Search Results (1,372)

Both Chicken anemia virus (CAV) and Gyrovirus galga 1 (GyVg1) belong to the family Anelloviridae and genus Gyrovirus. They are non-enveloped, single-stranded circular DNA viruses that can infect chicken populations, leading to similar clinical symptoms such as growth retardation and immunosuppression, making clinical differentiation challenging. This study established a duplex TaqMan real-time PCR assay to simultaneously detect CAV and GyVg1. Specific primers and probes were developed to target the VP3 gene of CAV (labeled with VIC) and the VP1 gene of GyVg1 (labeled with FAM). The optimization process involved fine-tuning reaction conditions, such as primer/probe concentrations and annealing temperature, through a systematic matrix approach. The assay demonstrated remarkable linearity under optimized conditions (0.4 μmol/L primers, 0.2 μmol/L probes, and an annealing temperature of 52 °C), yielding high R² values of 0.9973 for CAV and 0.9981 for GyVg1. The method exhibited high specificity, showing no cross-reactivity with other common avian pathogens. The limits of detection were 1.0 × 10⁰ copies/μL for CAV and 1.0 × 10¹ copies/μL for GyVg1, making it 10-fold more sensitive than conventional PCR. Intra-assay and inter-assay coefficients of variation were below 1.5% and 2.5%, respectively, indicating excellent reproducibility. When tested on 411 clinical samples from Shandong Province, CAV showed a positivity rate of 48.66% (200/411), GyVg1 18.73% (77/411), and a co-detection rate of 17.52% (72/411). Moreover, 25 complete genome sequences of GyVg1 (2376 bp each) were acquired from positive samples. These sequences exhibited a nucleotide identity ranging from 93.6% to 99.3% among the isolates and 92.2% to 99.6% with reference strains. The phylogenetic analysis, which was based on whole-genome sequences, categorized the Shandong isolates into four clusters (I–IV). Notably, the strains were dispersed across these clusters and were intertwined with references from various regions and hosts. This observation suggests the absence of distinct geographic clustering and implies frequent cross-regional and cross-species transmission. In summary, this duplex real-time PCR assay offers a sensitive, specific, and dependable method for concurrently detecting and distinguishing CAV and GyVg1. The study’s findings reveal a significant co-infection rate and genetic variation in GyVg1, underscoring the importance of increased surveillance and deeper exploration of its pathogenicity and transmission patterns. Full article

Non-small-cell lung cancer (NSCLC) is one of the most frequent cancer types and is responsible for the majority of cancer-related deaths worldwide. For this reason, initial diagnosis, prognosis, and targeted therapy of NSCLC represent very attractive areas of study. Aptamers are single-stranded nucleic acids (RNA or DNA) generated through Systematic Evolution of Ligands by Exponential Enrichment (SELEX); they are able to bind to a molecular target with high affinity and specificity. Thanks to their intrinsic nucleic acid properties, they can be easily modified and optimized to enhance target binding and their half-life; moreover, they exhibit no immunogenicity and toxicity. Due to this, many aptamers have just been selected against NSCLC biomarkers and provide specific imaging agents to improve the diagnosis of this type of cancer. However, despite the promising results in preclinical studies, the application of aptamers in NSCLC diagnosis is still in its early stages, mainly due to the limited literature in the research world, the dominance of antibodies in the pharmaceutical market and the challenge of target identification. Consequently, this appears to be a temporary issue, and aptamers could see increasing application in the future; thus, we performed a review aimed at summarizing current knowledge on the new promising DNA and RNA aptamer-based molecules for NSCLC diagnosis. All studies from 2000 were included and investigated. Our findings showed that several DNA and RNA aptamers are promising diagnostic tools for NSCLC management. Full article

