Search Results (407)

Eukaryotic genomic DNA is packaged into chromatin as nucleosomes, where it remains susceptible to reactive oxygen species (ROS) that generate the mutagenic lesion 8-oxo-7,8-dihydroguanine (8oxoG). While 8-oxoguanine DNA glycosylase 1 (OGG1) can initiate repair of 8oxoG base paired with C within the nucleosome core particle (NCP) in a position- dependent manner, it is unknown whether MutY homolog (MUTYH), the DNA glycosylase that excises misincorporated A opposite 8oxoG, can initiate repair of 8oxoG:A base pairs within NCPs. To address this, we combined cryo-EM, molecular dynamics (MD) simulations, and biochemical assays. We determined that MUTYH activity on nucleosomal 8oxoG:A is strongly suppressed, with detectable excision limited to the entry/exit region. Cryo-EM structures at four superhelical locations reveal that 8oxoG adopts the syn conformation and Hoogsteen base pairs with A, as in non-nucleosomal DNA, indicating that lesion presentation is not altered by the histone octamer. MD simulations further reveal that 8oxoG:A base pair dynamics and local DNA backbone perturbations are similar in nucleosomal and non-nucleosomal DNA. Together, these data establish that the NCP sterically excludes MUTYH from 8oxoG:A base pairs, making them largely inaccessible to MUTYH processing. This work ultimately provides mechanistic insight for the elevated G to T transversion rate observed in histone-bound DNA following oxidative stress. Full article

Diadromous fish provide key ecological and socio-economic services in European Atlantic catchments, yet their marine phase remains poorly understood and weakly integrated into management. Involving nine partners from Portugal, Spain, France and Ireland, the DiadSea Interreg Atlantic Area initiative aims to fill these critical knowledge gaps on the marine and estuarine phases and to translate this information into coordinated conservation and fisheries management tools. To do so, the project combines historical and newly collected fishery-dependent and -independent data (landings, by-catch, cooperative surveys with commercial and recreational fishers) with advanced microchemical, genetic and environmental DNA (eDNA) analyses to characterize marine distributions, mixing areas and connectivity for shads, Atlantic salmon, sea trout, European eel, sea lamprey and other diadromous species. It also includes innovative case studies on lamprey tagging and intestinal metabarcoding, coastal habitat suitability mapping for shads using river plumes and environmental variables, and joint otolith microchemistry–genomics approaches to reassess European eel panmixia and maternal origin at the Atlantic scale. Species distribution models under present and future climate scenarios, specifically RCP4.5 and RCP8.5, are used to identify priority marine areas for conservation, zones of high temporal turnover and key interfaces ensuring longitudinal (river–sea) and latitudinal connectivity, which will feed into an updated interactive web Atlas of diadromous species. In parallel, DiadSea establishes a transnational observatory of stakeholders to harmonize legislation, co-develop adaptive fisheries management plans and produce climate-aware policy guidelines, while capacity-building actions include an origin-labeling scheme for sustainably harvested diadromous fish, educational games and a comic book to raise awareness among younger generations and the wider public. Together, these work packages will deliver the first integrated, marine-focused, evidence-based and decision-support framework for diadromous fishes in the North-Eastern Atlantic, strengthening conservation, sustainable fisheries and stakeholder engagement under ongoing climate change. Full article

