We report the complete mitochondrial genome of the halotolerant green alga Dunaliella salina CS-265, isolated from a hypersaline lake in central Australia. The genome is a circular DNA molecule of 30,073 bp, encoding seven protein-coding genes, nine rRNAs, and three tRNAs. Four core genes (cox1, cob, nad1, and nad5) are fragmented by multiple introns, whereas others remain intact. The absence of ATP synthase subunits and ribosomal protein genes reflects ongoing reductive evolution in Dunaliella mitochondria. This genome adds a new organellar resource from an Australian isolate, complementing previous studies and providing further insight into mitochondrial genome dynamics in halotolerant green algae.

CEP290 is Associated With Chromatin Accessibility of Hepatitis B virus cccDNA.

We present the complete mitochondrial genome of Stonychophora maculata from Malaysia, which is a circular DNA molecule with a total length of 15,956 bp and is AT-rich (74%). It contains 37 mitochondrial genes, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a control region.

Extrachromosomal circular DNA (ecDNA) is frequently generated within the nucleus, contributing to genome dynamics and heterogeneity, thereby promoting cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here, using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes as key drivers of ecDNA production. Following DNA segmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively mediates ecDNA production by joining the free ends of the DNA fragments. Furthermore, ecDNA from patient tumors harbors junction sites with a LIG4 signature. Notably, disruption of either LIG4 or the BRCA1-A complex in cancer cells impairs ecDNA-mediated adaptation, hindering the development of resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.

Characterization of bacteriophage Henu4_2 lytic for Escherichia coli and its therapeutic efficacy in infection models.

Small extracellular vesicles (sEVs) hold immense potential for liquid biopsy given the wealth of biological information they carry. Currently, the clinical application of these methods is limited due to their low abundance and the complexities associated with traditional isolation techniques. To address this, we developed a strategy integrating cholesterol-mediated capture with a Self-Protected DNAzyme Walker for the rapid and simultaneous specific isolation and quantification of small extracellular vesicles (sEVs). Upon specific binding to CD63, the blocker strand is released, which activates the DNAzyme catalytic core, leading to substrate cleavage, which triggers the specific release of sEVs from magnetic beads and the generation of a fluorescent signal. Importantly, the circular DNA Shield design provides remarkable stability to the system by safeguarding the DNAzyme core from nuclease degradation. Furthermore, the cyclic cleavage mechanism allows for highly sensitive detection, achieving a limit of detection (LOD) as low as 361 particles per μL. In addition, by leveraging the lipid bilayer structure for sEV enrichment, this strategy effectively eliminates interference from free proteins. Furthermore, the clinical feasibility of this assay was validated by successfully distinguishing Stage I breast cancer patients from healthy individuals with high statistical significance (p < 0.001), highlighting its promise for early cancer diagnosis. This work presents a robust paradigm for sEV analysis and lays a solid foundation for their downstream biomedical applications.

Innate immunity constrains the hepatitis B virus (HBV) by sensing pathogen-associated molecular patterns (PAMPs) and inducing type I/III interferons and interferon-stimulated genes. This review synthesizes molecular mechanisms by which HBV nucleic acids and proteins are detected by pattern recognition receptors (PRRs) and how the virus evades such surveillance. At the DNA level, covalently closed circular DNA (cccDNA) persists as a chromatin-like episome with low immunogenicity; cGAS–STING signaling is functionally dampened, whereas nuclear interferon-inducible protein 16(IFI16) and cytoplasmic/nuclear ABCF1 bind cccDNA to repress transcription, and APOBEC3A-mediated deamination requires robust interferon signaling. At the RNA level, TLR3/7/8 and retinoic acid-inducible Gene I(RIG-I) sense circulating HBV RNA and 5′-triphosphate pregenomic RNA, respectively. HBV counteracts RIG-I-like receptor (RLR) pathways through ADAR1 editing, TIAR-dependent translational control, and a metabolic checkpoint involving lactate-MAVS/hexokinase, whereas spliced viral RNAs (svRNAs) have emerged as immunologically relevant species. At the protein level, Hepatitis B Surface Antigen (HBsAg) impairs interferons (IFN) induction by blocking the TAK1–TAB2–NF-κB/IRF axis; Hepatitis B Virus X Protein (HBx) sustains cccDNA transcription via DDB1-directed Smc5/6 degradation and broadly suppresses PRR/IFN signaling, with TRIM25 acting as a host restriction factor. These insights nominate combinatorial strategies—PRR agonists (TLR/STING), MAVS sensitization, metabolic disinhibition, pharmacological disruption of the HBx–DDB1 axis, and reinforcement of IFI16/ABCF1—to achieve functional control of cccDNA and advance curative hepatitis B virus (HBV) therapy.

