Search Results (637)

Hepatitis C Virus (HCV) is a plus-strand RNA virus that replicates its genome via a minus-strand intermediate, which in turn is the template for the synthesis of progeny plus-strand genomes. In order to characterize sequence elements in the HCV 5′-untranslated region (5′UTR) that are possibly involved in the regulation of minus-strand RNA synthesis starting at the genome’s 3′end, we used a replicon system in which a possible function of these sequences is uncoupled from other functions like translation regulation. For the specific detection by RT-qPCR of minus strands newly synthesized in the cells from the transfected replicon RNAs, we carefully eliminated the contaminating DNA and transfected RNA and avoided self-priming caused by hairpin formation. We found that the absence of any HCV sequences at the 5′end does not allow genome replication. Stem-loop I-II sequences only allow extremely low-level replication, whereas the presence of stem-loops I-III or the complete 5′UTR allows efficient replication. The mutation of sequences required for the binding of translation initiation factor 3 (eIF3) and the ribosomal 40S subunit in the 5′UTR of the plus strand severely impairs minus-strand synthesis. This suggests that eIF3 and the 40S subunit are involved in plus-strand 5′-3′-end communication and the regulation of minus-strand synthesis. Full article

Singleplex and multiplex real-time PCR-HRM (polymerase chain reaction with high resolution melting), both with specific and non-specific amplicon detection, are used for a wide range of applications, from clinical diagnostics to food authentication. However, their results can be influenced by the quality of the template DNA and composition of the reaction mixture. The methods used for the analysis of these results then influence the conclusions drawn. In this work we present an example from our laboratory practice, where the results of singleplex and multiplex real-time PCR differed, despite using the same reaction conditions, primers and analyzed plant material. We show the influence of a singleplex and multiplex PCR setup on the results, as well as the influence of template contamination on the melting behaviour of amplicons. We also discuss the usefulness of cluster analysis for the clarification of real-time PCR-HRM results which appear unclear when only melting and difference curves or similarity scores are used for the analysis of these results. We provide a discussion of problems which we encountered during an analysis of commercial teas and which should be considered by researchers new to PCR-based analysis of plant material, especially if the studied material is rich in various contaminants. Full article

Melanoma remains a highly aggressive malignancy, particularly in advanced metastatic stages where therapeutic options are limited. Natural compounds provide a structural basis for discovering novel anticancer agents. In this study, we employed an integrated in silico approach to evaluate the pharmacokinetic properties, toxicity profiles, and molecular targets of key alkaloids from Chelidonium majus, including berberine, sanguinarine, chelerythrine, chelidonine, protopine, umbelliferone and coptisine. ADME/T predictions (SwissADME and DeepPK) revealed favorable drug-likeness and oral bioavailability for most compounds, with berberine exhibiting the most balanced safety and absorption profile. All compounds demonstrated high intestinal absorption (>99%) and implicated key melanoma targets, including APE1/Ref-1, CXCR4, CCR2, TLR8, galectin-3, and VEGFR2. These molecules represent valuable templates for the development of melanoma therapies. Among the tested compounds, chelidonine emerged as a potential APE1 inhibitor, exhibiting the highest binding affinity and forming specific interactions within the enzyme’s catalytic site, suggesting its potential as a DNA repair-targeted agent in melanoma. These findings support the further exploration of natural alkaloids, including structural optimization or advanced formulation strategies, to enhance safety, bioavailability, and therapeutic efficacy in melanoma. Full article

