Search Results (122)

Background: Classical reverse transcriptase (RT) drug resistance mutations (DRMs) are primary determinants of antiretroviral treatment failure, but polymorphisms within the RT connection and RNase H domains may also influence RT inhibitor susceptibility. These regions remain poorly characterised in HIV-1 subtype C (HIV-1C), the predominant subtype in sub-Saharan Africa. This study investigated RT connection and RNase H polymorphisms in treatment-experienced people with HIV (PWH) experiencing virologic failure (VF) in Botswana. Methods: Seventeen plasma samples from the Botswana National HIV Drug Resistance Programme with documented VF (viral load >200 copies/mL) and prior Sanger resistance results were selected. One sample was excluded because of insufficient Oxford Nanopore Technologies (ONT) sequencing coverage, leaving 16 samples for analysis. Samples were amplified using the DeepChek^® HIV-1 Full PR/RT/INT assay and analysed using the HIVgenomeR™ v2.0 ONT pol DRM pipeline. ONT-derived RT resistance profiles were compared with historical Sanger results, and RT connection and RNase H polymorphisms were characterised. Results: ONT identified additional RT DRMs in five sequences previously classified as lacking classical RT DRMs. Of the remaining six sequences without classical DRMs, four harboured minority RT variants. RT connection polymorphisms were common, including G335D (13/16) and T377M (9/16). Recurrent mutation combinations included T377M–T470A, A371V–E399D, T377M–E399D, and T377M–A360T. Conclusions: The extended HIV-1 pol genotyping assay enabled characterisation of the RT connection and RNase H domains while improving detection of classical RT DRMs and minority variants. These findings support further investigation of extended RT sequencing for HIV-1C molecular surveillance. Full article

‘Genetic zipper’ technology—based on CUAD (Contact Unmodified Antisense DNA) biotechnology, briefly CUADb—represents a step forward in eco-friendly pest control. This unique innovative approach is based on a fundamentally new biological mechanism—a two-step DNA containment (DNAc) mechanism. DNAc employs short, unmodified antisense DNA molecules to selectively degrade target pre-rRNA and/or rRNA in insect pests recruiting up-regulated RNase H1 and RT-RNase H during DNAc, disrupting protein synthesis and causing the down-regulation of kinases due to ATP insufficiency and ultimately leading to high mortality rates. Demonstrating exceptional speed and precision, this technology enables the design of effective and selective DNA pesticides (oligonucleotide pesticides) for no less than 15% of known insect pests in a single day. In this review, we highlight the simplicity and global applicability of this technology using case studies involving 12 economically significant pest species, including hemipterans and one spider mite, from five continents. These oligonucleotide pesticides, computationally predicted via the DNAInsector web tool, are supposed to offer 80–90% efficacy against target pests within one–two weeks under laboratory or field conditions. Their action is primarily non-systemic, requiring direct contact. Oligonucleotide pesticides are environmentally safe, biodegradable, and highly specific, reducing risks to non-target organisms. The ‘genetic zipper’ technology not only provides a powerful tool for researchers and practitioners but also opens a new era in DNA-programmable pest management, where personalized, algorithm-driven pesticides can be easily created and applied for sustainable agriculture. Full article

(1) Background: The Hepatitis B virus (HBV) is characterized by extensive genetic diversity, including low-frequency variants that contribute to disease progression. We aimed to characterize low-frequency variants and evaluate their potential clinical impact. (2) Methods: We utilized 104 HBV near-full-length sequences generated using next-generation sequencing (NGS) from people living with HIV (PLHIV). We used an in-house bioinformatics suite (HBVgenomeR v5.9.7) to filter for low-frequency variants (5–50%), which were compared to escape and drug resistance mutations (DRMs) and hepatocellular carcinoma (HCC)-associated mutations reported at the consensus level. Unclassified variants were characterized by HBV open reading frames (ORFs) to determine mutation frequency per genomic region. (3) Results: A total of six escape mutations were detected in 8/104 (7.7%) sequences, with _surfaceN131T being the most prevalent (5/8). We also observed six DRMs in 30/104 (28.8%), with _rtV173L being the most prevalent (21/30). Truncation mutations were also observed with _rtA181T/_sW172* and _rtM204I/_sW196L being the most prevalent. A total of 8/104 (7.7%) sequences had four variants associated with HCC. The _xP46S was the highest observed HCC-associated mutation at 5/8. We report 1152 unique uncharacterized variants across all ORFs, and these were found in 94/104 (90.4%) sequences. The RNaseH domain had the highest burden (330/1152, 28.6%). (4) Conclusions: Deep sequencing results identified clinically significant mutations, including those below the 20% detection limit of traditional sequencing, that would go unreported. This highlights the possible underreporting of mutational burden in people living with HBV/HIV, indicating the importance of deep sequencing to aid in HBV/HIV understanding and management. Full article