Cardiac aging is associated with progressive cardiac fibrosis and dysfunction, yet the underlying mechanisms remain incompletely understood. Extrachromosomal circular DNA (eccDNA) has been reported to participate in tumor and age-related genomic instability, while its role in cardiac fibrosis during aging remains to be fully elucidated. In this study, circular DNA sequencing and RNA seqencing were performed to analyze eccDNA profiles in young and aged cardiac tissues. The number of eccDNAs in the cardiac tissue of aged mice is higher than that in young mice. Combining the annotation of eccDNAs and the key genes related to aging identified in the transcriptome, we identified sterile alpha and TIR motif containing 1 (Sarm1), a key regulator of NAD+ metabolism and neurodegeneration located in eccDNAs, as a novel driver of cardiac aging via pro-fibrotic signaling. In aged mice, Sarm1 knockdown significantly restored cardiac function and reduced fibrosis. Conversely, Sarm1 accelerated cardiac aging phenotypes in young Sarm1-overexpressing transgenic mice. Mechanistically, co-immunoprecipitation combined with mass spectrometry identified TGF-β-Smad2/3 as the dominant pathway, with pharmacological inhibition by SIS3 abolishing Sarm1-driven Smad2/3 phosphorylation. Our findings reveal that Sarm1-containing eccDNA drives cardiac aging by amplifying pro-fibrotic signaling through the TGF-β-Smad2/3 pathway, proposing eccDNAs clearance and Sarm1 inhibition as novel therapeutic strategies for aging-related cardiac fibrosis.

Circular Rep-encoding single-stranded DNA (CRESS-DNA) virus Rep proteins are multidomain enzymes that mediate viral DNA rolling-circle replication. Reps nick viral DNA to expose a 3' end for polymerase extension, provide an NTP-dependent helicase activity for DNA unwinding, and join nicked ends to form circular viral genomes. Here, we present the first structures of a Rep protein from the Redondoviridae family, a newly discovered family of human-associated CRESS-DNA viruses that replicates within the oral protozoan Entamoeba gingivalis. Using cryo-EM, we characterized the hexameric structures of a Redondovirus Rep helicase bound with ATPγS, representing the initial ATP-bound state, and with ADP, reflecting the protein state after hydrolysis. The ADP state, but not the ATP state of Rep shows a staircase arrangement of DNA-binding loops that plays a central role in current models for SF3 helicase function. Additionally, we determined a head-to-tail dodecameric structure of ATPγS-bound Rep, in which both the helicase and endonuclease domains are ordered. Conservation of residues involved in stabilizing the dodecamer suggest that this assembly may be functionally relevant for many CRESS-DNA viruses. The positioning of endonuclease domains in the Rep hexamer, combined with our biophysical analyses of Rep oligomerization, provide new insights into Rep function during viral replication.

The roles of extrachromosomal DNA in tumorigenesis and therapeutic resistance in cancer.

SOX6 is a novel host factor that promotes hepatitis B virus replication by enhancing the transcriptional activity of enhancer I.

Extrachromosomal DNA as a dynamic engine of cancer evolution: Structure, plasticity and therapeutic resistance.

Novel mechanism of Guizhi Jia Huangqi decoction disrupting HBV lifecycle via cAMP/PKC/PKB axis beyond NTCP blockade.

IntroductionTicks are globally recognised as the second most important vectors of infectious diseases, posing significant threats to human and animal health. Haemaphysalis parva (Acari: Ixodidae) is frequently reported infesting humans and domestic animals and has been experimentally demonstrated to transmit Babesia ovis, with field associations to ovine babesiosis during the colder months. It has also been reported to harbour several zoonotic pathogens, including Coxiella burnetii, Francisella tularensis, and various Rickettsia species. Here, we aim to report the complete mitochondrial genome of Haemaphysalis parva (Ixodida: Ixodidae), a zoonotic tick species with significant public health relevance in Türkiye. MethodsFor this purpose, we isolated total genomic DNA from H. parva and sequenced using Illumina HiSeq 2000 platform, raw reads were processed, and then the mitogenome was assembled using the Geneious R9 program with "map to reference" and verified via "de novo assembly" options.Results and DiscussionThe mitogenome of H. parva is a circular DNA molecule of 14,843bp, comprising the canonical 37 genes (13 PCGs, 22 tRNAs, and 2 rRNAs) and two major non-coding regions (312bp and 304bp). Strand-specific compositional bias revealed a strong A + T enrichment (77.8%) and pervasive negative AT- and GC-skew values, diverging from the typical skew profiles observed in most arthropods and possibly reflecting lineage-specific replication asymmetries. All PCGs exhibited AT-biased codon usage, preferentially encoding hydrophobic amino acids. Several genes (cox1, cytB, nd2, nd6) showed dN/dS ratios > 1, suggesting positive adaptive evolution. Comparative mitogenomic analysis of 27 Haemaphysalis species confirmed overall structural conservation but identified a rearranged nd1-rrnS gene block relative to the Ixodes reference genome. Collinearity and synteny analyses revealed multiple conserved sequence blocks, including a putative humanin-like ORF within the rrnL gene region, indicating potential dual-coding or regulatory elements within non-PCG regions.

