Search Results (2,871)

Alternaria blight, caused by fungi of the genus Alternaria, is one of the most common and damaging diseases affecting tomatoes, leading to significant yield losses. The intensive use of chemical fungicides faces the problems of pathogen resistance development and negative environmental impacts. This study investigated the possibility of using RNA interference technology based on exogenous double-stranded RNAs (dsRNAs) to protect tomatoes against the causal agent of early blight (EB), Alternaria alternata. Key genes of the pathogen A. alternata were selected as targets: Alt-a1 (a major allergen and virulence factor), TEF1a (translation elongation factor 1-alpha) and β-Tub (β-tubulin). Specific dsRNAs were synthesized in vitro and applied to tomato plants (Solanum lycopersicum L. cv. Micro-Tom) simultaneously with inoculation of A. alternata strain C7.24-T2-L-F1 spores. Visual assessment, measurement of chlorophyll A and B, and real-time quantitative PCR analysis showed that treatment with dsRNAs targeting the Alt-a1, TEF1a and β-Tub genes significantly suppressed infection development, reducing the amount of pathogen DNA in plant tissues by 7 to 27 times depending on the dsRNA type. The most effective was dsRNA to the Alt-a1 gene. Thus, the obtained results demonstrate the promise of spray-induced gene silencing (SIGS) as a strategy for protecting tomato plants against the pathogen A. alternata. Full article

Cancer represents a major global health challenge, contributing to an estimated 19 million new cases annually. While conventional chemotherapeutic approaches continue to advance, target-based therapeutic strategies are increasingly recognized as effective pathways in modern drug development. A prominent biological target in current anticancer research is the selective inhibition of Topoisomerase II alpha (TOP2A). TOP2A, a crucial DNA topoisomerase, is vital for maintaining genomic integrity by mediating the cleavage and re-ligation of double-stranded DNA during essential cellular processes, such as DNA replication and transcription. Inhibiting TOP2A effectively disrupts these processes, leading to cell death. This study employed computer-aided drug design approaches to virtually screen a library of 3000 xanthone derivatives against the TOP2A target, and the results were preliminarily validated through cytotoxicity assays on the A549 and HepG2 cancer cell lines. The computational methods utilized included molecular docking, pharmacological modeling, molecular dynamics simulations, and steered molecular dynamics simulations. The virtual screening identified two highly promising HIT compounds, CID162372098 and CID156619937, that exhibited the most favorable interactions and stability profiles in relation to the TOP2A active site. The experimental results demonstrated that both hit compounds effectively exhibited significant anti-proliferative activities against the HepG2 cell line, with IC50 values of 9.54 ± 0.26 µg mL⁻¹ (CID162372098) and 10.03 ± 0.36 12.69 ± 0.31 µg mL⁻¹ (CID156619937), respectively. Collectively, these findings demonstrate the potential of xanthone-based scaffolds as inhibitors of TOP2A and provide a rational framework for the screening and development of novel anticancer agents. Full article

Virophages are satellite viruses that depend on the replication machinery of giant double-stranded DNA viruses and influence the structure and dynamics of viral communities through multilayered interactions among giant viruses, their hosts, and virophages. Since the discovery of the Sputnik virophage in 2008, virophages have been increasingly recognized for their roles in regulating giant virus replication, contributing to host defense mechanisms, and shaping the evolution of mobile genetic elements. However, quantitative syntheses examining how virophage research has developed over time, particularly in marine environments, remain limited. Here, we conducted a bibliometric analysis of virophage research published between 2008 and 2025 using the Web of Science Core Collection. By comparing an overall virophage research corpus with a marine virophage sub-corpus, we assessed publication and citation trends, collaboration structures, and keyword-based intellectual and thematic evolution. Our results show that virophage research has gradually transitioned from an early phase dominated by landmark discoveries and experimental model systems to a data-intensive stage driven by genome- and metagenome-based analyses and computational approaches. Although marine virophage studies represent a relatively small proportion of the total literature, they exhibit sustained citation impact and form a distinct research axis within the field. In particular, marine-focused studies emphasize metagenomic discovery, genome sequence alignment, and the analysis of mobile genetic elements such as polinton-like viruses, highlighting the role of marine environments in accelerating the intellectual transition of virophage research. Collectively, these findings demonstrate that virophage research has moved beyond a “discovery and definition” phase toward data-driven integrative interpretation, with marine virophage research emerging as a key domain for understanding the structure and evolutionary dynamics of marine viral ecosystems. Full article

