Search Results (202)

Porcine epidemic diarrhea virus (PEDV) is a major pathogen responsible for severe diarrhea, dehydration, and high mortality in neonatal piglets, continually threatening global swine production. Rapid differentiation of its major genotypes (classical G1, variant G2, and recombinant S-INDEL) is vital for molecular epidemiology and effective disease control, yet existing approaches rely mainly on time-consuming sequencing and phylogenetic analysis of the S gene. To overcome this limitation, we developed a novel triplex TaqMan-based real-time PCR assay for rapid detection and differentiation of the three PEDV genotypes. The assay demonstrated high sensitivity, with the lowest detection limit of 10² copies/μL, and strong specificity, showing no cross-reactivity with six other common swine pathogens (TGEV, PDCoV, PoRV, PRRSV, CSFV, and PRV). It also exhibited excellent reproducibility, with both intra- and inter-assay coefficients of variation maintained below 1.5%. In clinical validation, the assay showed 100% concordance with results obtained from S gene sequencing and phylogenetic analysis. Furthermore, testing of 160 clinical samples revealed cases of co-infection involving G2 and S-INDEL strains. In conclusion, this rapid, specific, and reproducible assay provides a reliable tool for routine molecular diagnosis, facilitating large-scale epidemiological surveillance and enabling genotype-informed control strategies against PEDV. Full article

Triplexes (TRX) are a class of flipons that can form due to the interaction of RNA with B-DNA. While many proteins have been proposed to bind triplexes, structural models of these interactions do not exist. Here, I present AlphaFold V3 (AF3) models that reveal interactions between the high-mobility group protein B1 (HMGB1), HNRNPU (SAF-A), TP53, ARGONAUTE (AGO), and REL domain proteins. The TRXs result from the sequence-specific docking of RNAs to DNA via Hoogsteen base pairing. The RNA and DNA strands in apolar TRX are oriented in the opposite 5′ to 3′ direction, while copolar TRX have RNA and DNA strands pointing in the same 5′ to 3′ direction. TRXs can incorporate different RNA classes, including long noncoding RNAs (lncRNAs), short RNAs, such as miRNAs, piRNAs, and tRNAs, nascent RNA fragments, and non-canonical base triplets. Many pathways regulated by TRX formation have evolved to constrain retroelements (EREs), which are both an existential threat to the host and a source of genotypic variation. TRXs help set the boundaries of active chromatin, repressing the expression of most EREs, while depending on other flipons to modulate cellular programs. The TRXs help nucleate folding of intrinsically disordered proteins. Full article

Dorsal vein thrombophlebitis, or penile Mondor disease, is a rare benign penile condition presenting with cord-like induration at the dorsum of the penis. This induration is caused by an isolated thrombosis of the dorsal superficial vein of the penis. As symptoms are not typical and many patients are asymptomatic, it is often underdiagnosed. Causes include trauma, infection, sexual activity, genital surgery, and cancer. Differential diagnosis includes Peyronie’s disease and sclerosing lymphangitis, and diagnosis remains crucial as it facilitates the treatment plan and reassures the patient. Treatment consists of conservative measures, such as oral nonsteroidal anti-inflammatory medications (NSAIDs) and anticoagulation, and surgical management, with excision of the thrombosed vein. We present a case report of penile Mondor disease following circumcision, with the aim to provide educational ultrasound images of this rare entity. The patient, 32 years old, on the sixth postoperative day, developed a cord-like induration, along with pain, at the dorsum of the penis. Physical examination revealed a cord-like mass on the dorsal aspect of the penis. Penile triplex demonstrated a lack of endoluminal flow signals of the superficial dorsal veins, which were uncompressible. Triplex of the femoral and iliac veins showed no sign of thrombosis. Clinical presentation, along with imaging findings, facilitated the diagnosis of Mondor disease. The patient was treated conservatively with sexual abstinence and NSAIDs, and 6 weeks after the presentation, the patient was asymptomatic, without evidence of the disease in clinical examination. Full article

