Search Results (140)

Agrobacterium tumefaciens, a destructive pathogen causing crown gall disease, results in substantial agricultural losses. Traditional chemical and existing biocontrol methods are limited by environmental pollution, pesticide resistance, and low efficacy, while bacteriophages emerge as a promising alternative due to their high host specificity, environmental compatibility, and low resistance risk. In this study, we isolated and characterized a lytic phage (PAT-A) targeting A. tumefaciens, evaluating its biological traits, genomic features, and biocontrol potential. The host strain A. tumefaciens CL-1 was isolated from cherry crown gall tissue and identified by 16S rDNA sequencing. Phage PAT-A was recovered from orchard soil via the double-layer agar method, showing a tadpole-shaped morphology (60 nm head diameter, 30 nm tail length) under transmission electron microscopy (TEM). Nucleic acid analysis confirmed a double-stranded DNA genome, susceptible to DNase I but resistant to RNase A and Mung Bean Nuclease. PAT-A exhibited an optimal MOI of 0.01, tolerated wide pH and temperature ranges, but was sensitive to UV (titer declined after 15 min of irradiation) and chloroform (8% survival at a 5% concentration). Whole-genome sequencing revealed a 44,828 bp genome with a compact structure, and phylogenetic/collinearity analyses placed it in the Atuphduvirus genus (Autographiviridae). Biocontrol experiments on tobacco plants demonstrated that PAT-A significantly reduced crown gall incidence. Specifically, simultaneous inoculation of PAT-A and A. tumefaciens CL-1 resulted in the lowest tumor incidence (12.0%), while pre-inoculation of PAT-A 2 days before pathogen exposure achieved an incidence rate of 33.3%. In conclusion, PAT-A is a novel strictly lytic phage with favorable biological properties and potent biocontrol efficacy against A. tumefaciens, enriching phage resources for crown gall management and supporting phage-based agricultural biocontrol strategies. Full article

ATP hydrolysis drives essential processes across biology, from nucleic acid translocation and conformational switching to signal transduction. The GHKL ATPase family—DNA Gyrase B, Heat Shock Protein 90 (Hsp90), Histidine Kinases, and MutL homologs—shares a Bergerat-fold that couples nucleotide binding and hydrolysis to conformational changes, dimerization, and signaling. Despite their diverse roles, GHKL proteins rely on common ATP-dependent principles. Within this family, MutLα (MLH1-PMS2 in humans, Mlh1-Pms1 in yeast) is central to eukaryotic mismatch repair, where it provides the endonuclease activity needed for strand incision and coordinates interactions with other repair partners. MutLα exemplifies how the Bergerat-fold has been adapted to regulate DNA interactions, partner communication, and protein turnover on DNA. By examining MutLα through the lens of other GHKL proteins, we can clarify how ATP binding and hydrolysis drive its conformational dynamics, nuclease activation, and regulation within its pathway, highlighting how conserved mechanistic strategies are repurposed across biological systems. Full article

Leuconostoc mesenteroides is a key microorganism in food biotechnology, valued for its production of flavor-forming metabolites and exopolysaccharides, and its inclusion in starter cultures and biocatalytic systems. However, the application of advanced genetic tools to L. mesenteroides remains hindered by multiple barriers, including inefficient DNA transfer, elevated endogenous nuclease activity, and restriction–modification systems sensitive to plasmid methylation patterns. As a result, even widely accepted electroporation methodologies often yield inconsistent or irreproducible transformation results, limiting the strain’s amenability to metabolic engineering and synthetic biology applications. In this study, a reproducible electroporation protocol for the L. mesenteroides strain H32-02 Ksu is developed and experimentally validated. The protocol concept relies on the sequential optimization of key process steps: targeted weakening of the cell wall followed by osmotic protection, the development of a gentle electrical stimulus that ensures membrane permeability without critical damage, and the creation of recovery conditions that minimize loss of viability and degradation of incoming DNA. Matching plasmid methylation to the recipient’s restriction profile proved critical: choosing a source for plasmid DNA production with a compatible methylation pattern dramatically increased the likelihood of successful transformation. In our case, the selection of an E. coli strain with a more suitable methylation profile increased the yield of transformants by 3.5 times. It was also shown that reducing the pulse voltage increase transformant number by 3 times. The combined optimization resulted in an approximately 40-fold increase in transformation efficiency compared to the baseline level and, for the first time, provided consistently reproducible access to transformants of this strain. The highest transformation efficiency was achieved: 8 × 10² CFU µg⁻¹ DNA. The presented approach highlights the strain-specificity of barriers in Leuconostoc and forms a technological basis for constructing strains with desired properties, expressing heterologous enzymes, and subsequently scaling up bioprocesses in food and related industries. The methodological principles embodied in the protocol are potentially transferable to other lactic acid bacteria with similar limitations. Full article