The use of proton beam therapy (PBT), as a more precision-targeted radiotherapy technique, is increasing in the treatment of head and neck squamous cell carcinoma (HNSCC). PBT benefits from the precise delivery of the radiation dose to the tumour via the Bragg peak. However, challenges still remain in the treatment of HNSCC with radiotherapy, particularly with tumour radioresistance and recurrence, requiring strategies leading to radiosensitisation. There are added complexities with the use of PBT given the increase in linear energy transfer (LET) at and around the Bragg peak, which can cause an altered cellular response compared to low-LET radiation. Nevertheless, targeting the cellular DNA damage response is considered an important strategy to enhance tumour cell killing caused by radiotherapy. Therefore, using specific inhibitors against the protein kinases ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and the DNA-dependent protein kinase catalytic subunit (DNA-Pkcs), we investigated their impact in radiosensitising HPV-negative HNSCC cells to PBT of increasing LET. We demonstrate that inhibitors against ATR (AZD6738), and particularly ATM (AZD1390) and DNA-Pkcs (AZD7648), could significantly decrease clonogenic survival of HNSCC cell lines following PBT at both low and relatively high LET (~2 keV/µm and ~8 keV/µm, respectively). We confirmed that the inhibitors in combination with PBT led to DSB persistence through neutral comet assays and monitoring γH2AX/53BP1 foci. We also show that this strategy can enhance the sensitivity of patient-derived organoids of HNSCC to PBT of both low and high LET, highlighting this as a strategy which should be exploited further. Full article

The reaction of the HS^•/S^•− radical (pKa ~3.4), generated selectively from H₂S by γ-irradiated N₂-flushed aqueous solutions at pH 5, with purine nucleosides (dG or dA), a 10-mer double-stranded oligodeoxynucleotide (ds-ODNs), and calf thymus (ct) DNA was investigated, under various experimental conditions. Concurrent quantification of the four purine 5′,8-cyclo-2′-deoxynucleosides (cPu) and two 8-oxo-7,8-dihydro-2′-deoxypurines (8-oxo-Pu) by LC-MS/MS analysis using isotopomeric internal standards was achieved. The formation of 8-oxo-Pu is several tens of times larger than cPu. Mechanistic schemes for the formation of the two product groups are proposed. Hydrogen atom abstraction from C5′–H by S^•− produces the cPu via cyclization of the C5′ radical onto C8, forming a new covalent bond, C5′–C8. The unexpected formation of 8-oxo-Pu should be mechanistically more complex. We propose that an S^•− (coupled with H⁺) adds to the base rings, followed by the elimination of HS⁻ to form the corresponding radical cation; subsequent reactions with H₂O and radical disproportionation with another S^•− lead to 8-oxo-Pu. A comparison of S^•− with the available literature data for HO^• reactivity towards ct-DNA in de-oxygenated aqueous solutions is also presented. Before the present findings, cPu lesions were attributed exclusively to HO^• reactivity toward ct-DNA. The reaction of the thiyl radical (HOCH₂CH₂S^•) with ct-DNA was also investigated, yielding results similar to those of S^•− obtained under comparable experimental conditions. Our results contributed to a better understanding of DNA damage induced by reactive sulfur species (RSS), particularly the formation of purine lesions and the relative abundance of cPu versus 8-oxo-Pu. Full article

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, with anti-double-stranded DNA (anti-dsDNA) antibodies as its serological biomarkers. However, conventional anti-dsDNA antibody detection methods, which mainly rely on antibody-binding assays, often suffer from limited sensitivity and specificity, cumbersome procedures, and poor suitability for accurate clinical analysis. Herein, we developed an integrated detection system combining a circularly permuted fluorescent protein (cpFP)-based biosensor with a microfluidic chip for rapid and reliable anti-dsDNA antibody detection. The biosensor, cpR-dsAb-C1, was engineered from mApple by inserting an affinity peptide identified through phage display, enabling specific recognition of the variable region of anti-dsDNA antibodies. The biosensor exhibited good sensitivity, specificity, and anti-interference capability. Furthermore, integration of cpR-dsAb-C1 with a polydimethylsiloxane (PDMS)-based microfluidic chip yielded a microfluidic detection platform with good linearity for rapid antibody analysis. Clinical validation showed significantly higher anti-dsDNA antibody levels in patients with SLE than in healthy controls, and the results were consistent with those obtained using routine clinical methods, with an accuracy exceeding 95%. Overall, this system provides a promising low-cost, efficient, and accurate strategy for the early diagnosis and dynamic monitoring of SLE. Full article