The overlapping circulation of influenza (Flu), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; SC2), and respiratory syncytial virus (RSV) continues to challenge clinical laboratories, particularly in settings with limited automation and fragmented healthcare coverage. This study expanded the CDC Flu-SC2 assay by incorporating a laboratory-developed test (LDT) for RSV A/B detection into a fully automated quadruplex RT-qPCR (LDRA) on the Panther Fusion^® Open Access™ system. The design, based on more than 8000 RSV genomic sequences targeting the conserved M gene, achieved optimal amplification efficiencies (97–105%) and full multiplex compatibility. Analytical assessment established limits of detection between 9.6 and 37.8 copies per reaction, absence of cross-reactivity with 30 respiratory pathogens, and inclusivity for 32 viral variants. Commutability and diagnostic performance among the LDRA, CE IVD-marked Allplex™ SARS-CoV-2/FluA/FluB/RSV, and US IVD-marked Panther Fusion^® SARS-CoV-2/Flu A/B/RSV Assays were evaluated using 405 nasopharyngeal UTM-preserved swabs. The LDRA demonstrated excellent concordance (overall agreement ≥ 98%, κ > 0.95), strong diagnostic accuracy, and reliable detection of mixed infections. This quadruplex provides a fully automated, rapid, and accurate solution for the simultaneous detection of influenza A, influenza B, SARS-CoV-2, and RSV viruses, enhancing molecular diagnostic capacity and supporting equitable, timely clinical decision-making in middle-income healthcare systems such as that of the Dominican Republic. Full article

The role of alternative nucleic acid structures (ANS) in biology is an area of increasing interest. These non-canonical structures include the Z-DNA and Z-RNA duplexes (ZNA), the three-stranded triplex, the four-stranded G-quadruplex (GQ), and i-motifs. Previously, the biological relevance of ANS was dismissed. Their formation in vitro often required non-physiological conditions, and there was no genetic evidence for their function. Further, structural studies confirmed that sequence-specific transcription factors (TFs) bound B-DNA. In contrast, ANS are formed dynamically by a subset of repeat sequences, called flipons. The flip requires energy, but not strand cleavage. Flipons are enriched in promoters where they modulate transcription. Here, computational modeling based on AlphaFold V3 (AF3), under optimized conditions, reveals that known B-DNA-binding TFs also dock to ANS, such as ZNA and GQ. The binding of HLH and bZIP homodimers to Z-DNA is promoted by methylarginine modifications. Heterodimers only bind preformed Z-DNA. The interactions of TFs with ANS likely enhance genome scanning to identify cognate B-DNA-binding sites in active genes. Docking of TF homodimers to Z-DNA potentially facilitates the assembly of heterodimers that dissociate and are stabilized by binding to a cognate B-DNA motif. The process enables rapid discovery of the optimal heterodimer combinations required to regulate a nearby promoter. Full article

Sweet cherry (Prunus avium L.) cultivation in China covers an estimated area of 25,600 hectares, representing more than one-third of the global total. Viral diseases present a serious challenge to cherry production worldwide; however, the phytosanitary status of sweet cherry in China has remained poorly understood. In this study, 191 sweet cherry samples were collected from major growing regions and screened using RT-PCR combined with DNA sequencing for the presence of 14 viruses previously reported in China. Results revealed that 80.1% of the tested samples were infected with at least one virus, with mixed infections detected in 51.3% of the samples. Prevalent viruses included cherry virus A (CVA, 53.4%), prunus necrotic ringspot virus (PNRSV, 35.1%), cherry green ring mottle virus (CGRMV, 32.5%), plum bark necrosis stem pitting-associated virus (PBNSPaV, 31.4%), and prune dwarf virus (PDV, 10.5%). Cherry necrotic rusty mottle virus (CNRMV) was found at a very low frequency (0.5%), and the remaining eight viruses were not detected in any sample. Based on these findings, we developed multiplex RT-PCR assays for simultaneous detection of CVA, PNRSV, CGRMV, PBNSPaV, and PDV. Several dual and triplex RT-PCR systems were successfully established, including combinations such as PBNSPaV/PNRSV, CVA/PDV, CVA/CGRMV, PBNSPaV/PDV/CGRMV, and PBNSPaV/PNRSV/PDV. This study identifies CVA, PNRSV, CGRMV, PBNSPaV, and PDV as the prevalent viruses in the investigated Chinese sweet cherry orchards. Accordingly, multiplex RT-PCR assays were developed for their simultaneous detection. Our work advances the understanding of sweet cherry viral diseases in China and provides a valuable complementary tool for the existing diagnostic toolkit. Full article