Introduction: DNA aptamers are single-stranded nucleic acids capable of selectively binding target proteins and have emerged as potential alternatives to antibodies in molecular diagnostics. This study aimed to develop and characterize in silico designed DNA aptamers targeting Interferon alpha-inducible protein 6 (IFI6) and Radical S-adenosyl-L-methionine domain-containing protein 2 (RSAD2) for oral squamous cell carcinoma (OSCC). Methods: Genomic transfer RNA sequences from Homo sapiens, Mus musculus, and Escherichia coli were computationally truncated and optimized to generate DNA aptamer candidates ranging from 35–50 mers. Secondary and tertiary structures were generated using Mfold and RNAComposer, followed by molecular docking with AutoDock Vina and molecular dynamics simulations in GROMACS to evaluate docking interactions and structural behavior comparatively. Selected DNA aptamer candidates were synthesized and evaluated by aptahistochemistry (AHC) to qualitatively explore preliminary tissue reactivity in formalin-fixed paraffin-embedded OSCC tissues under optimized conditions. Results: Molecular docking demonstrated favorable comparative docking scores ranging from −15.6 to −18.7 kcal/mol. RMSD analysis demonstrated that selected IFI6 and RSAD2 DNA aptamer–protein complexes reached a plateau with relatively small fluctuations following maximum RMSD values. In contrast, RMSF analysis identified increased flexibility predominantly within loop regions. Cross-reactivity analysis explored preliminary target selectivity, with no interactions observed within the selected target-binding regions. During AHC optimization, the 50-IFI6 and 45-RSAD2 DNA aptamer candidates exhibited cytoplasmic brown granular staining in >50% of OSCC tumor cells under the optimized experimental conditions. Conclusions: The findings support the applicability of an integrated in silico workflow for generating structurally optimized DNA aptamer candidates and provide preliminary proof-of-concept for their exploratory histopathological application in OSCC. Full article

Background/Objectives: Non-invasive prenatal testing for monogenic disorders (NIPT-MD) provides a safe alternative to invasive procedures. However, its clinical utility is often limited by challenges such as low fetal fractions (FF), technical artifacts, and the difficulty in resolving maternally inherited pathogenic variants from the overwhelming background of maternal cell-free DNA (cfDNA). This study aimed to develop and validate a robust NIPT-MD platform for monogenic disorders, applicable across major common Mendelian inheritance patterns, by enhancing the accuracy of fetal variant detection in cfDNA. Methods: We developed a novel NIPT-MD platform validated using both simulated samples and a retrospective clinical cohort. The workflow involves a single-strand capture library preparation incorporating unique molecular identifiers (UMIs) to mitigate amplification artifacts. FF was precisely quantified using a fixed panel of high-minor-allele-frequency single-nucleotide polymorphisms (SNPs). Fetal genotypes were then inferred by resolving parental haplotypes through a statistical model that integrates weighting of variant allele frequency (VAF) and haplotype-informative SNP counts. Results: The single-strand capture protocol incorporating UMIs significantly reduced amplification biases. Validation of the fetal DNA fraction estimation algorithm revealed strong concordance with a Y-chromosome-derived method and expected spike-in samples. Optimization of the FF estimation panel conferred greater experimental stability. The platform reliably detected both paternally and maternally inherited pathogenic variants. In a retrospective cohort of 35 clinical cases, the NIPT-MD platform achieved high concordance with genotypes determined by invasive testing. Conclusions: These findings present an accurate and robust NIPT-MD platform for monogenic disorders, with high concordance to invasive testing validated in a retrospective cohort of 35 clinical cases. This method holds promise for clinical application in managing families at high risk of monogenic diseases. Full article

Replication slippage at repetitive DNA sequences generates insertions and deletions that drive genomic instability. Although magnesium ions are essential cofactors for DNA polymerase activity, their role in modulating slippage fidelity remains unclear. Using an in vitro primer extension assay on a single-stranded template carrying direct repeats flanking a hairpin-forming inverted repeat, we investigated the effect of Mg²⁺ concentration on slippage produced by mesophilic (T4 Pol, T7 Pol, E. coli pol I Klenow fragment, pol I KF exo⁻, and pol III holoenzyme) and thermophilic (Taq pol and Pfu pol) DNA polymerases. We show that Mg²⁺ modulates slippage frequency in a polymerase-dependent manner, as follows: low concentrations suppress slippage in T7 Pol, pol I KF, pol III HE, and Taq Pol, whereas T4 Pol and Pfu Pol slip at all productive concentrations. Mechanistically, Mg²⁺ modulates strand displacement activity, and polymerases that acquire enhanced strand displacement at intermediate concentrations show a corresponding reduction in slippage. Proofreading activity had no detectable effect on slippage frequency. These findings reinforce the inverse correlation between strand displacement activity and slippage propensity and suggest that physiological free Mg²⁺ levels may help suppress slippage in vivo. Full article