Hepatitis B surface antigen (HBsAg) seroclearance is widely regarded as a functional remission of chronic hepatitis B virus (HBV) infection. However, HBV covalently closed circular DNA (cccDNA) may persist in hepatocytes, posing a risk of reactivation even years after seroclearance. We aimed to determine the incidence, clinical context, and virological features of HBV reactivation in a large, population-based cohort from a geographically isolated island region of Japan with a high HBV prevalence. Between 1978 and 2008, 34,517 residents of the northern Goto Islands underwent community-based HBsAg screening, identifying 1045 individuals with chronic HBV infection (3.0% prevalence). Among them, 177 achieved spontaneous HBsAg seroclearance and were followed for a median of 6.3years (1500 person-years). Age- and sex-matched controls were selected from HBsAg-negative residents (3043 person-years). Reactivation-related events were defined as reappearance of HBsAg after confirmed seroclearance. HBV reactivation-related events occurred in 4 of 177 patients (2.3%), corresponding to 27 per 10,000 person-years, whereas none occurred among 354 matched controls. All HBsAg reappearance cases had cirrhosis or hepatocellular carcinoma at baseline. One patient developed a hepatitis flare with high-level viremia and HBeAg reappearance. Viral sequencing demonstrated 99.1% similarity between baseline and reactivated strains, confirming reactivation rather than reinfection. Reactivation-related events after HBsAg seroclearance were rare in this population-based cohort. These findings suggest that HBsAg seroclearance may not necessarily indicate complete viral eradication and that careful long-term monitoring may be warranted, particularly in patients with advanced liver disease.

Circular nucleic acids such as extrachromosomal circular DNA (eccDNA) and circular RNA (circRNA) are increasingly recognized for their biological relevance and potential as biomarkers in disease contexts. Despite their growing importance, their detection remains challenging due to tool-specific biases, limited validation frameworks, and high variability in performance across datasets. We benchmarked 10 circle detection tools across diverse conditions using both simulated and biological datasets. Our evaluation included classical performance metrics and a novel internal measure of read distribution symmetry (ΔCJ) to assess circle prediction confidence. We explored the impact of sequencing protocols, filtering strategies, and combined tool consensus. We found that detection accuracy was highly influenced by sequencing depth, alignment algorithm, and experimental enrichment protocols. ΔCJ proved effective in flagging potential false positive circles, showing improved accuracy of Intersect (circles detected by all tools) and Rosette (circles detected by ≥2 tools) combinations. This study offers a broad evaluation of circular detection tools, suggesting that the combination of ≥ 3 tools is necessary for a correct prediction. These insights will inform future experimental design and data analysis pipelines in both experimental and clinical settings.

Although far less well-known and understood than chromosomal DNA, extrachromosomal circular DNA (eccDNA) are a pervasive and dynamic component of eukaryotic genomes. eccDNA are nuclear-localized, double-stranded DNA circles that exist independently of the main chromatin body. They share many sequence features with chromosomal DNA, including encoding functional genes; however, unlike chromosomes, eccDNAs are highly heterogenous, capable of autonomous replication and ultra-high gene expression, and do not necessarily segregate evenly or follow Mendelian inheritance during cell division. Although several recent reviews have focused on their roles in human health, emerging research in plants shows that eccDNAs are intricately associated with rapid adaptation to stress, particularly in weedy and invasive plants. This plant-centric review synthesizes evidence that eccDNAs carry full-length genes, regulatory elements, and transposable sequences, that collectively enable gene amplification, novel protein variants, and context-specific expression. We propose that eccDNAs function as "genomic shock absorbers": stress-inducible, non-Mendelian reservoirs of genetic diversity that buffer genomes against environmental challenges such as nutrient limitation and xenobiotic exposure. Drawing parallels with bacterial plasmids, we argue that eccDNA facilitate novel and important genome-environment interactions beyond those mediated by chromosomes. Harnessing these elements as non-Mendelian vehicles for genetic innovation could offer a route to translate weed-derived resilience into novel crop improvement strategies, enabling the design of climate-ready, stress-resilient agriculture grounded in weed inspired mechanisms of adaptability and tolerance.