Antisense inhibition of gene expression is usually achieved using nuclease-resistant oligonucleotide analogs that promote mRNA degradation through RNase H or RNase P, or by steric hindrance of translation. Bridge nucleic acids (BNAs) are nucleotide analogs available in a few chemical variants. We evaluated gapmers composed of an oligodeoxynucleotide flanked by BNA residues in a BNA₅-DNA₈-BNA₄ configuration, using the available variants: the original locked nucleic acid (LNA; 2′-O,4′-methylene bridge), cET (2′-O,4′-ethyl bridge), cMOE (2′-O,4′-methoxyethyl bridge), and BNANC (2′-O,4′-aminomethylene bridge). These gapmers were tested in vitro for their ability to induce cleavage of the model aac(6′)-Ib mRNA. All gapmers complementary to a previously identified region suitable for interaction with antisense oligomers induced RNase H-mediated degradation. Instead, only the LNA-containing gapmer also elicited RNase P-dependent cleavage, demonstrating dual RNA- and DNA-mimicking capability. In vitro coupled transcription–translation assays using cell lysates or reconstituted systems confirmed inhibition of expression and ruled out steric hindrance as the mechanism. In contrast, gapmers targeting the ribosome-binding site strongly inhibited expression by steric hindrance. These findings demonstrate that LNA-containing gapmers can exert their effects through multiple mechanisms, depending on the targeted mRNA region, thereby supporting their potential for synergistic inhibition of gene expression. Full article

Background/Objectives: Heterofunctional cationic polyester dendrimers derived from a 2-(bromomethyl)-2-(hydroxymethyl)propane-1,3-diol (BHP-diol) based AB₂C monomer were evaluated as efficient and biodegradable nonviral carriers for siRNA delivery. Methods: These dendrimers feature dual internal and external charge architectures, enabling precise control of charge distribution and siRNA interaction strength. Results: They achieved complete siRNA complexation at nitrogen-to-phosphate (N/P) ratios of 0.50–2.14 and provided up to 93% RNase protection, outperforming amino-functional scaffolds based on 2,2-bis(methylol)propionic acid (bis-MPA). In human (T98G) and murine (GL261) glioblastoma cells, the dendrimers exhibited minimal cytotoxicity while achieving 52–61% target protein knockdown, a two- to three-fold improvement over conventional polyester dendrimers, and approaching the silencing efficiency of the commercial Interferin^® reagent. Conclusions: The combination of high complexation efficiency, strong nuclease resistance, and excellent biocompatibility establishes these heterofunctional dendrimers as a new generation of precisely tunable, biodegradable vectors for therapeutic siRNA delivery. Full article