Clustering and classification of RNA secondary structures are central to understanding RNA function. However, widely used alignment methods, such as LocARNA and bpRNA-align, are not explicitly designed to exploit the hierarchical relationships among RNA structural elements, limiting their applicability to complex, multi-branched structures. In this study, we introduce Tree_RNA-Align, a novel method for RNA secondary structure clustering and classification based on a tree-structure alignment algorithm. The method transforms dot–bracket structures into tree representations, in which multibranch loops and stems serve as nodes, thereby preserving the hierarchical relationships among structural elements. It integrates a bottom–up hierarchical comparison for clustering with a top–down comparison for classification and prediction. Notably, classification experiments on five RNA families (16S rRNA, group_I_intron, RNase P, SRP, and tmRNA) achieved a micro-averaged F1-score of 0.933 (range across families: 0.760–0.981) and effectively identified representative structures within each family. Overall, these results suggest that incorporating classification can further improve RNA secondary structure prediction, demonstrating the utility of Tree_RNA-Align for structural analysis and functional annotation. Full article

Crimean–Congo hemorrhagic fever (CCHF) is a severe tick-borne viral disease with high mortality rates and no approved specific antiviral therapy or vaccine. Circular RNAs (circRNAs) are emerging regulators of gene expression and have been implicated in various infectious and inflammatory diseases; however, their role in CCHF remains unknown. In this study, we investigated circRNA expression profiles in peripheral blood samples from CCHF patients and healthy controls. Total RNA was isolated, enriched for circRNAs using RNase R treatment, and analyzed with the Arraystar Human circRNA Array V2 platform. Differential expression analysis was performed using the limma package in R, followed by RT-PCR validation and bioinformatic analyses. Compared with healthy controls, 15 circRNAs were significantly upregulated and 264 were significantly downregulated in CCHF patients. The most highly upregulated circRNAs included hsa_circ_011083, hsa_circ_092556, hsa_circ_001766, hsa_circ_001569, and hsa_circ_027934, whereas hsa_circ_405788, hsa_circ_104982, hsa_circ_002008, hsa_circ_406768, and hsa_circ_037139 were among the most downregulated. Additionally, 641 and 455 differentially expressed circRNAs were identified in fatal–control and fatal–nonfatal comparisons, respectively. Functional enrichment analyses revealed associations with immune response, inflammatory signaling, and host–virus interaction pathways. To our knowledge, this is the first study to characterize circRNA expression patterns in CCHF, providing novel insights into disease pathogenesis and identifying potential biomarkers associated with disease severity and mortality. Full article

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/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

Chagas disease (CD) is a neglected tropical disease caused by the flagellate protozoan Trypanosoma cruzi, representing a major socioeconomic challenge. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression, and several pathogens, including T. cruzi, can modulate host miRNA networks. In this context, we hypothesized that host-derived miRNAs could serve as biomarkers in chronic CD. Given the intrinsic lability of RNA, we evaluated the efficacy of a 6 M guanidine-HCl/0.2 M EDTA solution, widely used in the molecular detection of T. cruzi DNA, in preserving mRNAs and miRNAs when mixed in a 1:1 ratio with human blood. Samples with or without guanidine were enriched with exogenous miRNAs (cel-miR-39 and cel-miR-54) and stored at 4 °C. RNase P expression was also evaluated in blood samples stored for up to 120 days and in samples from patients with CD, allowing direct comparison of mRNA stability over time. Samples preserved with guanidine-EDTA showed Ct values that were 4 to 5 cycles lower for all targets analyzed and demonstrated greater RNA stability over time. Taken together, these findings demonstrate that guanidine-EDTA robustly preserves mRNA and miRNAs in human blood, expanding the feasibility of molecular analyses in retrospective samples and corroborating its potential application in the studies of biomarkers of therapeutic response and prognosis in CD. 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

Milk fat composition and content are crucial indicators that influence the taste, flavor and nutritional value of milk, and they are regulated by various non-coding RNAs. To enhance milk quality, it is essential to investigate the regulatory mechanisms underlying milk fat metabolism. This study analyzed significantly up-regulated circ_0009058 and circ_0004021 and significantly down-regulated circ_0011934 and circ_0008056 in bovine mammary epithelial cells (BMECs) with high and low milk fat percentages (MFP). RNase R digestion assays and Sanger sequencing were conducted to confirm their presence and high expression in the mammary tissue of dairy cows. Subcellular localization indicated that these RNA may exhibit regulatory functions through the competing endogenous RNA (ceRNA) network. Consequently, a ceRNA regulatory network was constructed, identifying six hub target genes in the ceRNA network (VAV1, PTPN6, PIK3R1, RHOA, ERBB3, PIK3CG) using the CytoHubba and MCODE plugins in Cytoscape (version 3.9). Quantitative real-time PCR (RT-qPCR) and dual luciferase reporter gene assays validated the existence of the interactive regulatory networks circ_0004021/bta-miR-541/PTPN6, circ_0008056/bta-miR-2309/ERBB3, and bta-miR-10175-3p/RHOA in BMECs. Functional validation of miRNAs within the ceRNA network demonstrated that bta-miR-541 and bta-miR-10175-3p reduced triglyceride and cholesterol levels while inhibiting lipid droplet secretion in BMECs. Conversely, the function of bta-miR-2309 was found to be opposite to that of bta-miR-541 and bta-miR-10175-3p. This study identified several critical candidate ceRNA networks involved in fatty acid metabolism in dairy cows through data analysis and a series of experiments. These functional and mechanistic studies provide a theoretical foundation for improving milk quality. Full article