Spodoptera litura is a highly destructive agricultural pest with increasing reliance on chemical insecticides. We aimed to develop nanotechnology-enabled strategies that enhance insecticide efficacy against S. litura and reduce chemical inputs. To this end, SlChitinase5 was identified and characterized as a potential RNAi target. This gene contains conserved domains typical of lepidopteran chitinases and is highly expressed during key developmental stages, including larval molting and the prepupal phase. RNAi-mediated suppression of SlChitinase5 through larval injection of double-stranded RNA (dsSlChitinase5) significantly reduced body weight, increased mortality, and disrupted molting. When RNAi-treated larvae were exposed to sublethal concentrations of emamectin benzoate (EB) or an emamectin benzoate–tebufenozide mixture (EBT), larval mortality reached 96.7% on day 5. To evaluate an alternative formulation and exposure approach, dsSlChitinase5 was incorporated into chitosan nanoparticles (CS) and applied topically. This treatment induced SlChitinase5 knockdown and, in combination with sublethal EB or EBT, resulted in complete larval mortality within five days under the conditions tested. These findings validate SlChitinase5 as a molecular target and suggest that CS-dsSlChitinase5 nanocarriers have the potential to enhance insecticide performance, which may support integrated pest management and future efforts toward reduced-input crop protection strategies. Full article

Duchenne muscular dystrophy (DMD) remains a major challenge in genetic medicine due to the difficulty of achieving durable, body-wide restoration of dystrophin in post-mitotic muscle tissues. Although current therapies—including exon skipping and micro-dystrophin gene replacement—have demonstrated clinical feasibility, their benefits are limited by incomplete efficacy, mutation specificity, and the need for repeated or high-dose interventions. These limitations highlight the need for strategies capable of directly and permanently correcting the underlying genetic defect. Recent advances in genome editing have positioned CRISPR-based technologies as promising candidates for this objective. Rather than functioning as a single approach, gene-editing platforms encompass a spectrum of strategies—including exon deletion, exon reframing, base editing, and prime editing—each with distinct advantages depending on the mutational context. In particular, the emergence of precision editing tools has enabled controlled nucleotide-level modifications, expanding the range of correctable mutations while reducing reliance on double-strand DNA breaks. In this review, we adopt a comparative and translational perspective to evaluate gene-editing strategies for DMD. We examine how different approaches align with specific mutation types, summarize key findings from preclinical studies, and analyze the major barriers to clinical implementation, including delivery efficiency, immune responses, editing durability, and genomic safety. We further discuss emerging innovations in editing technologies and delivery systems that aim to address these limitations. Collectively, this work reframes gene editing as a decision-oriented and application-driven therapeutic framework. Continued integration of advances in genome engineering, delivery platforms, and muscle biology will be essential to translate these technologies into safe, effective, and durable treatments capable of altering the clinical trajectory of DMD. Full article

This study aimed to establish an efficient and precise cytosine base editor (CBE)-mediated knockout system in chicken somatic cells and primordial germ cells (PGCs). PGCs are pivotal for generating genome-edited chickens, but low transfection efficiency limit their application. Unlike CRISPR/Cas9, CBEs achieve precise C-to-T conversion without DNA double-strand breaks or donor templates, making them safer for avian genome engineering. We used CBEs to introduce premature stop codons in exon 1 of the sex-determining AR and DMRT1 genes for targeted knockout. Among 12 screened sgRNAs, sgRNA6 (AR, 94.67 ± 6.66%) and sgRNA9 (DMRT1, 6.67 ± 6.51%) performed best in DF-1 cells; in PGCs, their editing efficiencies reached 51.0% and 91.0%, respectively. No off-target mutations were detected in edited DF-1 cells. These findings confirm that CBE-mediated knockout is highly efficient and safe in chicken somatic and germ cells, providing a robust tool for avian genome editing. Full article