Background: Ancient DNA (aDNA) research is one of the biological fields that has been transformed by the development of next-generation sequencing (NGS). To date, Illumina sequencing has dominated aDNA research. However, its high costs are driving the adoption of alternative sequencing platforms. Combining data generated on different platforms may introduce artifacts arising from platform-specific errors. This study systematically compared the performance of four sequencing platforms (Illumina NovaSeq 6000, GeneMind SURFSeq 5000, Salus Evo, and MGI DNBSEQ-G400) for whole-genome aDNA sequencing using the same set of six samples and libraries. Methods: Single-stranded libraries, prepared with and without enzymatic damage repair, were generated from six human aDNA specimens recovered from Phanagoria polis and sequenced on all four platforms. Data were subsampled to equal read counts per library, mapped to the human reference genome, and compared using standard NGS quality metrics. Population genetic analyses, including principal component analysis (PCA) and ADMIXTURE, were performed to assess potential platform-specific biases. Results: All platforms produced raw sequencing data of acceptable quality. Only minor differences were observed among platforms in standard NGS metrics. The MGI platform showed a shift toward longer sequenced fragment lengths compared with Illumina and the Illumina-like platforms. Post-mortem damage patterns, particularly C>T substitutions, were highly consistent across all platforms. PCA and ADMIXTURE analyses revealed no evidence of platform-specific bias: results from all platforms clustered tightly together, and platform choice had no significant effect on ancestry component estimates. Conclusions: Our findings demonstrate that the GeneMind, Salus, and MGI sequencing platforms are comparable to Illumina for paleogenomic research. Moreover, aDNA datasets generated on these platforms can be combined for downstream analyses without introducing detectable bias. However, the fragment-size shift observed on the MGI platform warrants caution and adaptation when working with highly degraded or low-endogenous-content samples. Full article

Background: The computation of DNA is a field of growing interest, primarily focused on studying its individual constituents, but a few authors have gone further by analyzing standard microhelices with DFT methods. Objective: Because these studies did not consider that, at the atomic level, other possible DNA and RNA helices with the WC-type arrangement are also stable, this is the main objective here. Methods: Therefore, forty microhelices were optimized in a simple model with three WC base pairs composed of nucleosides using the M06-2X DFT method. Results: Based on the spatial arrangement of the intramolecular H-bond with the 2′-OH group, five types of RNA:RNA double microhelices were obtained; by this arrangement, they were related to the corresponding DNA:DNA ones. In addition to A-type and B-type microhelices, two new types were found, conveniently named B1-type and C-type. The structural geometric parameters of all these optimized microhelices in an isolated state were compared and analyzed in detail. The total counterpoise-corrected interaction energies were determined in these microhelices. RNA microhelices with different types of intramolecular H-bond arrangements involving the 2′-OH group in each single strand were also optimized, as well as their corresponding DNA helices having the same spatial arrangement. Different DNA:RNA hybrid microhelices were also considered, and their structural geometric parameters were compared to those of other microhelices. Conclusions: the identification and detailed characterization, for the first time, of ten main stable spatial geometric shapes with different exocyclic and endocyclic torsional angles. Several relationships among the structural geometric parameters of these microhelices were also established. Although the new DNA/RNA helix types containing theoretically obtained WC pairs have not been found in biological helices, they could be synthesized and they may open the possibility of being used for other purposes. Full article