This study reports the synthesis, spectroscopic characterization, and biological evaluation of a novel moxifloxacin hydrazide derivative (MOX-H) and its metal complexes with Co(II), Ni(II), Cu(II), VO(IV), and Gd(III). The ligand was synthesized by hydrazinolysis of moxifloxacin hydrochloride, and the resulting hydrazide was subsequently complexed with the respective metal salts. The interaction between MOX-H and the metal ions yielded the corresponding complexes, formulated as [Co(H₂O)Cl(MOX-H)₂]Cl·2.5H₂O, [Ni(H₂O)Cl(MOX-H)₂]Cl.4.5H₂O, [VO(MOX-H)₂]SO₄.3.5H₂O, [Gd (H₂O)(MOX-H)₂(NO₃)₂]NO₃.2H₂O, and [Cu(MOX-H)₂(H₂O)Cl]Cl·xH₂O (where x = 2, 2.5, 0.5, for products synthesized via template, microwave-assisted, and hydrothermal methods, respectively). The synthesized analogues were characterized by elemental analysis (CHN), FT-IR, UV-visible, and ¹H NMR spectroscopy, and mass spectrometry, as well as thermogravimetric (TG/DTG) and magnetic measurements. FT-IR spectra confirmed coordination through the hydrazide carbonyl and amine groups, while UV–visible and magnetic data indicated predominantly octahedral geometries. The thermal behavior exhibited multistep decomposition with activation parameters supporting exothermic processes. When compared to the free ligand, the metal complexes showed increased antimicrobial activity against both Gram-positive and Gram-negative bacteria and fungus species, particularly for the Co(II) and Cu(II) complexes, which showed the largest inhibition zones. The Cu(II)–MOX-H complex exhibited the lowest MIC values (4.88–9.76 µg/mL) among all tested compounds, confirming its outstanding antibacterial potency and high sensitivity compared to the free ligand and standard drug. Cytotoxicity assays demonstrated selective anticancer activity, with the Cu(II)–MOX-H complex showing the highest potency (IC₅₀ ≈ 2.95 µM against MCF-7 and IC₅₀ ≈ 0.98 µM against HepG-2), while maintaining minimal toxicity toward normal cells. These findings were corroborated by molecular docking investigations, which showed that the MOX-H complexes had substantial binding affinities (−9 to −10 kcal/mol) toward DNA topoisomerase II, consistent with their observed biological effects. Full article

The poly(A) tail of mRNA plays a vital role in mRNA transcript stability, translational efficiency, and immunogenicity. Co-transcriptionally polyadenylated in vitro transcribed (IVT) mRNAs typically contain poly(A) tails of 50–120 nucleotide tail length due to limitations in production of template pDNA with longer poly(A) sequences. In contrast, post-transcriptional enzymatic polyadenylation of mRNA with poly(A) polymerase (PAP) presents a modular alternative to increase the tail length. However, the lack of real-time control strategies for PAP-mediated tailing has limited its broader applicability in mRNA production. Here, we develop a methodology for controlling poly(A) tail length in post-transcriptional polyadenylation of mRNA that uses adenosine triphosphate (ATP) consumption measured at-line to predict the poly(A) tail length. We establish a novel analytical method based on monolith reverse-phase chromatography to validate the poly(A) predictions. We were able to produce longer poly(A) tails and accurately determine their length in 300–700 nt range. The resulting longer poly(A) tailed reporter mRNAs outperformed the encoded and shorter poly(A) tailed mRNAs in cell-based assays. This work presents a new strategy for controlled post-transcriptional polyadenylation using ATP consumption as a process control metric, an approach which may in future be expanded to other NTP-dependent enzymatic conversions. Full article

Ribosome biogenesis is a fundamental process underlying plant growth, development, and environmental adaptation, and processing of precursor rRNA (pre-rRNA) represents one of its most critical regulatory steps. This review provides a systematic overview of the multi-layered regulatory mechanisms controlling pre-rRNA processing in plants, with Arabidopsis thaliana as the primary model system. We focus on the genomic organization of ribosomal DNA (rDNA) and its epigenetic regulation, illustrating how highly repetitive and sequence-diverse rDNA arrays maintain genomic stability while enabling tissue-specific expression of distinct rDNA variants. We further summarize the dynamic pathways of pre-rRNA processing and their plastic regulation under environmental conditions such as elevated temperature. In addition, we review the quality control systems that monitor pre-rRNA maturation, including non-templated tailing and exonuclease-dependent degradation pathways, which play essential roles in removing aberrant processing intermediates. We further examine how perturbations in pre-rRNA processing give rise to plant ribosomopathies and discuss complementary models of ribosome homeostasis and ribosome heterogeneity as frameworks for interpreting shared developmental phenotypes. Finally, by synthesizing genetic and molecular evidence, we highlight the pivotal role of pre-rRNA processing in orchestrating plant development and propose directions for future research. Full article