The vertebrate mitochondrial genome (mitogenome) is a small, circular DNA molecule typically ~16–17 kb in length, encoding 37 genes that are essential for the electron transport chain, the mechanism that drives mostly all the ATP synthesis in cells. Owing to its central role in energy metabolism, its structure is highly conserved across vertebrate lineages in both the number and relative position of each gene in the genome. Nevertheless, different variations have been found in several teleost lineages, including antarctic fishes (Nototheniidae), gadiforms, hatchetfishes (Sternoptychidae), and Batrachoidiformes. The explanation for these phenomena remains unknown yet may reflect shifts in functional constraints and can provide insights into lineage-specific and/or coevolutionary processes. This raises the possibility that mitogenome structure is related to habitat selection, potentially reflecting environmental influences on energetic regulation. To further test this hypothesis, we studied more than 400 teleost species across all major teleost lineages. The mitogenome sequences were downloaded from NCBI and annotated using two independent algorithms (MITOZ and MITOS) and then compared with a reference (Danio rerio) to find any deviation from the standard structure. Similarly, ecological data was downloaded from FishBase using the R Package “rfishbase” 5.0.3. Two independent ancestral reconstruction analyses were carried out for both traits, “Mitogenome” and “Habitat”, using a reference evolutionary tree for teleosts to unravel both evolutionary histories. The possible association between mitogenome and habitat was then assessed using a suite of phylogenetic comparative methods, including Pagel’s correlation test (corHMM) to evaluate whether both traits evolved in a correlated fashion, branch-level co-transition analysis to identify lineages where structural changes and habitat shifts co-occurred, and node-by-node comparisons of ancestral state probabilities across the phylogeny. Preliminary results suggest a correlation between some deep-sea environments and a modified mitogenome structure, with structural deviations tending to cluster in lineages inhabiting greater depths. These exploratory findings raise the possibility that changes in mitogenome architecture may be linked to adaptations in energetic metabolism required for life in extreme low-energy environments. Further analyses are underway to clarify the functional significance of these genomic changes and their relationship to ecological and metabolic pressures in teleost evolution. Full article

Stable cell lines have recently achieved recombinant adeno-associated virus (rAAV) titers comparable to the standard triple transfection approach, making them a promising alternative to plasmid-based production systems. However, whether integration of the rAAV transgene into the host genome influences packaging efficiency and vector quality remains unclear. In this study, we generated stable HEK293 cell lines carrying the rAAV transgene in their genome. rAAV production was enabled by supplying the rep/cap and helper genes on two plasmids, rendering vector genome generation dependent on the chromosomally integrated transgene. Although the stable cell lines produced a 4.5-fold lower titer of viral genomes (VGs) compared to the standard triple transfection method, VG-normalized potency was four times higher. Detailed particle characterization further revealed 3-fold lower plasmid backbone DNA packaging in rAAVs produced by stable cell lines relative to triple transfection. Consistent results were obtained from mass photometry and ELISA/ddPCR analyses for the double transfection condition, while discrepancies emerged under triple transfection. These findings emphasize the importance of functional and qualitative assessments for evaluating different rAAV production approaches. Full article

Background: Escherichia coli remains a critical multidrug-resistant nosocomial pathogen, driving interest in bacteriophage-based biocontrol. The genus Kayfunavirus (family Autotranscriptaviridae) exhibits obligately lytic replication cycles and favorable biosafety profiles, yet each new phage requires comprehensive genomic characterization to expand therapeutic candidate pools. This study aimed to isolate and genomically characterize a novel Kayfunavirus from an environmental reservoir in Vietnam. Methods: Escherichia phage vB_EcoA-Sparklingdew was isolated from Can Tho River water using host E. coli AgE9. The genome was assembled using SPAdes. The termini were resolved with PhageTerm. The annotation was done via the Pharokka pipeline and HHpred. Taxonomic classification was performed using taxMyPhage, VIRIDIC intergenomic comparisons, and maximum likelihood phylogeny of concatenated structural proteins. Results: The complete genome comprises a 37,944 bp linear dsDNA molecule (49.9% GC), encoding 51 open reading frames in a predominantly unidirectional arrangement. Key features include a virion-encoded T7-like RNA polymerase, a 723-residue T7-like DNA polymerase, a canonical lysis triad, and two putative tailspike proteins. A 212 bp direct terminal repeat and coverage profiles support a headful (pac) packaging mechanism. Comprehensive screening confirmed the absence of lysogeny, virulence, and antibiotic resistance determinants. A single synonymous SNP indicated high clonal purity. Intergenomic identity peaked at 87.7% against ICTV references, confirming placement in a novel species. Conclusions: Phage Sparklingdew represents a strictly lytic Kayfunavirus with a compact genomic architecture. Its favorable safety profile and absence of temperate markers support further evaluation for targeted therapeutic applications against pathogenic E. coli. Full article