Hosta ventricosa, also known as blue plantain lily, is an important traditional herbal medicinal and ornamental plant in China. Prior to this study, no virus has been reported to infect H. ventricosa plants. Based on RNA-seq, transmission electron microscopy, and RT-PCR analyses, we have demonstrated that the H. ventricosa plant showing leaf chlorosis, mottle, mosaic, and crinkling symptoms was co-infected with hosta virus X (HVX) and a novel luteovirus, which we tentatively named hosta ventricosa luteovirus (HVLV). The genome of HVLV is a 5723 nt long, positive-sense, and single-stranded RNA with seven open reading frames (ORFs). Phylogenetic analysis based on the amino acid (aa) sequence of the viral RNA-dependent RNA polymerase (RdRp) revealed that HVLV is clustered within the genus Luteovirus. The HVLV RdRp shares 9.75–45.17% aa sequence identity with the 14 closely related luteoviruses. The P1–2 and P3–5 proteins of HVLV were identified as potential viral pathogenicity determinants through the PVX heterologous expression in Nicotiana benthamiana plants. Moreover, P2, P5 and P3–5 proteins of HVLV have been found to exhibit RNA silencing suppression activities. Additionally, we have successfully constructed an infectious cDNA clone of HVLV and uncovered that this infectious cDNA clone can infect N. benthamiana plants through agroinfiltration. These findings have expanded our understanding of luteoviruses and their host range. Full article

Boron neutron capture therapy (BNCT) is a radiotherapeutic modality that enables tumor-targeted cell killing. The nuclear capture reaction between boron-10 (¹⁰B) and low-energy thermal neutrons produces high linear energy transfer (LET) particles (α-particles and recoiling lithium nuclei), each of which have short path lengths within the diameter of a single mammalian cell. The deposited energy creates clustered DNA double-strand breaks that are cytotoxic in these tumor cells while sparing the surrounding healthy tissues. This advantage makes BNCT a highly attractive treatment modality compared to conventional radiotherapy. Nevertheless, despite its theoretical precision, the clinical translation of BNCT remains constrained by suboptimal tumor-selective boron delivery; insufficient intracellular accumulation; and heterogeneous biodistribution profiles associated with conventional small-molecule-based boron agents, such as boronophenylalanine (BPA) and sodium borocaptate (BSH). While the development of new accelerator-based neutron sources (ABNSs) has renewed interest in BNCT, effective ¹⁰B delivery remains a major challenge. To address this, nanomedicine has been steadily on the rise in cancer research. In recent years, nanocarrier-based delivery systems have emerged as a transformative alternative delivery strategy. Nanodrugs offer several advantages over conventional small-molecule drugs, such as improved solubility, increased plasma half-life, enhanced permeability and retention in tumors, and active targeting, as well as decreased systemic toxicity and drug resistance. In recent years, nanocarrier-based delivery systems have emerged as a transformative strategy for ¹⁰B delivery. In this focused review, we will discuss various types of nanocarriers used for boron drug delivery that enhance boron loading efficiency and evaluate what enables their selective delivery to and accumulation within tumor cells. Full article

This prospective single-center study investigated the impact of iodinated contrast agents (Ultravist/iopromide and Visipaque/iodixanol) on DNA double-strand breaks during pediatric chest computed tomography (CT). Twenty-six children underwent medically indicated chest CT between March 2022 and November 2024: unenhanced (n = 7), Ultravist-enhanced (n = 10), and Visipaque-enhanced (n = 9). Blood samples were collected before and 30 min post-CT. DNA damage was quantified by analyzing γ-H2AX and 53BP1 foci formation in peripheral lymphocytes using immunofluorescence microscopy. All groups showed significant increases in DNA damage following CT. The absolute increases were 0.915 ± 0.250 foci/cell (unenhanced), 0.972 ± 0.279 (Ultravist), and 1.261 ± 0.251 (Visipaque), representing 6.2% and 37.8% higher values in contrast groups. However, these differences between groups were not statistically significant (p = 0.579, Kruskal–Wallis test), with small effect sizes (Cohen’s d < 0.5 for all comparisons). Contrast-enhanced protocols employed higher radiation doses than unenhanced scans (CTDIvol: 4.04–4.57 mGy vs. 2.66 mGy). When DNA damage was normalized for CTDIvol, no significant differences were observed between groups (p = 0.659). DNA damage showed no significant correlation with dosimetric parameters. When accounting for radiation dose differences, iodinated contrast agents do not enhance radiation-induced DNA double-strand breaks in pediatric chest CT, confirming the safety of contrast-enhanced CT. Full article

