Search Results (162)

Background/Objectives: RNA virus metagenomic sequencing is a core technology for emerging infectious disease prevention and control, as well as for rapid pathogen identification. However, two major bottlenecks hinder its clinical application: the low fraction of informative sequencing reads caused by host rRNA contamination, and insufficient viral genome coverage. This study aimed to optimize the experimental parameters of RNA virus metagenomic sequencing, address the above bottlenecks, and establish a standardized workflow. Methods: Forty-five clinically positive samples (20 influenza virus-positive; 25 SARS-CoV-2-positive) were investigated in three parallel comparative experiments: rRNA depletion versus no depletion; probe-mediated RNase H digestion versus rRNA blocking; and two fragmentation timing strategies (fragmentation before versus after reverse transcription). Sequencing was performed on the GeneMind platform, and key performance metrics were systematically analyzed. Results: Following rRNA depletion, the host sequence proportion in the influenza virus and SARS-CoV-2 samples decreased from 39.5 to 90.5% to 3.6 to 32.2%, while the 10× genomic coverage increased from 0 to 99.4% to 98.1 to 100.0%. The proportion of host sequences captured by probe capture depletion (0.3–16.2%) was significantly (p < 0.05) lower than that captured by rRNA blocking module (14.3–92.3%). No significant differences were observed in the 10× genomic coverage (96.5–100.0%) or the fraction of effective viral reads between the two fragmentation strategies (p > 0.05). rRNA depletion is key to improving library quality, with post-capture probe digestion being optimal. Conclusions: The suggested optimization process will enhance sequencing efficiency and support the standardization of clinical RNA virus identification. Full article

Background: The m⁷G cap structure, which mimics the natural cap of eukaryotic mRNA, is a critical determinant of mRNA vaccine efficacy, safety, and stability. However, its precise quantification remains challenging due to complex impurity profiles and the high physicochemical similarity between the target cap and related impurities. Although liquid chromatography mass spectrometry (LC-MS) is widely employed for this purpose, current methodologies still face significant limitations, including labor-intensive sample preparation, low analytical throughput, poor reproducibility in quantifying low-level impurities, and a lack of universally applicable strategies across diverse mRNA vaccine platforms. Methods: We systematically optimized sample preparation and LC-MS detection workflows. RNase H-mediated cleavage was compared with DNAzymes, guide DNA probes were rationally designed, and thermostable RNase H was introduced for one-step denaturation and cleavage. To establish an accurate, efficient, and universal sample preparation workflow. Chromatographic conditions were optimized using an ion-pairing reagent system to suppress ESI-MS metal adducts. Eliminating sample purification improves recovery, reduces manual handling errors, and boosts assay efficiency. Results: Through optimally designed guide DNA probes, RNase H cleavage specificity reached ≥98% with high cleavage efficiency, offering higher efficiency than DNAzyme. Furthermore, the incorporation of thermostable RNase H enabled a single-step workflow combining high-temperature denaturation and site-specific cleavage, substantially streamlining sample preparation. On the chromatographic side, optimization of the ion-pairing reagent system effectively suppressed metal adduct formation in electrospray ionization mass spectrometry (ESI-MS). This advancement enabled direct injection of the 5′ cap fragments without purification, achieving high-recovery quantification while demonstrating broad compatibility across mainstream LC-MS platforms. The optimized assay reduces the total analytical workflow from 4~6 h to under 1.5 h. Conclusions: Combining high accuracy, robustness, and broad platform compatibility, this method offers a universal, high-throughput analytical solution for mRNA vaccine quality control and continuous process development. Full article

Antisense oligonucleotides (ASOs) are a class of nucleic acid therapeutics that modulate gene expression through diverse mechanisms. Since their initial demonstration in inhibiting viral genes, advances in medicinal chemistry, pharmacology, and delivery have enabled robust and durable target engagement across multiple tissues. Chemical modifications to the backbone, ribose, and nucleobases have improved nuclease resistance, binding affinity, and pharmacokinetics, while conjugation and delivery technologies have expanded tissue accessibility. Beyond classical RNase H–mediated RNA degradation, ASOs regulate gene expression via splicing modulation, microRNA inhibition, transcriptional activation, and translation modulation, supporting both gene silencing and upregulation strategies. Multiple ASO drugs are now approved, particularly for genetic diseases, with many more in clinical development. This review outlines the evolution of antisense technology, key chemical and delivery innovations, ASO pharmacokinetics and intracellular trafficking, the mechanisms underlying gene regulation, and current clinical applications and future opportunities. Full article