Background: The relationship between neonatal DNA methylation (DNAm) and childhood atopy, particularly its temporal dynamics, remains inadequately characterized. Establishing this will provide insights into the epigenetic mechanisms underlying atopy development. Methods: Skin prick tests (SPT) for 11 common allergens were performed in the Isle of Wight third-generation birth cohort (IOWF2) at ages 1, 3, and 6 years. Atopy was defined as a positive response to one or more allergens on SPT. DNAm at birth at 294,265 CpGs from umbilical cords (n = 192) or Guthrie cards (n = 107) was screened (through R package ttscreening) for potential association with atopy. Pathway enrichment analysis of screened CpGs was performed using the missMethyl package in R. Associations between CpGs that passed screening and atopy status were assessed via logistic regressions with repeated measures, adjusting for age, sex, and birth weight. Age-specific associations were examined via DNAm × age interactions. CpGs showing age-specific association were further tested in the parental cohort (IOWBC (F1); n = 717). Multiple testing was controlled using FDR-adjusted p-values at 0.05. Results: In total, 601 CpGs passed screening. Pathway enrichment analysis identified enrichment of the cell activation pathway (GO:0001775; FDR-adjusted p-value = 0.017). DNAm at 502 CpGs in F2 showed age-specific associations with atopy. Among these, 102 CpGs showed consistent directions in F1, and 14 were statistically significant (p-value < 0.05). Except for cg01519508 (FOXF1), DNAm–atopy associations weakened over time at the remaining 13 CpGs. Conclusions: At certain CpGs, DNAm at birth is associated with childhood atopy in an age-dependent manner, and for CpGs showing association at an earlier age, such associations weaken at later ages. Full article

Modification of PLA with functional nanoparticles is a promising approach for imparting new properties to the material. In this work, titanium nanoparticles (Ti NPs) and titanium dioxide nanoparticles (TiO₂ NPs) were synthesized by laser ablation and characterized by dynamic light scattering, spectrophotometry, and transmission electron microscopy. The average hydrodynamic diameter of Ti NPs was 12 nm, while that of TiO₂ NPs was 24 nm; both dispersions possessed a positive zeta potential (23–27 mV) and spherical morphology. L-PLA composite films containing 0.1 wt.% Ti NPs or TiO₂ NPs were obtained by solution casting. Atomic force and modulation-interference microscopy confirmed the uniform distribution of nanoparticles within the polymer matrix, although partial aggregation was observed. The introduction of TiO₂ NPs increased the water contact angle. Mechanical testing revealed a significant reinforcing effect: the addition of 0.1 wt.% NPs increased the Young’s modulus by 62–68% and the ultimate tensile strength by 16–18% while maintaining a ductile fracture pattern with elongation at break up to ~8%. Both types of composites generated reactive oxygen species (ROS) in aqueous solutions: Ti NPs increased H₂O₂ production by 5.5 times and TiO₂ NPs by 4.9 times, and they also induced the formation of hydroxyl radicals. The accumulation of 8-oxoguanine in DNA and long-lived oxidized protein species confirmed the materials’ ability to cause oxidative damage to biomacromolecules. For E. coli, growth inhibition reached 40.5% (for composites with Ti NPs) and 71% (for composites with TiO₂ NPs). The effect was even more pronounced for S. aureus, where inhibition levels were approximately 70% and 80%, respectively; flow cytometry confirmed the strong bactericidal effect, showing that materials containing TiO₂ NPs increased the proportion of dead cells to 25% for E. coli and ~68% for S. aureus. Cytotoxicity assessment on human fibroblasts (HSF) demonstrated the high biocompatibility of neat L-PLA and composites with Ti NPs (viability > 95%) and with TiO₂ NPs (viability ~93%). The obtained results indicate that L-PLA-based composites with Ti NPs and TiO₂ NPs exhibit pronounced ROS-mediated antibacterial activity without additional UV irradiation. These findings position these materials as highly promising candidates for active biodegradable food packaging to extend shelf-life and for biomedical devices, such as wound dressings and implants, where reducing the risk of bacterial colonization is critical. Full article