The human mitochondrial Lon protease (LonP1) is a central regulator of mitochondrial DNA copy number and metabolic reprogramming. However, the structural basis for how LonP1 recognizes native physiological substrates remains elusive. Here, we present the high-resolution cryo-EM structure of the human LonP1 hexamer actively engaging its native substrate, TFAM. The reconstruction reveals a distinct bipartite search-and-shred mechanism. Unlike its bacterial homologs, the human N-terminal domain (NTD) adopts a compact architecture acting as a selective vestibule to recruit and initially unfold the substrate tertiary structure. Subsequently, the polypeptide is threaded through the central channel via a hand-over-hand mechanism driven by a spiral array of aromatic pore-loops. This structural framework provides a mechanistic rationale for the spatial segregation of LonP1 and offers a template for targeting mitochondrial proteostasis in human diseases. Full article

Spotted fever group (SFG) rickettsioses are tick-borne infectious diseases caused by more than 30 Rickettsia species. As ticks may harbor and transmit multiple pathogens during a single blood meal, sensitive and specific molecular detection methods are essential for early diagnosis. Conventional nested PCR is commonly used but is time-consuming and prone to cross-contamination due to multiple amplification steps. This study evaluated a dual-target one-step nested PCR assay developed as a rapid alternative to conventional nested PCR for SFG Rickettsia detection. Gene-specific primers targeting the Rickettsia outer membrane protein A (ompA) gene and the 17 kDa antigen gene were designed, with a Plasmodium falciparum thrombospondin-related anonymous protein (TRAP) gene included as an internal amplification control. Primer specificity was verified in silico, and assay performance was assessed using synthetic DNA templates. The dual-target one-step nested PCR achieved detection limits of 10 gene copies for the 17 kDa gene and 1000 gene copies for ompA, compared with 10 and 100,000 gene copies, respectively, using conventional nested PCR. Screening of 184 tick specimens identified one positive sample (0.54%) for the Rickettsia 17 kDa gene. Overall, the dual-target one-step nested PCR demonstrated comparable sensitivity to conventional nested PCR while reducing assay time and contamination risk, indicating its potential as a reliable tool for SFG Rickettsia detection. Full article

In this work, we analyzed the ability to incorporate 8-oxo-dATP by several human DNA polymerases: replicative Pol ε (exo-) from Family B; base excision repair (BER) enzymes Pol β and Pol λ from Family X; and translesion Pol η, Pol ι, and Pol κ from Family Y. We demonstrated that human DNA polymerases differ in their abilities to discriminate against 8-oxo-dATP. Among the tested DNA polymerases, Pol λ exhibited the worst ability to discriminate against 8-oxo-dATP opposite template T on DNA substrates with a protruding single-stranded 5′-end and a double-stranded DNA with a 1 nt gap. Pol β and DNA polymerases of Family Y showed relatively high accuracy. Pol η demonstrated the most effective discrimination against 8-oxo-dATP on templates T and G. Pol ι exclusively incorporated 8-oxo-dATP opposite template G but not T. Unexpectedly, the catalytic subunit of high-fidelity Pol ε (exo-) incorporated 8-oxo-dATP opposite templates T and G with higher efficiency compared with the error-prone polymerases of Family Y and Pol β. While the structures of human polymerases with incoming 8-oxo-dATP are not available, we speculate on a possible mechanism of 8-oxo-dATP discrimination. Full article

