showed that cleavage-defective Top1 mutants strongly elevate the rate of recombination at G4 DNA, which involves binding to G4 DNA and interaction with the protein nucleolin (Nsr1 in yeast). Here, we further explored the mechanism of genome instability induced by the yeast Top1Y740* mutant, analogous to the human Top1W765Stop mutant conferring resistance to CPT. We show that yTop1Y740* elevates duplications as well as recombination specifically at G4-forming sequences. Interestingly, SUMOylation of yTop1Y740*, which does not affect the G4 DNA-binding or Nsr1-interaction by this mutant, is necessary for such elevated G4-specific genome instability. Many tumors with mutations at the C-terminal residues of Top1, particularly W765, have significantly high G4-associated mutations, underscoring the importance of further investigation into how SUMOylation affects the function of these Top1 mutants at G4 DNA. Full article

Nucleic acid-based gene-interfering molecules, such as antisense oligonucleotides, ribozymes, and small interfering RNA (siRNA), represent exciting gene-targeting agents for therapeutic applications. RNase P ribozymes derived from M1 RNA, the catalytic RNA subunit of RNase P in Escherichia coli, have shown great promise as a novel nucleic acid-based gene interference approach to modulate gene expression. When M1 RNA is covalently linked to a guide sequence (GS), it can be engineered into a sequence-specific endonuclease M1GS ribozyme, which can hydrolyze any mRNA that base-pairs with the guide sequence. M1GS activity enhancement has been achieved through an in vitro selection process that introduced mutations into M1 RNA. This selection process generated ribozyme variants with improved cleavage efficiency and substrate affinity. Hepatitis B virus (HBV) chronically infects more than 250 million people worldwide and is the leading cause of cirrhosis and liver cancer globally. Current FDA-approved drugs cannot completely eliminate HBV chronic infections. RNase P ribozymes have recently been demonstrated to effectively inhibit HBV gene expression and replication in human cells. This review summarizes the recent progress in using RNase P ribozymes to inhibit HBV infection and discusses prospects for developing engineered RNase P ribozymes for therapeutic applications against HBV infection and associated diseases. Full article

The rapid evolution of CRISPR technology has revolutionized molecular biology, and among the various systems, CRISPR/Cas12a stands out for its high specificity and efficient collateral cleavage activity. This review article focuses on the recent advancements and applications of split technology within the CRISPR/Cas12a framework, highlighting its transformative role in molecular diagnostics and biosensing. Split technology innovatively divides functional nucleic acid components into modular segments that are activated by specific targets, significantly enhancing the specificity and sensitivity of biosensors. This design addresses the inherent limitations of traditional sensor systems, enabling the direct detection of ultrashort nucleic acids and improved discrimination of single-nucleotide variants, thereby facilitating the simultaneous detection of multiple biomolecules. The versatility of split-enabled biosensors extends beyond genetic testing, making them valuable tools in diagnostics, therapeutics, and environmental science. Despite challenges such as crRNA degradation and reassembly kinetics, ongoing research and engineering solutions continue to enhance the stability and performance of these systems. This review synthesizes the foundational principles, recent advancements, and potential applications of split technology while also identifying challenges and opportunities for future exploration. Ultimately, our insights provide a comprehensive resource to leverage the full potential of CRISPR/Cas12a-based split technology in advancing biosensing methodologies and clinical applications. Full article

Chitosanases are glycoside hydrolases (GHs) that catalyze the endo- or exo-type cleavage of β-1,4-glycosidic linkages in chitosan, enabling the selective production of chitooligosaccharides (COSs) with well-defined structures and diverse bioactivities. Owing to their substrate specificity and environmentally friendly catalytic action, chitosanases have garnered increasing attention as sustainable biocatalysts for COS production, with broad application potential in agriculture, food, medicine, and cosmetics. This review provides a comprehensive overview of recent advances in chitosanase research, focusing on the catalytic mechanisms and structure–function relationships that govern substrate selectivity and functional divergence across different GH families. Microbial diversity and heterologous expression systems for chitosanase production are discussed in parallel with biochemical characterization to support the rational selection of enzymes for specific biotechnological applications. Advances in protein engineering and computational approaches are highlighted as strategies to improve catalytic efficiency, substrate range, and stability. In addition, bioprocess optimization is addressed, with emphasis on fermentation using low-cost substrates and the application of immobilized enzymes and nano-biocatalyst systems for green and efficient COS production. Summarizing and discussing previous findings are essential to support future research and facilitate the development of next-generation chitosanases for sustainable industrial use. Full article

Type I restriction-modification (RMI) systems play a crucial role in bacterial defense against mobile elements by distinguishing self and foreign DNA through sequence-specific methylation and cleavage. Here, we characterize BlihIA, a novel RMI system from Bacillus licheniformis DSM13 which features redundancy in its hsdS gene copies. Using ONT sequencing, we identify the bipartite recognition site of BlihIA as RTAC(N)₅GCT. We demonstrate the system’s activity both in vivo through efficiency of plaquing (EOP) assay and in vitro in a nuclease reaction with purified BlihIA complex. Notably, mutation of the recognition site abolished in vitro DNA cleavage, confirming sequence specificity. Furthermore, we show that the antirestriction protein ArdB from plasmid R64 effectively prevents DNA cleavage by BlihIA, suggesting a direct mechanism of inhibition. This study provides the first functional characterization of a novel RM system BlihIA, extending the diversity of RM systems in Bacillus species and suggesting potential applications for improving genetic transformation in industrial strains. Full article