Bacillus and Paenibacillus species are common and widely distributed microorganisms in food systems, often implicated in food spoilage and quality issues. Bacillus subtilis, in particular, has been associated with gas production and package bulging in seasoned foods. In this study, we developed a rapid and visual detection method for Bacillus subtilis by integrating (Recombinase Polymerase Amplification) RPA with (Clustered Regularly Interspaced Short Palindromic Repeats) CRISPR/Cas12a technology (designated as RPA-CRISPR/Cas12a). Specific RPA primers and probes were designed based on the conserved gyrB gene of Bacillus subtilis. Two sets of crRNA were designed according to the number of T-rich PAM sites on the RPA-amplified target sequence, and the reaction conditions were optimized in combination with the CRISPR/Cas12a trans-cleavage detection technology. Under optimized conditions, the crRNA3 guide (with a TT-rich PAM site) demonstrated superior cleavage efficiency compared to crRNA2 (TTT-rich PAM), while crRNA1 (TTTT-rich PAM) showed no activity. The assay achieved a detection limit of 150 pg/μL for genomic DNA and 5.5 CFU/mL for bacterial suspensions within 10 min at 37 °C. The method exhibited high specificity and sensitivity, providing a robust tool for early and on-site detection of Bacillus subtilis in food products. Full article

Apolipoprotein B mRNA editing enzyme catalytic polypeptide 3s (APOBEC3s, A3s) are single-stranded DNA cytidine deaminases with antiviral activity against diverse DNA and RNA viruses. The human APOBEC3 locus encodes seven members: A3A, A3B, A3C, A3D, A3F, A3G, and A3H. Of these, A3C, A3D, A3F, A3G, and A3H are packaged into HIV-1, lacking the viral infectivity factor (VIF, HIV-1Δvif), while A3D, A3F, A3G, and A3H hap II exhibit strong antiviral activity. Packaging of A3s into virions is critical for viral restriction, yet the underlying mechanisms remain incompletely understood. A3 incorporation requires interactions with the GAG polyprotein, especially the matrix (MA) and nucleocapsid (NC) domains, and binding to cellular or viral RNAs. Specific amino acid residues within A3 proteins mediate these contacts, and A3G localization to lipid rafts facilitates packaging. While A3F and A3G incorporation have been extensively characterized, mechanisms for other A3s remain poorly defined. This review synthesizes current knowledge on A3 packaging, emphasizing the interplay of protein, RNA, and membrane determinants in efficient virion incorporation. Full article

The order Herpesvirales contains three families, Orthoherpesviridae, Alloherpesviridae, and Malacoherpesviridae. The time since divergence of families from the common ancestor makes protein primary sequence comparison an insensitive tool for identifying common genes. Comparison of three-dimensional protein structures can reveal similarities that are not evident in primary sequences. Salmonid herpesvirus 1 (SalHV-1) is an alloherpesvirus. Complete sequencing of SalHV-1 VR-868 strain Winthrop by a combination of short- and long-read methods revealed 120 putative open reading frames (ORFs). BLAST search for similar protein sequences discovered five ORFs that encoded proteins with homologs in the orthoherpesviruses, including the major capsid protein, capsid triplex subunit 2, the catalytic subunit of the DNA polymerase, the helicase subunit of the helicase/primase complex, and the terminase ATPase subunit. An annotation of the ORFs of SalHV-1 was performed in which ORFs of SalHV-1 were modeled using AlphaFold3, and the models were used as prompts for structural similarity search using DALI and FoldSeek. Completion of this search strategy for the entire genome expanded the set of genes shared among the Herpesvirales to include additional proteins related to DNA replication and genome integrity, capsid assembly and genome packaging, and capsid nuclear egress. No homologs for any tegument proteins or proteins of the conserved entry apparatus of the Herpesviridae (gB, gH or gL) were discovered. Full article