Background: Vitamin D is involved not only in calcium–phosphate homeostasis but also in immune and endothelial regulation. Vitamin D deficiency has been suggested to worsen disease activity in systemic lupus erythematosus (SLE). Environmental and lifestyle factors, including seasonal sun exposure, smoking, diet, and supplementation, may influence vitamin D status and disease manifestations. This study aimed to evaluate the association between serum 25-hydroxyvitamin D [25(OH)D] levels, disease activity, and anti-double-stranded DNA (anti-dsDNA) antibody titers in patients with SLE, taking selected lifestyle and environmental factors into account. Methods: Serum 25(OH)D concentrations, SLE disease activity assessed by the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score, and anti-dsDNA antibody titers were measured in patients with SLE and healthy controls. Blood samples were collected during sunny (April–September) and non-sunny (October–March) months. Information on vitamin D supplementation, smoking status, and dietary habits was obtained using a structured questionnaire. Associations between vitamin D status, disease activity, anti-dsDNA seropositivity, season of blood collection, supplementation, smoking, and diet were analyzed statistically. Results: Patients with SLE had significantly higher mean serum 25(OH)D levels than controls, mainly due to frequent vitamin D supplementation. No significant associations were observed between serum 25(OH)D levels and SLEDAI-2K scores or anti-dsDNA antibody positivity. Seasonality, smoking status, and adherence to special diets were not significantly related to disease activity or anti-dsDNA seropositivity. Vitamin D supplementation was strongly associated with sufficient 25(OH)D levels but did not translate into reduced disease activity or lower anti-dsDNA prevalence. Conclusions: Serum 25(OH)D concentration was not associated with clinical or immunological activity of SLE in this cross-sectional study, despite effective correction of deficiency through supplementation. These findings likely reflect the heterogeneity of SLE and the limitations of single time-point assessments, although regular monitoring and individualized vitamin D supplementation may still be considered in SLE care, particularly in the context of recommended photoprotection. Full article

Ribonucleotides are frequently incorporated into DNA during the replication of genetic information and, if missed during ribonucleotide excision repair, they may undergo phosphodiester bond rearrangement or cleavage. These changes can in turn lead to deformation of the spatial geometry of the local double helix and potentially interfere with charge transfer through ds-DNA. This process is believed to support long-range communication between proteins involved in genome replication and repair. This study theoretically explores how a single embedded riboadenosine (A₃) affects the structure, electronic properties, and charge-transfer properties of double-stranded DNA ([A₁G₂A₃G₄A₅]*[T₅C₄T₃C₂T₁]). In particular, the study focuses on four products formed at the ribonucleotide site: native 3′,5′-linkage (R-DNA), the 2′,3′-cyclic phosphate intermediate (IM-R-DNA), rearranged 2′,5-linked (RE-R-DNA), and the single-strand-break cleavage product (SSB-R-DNA). This theoretical investigation was performed at the M06-2X/6-31++G**//M06-2X/D95** level of theory in the aqueous phase. Significant spatial geometry perturbations were found at the central part of ds-oligonucleotides, i.e., the A₃T₃|G₄C₂ region, where the modified linkage affected the base overlap and stacking interactions most strongly; in the rearranged and cleaved forms, stacking at this site decreased by about 7 kcal•mol⁻¹ relative to native DNA. Global electronic analysis showed that R-DNA had the highest ionisation potential and the lowest electron affinity, whereas SSB-R-DNA displayed the lowest adiabatic ionisation potential and the highest adiabatic electron affinity, indicating a much greater tendency to stabilise excess charge. At the base-pair level, G₂C₄ was usually the preferred hole sink, except in RE-R-DNA, where G₄C₂ was favoured. In contrast, electron localisation was generally favoured at G₄C₂, while, in SSB-R-DNA, the A₃T₃ pair became the most favourable electron-accepting site. Overall, the results show that even a single ribonucleotide, depending on its linkage chemistry, can substantially reshape charge migration through ds-DNA and may therefore influence lesion recognition, repair efficiency, and genome stability. Full article