Computational and machine learning approaches play a pivotal role in identifying, characterizing, and targeting noncanonical DNA structures, including G-quadruplexes, Z-DNA, hairpins, and triplexes. These configurations play critical roles in maintaining genomic stability, facilitating DNA repair, and regulating chromatin organization. Although the human genome predominantly adopts the B DNA conformation, evidence indicates that non-B DNA forms exert significant influence on gene expression and disease development. This highlights the need for dedicated computational frameworks to systematically investigate these alternative structures. Machine learning model, encompassing supervised and unsupervised algorithms such as K Nearest Neighbors, Support Vector Machines, and deep learning architectures including Convolutional Neural Networks, have shown considerable potential in predicting sequence motifs predisposed to forming non-B DNA conformations. These predictive tools contribute to identifying genomic regions associated with disease susceptibility. Complementary bioinformatics platforms and molecular docking tools, notably Auto Dock, along with chemical libraries like ZINC, facilitate the virtual screening of small molecules targeting specific DNA structures. Stabilizers of G quadruplexes, exemplified by CX 5461, have demonstrated therapeutic promise in BRCA-deficient cancers, highlighting the translational impact of computational methods on drug discovery. Anticipating DNA structural shifts opens new avenues in personalized medicine for complex diseases, with computational chemistry and machine learning deepening our understanding of DNA topology and guiding smarter ligand design. The integrated approach proposed in this review addresses the previous studies performed in this field and highlights the current limitations in structural genomics and advances the development of precision therapeutics aligned with individual genomic profiles. Full article

This study offers a comprehensive overview of the scientific landscape surrounding computer-aided drug design (CADD) for triplex-forming oligonucleotides (TFOs) within antisense and antigene therapeutic strategies. A dual-method approach was used, combining bibliometric mapping of 6154 Scopus-indexed articles (1980–2023) to identify publication trends and intellectual networks, with a PRISMA 2020-guided systematic review of 62 experimental studies (2015–2024) from Scopus and Web of Science, after removing duplicates using AteneaSIRES. Results show the strong dominance and clinical maturity of antisense strategies, supported by 18 FDA/EMA/MHLW-approved drugs, whereas antigene approaches remain technically limited and underdeveloped. Antigene research has focused on triplex stability modeling and biophysical feasibility but faces challenges with poor biochemical stability, limited in vivo validation, and outdated methods. Meanwhile, antisense design benefits advanced CADD pipelines, including molecular dynamics and docking modeling. Based on these insights, we propose a practical, narrative roadmap as a methodological guide: integrating proven antisense design practices and providing actionable strategies to enhance antigene research, ultimately increasing the translational potential of therapeutic TFOs with solid mechanistic and translational support. Full article

Photocatalytic oxidation of microorganisms is a powerful alternative to established disinfection approaches, applicable to a variety of water matrices. Bacterial vegetative cells, spores, fungi, and viruses, represent potential biopathogens and photocatalysis targets. Inactivation efficiency is usually evaluated by assessing viability through culture. However, additional inactivation assessment approaches are needed, as some microbes, despite being unculturable, remain metabolically active and pathogenic. Nucleic acid quantification approaches (qPCR) can assess nucleic acid release and degradation during photocatalysis. We developed a novel multiplex qPCR assay for simultaneous detection/quantification of genomic DNA from different bacterial and fungal species and of MS2 bacteriophage load. Following small-scale solar titanium dioxide photocatalysis on a microbial suspension mixture containing different biopathogen classes, we assessed photocatalytic efficiency by conventional microbiological assays (culture) and our novel molecular assay. Microbiological assays show a significant reduction in microbe viability within one hour of processing, following previously reported patterns of microbial species resistance. Molecular analysis data show that nucleic acids released in solution due to microbial oxidative damage were significantly reduced due to oxidative degradation within six hours. Through targeting different biopathogen classes, our assay could be a useful tool for assessment of photocatalytic microbe inactivation both in laboratory and real-wastewater applications. Full article