Although large-scale studies and potential pathways of genes on intramuscular fat (IMF) in livestock have been reported, research on circRNAs in yaks—a unique, low-IMF-content animal species that is native to the Qinghai–Tibetan Plateau—is still lacking. Based on previous high-throughput sequencing results on longissimus dorsi with different IMF content, a novel circRNA encoded by the VPS13C gene (designated as circVPS13C) was found to exhibit significant differential expression. Here, we systematically characterized the function and mechanism of circVPS13C on IMF deposition in yaks by adopting a series of experiments. Sequencing, RNase R processing, and nucleoplasmic separation experiments confirmed the circular structure feature of circVPS13C, and it was predominantly distributed in the cytoplasm. Furthermore, these experiments demonstrated that circVPS13C was mainly distributed in the cytoplasm. The circVPS13C/miR-5606-x/ECHDC3 axis was constructed through ceRNA network analysis and validated by dual-luciferase reporter and rescue experiments. Furthermore, the function of these three potential regulators during IMF deposition was investigated through CCK-8, BODIPY, Oil Red O staining, and qRT-PCR analyses, and results showed that both circVPS13C and miR-5606-x promoted the differentiation and inhibited the proliferation of yak intramuscular preadipocytes, while the function of ECHDC3 was the opposite. In conclusion, circVPS13C could act as a competitive endogenous RNA (ceRNA) sponge to sequester miR-5606-x, thereby relieving the inhibitory effect of miR-5606-x on ECHDC3. 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

Transcription–replication conflicts (TRCs) often occur in cells and cause DNA replication fork stalling. In this study, we investigated the interplay of RNA polymerase (RNAP), DNA polymerase, and RNase H1 (RH1) during head-on TRC in vitro with precise control over the reaction conditions. We show that it is a catalytically competent transcription elongation complex (TEC) that interferes with the action of both the Klenow fragment and full-length DNA Pol I. An incompetent RNAP complex with an R-loop stimulates the 3′→5′ exonuclease activity and pauses the DNA polymerase during head-on TRC. As RNAP advances along the DNA template, elongating the RNA, the head-on TRC is slowly overcome in our model system, likely through the reassociation of the displaced DNA polymerase with the nontemplate DNA strand upstream of RNAP. An isolated R-loop containing an 11-nt heteroduplex (R-loop-11) does not interfere with DNA replication by the Klenow fragment. For DNA Pol I, such an R-loop also does not stall replication but stimulates its 3′→5′ exonuclease activity. We demonstrate that a stalled Klenow fragment does not interfere with transcription, whereas a Klenow fragment moving along the TRC substrate towards RNAP alters the kinetics of RNAP. Stalled DNA Pol I does not stop RNAP but stimulates its endonuclease activity. We find that RH1 alone does not displace stalled RNAP from a competent TEC containing R-loop-11 and does not resolve the head-on TRC. On the other hand, RH1 displaces RNAP from the incompetent complex with the TRC substrate. This eliminates the stimulation of the 3′→5′ exonuclease activity of DNA polymerase during head-on TRC. Full article

R-loops are three-stranded nucleic acid structures implicated in genome regulation and stability. In Arabidopsis thaliana, the chloroplast-localized RNase H1 enzyme (AtRNH1C) is important for chloroplast development and genome integrity; however, its molecular activity has not been experimentally verified. In the present study, we characterized the enzymatic activity of recombinant AtRNH1C toward model R-loops of various structures. Using a set of synthetic R-loop substrates, we demonstrate that AtRNH1C cleaves the RNA within DNA/RNA hybrids with a strong preference for purine-rich sequences, most notably at G↓X dinucleotides. Kinetic assays showed that the enzyme’s efficiency is highly dependent on the length of the hybrid duplex but is not affected by a G-quadruplex structure in the single-stranded DNA flap of the R-loop. The most rapid degradation was observed for an R-loop with an 11 nt DNA/RNA hybrid region. This study provides a comparative analysis of chloroplast-localized RNase H1 activity and elucidates its substrate preferences, suggesting that an R-loop with a heteroduplex length closest to the native size found in transcription elongation complexes is the most efficient substrate. These findings suggest that the enzymatic activity of AtRNH1C is sufficient to perform its function in maintaining chloroplast genome stability by the degradation of R-loops in DNA. Full article