Coronary artery disease presents heterogeneous clinical manifestations ranging from stable coronary syndrome (SCS) to acute coronary syndrome (ACS). Epigenetic mechanisms, particularly DNA methylation, may contribute to both chronic disease progression and acute plaque destabilization. However, genome-wide methylation differences between ACS, SCS, and healthy individuals remain incompletely characterized. Genome-wide DNA methylation analysis was performed in patients with ACS, patients with SCS, and healthy controls using pairwise comparisons (ACS vs. control, SCS vs. control, and ACS vs. SCS). Differentially methylated regions were identified using logistic regression implemented in the methylKit package in R. Regions with a false discovery rate-adjusted q-value < 0.05 and an absolute methylation difference (|Δβ|) > 20% were considered significant. Unsupervised hierarchical clustering revealed clear separation between ACS, SCS, and control samples, indicating distinct epigenetic profiles. ACS showed the most pronounced methylation alterations compared to controls, whereas SCS exhibited more moderate changes consistent with chronic epigenetic remodeling. Direct comparison between ACS and SCS identified dynamic, state-dependent methylation differences. Pathway analysis demonstrated enrichment of stress response, apoptotic signaling, and cell adhesion pathways in ACS, while SCS was primarily associated with pathways related to intercellular communication and vascular signaling. Our findings demonstrate that acute and stable coronary syndromes are characterized by distinct DNA methylation landscapes and pathway signatures. Epigenetic regulation of stress, adhesion, and signaling pathways may contribute to disease acuity and progression, highlighting DNA methylation as a potential molecular marker in coronary artery disease. Full article

Background: Once considered a sterile organ, the human stomach is now known to harbor a diverse microbial community that may influence both gastric homeostasis and disease. While extensive research has been conducted worldwide, regional variation in the gastric microbiome remains insufficiently characterized. This study aimed to describe the gastric antrum microbiome of Helicobacter pylori-negative Greek adults using 16S rRNA next-generation sequencing (NGS). Methods: Samples of gastric biopsies were obtained from patients undergoing gastroscopy at a tertiary hospital in Greece. H. pylori infection was excluded through a combination of bacterial culture and patient medical history. The final study group consisted of 9 subjects. Following DNA extraction, the 16S rRNA gene was sequenced on the Ion Torrent™ platform. Bioinformatic processing and statistical analyses were performed using the phyloseq, vegan, and ggplot2 R packages. Microbial composition, relative abundance, and alpha diversity (Shannon and Inverse Simpson indices) were evaluated at the genus level. Results: The gastric microbiome comprised 19 phyla, 150 families, 213 genera, and 391 species. The predominant phyla were Proteobacteria (36.92%), Firmicutes (34.21%), and Bacteroidetes (12.97%). The most prevalent families were Streptococcaceae, Helicobacteraceae, Prevotellaceae, and Pasteurellaceae. At the genus level, Streptococcus (21.71%), Helicobacter (18.39%), and Prevotella (9.99%) accounted for nearly half of the total relative abundance. Alpha diversity indices indicated moderate richness and evenness across samples. Conclusions: The gastric antrum microbiome of H. pylori-negative Greek individuals exhibits substantial taxonomic diversity dominated by Proteobacteria and Firmicutes. The microbial community structure aligns closely with profiles reported in other global populations. These findings provide a reference baseline for future comparative analyses involving H. pylori-positive individuals to better understand microbiome shifts associated with colonization and gastric disease. Full article

Acute myeloid leukemia (AML) is a biologically heterogeneous hematologic malignancy arising from the oncogenic transformation of hematopoietic stem and progenitor cells, resulting in clonal expansion and progressive subclonal diversification. Although considerable advances have deepened our understanding of AML pathogenesis, major challenges persist, particularly regarding relapses and therapeutic resistance. In recent years, exosomes—extracellular vesicles of 30–150 nm in diameter of endosomal origin—have emerged as critical mediators of intercellular communication within the AML tumor microenvironment. These vesicles transport a diverse cargo of proteins, metabolites, and nucleic acids, including mRNA, non-coding RNA species, and DNA, which is selectively packaged during their biogenesis. Circulating exosomes have garnered attention as promising liquid biomarkers for diagnosis, prognosis, and monitoring minimal residual disease, while also representing potential therapeutic targets or delivery platforms. Nonetheless, significant knowledge gaps remain regarding the mechanisms governing exosome biogenesis, cargo selection, and the functional impact on leukemia progression and immune modulation. This review focuses on the role of exosomes in acute myeloid leukemia, with an emphasis on the molecular mechanisms underlying their involvement in pathogenesis, tumor communication, and resistance to therapies, as well as their potential as diagnostic biomarkers. Full article