Common bean (Phaseolus vulgaris) is an important crop for human nutrition due to its high protein content and capacity to fix atmospheric nitrogen. However, crop productivity is frequently compromised by biotic and abiotic stresses, among which wounding represents a highly prevalent challenge. Thus, understanding early molecular and biochemical responses to tissue damage is essential for improving plant stress resilience. We have investigated the effects of mechanical wounding on nucleic acid-degrading activities in the common bean. Mechanical wounding of leaves rapidly induced ribonuclease activity, whereas nuclease activities remained unchanged. Gel activity assays revealed a predominant ribonuclease, which was identified by proteomic analysis as PvRNS2, a member of the S-like RNase T2 family. This wound-induced ribonuclease was inhibited more strongly by nucleoside di- and triphosphate than by the corresponding nucleoside monophosphate. The increase in ribonuclease activity correlated with a rapid and transient induction of PvRNS2 expression, which peaked at 2 h after injury (600-fold increase). A similar transcriptional response was observed in radicles subjected to mechanical damage (55-fold increase), indicating that PvRNS2 responds to wounding in both aerial and subterranean tissues. In contrast, the wound-induced increase in PvRNS2 expression was not associated with a coordinated upregulation of genes encoding enzymes involved in downstream nucleotide degradation. Together, these results identify PvRNS2 as a major contributor to wound-induced RNA turnover in the common bean and support the involvement of RNA metabolism in early responses to mechanical damage. The participation of ribonucleases in the wound response of economically vital legumes remains unexplored. This work addresses this knowledge gap, establishing a new framework for understanding nucleic acid degradation during legume defense. Full article

Mayaro virus (MAYV) is an emerging arbovirus from the Togaviridae family where inflammation plays a central role in disease development. As the cause of Mayaro fever, MAYV triggers strong production of pro-inflammatory cytokines, which can result in long-lasting arthralgia in affected individuals. Macrophages are both targets for viral infection and key regulators of inflammatory responses. Human monocyte-derived macrophages (MDMs) are susceptible to MAYV infection in vitro and support productive viral replication. With no approved antivirals or vaccines, finding host-directed therapies is an urgent priority. Cannabinoids are compounds with antiviral and immunomodulatory properties, suggesting potential against MAYV infection. Here, we examined the effects of cannabidiol (CBD) and the synthetic cannabinoid WIN 55,212-2 on MAYV-infected MDMs in pre- and post-treatment conditions. Cells and supernatants were collected at 6 and 24 h post-infection (h.p.i). To understand the mechanisms involved, transcriptomic and functional analyses were performed at 24 h.p.i in the post-treatment setting, focusing on inflammatory, antiviral, and endoplasmic reticulum (ER) stress pathways. WIN 55,212-2 post-treatment significantly decreased viral replication at 24 h.p.i without any direct virucidal activity and was independent of type I interferon activation or interferon-stimulated gene induction, instead being linked to the modulation of ER stress signaling. Specifically, WIN 55,212-2 increased IRE-1α RNase activity, promoting the alternative splicing of sXBP1, while the integrated stress response appeared central to its antiviral effect. Additionally, WIN 55,212-2 downregulated inflammation-related genes and altered cytokine and chemokine production, counteracting the strong inflammatory response caused by MAYV. Remarkably, it also exerted broader immunomodulatory effects independent of infection. Full article

Residual cardiovascular risk arises from dysregulated expression of genes encoding apolipoprotein(a) (LPA), apolipoprotein C-III (APOC3), angiopoietin-like gene 3 (ANGPTL3), and proprotein convertase subtilisin/kexin type 9 (PCSK9). RNA-based therapies, small interfering RNAs (siRNAs), and antisense oligonucleotides (ASOs) modulate these targets at the post-transcriptional level through RNA interference and RNase H-mediated degradation, respectively. This scoping review maps the molecular mechanisms, target involvement, and pharmacodynamic outcomes of RNA therapies for managing residual cardiovascular risk, with contextual comparison to traditional lipid-lowering agents. A systematic search of PubMed, Embase, Web of Science, and Scopus was performed from 2020 to February 2026. Of the 1088 records identified, 30 studies met the inclusion criteria. RNA therapies have demonstrated potential for engagement, with 80–98% reductions in Lp(a) (pelacarsen, olpasiran, zerlasiran, lepodisiran), 50–80% reductions in triglycerides (olezarsen, plozasiran, volanesorsen), and 36–44% reductions in low-density lipoprotein cholesterol (LDL-C). Mechanistically, siRNAs achieve gene silencing through RISC-mediated mRNA cleavage, with sustained pharmacodynamic effects (3–6 months) because of Argonaute-2 stability, while gapmer ASOs recruit RNase H1 for mRNA degradation. Conjugation with GalNAc allows for hepatocyte-specific delivery with a subcutaneous bioavailability of 70–85%. Safety profiles were favorable, with injection site reactions (4–12%) being the most common adverse event. This analysis maps the emerging molecular landscape of RNA therapies, highlighting their substantial precision for targeting residual cardiovascular risk pathways that cannot be addressed by traditional agents. Full article