Long-duration space missions expose astronauts to galactic cosmic radiation (GCR), a complex spectrum of high-charge, high-energy (HZE) ions that pose significant risks of chronic tissue injury. To model these effects, we examined intestinal outcomes in wild-type mice 5 months after low-dose (50 cGy) 33-ion mixed-field GCR simulation (GCRsim). GCRsim induced sustained DNA double-strand breaks (DSBs) and oxidative stress, as shown by elevated γH2AX foci and 4-HNE staining. Intestinal epithelial cells (IECs) exhibited pronounced senescence, marked by increased SA-β-gal activity, p16 upregulation, LaminB1 loss, and induction of senescence-associated secretory phenotype (SASP) cytokines (Cxcl10, IL-6, IL-1β, Icam1). GCRsim also elevated circulating LINE-1 DNA and reduced expression of DNA-degrading nucleases (DNase2, TREX1), indicating impaired extracellular DNA clearance. Targeted molecular study revealed persistent activation of the cGAS–STING pathway, with elevated cGAS, STING, pTBK1, pIKKα/β, and nuclear pIRF3, pIRF7, and p65, consistent with chronic innate immune signaling. Functionally, GCRsim altered nutrient absorption gene expression—upregulating glucose transporters (Slc2a2, Slc2a5, Slc5a1) and gut hormones (Cck, Gip), while downregulating cholesterol/fat transporters (Npc1, Npc1l1). Biochemical markers supported intestinal injury, with decreased serum citrulline and increased intestinal fatty acid-binding protein (I-FABP), indicating barrier compromise. Collectively, these findings demonstrate that GCRsim drives sustained intestinal dysfunction, highlighting the need for countermeasures to protect GI health during deep-space missions. Full article

The cGAS-STING pathway initiates the core cascade of innate immune defense by recognizing pathogen-associated and self-derived abnormal nucleic acids, and key molecules (such as cGAS, STING, downstream IFN-β, IL-6, etc.) may serve as biomarkers in various diseases. The diverse mechanisms by which distinct nucleic acids activate this pathway provide novel insights for therapeutic strategies targeting infectious diseases, cancer, and autoimmune disorders. To prevent aberrant cGAS-STING pathway activation, cells employ multiple regulatory mechanisms, including restricting self-DNA recognition and terminating downstream signaling. Strategies to mitigate pathological activation involve reducing nucleic acid accumulation through nuclease degradation (e.g., of mitochondrial DNA or neutrophil extracellular traps, NETs) or directly inhibiting cGAS or STING. This review elucidates the molecular mechanism of nucleic acid-mediated regulation of cGAS-STING and its role in disease regulation. Full article

Pest management is undergoing a transformative shift with the development of the cutting-edge antisense technologies: RNA interference (RNAi), contact unmodified antisense DNA biotechnology (CUADb), and the CRISPR-associated proteins (CRISPR/Cas). These approaches function by facilitating sequence-specific pairing of nucleic acids followed by nuclease-mediated cleavage, offering exceptional precision for targeted pest control. While RNA-guided mechanisms such as RNAi and CRISPR/Cas were initially characterized in non-insect systems, primarily as innate defenses against viral infections, the DNA-guided CUADb pathway was first identified in insect pests as a functional pest control strategy. Its broader role in ribosomal RNA (rRNA) biogenesis was recognized later. Together, these discoveries have revealed an entirely new dimension of gene regulation, with profound implications for sustainable pest management. Despite sharing a common principle of sequence-specific targeting RNAi, CUADb, and CRISPR/Cas differ in several key aspects, including their mechanisms of action, target specificity, and applicability. Rather than serving as universal solutions, each technology is likely to be optimally effective against specific pest groups. Moreover, these technologies allow for rapid adaptation of control strategies to overcome target-site resistance, ensuring long-term efficacy. This review summarizes the core functional characteristics, potential applications, and current limitations of each antisense technology, emphasizing their complementary roles in advancing environmentally sustainable pest control. By integrating foundational biological discoveries with applied innovations, this work provides a new perspectives on incorporating antisense-based strategies into next-generation integrated pest management systems. Full article

Aptamers have high specificity and affinity to target analytes, along with good stability and low cost, making them widely used in the detection of target substances, especially in the increasingly popular aptamer-based electrochemical biosensors. Aptamer-based electrochemical biosensors are composed of aptamers as the biorecognition elements and sensors that convert the biological interactions into electrical signals for the quantitative detection of targets. To detect low-abundance target substances, the improvement of the sensitivity of biosensors is a pursuit of researchers. Therefore, different amplification strategies for significantly enhancing the detection sensitivity of biosensors have been explored. Thus, this paper reviews the different amplification strategies with various functional materials to amplify the detection signals. Currently, such strategies commonly use gold nanoparticles to construct electrodes that facilitate the transfer of biological reactions or to obtain enhanced signals through nucleic acid amplification. Some strategies use nucleases for target recycling to further enhance the signals. This review discusses the recent progress in signal amplification methods and their applications, and proposes future directions of study to guide subsequent researchers in overcoming the limitations of previous approaches and to produce reproducible biosensors for clinical applications. Full article