Background/Objectives: Antigen presenting cells (APCs) and immune cells have unique properties to drive or suppress immune responses. They are therefore key targets for the expression of vaccine antigens or transgene proteins. To better determine the utility of different molecular therapies to modify these cells, mRNA and DNA-based molecular therapy vectors were compared for their ability to genetically modify immune cells after intradermal injections in mice. DNA-based vectors included naked plasmid DNA, plasmid packaged in lipid nanoparticles (LNPs), and replication-defective adenovirus (Ad) vectors. mRNA delivery was mediated by packaging into LNPs like those used in COVID-19 vaccines. Methods: Each vector was used to deliver Cre recombinase into Cre reporter mice whose cells were activated to express green fluorescent protein (GFP) and firefly luciferase after Cre recombination. The mice were injected intradermally (ID) near the base of their tail at a site that drains into the inguinal lymph node. Luciferase activity was imaged in the living mice 1 or 4 days after vector injection. The animals were then euthanized, and luciferase activity was imaged in the draining inguinal lymph node. Cells were prepared from the intradermal injection site and from the draining lymph node to determine which immune cells were genetically modified by phenotyping CD45, CD3, and CD11b GFP-positive cells by flow cytometry. Given that the skin uniquely contains Langerhans dendritic cells, these CD207⁺ cells were also phenotyped in skin samples and in the draining lymph node. Results: In both the skin and in the draining lymph node, the rank order of luciferase and GFP activation by the vectors were: (1) Ad; (2) mRNA-LNP; (3) DNA-LNP; and (4) naked DNA. Only mRNA-LNP and Ad vectors mediated obvious luciferase activity in the living animals and in the draining lymph nodes by imaging. Notably, both vectors appeared to leak from the ID injection site and not only modify the draining lymph node but also strongly modify the livers of the mice. Naked DNA and DNA-LNP mediated detectable GFP activation in the skin and draining lymph node in some mice, but this activity was low and did not reach statistical significance when compared to PBS-treated animals. mRNA-LNPs and Ad both mediated significant Cre delivery in CD45⁺, CD3⁺, CD11b⁺, and CD207⁺ immune cells in the skin and in the lymph node, with adenovirus mediating consistently higher levels of expression in all of the tested cells. Conclusions: These data indicate that mRNA-LNP and Ad vectors mediate stronger modification of skin and lymph node immune cells after intradermal injections. Naked DNA and DNA-LNPs were markedly less potent at this activity than the other vectors. These data are consistent with the higher vaccine potency of mRNA-LNP and Ad vectors and suggest that approaches that increase targeting of immune cell subsets may have utility to increase efficacy while also reducing off-target modification of tissues like the liver. Full article

Background: Advanced paternal age is increasingly encountered in assisted reproduction as parenthood is deferred. The clinical question is whether paternal age from about 40 to 45 years and older affects embryo development or outcomes, and to what extent any effect relates to the sperm epigenome. Methods: This narrative review synthesized PubMed-indexed evidence on sperm aging biology, including DNA methylation, chromatin packaging and nucleosome retention, small non-coding RNAs, telomere dynamics, DNA fragmentation, and oxidative and mitochondrial stress, and their potential clinical impact on assisted reproduction outcomes. Results: Maternal age remains the principal determinant of embryo aneuploidy. After multivariable adjustment, independent paternal-age effects on fertilization, blastocyst formation, and preimplantation genetic testing for aneuploidy are small or not detected. At very advanced paternal ages near or above 50 years, some studies report higher miscarriage and lower live birth, without a consistent change in early embryo morphology. Aging in men is linked to higher DNA fragmentation and oxidative and mitochondrial signatures, together with reproducible sperm-epigenome changes, including age-linked DNA methylation, altered histone retention, and small-RNA shifts. These molecular findings support modest intergenerational influences on early development, while stable transgenerational inheritance in humans is not supported. Conclusions: Advanced paternal age should be regarded as a risk modifier rather than a primary driver of preimplantation failure. Counseling should emphasize realistic effect sizes and the predominance of maternal age. Laboratory workflows should minimize oxidative stress. Selective DNA-fragmentation testing may be appropriate in recurrent ART failure or recurrent loss. Sperm-epigenome assays remain investigational and should undergo prospective, standardized validation before use in routine care. Full article