RNA-based therapeutics are reshaping the treatment landscape for skeletal muscle disorders by enabling modulation of RNA processing or direct correction of disease-causing alleles. In Duchenne muscular dystrophy (DMD), four antisense oligonucleotides—eteplirsen, golodirsen, viltolarsen, and casimersen—have received FDA approval; these phosphorodiamidate morpholino oligomers (PMOs) induce exon skipping to restore the reading frame and enable expression of internally truncated dystrophin. Beyond splice switching, RNA therapeutics include RNase H-active gapmers and steric-blocking antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) that mediate post-transcriptional gene silencing, and RNA-guided gene-modifying technologies such as CRISPR systems that can reframe or repair endogenous alleles. Despite major progress in DMD, broader clinical impact remains constrained by inefficient delivery to skeletal and especially cardiac muscle, the need for repeat administration for most modalities, and safety considerations that limit dose escalation and durability. Next-generation approaches aim to overcome these barriers through peptide- or antibody-conjugated oligonucleotides that enhance cellular uptake and tissue distribution, alternative chemistries with improved stability and potency, and viral or non-viral platforms for durable splice modulation. In parallel, CRISPR-based strategies—including base and prime editing—offer the prospect of one-time correction, while raising important questions regarding delivery, immunogenicity, editing specificity, and long-term safety. This review synthesizes recent advances in antisense and gene-modifying strategies for skeletal muscle and highlights practical priorities for translation, including improved muscle/heart delivery, controllable safety mechanisms, scalable manufacturing, and standardized biomarker-to-clinical outcome relationships. Full article

Beyond its function as a carrier of hereditary information, recent research has uncovered novel properties of extracellular DNA, including its role in the adaptation to the environment when released from plants. The secreted DNA has been shown to exert insecticidal effects against insect pests, which play an adaptive role in plant-insect interactions, particularly in regulating populations of economically important sap-feeding insects. The molecular mechanisms underlying this insecticidal effect are underinvestigated and remain largely unknown. Therefore, there is a need for more efforts to uncover these mechanisms to better understand plant–pest interactions, which would provide new insights into natural pest control strategies and inspire biotechnological applications. In the current study, we show that Pittosporum tobira (P. tobira) secretes single-stranded DNA (ssDNA) that exerts an insecticidal effect on Coccus hesperidum (C. hesperidum). We collected extracellular DNA from P. tobira leaves and tested its potential insecticidal effect by applying it to C. hesperidum, which is a well-known pest that causes damage to P. tobira. Our results revealed that the outermost layer of the leaf cuticle of P. tobira predominantly contains ssDNA of approximately 100 nt in length, originating from both chloroplast and nuclear genomes. This DNA exhibited pronounced insecticidal activity against C. hesperidum, with chloroplast-derived sequences significantly enriched compared to the total DNA in intact plant cells. These findings suggest that the microevolution of the P. tobira nucleome and plastome contributed to the formation of extracellular DNA with insecticidal properties (eci-DNA), which is part of its defense strategy against insect pests. Moreover, in this article, for the first time, we show that antisense DNA (illustrated with oligonucleotide insecticide Coccus-11) is capable of activating insect retrotransposons and upregulating their RT-RNase H, a crucial enzyme for the DNA containment mechanism and successful action of oligonucleotide insecticides. Notably, the laboratory-developed ssDNA-based ‘genetic zipper’ technology, designed for sustainable pest management, possesses characteristics similar to eci-DNA found in nature, highlighting a potential natural parallel to this biotechnological approach for sustainable pest management. Full article