In recent years, global public health security has encountered significant challenges, with infectious diseases accounting for approximately 25% of global mortality annually. The worldwide pandemic instigated by the novel coronavirus, alongside the persistent threats posed by Ebola, influenza, and multidrug-resistant bacteria, has severely compromised human health, economic development, and social stability. Within this context, the development of rapid and precise pathogen detection technologies has emerged as a critical frontline defense for epidemic prevention and control, serving as a pivotal component in the implementation of the “early detection, early isolation, and early treatment” strategy. The Argonaute (Ago) protein, recognized as a programmable and target-specific activated nuclease, has demonstrated substantial potential in the realm of nucleic acid detection due to its distinctive biological properties, garnering considerable attention. In this study, we delineate the structural characteristics of Ago proteins and elucidate the mechanism underlying their nuclease activity. Furthermore, we review the principles of nucleic acid detection based on Argonaute and provide a comprehensive analysis of recent advancements in related detection systems. Additionally, we compare the advantages of detection based on Argonaute with other detection methodologies. Through a comprehensive analysis, we aim to provide a robust theoretical foundation and an advanced technical reference for the development of new-generation nucleic acid detection platforms with high sensitivity and high specificity. Full article

Nucleases are critical metabolic enzymes expressed by mycoplasmas to acquire nucleic acid precursors from the host for their parasitic existence. Certain nucleases, either membrane-bound or secreted, not only contribute to the growth of mycoplasmas but also serve as key virulence factors due to their unique spatial structures and physiological activity. The pathogenesis includes, but is not limited to, degradation of host DNA and RNA, leading to disruptions of nucleic acid metabolism and the induction of host cell apoptosis; degradation of neutrophil extracellular traps (NETs), allowing escape from neutrophil-mediated killing; and upregulation of inflammatory molecules to modulate the immune response of the host. Understanding the biological functions of nucleases is essential for gaining deeper insights into the virulence and immune evasion strategies of mycoplasmas, which can inform the development of novel approaches for the prevention, diagnosis, and treatment of mycoplasma infections. Full article

The Saccharomyces cerevisiae chromosomal architectural protein Hmo1 is categorized as an HMGB protein, as it contains two HMGB motifs that bind DNA in a structure-specific manner. However, Hmo1 has a basic C-terminal domain (CTD) that promotes DNA bending instead of an acidic one found in a canonical HMGB protein. Hmo1 has diverse functions in genome maintenance and gene regulation. It is implicated in DNA damage tolerance (DDT) that enables DNA replication to bypass lesions on the template. Hmo1 is believed to direct DNA lesions to the error-free template switching (TS) pathway of DDT and to aid in the formation of the key TS intermediate sister chromatid junction (SCJ), but the underlying mechanisms have yet to be resolved. In this work, we used genetic and molecular biology approaches to further investigate the role of Hmo1 in DDT. We found extensive functional interactions of Hmo1 with components of the genome integrity network in cellular response to the genotoxin methyl methanesulfonate (MMS), implicating Hmo1 in the execution or regulation of homology-directed DNA repair, replication-coupled chromatin assembly, and the DNA damage checkpoint. Notably, our data pointed to a role for Hmo1 in directing SCJ to the nuclease-mediated resolution pathway instead of the helicase/topoisomerase mediated dissolution pathway for processing/removal. They also suggested that Hmo1 modulates both the recycling of parental histones and the deposition of newly synthesized histones on nascent DNA at the replication fork to ensure proper chromatin formation. We found evidence that Hmo1 counteracts the function of histone H2A variant H2A.Z (Htz1 in yeast) in DDT possibly due to their opposing effects on DNA resection. We showed that Hmo1 promotes DNA negative supercoiling as a proxy of chromatin structure and MMS-induced DNA damage checkpoint signaling, which is independent of the CTD of Hmo1. Moreover, we obtained evidence indicating that whether the CTD of Hmo1 contributes to its function in DDT is dependent on the host’s genetic background. Taken together, our findings demonstrated that Hmo1 can contribute to, or regulate, multiple processes of DDT via different mechanisms. Full article