Accurate analysis of prostate cancer (PC)-related biomarkers requires sensing platforms capable of sensitive and multiplex detection in complex biological environments. Herein, we propose a signal-on electrochemical aptamer-based sensor (E-AB) for the simultaneous detection of L-lactate (L-Lac) and prostate-specific antigen (PSA). To maximize analytical performance, two Lac aptamer sensing configurations, single-stranded (ssLac201) and double-stranded (dsLac201), were constructed and comparatively evaluated. The dsLac201 structure displayed more effective background suppression and enhanced target induced signal response. Under optimized conditions, the dsLac201-based sensor exhibited a wide linear range from 500 nM to 10 mM for L-Lac, with a low detection limit of 157 nM and high selectivity. Based on this optimized design, a dual-aptamer electrochemical platform was further engineered through programmable nucleic acid assembly, enabling simultaneous detection of L-Lac and PSA via dual-input signal integration. The dual-target sensor showed broad analytical ranges for both biomarkers (L-Lac: 500 nM–10 mM; PSA: 10 pg mL⁻¹–500 ng mL⁻¹) and retained promising performance in serum samples. This work demonstrates a simple and versatile strategy for multiplex electrochemical biosensing and provides a promising platform for PC-related biomarker monitoring and clinical biomedical analysis. Full article

Background: Long noncoding RNAs (lncRNAs) have emerged as critical regulators of hepatic metabolism and disease progression. The hepatocyte nuclear factor 1 alpha antisense 1 (HNF1A-AS1) lncRNA modulates liver-specific transcription factors; however, its physiological role in diet-dependent lipid homeostasis remains poorly defined. Methods: In this study, we investigated the mouse ortholog, Hnf1a opposite strand 1 (Hnf1aos1), using AAV-mediated knockdown in C57BL/6J mice fed either a chow diet (10% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) for 12 weeks. Metabolic phenotyping included hepatic lipid quantification, histological analysis, serum biochemistry, and quantitative gene expression profiling. Results: Loss of Hnf1aos1 produced distinct, diet-dependent alterations in hepatic lipid handling. Under chow conditions, knockdown mice exhibited selective hepatic cholesterol accumulation (6.10 ± 2.9 mg/g tissue vs. 3.51 ± 1.1 mg/g in controls), accompanied by dysregulation of cholesterol clearance pathways. In contrast, under HFD conditions, knockdown precipitated severe macrovesicular degeneration, with hepatic triglyceride levels approximately doubled relative to HFD-fed controls (51.72 ± 19.8 mg/g vs. 26.34 ± 11.9 mg/g) and a numerically elevated triglyceride-to-cholesterol ratio (TG:TC ≈ 6.1:1; p = 0.0621, trend). Chow/Kd mice gained significantly less weight than chow-fed controls, whereas HFD/Kd mice exhibited weight gain comparable to HFD controls despite severe hepatic steatosis. This paradoxical phenotype suggests impaired metabolic feedback at the post-transcriptional level, in which compensatory upregulation of Hnf1a mRNA is insufficient to suppress lipid-associated genes such as Cd36, despite profound lipid overload; however, HNF1A protein levels were not directly measured in this study. Conclusion: Collectively, these findings identify Hnf1aos1 as a regulator of hepatic lipid homeostasis whose loss produces a phenotype consistent with inappropriate lipid accumulation during nutrient excess, without defining the underlying molecular mechanism. Our results support a role for Hnf1aos1 in shaping hepatic metabolic plasticity and provide insight into lncRNA-associated MASLD phenotypes. Full article

We aimed to clarify how the priming dose and the experimental design could affect the development of an adaptive response (AR) induced by low-dose Zeocin (Zeo) in Saccharomyces cerevisiae strains with differing genetic constitution. Constant-field gel electrophoresis was used for measuring double-strand breaks (DSBs) induction and DNA rejoining; for microbiological experiments, Zimmermann’s test was used for measuring survival fraction and genetic events. Favorable experimental conditions for the induction of AR in both D7ts1 and 551 strains were determined: the priming dose inducing about 20% lethality or at least a 1.5-fold increased DSB level, 45 min inter-treatment time, and recovery time of 30–45 min. Both strains developed well-expressed AR, measured by increased cell survival, but differed in their ability to develop AR, measured by reduction in DSBs. This discrepancy could be due to different DSBs rejoining rather than different DNA susceptibility, and the partial contribution of DSB repair to cell survival in the split-dose experiments. The frequency of mutagenic and recombinogenic events and DSB levels were lower in split-dose treatment. The development of AR depends on several factors: the magnitude of the priming dose, DNA susceptibility, the duration of the ITT window, the duration of recovery time, as well as genetic constitution of strains. Full article

