Identification and analysis of extrachromosomal circular DNAs in pancreatic islets during the early and late stages of T2DM mice.

Prospects for leveraging knowledge on ISG and NF-kB effector functions into anti-HBV therapies.

Escherichia coli is a major cause of hospital-acquired infections in China, including urinary tract, bloodstream, and intestinal infections. Given the rising prevalence of antibiotic-resistant E. coli, phages are increasingly regarded as promising alternatives to conventional antibiotics. Henuyfy11N was isolated using the double-layer agar method and characterized via transmission electron microscopy in this study. Biological assays included stability under varying pH and temperature, UV sensitivity, host range, optimal multiplicity of infection, adsorption rate, and one-step growth curve. In vitro lytic activity against extended-spectrum β-lactamase (ESBL)-producing E. coli and biofilm eradication capacity was assessed. Whole-genome sequencing enabled phylogenetic, synteny (the analysis of conserved blocks of genetic sequence between different genomes), and functional annotation analyses. In vivo, the therapeutic efficacy was evaluated in a mouse infection model. Phage Henuyfy11N has not yet been classified. It demonstrated high lytic activity, a short latent period, and a burst size of 57.1 PFU/cell. The phage remained stable across a broad pH range (3-11) and temperatures up to 70℃. Its circular double-stranded DNA genome (41,103 bp, G + C% 50.88) contains 54 open reading frames, with no tRNA, virulence, or antibiotic resistance genes. Genomic and phylogenetic analyses revealed close relatedness to phage BUCT789. Henuyfy11N effectively lysed ESBL-producing E. coli, disrupted biofilms, and significantly improved survival in the mouse infection model. Henuyfy11N shows high host specificity, efficient lytic activity, rapid replication, and a safe genomic profile, demonstrating some potential as a therapeutic agent against ESBL-producing E. coli infections.IMPORTANCEThe widespread use of antibiotics has led to increasing antibiotic resistance, which is a growing global health concern. Therefore, the development of novel antimicrobial therapy that can cure drug-resistant bacteria-induced infections is imperative. Phages are of increasing interest as natural enemies of bacteria, with clear advantages in antibacterial applications. In this study, by using extended-spectrum β-lactamase (ESBL)-producing Escherichia coli 2025011N as a host, we successfully isolated and purified Escherichia phage Henuyfy11N and conducted a series of experiments to verify its genomic character and biological character. Our findings revealed that the phage exhibited excellent tolerance to a broad spectrum of pH and wide temperature range. Phage Henuyfy11N was effective in disrupting mature biofilm, and no genes for virulence, lysogenic, integrase, or AMRs were found in the genome. Besides, Henuyfy11N showed promising antibacterial effects in vivo and in vitro, indicating potential as a therapeutic agent against ESBL-producing E. coli infections.

The rapid, noninvasive detection of circulating tumor DNA (ctDNA) is vital for the diagnosis and staging of breast cancer (BC). In this study, we developed a homogeneous CRISPR/Cas12a fluorescent platform using a hierarchical grape-cluster rolling circle amplification (GCRCA) nanomaterial to detect the PIK3CA E542K mutation. A pivotal discovery of this study is that activated Cas12a efficiently cleaves metal ion-mediated cytosine-Ag+-cytosine base pairs, which enables direct coupling between enzymatic activity and signal transduction. GCRCA, assembled by precise hybridization of long-chain RCA concatemers with auxiliary circular DNA, features Ag+-bridged dual-ring units that sequester both target sequences and Ag+ reporters within a self-shielding framework. Upon target recognition, activated Cas12a dismantles the GCRCA architecture, initiating an autocatalytic feedback loop that releases caged Ag+ to quench the quantum dot fluorescence. This label-free assay achieved attomolar sensitivity within 30 min without enzymatic preamplification or complex nucleic acid extraction. Importantly, the platform exhibits excellent sequence selectivity, enabling precise discrimination of single-base mutations against closely related sequences. Validation of 42 clinical plasma samples achieved 100% diagnostic specificity for BC. For staging, the platform yielded a sensitivity of 100%, a specificity of 92.3%, and an area under the curve of 0.978. With its exceptional sensitivity and operational simplicity, this platform offers a promising approach for precise ctDNA-based BC detection and staging, demonstrating significant translational clinical potential.