Marine algal viruses exhibit a high level of diversity, and closely related viruses targeting the same algal host species can stably coexist. Here we report an example of a single virus–host system concealing hidden complexity. We discovered two double stranded (ds) DNA viruses infecting the Arctic picophytoplankter Micromonas polaris coexisting in culture for over a decade. Genomic sequencing of the lysate originally characterized as MpoV-44T revealed that it comprises two distinct prasinoviruses with ~203–204 kb genomes (MpoV-44T.A and MpoV-44T.B), of which conserved regions only accounted for 36% (the nucleotide level). The viruses were subsequently separated and compared at both genomic and phenotypic levels. In dual infection studies using a single host strain under nutrient-replete conditions, MpoV-44T.A outcompeted MpoV-44T.B. Yet MpoV-44T.B-like viruses were more abundant than MpoV-44T.A-like ones in natural Arctic metagenomes. This apparent paradox may be explained by differences in host strain specificity and/or possible resilience to nutrient stress by MpoV-44T.B, which we hypothesize based on genomic data. This work unveils hidden virus diversity, illustrating that the dynamics of viral coexistence are not always easily predictable, and underscores the importance of studying the underlying mechanisms at play. Full article

Background/Objectives: Single-stranded oligonucleotides (ssODNs) are used as donor templates for therapeutic gene editing by CRISPR-Cas9 cleavage and homology-directed repair (HDR). Although ssODN sequence fidelity is critical to the safety and efficacy of editing, standard quality control methods cannot resolve individual nucleotide errors. Methods: We performed deep sequencing of ssODNs from three manufacturers and amplicons from edited hematopoietic stem/progenitor cells. Results: We find that synthesis errors are present in all ssODNs tested at rates that vary more than two-fold among manufacturers, at positions that are dependent on sequence context. These synthesis errors are propagated into the genome by HDR at frequencies proportional to their abundance in the ssODN. In our sickle cell mutation correction protocol, the most prevalent SNEs are predicted to produce benign β-globin variants, while the less frequent frameshift deletions are predicted to generate β-thalassemia-like alleles. Conclusions: Current quality control standards are insufficient to detect these errors, and deep sequencing of ssODNs should be incorporated into regulatory submissions for clinical gene editing programs. Full article

Chromosomal rearrangements that lead to the formation of oncogenic gene fusions, such as EML4-ALK, are thought to arise from incorrect repair of double-strand breaks in DNA. However, the mechanisms and factors driving rearrangement formation remain poorly understood, and analysis of these processes is limited by detection methods that are labor-intensive, low-throughput, and not readily quantitative at single-cell resolution. Here, we developed a genetically encoded ALK reporter based on A549 lung adenocarcinoma cells, created by inserting an ALK-P2A-mCherry cassette into the endogenous ALK locus, so that induced EML4-ALK fusion activated mCherry fluorescence. Reporter activation yielded a readily quantifiable mCherry-positive subpopulation that could be measured and enriched by flow cytometry and correlated with EML4-ALK levels. Using this platform, we combined CRISPR-mediated rearrangement induction with knockdown of DNA repair factors using RNA interference. Of the factors involved in base excision repair, homologous recombination-related pathways and canonical non-homologous end joining, knockdown of the APEX1 gene encoding apurinic endonuclease 1 (APE1) selectively increased EML4-ALK levels both in the reporter cell line and in parental A549 cells. Together, this work provides a sensitive, single-cell A549-based ALK reporter platform and a framework for future studies aimed at identifying cellular and environmental factors that modulate oncogenic EML4-ALK rearrangement formation. Full article