Background/Objectives: Bisulfite-based cell-free DNA (cfDNA) methylation assays enable the detection of clinically valuable epigenetic biomarkers but often cause DNA degradation and inconsistent conversion efficiency, limiting performance in low-input liquid biopsy samples. We aimed to develop and evaluate a fully enzymatic cfDNA methylation workflow that preserves DNA integrity and supports quantitative clinical detection. Methods: The assay integrates TET2-mediated oxidation and APOBEC3A deamination with RNase H2-guided primer design, uracil-DNA glycosylase error suppression, and dual-probe detection compatible with quantitative PCR (qPCR) and digital PCR (dPCR). Performance was assessed using serial dilutions of methylated HT29 DNA, unmethylated controls, and plasma cfDNA from colorectal cancer (CRC) patients and healthy donors. Analytical sensitivity, linearity, and concordance between platforms were evaluated. Results: The 40-marker panel demonstrated higher cumulative methylation scores and more frequent methylation-positive signals in CRC cfDNA compared to controls. dPCR confirmed single-molecule resolution and clear discrimination between methylated and unmethylated templates, with occasional double-positive partitions consistent with mixed allelic methylation. Signal intensity across the dilution series followed a four-parameter logistic model, achieving detection sensitivity below 0.2% methylated DNA. qPCR and dPCR results showed strong correlation across the HT29 dilution series (R² = 0.80) and high concordance in classifying CRC and healthy samples. Conclusions: This TET2–APOBEC-based enzymatic cfDNA assay enables sensitive, quantitative, sequencing-free methylation detection under gentle conditions, supporting its application in early colorectal cancer screening and routine clinical liquid biopsy workflows. Full article

Nucleocapsids protect viral genomes and play fundamental roles in viral assembly and infection. While many viruses adopt icosahedral or helical symmetries, negative-strand RNA viruses (NSVs) assemble their nucleocapsids with a distinct translation-based symmetry that is often considered helical because of their curvature. Our study analyzes the structural basis, assembly principles, and functional implications of the linear nucleocapsids. Structural coordinates of viruses were obtained from the Protein Data Bank (PDB) and examined using PyMOL version 1.3 to compare protein folds, RNA–protein interactions, inter-subunit contacts, and curvature properties across multiple nucleocapsids. We found that linear nucleocapsids share a similar 5H + 3H fold in their capsid proteins and encapsidate a fixed number of nucleotides per subunit, though the degree of nucleotide sequestration varies. Their architecture differs in inter-subunit interactions, determining whether empty capsids can assemble and influencing RNase sensitivity. Although these nucleocapsids may appear helical, they lack strict helical symmetry and instead display variable curvature that is modulated by environmental conditions. Relaxation of this curvature is likely required for viral RNA-dependent RNA polymerase to access the sequestered RNA genome during transcription/replication. In conclusion, linear nucleocapsids constitute a class of RNA–protein assemblies with variable curvature. The topologically conserved fold of the capsid protein enables genome protection while regulating exposure of RNA during viral RNA synthesis. Full article

Emerging evidence suggests that polymorphisms in the reverse transcriptase connection (RT-conn) and RNase H domains may contribute to resistance to reverse transcriptase inhibitors (RTIs). Here, we characterised the polymorphic landscape of the RT-conn and RNase H domains in HIV-1 subtype C (HIV-1C) from Botswana across the pre-ART and post-ART eras, including treatment-naïve (TN) and treatment-experienced (TE) individuals. A total of 1571 HIV-1C sequences were analysed: 76 pre-ART (≤2002) and 1495 post-ART (>2002) sequences were obtained from the Los Alamos database and the Botswana Combination Prevention Project (2013–2018). Post-ART sequences were stratified into TN (n = 1282) and TE individuals with virologic failure (TEVF, n = 213). Naturally occurring and ART-associated polymorphisms within RT-conn (aa 321–440) and RNase H (aa 441–560) were assessed. Among TN individuals, 12 polymorphisms exceeded 5% pre-ART, including R461K and L491P, while 31 polymorphisms were observed post-ART, indicating a temporal shift. Several substitutions were significantly higher in TEVF and showed a history of thymidine analogue-, tenofovir- and lamivudine/emtricitabine-based exposure. Covariant analysis identified significant co-occurrence of polymerase mutations (M184V/I, D67N) with RT-conn/RNase H substitutions (p < 0.05). These findings demonstrate HIV-1C evolution within the extended RT domains under ART pressure and support their inclusion in molecular surveillance frameworks in Botswana. Full article