Background: The development of plasmonic photothermal therapy (PPTT) to trigger cancer cells is often hindered by uncontrolled overheating and the lack of real-time feedback. Methods: In this study, we report the synthesis of gold nanorod-embedded mesoporous silica nanoshells (AuNR@Si) as a multifunctional theranostic platform designed for controlled hyperthermia and surface-enhanced Raman spectroscopy (SERS) monitoring. Using a layer-by-layer templating strategy, AuNRs were successfully obtained within a hollow silica architecture. Results: While AuNRs alone exhibited rapid photothermal spikes reaching 64 °C, the AuNR@Si platform moderated the photothermal response, maintaining a stable therapeutic window (41–45 °C). In vitro assays using T47D breast cancer cells demonstrated a 33% reduction in viability following irradiation. Furthermore, the structural stability of the AuNR@Si platform enabled SERS monitoring of cellular damage, identifying specific biochemical fingerprints of protein denaturation, cytochrome c release and DNA fragmentation. Conclusions: These results suggest that AuNR@Si nanoshells provide a safer, regulated approach to photothermal ablation with the added benefit of molecular detection, demonstrating proof-of-concept theranostic functionality in a luminal breast cancer model. Full article

Here we discuss three veterinary alphaherpesviruses—pseudorabies virus, equid alphaherpesvirus 1, and bovine alphaherpesvirus 1—that were instrumental in uncovering the true extent of transcriptome complexity through long-read RNA sequencing, which earlier short-read approaches could not resolve. We focus on three major transcriptomic features whose discovery and characterization relied heavily on these viral models: (i) widespread transcriptional overlaps that complicate read assignment and may drive transcriptional interference; (ii) diverse transcript isoforms arising from alternative 5′ and 3′ transcript termini, as well as splicing; and (iii) non-coding RNAs clustered near replication origins that illuminate potential replication–transcription interactions on a shared nuclear template. Long-read viromics in these veterinary systems has additionally served as a stringent benchmark for transcript callers and annotation pipelines, because the extreme density of overlaps and co-terminal transcript families exposes errors that often go unnoticed in typical mammalian transcriptomes. This has made veterinary herpesvirus datasets disproportionately influential in shaping best practices for full-length isoform calling, transcript end mapping, and artifact-robust cDNA library handling. We also discuss animal gammaherpesviruses as proxies for human gammaherpesviruses, allowing experimental investigation of viral programs difficult to study in human infection. Finally, we describe pseudorabies virus applications as a retrograde transneuronal tracer. Full article

Autosomal short tandem repeat (STR) markers remain the cornerstone of modern forensic genetics, providing exceptional power for individualization, kinship verification, and reconstruction of complex investigative cases. Over the last decade, the field has undergone a major technological transition from length-based capillary electrophoresis (CE) toward sequence-level characterization using massively parallel sequencing (MPS), enabling detection of internal sequence variants (isoalleles) and flanking-region polymorphisms that substantially increase discriminatory power in many forensic contexts. Although MPS is increasingly adopted in forensic laboratories, implementation remains dependent on infrastructure, cost considerations, validation requirements, and jurisdiction-specific legal frameworks. This review synthesizes the molecular mechanisms underlying STR variability, including replication slippage and mutation processes, and critically evaluates the transition to sequencing-based analysis. Particular attention is given to analytical challenges such as stochastic effects in ultra-low-template DNA and PCR inhibition in degraded samples. Special emphasis is placed on identification of skeletal remains from mass graves and historical contexts, where hierarchical analytical strategies—from mini-STR approaches to MPS-based workflows—enable recovery of highly fragmented DNA. The review also examines the evolution of probabilistic genotyping (PG), highlighting the importance of algorithmic transparency and reproducible analytical frameworks for judicial applications. By integrating technological advances with practical forensic challenges, this review outlines a comprehensive framework for implementing high-resolution STR analysis in contemporary genomic casework. As a narrative synthesis, the conclusions reflect currently available published evidence and acknowledge variability in validation status, implementation practices, and regional forensic infrastructures. Full article