Nucleic-acid-based therapies have emerged as a pivotal domain within contemporary biomedical science, marked by significant advancements in recent years. These innovative treatments primarily operate through the precise binding of DNA or RNA molecules to discrete target genes, subsequently suppressing the expression of the target proteins. The spectrum of nucleic-acid-based therapies encompasses antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs), etc. Compared to more traditional medicinal approaches, nucleic-acid-based therapies stand out for their highly targeted action on specific genes, as well as their potential for chemical modification to improve resistance to nucleases, ensuring sustained therapeutic activity and mitigating immunogenicity concerns. Nevertheless, these molecules’ limited cellular permeability necessitates the deployment of delivery vectors to enhance their intracellular uptake and stability. As nucleic-acid-based therapies progressively display promising pharmacodynamic profiles, there has been a burgeoning interest in these treatments for applications in clinical research. This review aims to summarize the variety of nucleic acid drugs and their mechanisms, evaluate the present status in research and application, discourse on prospective trends, and potential challenges ahead. These innovative therapeutics are anticipated to assume a pivotal role in the management of a wide array of diseases. Full article

CRISPR-Cas is an adaptive immune system found in bacteria and archaea that provides resistance against invading nucleic acids. Elements of this natural system have been harnessed to develop several genome editing tools, including CRISPR-Cas9. This technology relies on the ability of the nuclease Cas9 to cut DNA at specific locations directed by a guide RNA. In addition, the nuclease activity of Cas9 requires the presence of a short nucleotide motif (5′-NGG-3′ for Cas9 from Streptococcus pyogenes) called PAM, flanking the targeted region. As the reliance on this PAM is typically strict, diverse Cas9 variants recognising different PAM motifs have been studied to target a broader range of genomic sites. In this study, we assessed the potential of Cas9 from Streptococcus mutans strain P42S (SmutCas9) in gene editing. SmutCas9 recognises the rarely targeted 5′-NAA-3′ and 5′-NGAA-3′ PAMs. To test its efficacy, two genes of the virulent lactococcal phage p2 were edited, thereby demonstrating the potential of SmutCas9 for gene editing purposes, particularly in AT-rich genomes. Sequencing of total RNA also revealed the RNA components of this system, allowing further molecular characterisation of the type II-A CRISPR-Cas system of S. mutans. Full article

The extracts from fruits of Chaenomeles japonica (Thunb.) Lindl. ex Spach (CJE), Cornus mas L. (CME), and Hippophaё rhamnoides L. (HRE) are known inhibitors of a variety of eukaryotic hydrolases, engaged in the digestion of fats and polysaccharides. However, there are no data on their potential interaction with the bacterial hydrolases participating in the replication of microbial nucleic acids. This analysis predicted the interaction of the most abundant constituents of HRE, CJE, and CME with the bacterial nucleases. The analysis covered the molecular docking of isorhamnetin glycosides, procyanidins C1 and B2, epicatechin, loganic acid, and cornuside with bacterial enzymes (Escherichia coli endonuclease 1, colicin E9, and ribonuclease H; or Staphylococcus aureus thermonuclease and nuclease SbcCD). The suggested complexes have been subjected to molecular mechanics with generalized Born and surface area solvation (MM/GBSA) calculations. The second aim was the in vitro evaluation of the influence of the CJE, HRE, and CME on the metabolic activity of bacterial biofilm of selected microbial strains, as well as fibroblasts (L929) and adenocarcinoma intestinal cells (Caco-2) toxicity. Among all extracts, CME showed the most relevant effect on the survival of planktonic cells and biofilm of E. coli and Pseudomonas aeruginosa. As a result of in silico studies, most virtual hits were predicted to inhibit the proteins under investigation, except for procyanidin C1. Further research on the direct interaction of phytochemicals and selected enzymes in vitro is required and challenged. Full article