Next-generation sequencing (NGS) and antisense oligonucleotide (ASO) technologies are converging to transform the diagnosis and treatment of rare monogenic disorders. NGS enables comprehensive, single-test molecular diagnoses through targeted panels, whole-exome sequencing, and whole-genome sequencing, which together reveal pathogenic variants across coding, intronic, and structural domains. Integration with transcriptomic analyses, including RNA sequencing, further refines genotype–phenotype correlations and identifies splicing aberrations amenable to correction by ASOs. Therapeutic advances now span RNase H1-dependent gapmers for transcript knockdown, splice-modulating phosphorodiamidate morpholino oligomers (PMOs), and peptide/antibody-conjugated PMOs that enhance muscle and cardiac delivery. These platforms underpin the rise in N-of-1 ASO therapies—customized drugs developed for individual patients with unique pathogenic variants. Landmark cases such as Milasen and Atipeksen illustrate the clinical feasibility and ethical complexities of personalized RNA therapeutics, while updated FDA guidance supports expedited, patient-specific investigational pathways. Despite progress, challenges persist in delivery efficiency, long-term efficacy, and equitable access. Emerging approaches—including long-read sequencing, AI-driven oligo design, and improved delivery—promise to extend ASO precision and reach. This review synthesizes current advances linking genomic diagnosis to individualized RNA-targeted interventions, outlining how integrated NGS-ASO pipelines are reshaping the therapeutic landscape for rare genetic diseases. Full article

Self-incompatibility (SI) is a key reproductive mechanism in angiosperms that prevents self-fertilization and promotes genetic diversity while limiting breeding efficiency. Akebia trifoliata is a recently domesticated economic crop native to East Asia with medicinal, edible, and oil-producing value. However, its late-acting self-incompatibility (LSI) severely limits genetic improvement and commercial development. To investigate the molecular basis of LSI, we conducted comparative transcriptomic analyses of pistils at 48, 96, 144, 192, and 240 h after self- and cross-pollination, identifying 1552, 2954, 1302, 814, and 1978 differentially expressed genes (DEGs), respectively. DEGs were consistently enriched in mitogen-activated protein kinase (MAPK) signaling, plant hormone signal transduction, and ubiquitin-mediated proteolysis pathways, with clear transcriptional differences before and after 96 h. Compared with cross-pollinated pistils, self-pollinated pistils showed restricted pollen tube spread, and genes related to pollen recognition and tube development showed differential expression at 48 and 96 h, indicating that LSI probably occurs within the pollen tube. Collectively, these results indicate that pistils of A. trifoliata exhibit distinct early responses to self- and cross-pollination, and that DEG-enriched pathways are similar to those involved in S-RNase-mediated SI. These results provide new insights into the molecular basis of LSI and suggest potential targets for overcoming the SI barrier. Full article

Background. Eriobotrya seguinii (Lév.) Cardot ex Guillaumin (Rosaceae, Maleae) is native to China and inhabits various altitudes within the subtropical biome of the Yunnan-Guizhou Plateau. The complexity of the plant mitogenome has impeded a systematic description of this species, leading to a limited understanding of its evolutionary position. Methods. In this study, we constructed, annotated, characterized, and compared the complete E. seguinii mitogenome with previously reported Eriobotrya japonica. Results. The E. seguinii mitogenome exhibited a typical circular architecture, spanning 372,899 bp in length, with a GC content of 46%, making it the smallest and highest GC content of any known Eriobotrya species. It encodes 71 unique genes, comprising 47 protein-coding genes, 20 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. The genome contains rich repetitive sequences, with mononucleotides, A/T bias, and forward and palindromic repeats being the most prevalent. The predominant codons were GCU (Ala) and UAU (Tyr), with frequencies of 1.54 and 1.53, respectively. Thirteen genes (atp9, atp6, atp1, rps14, sdh4, sdh3, rps12, rnaseH, nad1, nad6, nad7, rpl16, and mttB) demonstrated high Pi values, ranging from 0.84 to 1. The evolutionary lineage of E. seguinii was explored using mitogenome data from 19 genera within the Rosaceae family, revealing that Eriobotrya species are monophyletic and closely related to E. japonica (MN481990). Conclusions. Understanding the mitogenome characteristics of E. seguinii enhances our understanding of its genesis and classification based on mitochondrial genome data. This study provides additional evidence for future research on the evolutionary relationships among species in the Rosaceae family. Full article

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