Interest in non-canonical DNA structures continues to grow, in part fueled by the recent discovery of a new structure, G-triplex DNA. Originally proposed as folding intermediates for G-quadruplex DNA, G-triplex DNA has more recently been shown to form from truncated G-quadruplex sequence oligonucleotides and other, specifically designed sequences. In this review, we provide the first, comprehensive survey of G-triplex DNA and RNA, covering the literature up to 2024. We include reports of G-triplex DNA from bulk solution and single-molecule approaches, the structural characterization of G-triplex DNA, and the breadth of oligonucleotide sequences that have been reported to form these structures. The formation of G-triplex RNA structures is also reviewed. The evolving understanding of sequence and environmental effects on G-triplex formation are presented together with challenges due to structural polymorphism and competing formation of multimeric G-quadruplex structures. Hints of the biological relevance of G-triplexes are provided by reports of protein recognition of these structures and their effects on DNA replication in vitro. Interaction of G-triplex DNA with a variety of ligands has been reported, although the search for selective ligands that can distinguish G-triplex from G-quadruplex is on-going. The vast majority of publications in the area have focused on the utilization of G-triplex in biosensing applications, which has shown some advantages compared to G-quadruplex-based systems. These results highlight the potential utility of G-triplex structures in a variety of domains and show its promise in applications in biotechnology, medicine, and research. Full article

RNA triple helices are relatively understudied, including their interactions with small molecules. In this study, we evaluated eight previously reported triplex-binding molecules (TBMs) for their functional effects on the premature and mature MALAT1 triple helix. Based on UV thermal denaturation experiments, the TBMs berberine, coralyne, sanguinarine, berenil, and neomycin selectively stabilize the Hoogsteen interface of the MALAT1 triple helix. Moreover, fisetin, luteolin, and quercetin were more sensitive to nucleotide composition, whereas berberine, coralyne, sanguinarine, and berenil were more sensitive to changes in the length of the major-groove triple helix. Most TBMs could not outcompete MALAT1 triple helix-binding proteins, except for neomycin. Surface plasmon resonance experiments demonstrated that berberine and sanguinarine display relatively quick association and dissociation binding profiles. Treating human colorectal carcinoma cells with each of the TBMs reduced MALAT1 levels by ~20–60%. This study demonstrates that TBMs broadly recognize the premature and mature MALAT1 triple helix but exhibit subtle sensitivities, suggesting that TBMs can be designed to selectively bind triple helices based on nucleotide composition, length, and structural context. Full article

The current study presents a multi-class, image-based classification of eight morphologically similar macroscopic Earthstar fungal species (Astraeus hygrometricus, Geastrum coronatum, G. elegans, G. fimbriatum, G. quadrifidum, G. rufescens, G. triplex, and Myriostoma coliforme) using deep learning and explainable artificial intelligence (XAI) techniques. For the first time in the literature, these species are evaluated together, providing a highly challenging dataset due to significant visual overlap. Eight different convolutional neural network (CNN) and transformer-based architectures were employed, including EfficientNetV2-M, DenseNet121, MaxViT-S, DeiT, RegNetY-8GF, MobileNetV3, EfficientNet-B3, and MnasNet. The accuracy scores of these models ranged from 86.16% to 96.23%, with EfficientNet-B3 achieving the best individual performance. To enhance interpretability, Grad-CAM and Score-CAM methods were utilised to visualise the rationale behind each classification decision. A key novelty of this study is the design of two hybrid ensemble models: EfficientNet-B3 + DeiT and DenseNet121 + MaxViT-S. These ensembles further improved classification stability, reaching 93.71% and 93.08% accuracy, respectively. Based on metric-based evaluation, the EfficientNet-B3 + DeiT model delivered the most balanced performance, with 93.83% precision, 93.72% recall, 93.73% F1-score, 99.10% specificity, a log loss of 0.2292, and an MCC of 0.9282. Moreover, this modeling approach holds potential for monitoring symbiotic fungal species in agricultural ecosystems and supporting sustainable production strategies. This research contributes to the literature by introducing a novel framework that simultaneously emphasises classification accuracy and model interpretability in fungal taxonomy. The proposed method successfully classified morphologically similar puffball species with high accuracy, while explainable AI techniques revealed biologically meaningful insights. All evaluation metrics were computed exclusively on a 10% independent test set that was entirely separate from the training and validation phases. Future work will focus on expanding the dataset with samples from diverse ecological regions and testing the method under field conditions. Full article