Extrachromosomal circular DNAs (eccDNAs) are closed circular DNA molecules widespread across eukaryotic cells, with emerging roles in gene regulation and tumor progression. Experimental assays remain costly and incomplete, underscoring the need for computational approaches. To address this, a deep learning framework termed DeepECC has been established to overcome the challenges posed by eccDNA heterogeneity and its complex biogenesis. Through a two-stage training strategy, DeepECC models the local sequence context flanking both the start and end breakpoints, thereby capturing mechanistically informative features that are often overlooked when analyses focus solely on eccDNA body sequences. Applied to multi-species (human, mouse, gallus) datasets, DeepECC robustly captures conserved breakpoint features, with a marked preference for GC-rich and transcriptionally active regions. Genome-wide scanning reveals non-uniform distributions of human cancer eccDNAs enriched in enhancers, expression quantitative trait loci, and CTCF sites, suggesting regulatory functions in tumor progression. Motif analysis further implicates ribosomal activity, translational regulation, and DNA damage response. Furthermore, genome-wide eccDNA predictions are integrated into the UCSC Genome Browser, enabling convenient querying and visualization of cancer-related eccDNAs associated with specific genes or genomic regions, facilitating functional interpretation for experimental research. Collectively, DeepECC provides a generalizable framework for systematic eccDNA discovery and insights into their functional significance in cancer.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) constitutes a viral persistence reservoir that sustains chronic infection. Although the DNA damage response (DDR) facilitates cccDNA biogenesis, its role in regulating cccDNA stability remains unclear. By intersecting published cccDNA-associated proteomic datasets with known DDR-related host factors, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) as a novel restriction factor that binds cccDNA and suppresses HBV replication by promoting cccDNA degradation. Mechanistically, hnRNPA2B1 interacted with the G-quadruplex (G4) structure of cccDNA, with preference for G4-1, G4-7, and G4-10, and leads to the recruitment of the cytidine deaminase APOBEC3B by its prion-like domain (PrLD), thereby inducing C>T and G>A hypermutations and initiating cccDNA decay. Notably, HBV counteracts this defense mechanism through HBx-mediated hnRNPA2B1 polyubiquitination and proteasomal degradation, revealing a viral evasion strategy that perpetuates cccDNA persistence. These findings reveal a G4-dependent surveillance axis wherein hnRNPA2B1 directs APOBEC3B-mediated cytidine deamination to destabilize cccDNA while identifying HBx-induced hnRNPA2B1 ubiquitination as a viral countermeasure. This mechanistic duality not only elucidates a critical virus-host interaction governing cccDNA persistence but also provides a promising therapeutic target for the treatment of HBV infection.

Assisted reproductive techniques are widely used to produce domestic animal embryos for commercial or research purposes. In cattle, abnormal trophoblast cell differentiation during embryo development causes pregnancy and placentation failures. The CRISPR-on system has been successfully used in bovine embryos to individually activate early trophoblast lineage genes TFAP2C and SMARCA4. This study evaluates the effect of CRISPR-on activation of early and advanced trophoblast-related genes in bovine zygotes to promote trophectoderm differentiation. In one experiment, RNA was microinjected to simultaneously activate TFAP2C and SMARCA4; in another, circular DNA was microinjected to induce CDX2 or GATA3 expression separately. Controls included groups without small guide RNAs (sgRNAs, SHAM) and non-injected embryos (IVF). RNA microinjection transiently and simultaneously increased TFAP2C, SMARCA4, and downstream gene expression, producing a prolonged effect beyond individual gene activation. This led to a significant increase in trophectoderm cells at the blastocyst stage. Individual activation of CDX2 and GATA3 was effective, also inducing upstream genes without altering trophectoderm cell percentages. Neither RNA nor DNA microinjection affected blastocyst production compared to IVF. This work demonstrates the successful increase of trophoblast cells in bovine embryos using CRISPR-on, offering a useful strategy for IVF or SCNT embryos. The CRISPR-dCas9VP160 system may also aid understanding of trophoblast lineage signaling during development.