Background: Currently marketed hepatitis B vaccines are primarily recombinant protein vaccines. However, their antigen immunogenicity is relatively weak, requiring combination with effective adjuvants to enhance the immune response. The development of novel, highly effective adjuvants is a key strategy for optimizing vaccine performance. Polyinosinic-polycytidylic acid (PolyI:C), a synthetic double-stranded RNA analog, activates TLR3/RLR pathways to enhance T-cell priming and cellular immunity. However, its utility as a sole adjuvant is limited by rapid nuclease degradation and poor cytosolic delivery. Lipid nanoparticles (LNPs), a mature delivery platform, enable high encapsulation efficiency, efficient cellular uptake, and endosomal escape. Objectives: This study aimed to evaluate the adjuvant effect of LNP-encapsulated PolyI:C (LNP-PolyI:C) on the immunogenicity of hepatitis B surface antigen (HBsAg) in vivo. Methods: The colloidal stability of LNP-PolyI:C stored at 2–8 °C for 9 months was monitored using dynamic light scattering (DLS) on a Zetasizer Lab instrument. Serum levels of HBsAg-specific IgG, IgG1, and IgG2a antibodies in immunized Kunming mice were measured by enzyme-linked immunosorbent assay (ELISA). The secretion of HBsAg-specific cytokines by splenocytes was analyzed using flow cytometry and enzyme-linked immunospot (ELISpot) assay. Results: The results demonstrated that the LNP-encapsulated PolyI:C adjuvant significantly increased the secretion of HBsAg-specific IFN-γ, IL-2, and TNF-α by splenocytes, indicating a Th1-biased and cytotoxic T lymphocyte (CTL)-mediated cellular immune response. In addition, this formulation markedly elevated serum titers of HBsAg-specific IgG, IgG1, and IgG2a. Conclusions: These findings underscore the advantages of the LNP-PolyI:C adjuvant in enhancing both humoral and cellular immunity, demonstrating its considerable potential as a novel adjuvant. Full article

In biological systems, DNA serves as the primary carrier of genetic information, and the stability of its structure is fundamental to cellular function. Metal ions and temperature are critical environmental factors that modulate DNA conformation and activity. However, the differential morphological effects of alkali, alkaline earth, and transition metal ions, especially when combined with thermal treatment, have not been systematically visualized and quantified. In this work, atomic force microscopy (AFM) was employed to investigate the effects of different metal ions (Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu²⁺) and temperature on DNA structure. The results demonstrated that monovalent ions (Na⁺ and K⁺) neutralized the negative charges on the DNA backbone, thereby reducing intermolecular electrostatic repulsion and promoting DNA aggregation into dendritic structures. Divalent ions (Mg²⁺ and Ca²⁺) not only provided more effective charge screening but also formed ion bridges between DNA strands, leading to more compact and cross-linked networks. In contrast, Cu²⁺ ions directly coordinated with DNA bases, causing local structural distortion and strand scission. Elevated temperatures induced DNA melting, with distinct morphological transitions from extended double strands to condensed single-stranded globules observed at temperatures exceeding the melting point ($T_m$). These findings elucidate the mechanisms by which environmental factors govern DNA morphology, providing insights relevant to nanotechnology and molecular biology applications. Full article

The detailed mechanism and sequence by which tick-borne flaviviruses (TBFVs), such as Langat virus (LGTV), infect and disseminate in arthropod hosts remain undefined. To begin characterizing these processes, we used artificial membrane feeding chambers to feed adult Ixodes scapularis ticks with blood containing LGTV. At 24, 48, 72, and 96 hours (h) after attachment, we removed and dissected the partially fed ticks to obtain the midgut and salivary glands. Histology confirmed infection in cells of the digestive epithelium lineage; infection was noted in midgut generative cells and the more differentiated functional digestive cells over the course of feeding. The viral envelope (E) protein, nonstructural protein 3 (NS3), and double-stranded RNA (dsRNA) were readily detected in these cells by 48 h after infection. Parallel analysis indicated that cells in salivary gland acini were also infected by 48 h, where virus target cells appeared to be the granular cells in acini types II and III. Thus, both salivary glands and midgut showed direct evidence of infection by 48 h. Although viral staining was not observed at 24 h, when organs were removed at 24 h and individually cultured ex vivo, the virus was detected. Taken together, our results provide evidence of LGTV infection in both the salivary glands and midgut within the first 24 h of a blood meal. The findings should prompt a reevaluation of the systemic dissemination of TBFV in infected ticks. Full article