Herpesviruses are prevalent infectious agents in humans, with complex structures and life cycles. The viability and detail of a model of capsid assembly and maturation can now be examined against the recently available mature herpesvirus capsids structures. The first large assembly product is the icosahedral procapsid with an outer shell composed of major capsid proteins (MCPs) connected by triplexes (heterotrimers composed of one Tri1 protein and two Tri2 proteins), and an inner shell of scaffold proteins. The asymmetric triplexes have specific and conserved orientations, suggesting a key role in assembly. In the mature capsid structures, triplexes bound to three MCPs may represent an assembly unit where, in most cases, the N-terminus of one MCP wraps around the E-loop of another MCP. The model accommodates the incorporation of a portal into capsid, required for genome encapsidation and viral viability. Cleavage of the scaffold triggers maturation of procapsid. Each of the MCPs rotates mostly as a rigid body, except for the flexible peripheral parts that remodel to close the capsid inner surface. Angularization of the capsid shifts the portal outward to a better contact with the capsid shell. Understanding these events in the herpesvirus life cycle to atomic detail could facilitate the development of drugs that uniquely target assembly and maturation. Full article

Wastewater can serve as both a source of pathogens that pose risks to human health and a valuable resource for tracking and predicting disease prevalence through wastewater-based surveillance (WBS). In WBS for SARS-CoV-2, both nucleocapsid-specific (N1 and N2) and the envelope (E) genes are common targets for primer design, but ambiguity remains regarding differences in results depending on the gene target chosen. This study investigated how and why two SARS-CoV-2 gene targets (N2 and E) varied when analyzed in a multiplex RT-ddPCR assay for a COVID-19 wastewater monitoring study. From December 2021 to June 2022, over 700 raw wastewater samples were collected from thirteen manholes in the University of California, Irvine sewer system. Murine hepatitis virus (MHV) was used as a matrix recovery and process control in the triplex RT-ddPCR assay. Water quality tests (TSS, COD, pH, turbidity and NH₃-N) were performed on all samples. Analyses showed that in over 10% of samples, the E gene concentration exceeded N2 by more than one order of magnitude. To evaluate matrix effects on amplification efficiency for N2 and E genes, multiple regression analysis was performed to explore whether water quality variables and MHV recovery efficiency could predict variance in gene concentrations, but no clear relationship was identified. However, viral recovery, as indicated by MHV recovery efficiency, was negatively impacted in samples with higher TSS and COD, suggesting PCR inhibition. These findings contribute to methodological standardization efforts in WBS and emphasize the importance of primer selection for large-scale monitoring. Full article

Background/Objectives: Temozolomide is an important drug used for the treatment of glioblastoma multiforme. Covalent conjugation of temozolomide to triplex-forming oligonucleotides could facilitate better sequence discrimination when targeted to DNA to lessen off-target effects and potentially reduce side-effects associated with conventional chemotherapy. The base sensitivity of temozolomide precludes use of basic deprotection conditions that typify the solid-supported synthesis of oligonucleotides. Methods: A novel di-iso-propylsilylene-linked solid support was developed and used in solid-supported synthesis of oligonucleotide conjugates. Results: Conditions were established whereby fully deprotected, solid-supported oligonucleotides could be prepared for derivatisation. Cleavage of the di-iso-propylsilylene linker was possible using mild, acidic conditions. Conclusions: The di-iso-propylsilylene-linked solid support was developed and shown to be compatible with base-sensitive oligonucleotide conjugate formation. The DNA triplex formation exhibited by a temozolomide oligonucleotide conjugate was equal in stability to the unconjugated control, opening new possibilities for sequence selective delivery of temozolomide to targeted DNA. Full article