Aspergillus fijiensis is an industrially important filamentous fungus, whose genetic analysis has been limited by the absence of species-specific tools. This study establishes an optimized CRISPR–Cas9 genome editing platform for A. fijiensis, from protoplast preparation to DNA repair pathway engineering. Antibiotic screening first identified hygromycin B and 5-FOA (5-fluoroorotic acid) as effective positive and counter-selection markers. A high-efficiency protoplast regeneration protocol was developed depending on specific osmotic stabilization and mycelial competence. Evaluation of a plasmid-based CRISPR system revealed that while autonomous replication was feasible, gene editing was constrained by low efficiency and a predominant bias toward NHEJ (non-homologous end joining). We implemented a Cas9–sgRNA RNP (ribonucleoprotein) delivery approach, with RNP delivery alone producing frequent indels. However, targeted integration remained inefficient when using conventional MMEJ (Microhomology-mediated end joining) donors. By employing donors containing short (5 bp) microhomology arms between cleavage sites, we effectively engaged the MMEJ pathway, enabling precise insertions and large-fragment deletions in 92% of the analyzed transformants. Donor templates containing minimal 5 bp microhomology sequences could effectively shift the predominant repair pathway from NHEJ to MMEJ. These findings demonstrate that MMEJ is the superior pathway with a unique mechanism for genome engineering in A. fijiensis, providing a versatile toolkit for unlocking the biotechnological potential of this recalcitrant species and a successful paradigm for establishing genetic systems in other species. Full article

In order to establish a rapid and sensitive LAMP visual detection method for Cherry Virus A on-site, this study used the conserved fragment of the CVA coat protein (CP) sequence as a template for primer design. The rapid visual LAMP detection method for Cherry Virus A was successfully established by optimizing the reaction system components (concentration ratio of internal and external primers, and concentrations of loop primers, Bst DNA, Mg²⁺, dNTPs and betaine) and reaction conditions (temperature and time). This method enables specific detection of Cherry Virus A and facilitates visual inspection of crude nucleic acid extracts within 40 min, significantly reducing the diagnostic turnaround time. The limit of detection is 67.54 pg μL⁻¹ (cDNA), which is 100 times more sensitive than PCR. Analysis of 70 field sweet cherry samples revealed an RT-LAMP positivity rate of 91.42%, significantly surpassing the 71.42% achieved by RT-PCR. This method is suitable for the rapid on-site detection of Cherry Virus, and can also provide a theoretical reference for the early diagnosis of cherry viral diseases. Full article

This critical and selective review synthesizes the accumulating body of biological evidence supporting a process we term epigenetic–genetic coupling as a mechanistic basis for Lamarckian inheritance of somatically acquired adaptations. We propose that evolutionary processes in mammals and higher vertebrates can involve deaminase-driven, reverse transcriptase-mediated, RNA-templated targeted homologous recombination. We contrast well-established examples of “Soft”, reversible epigenetic inheritance with historical and contemporary evidence suggestive of stable, DNA-integrated “Hard” Lamarckian transgenerational inheritance. Our analysis indicates that the establishment of “Hard” Lamarckian inheritance may require specific population dynamics, including inbreeding or interbreeding among phenotypically affected offspring, together with sustained and defined environmental stimuli over one or more generations to consolidate the acquired traits at the genomic level. We also present molecular and cellular evidence supporting RNA-to-DNA genetic feedback mechanisms involving targeted genomic integration, primarily mediated by the DNA repair–associated reverse transcriptase activity of DNA polymerase η. Finally, we review diversification mechanisms in molecular and cellular immunology that now routinely employ single-molecule, real-time, long-read genomic sequencing (6–8 kb). We recommend the broader application of these technologies in future breeding and experimental programs across other somatic systems. Their deployment offers a robust strategy for securing definitive “Hard” molecular evidence of Lamarckian acquired inheritance in diverse biological contexts; including somatically acquired immunity, as well as adaptive behavioral and central nervous system phenotypes. This is compatible with our over-arching goal—to provide an experimental road map of conceptual options to drive future experimentation in acquired inheritance breeding programs. Full article