Chronic hepatitis B virus (HBV) infection affects nearly 300 million people worldwide. Progress in understanding HBV immunopathogenesis and developing curative therapies has been hindered by the lack of suitable small animal models. HBV exhibits strict host and tissue tropism, with productive infection largely restricted to human and chimpanzee hepatocytes. Murine hepatocytes are resistant to HBV infection, even with ectopic expression of the human HBV entry receptor sodium taurocholate cotransporting polypeptide (huNTCP), because they apparently fail to form covalently closed circular DNA (cccDNA), the viral episome required for productive infection and persistence. To investigate the mechanisms restricting HBV infection in murine cells, we developed a piggyBac transposon-based system that efficiently generates inducible stable cell lines supporting HBV replication and cccDNA formation via intracellular amplification-a pathway that shares similar nucleocapsid uncoating and nuclear import of relaxed circular DNA (rcDNA) that occur during de novo infection. Remarkably, all tested murine hepatocyte and hepatoma cell lines, across multiple mouse genetic backgrounds, supported cccDNA formation at levels comparable to human cells, indicating that nucleocapsid uncoating and rcDNA nuclear import are not limited in mice. Given that huNTCP-expressing murine hepatocytes support hepatitis D virus infection, which shares early entry events with HBV, our findings reveal that the dominant restriction to HBV infection in huNTCP-expressing murine hepatocytes lies at a late entry step preceding nucleocapsid uncoating. By defining this mechanistic block, our study advances understanding of HBV host tropism and provides a foundation for facilitating development of fully HBV-susceptible mouse models.

Cancer is characterized not only by malignant transformation driven by genomic alterations but also by remarkable cellular plasticity that enables adaptation to environmental and therapeutic stress. Cancer cell plasticity frequently involves extensive remodeling of gene regulatory and transcriptional networks arising from complex interactions between genetic alterations and nongenetic regulatory mechanisms. The regulatory sources of cancer cell plasticity include changes in RNA molecules, such as microRNAs, alterations in RNA processing, epigenetic reprogramming, and higher-order cellular organization. In particular, biomolecular condensates-membraneless organelles formed through phase separation-and extrachromosomal DNA (ecDNA) have gained attention as important regulators of gene expression and cellular heterogeneity in cancer. Alterations in biomolecular condensates in cancer cells are frequently associated with dysregulated gene regulation and cell signaling. In addition, ecDNA represents an emerging mechanism that mediates high-level oncogene transcription, tumor heterogeneity, and drug resistance in cancer. This review summarizes recent advances in understanding how biomolecular condensates and ecDNA contribute to the regulation of cancer cell plasticity and discusses their implications for tumor evolution and therapeutic resistance. We also outline the usefulness of several CRISPR-based methods to test the roles of ecDNA, including generation of ecDNA-like circular DNA as model systems, optimization of Cas9 activity for prevention of Cas9-induced ecDNA loss and efficient ecDNA knock-in, and CRISPR interference-based perturbations.

The global escalation of bacterial antimicrobial resistance, particularly in Escherichia coli, constitutes an urgent public health threat. Bacteriophages represent a promising therapeutic arsenal against multidrug-resistant pathogens, underscoring the necessity of acquiring additional phages to expand phage libraries. In response to multidrug-resistant E. coli, this study isolated a lytic phage, designated E. coli phage vB_EcoS_GZMU_E2010. This phage demonstrated effective lytic activity against pathogenic E. coli strains, that included Extended-Spectrum β-Lactamase-producing E. coli (ESBL-E. coli) strains. The time-kill kinetics and terminal growth suppression data illustrate that, bacteriophage vB_EcoS_GZMU_E2010 exhibits potent antimicrobial action across a wide range of MOIs (from 0.001 to 1000). Transmission electron microscopy revealed that phage exhibits a siphovirus-like morphology within class Caudoviricetes, featuring a long noncontractile tail and icosahedral capsid. Whole-genome sequencing identified a 44,255 bp circular double-stranded DNA genome with 50.81% GC content. Comparative genomics revealed <95% whole-genome nucleotide identity to all reported phages, with the closest relative (Escherichia phage hz69, GenBank accession: ON556632.1) sharing only 90.7% identity, establishing its taxonomic novelty. Following a 60 min latent period, the phage entered a rapid burst phase characterized by exponential titer increase. Viral progeny release plateaued at 80 min postinfection. Bacteriophage vB_EcoS_GZMU_E2010 exhibited high stability across temperature (4-50 °C) and pH (4-10) gradients. Comprehensive screening confirmed the absence of antibiotic resistance genes and virulence factors. Besides, the maximum inhibition rate of this bacteriophage on biofilm reached 73.5%. Collectively, these findings position phage vB_EcoS_GZMU_E2010 as a promising candidate for combating drug-resistant infections, providing a potential strategy against multidrug-resistant E. coli pathogenesis.

Chronic hepatitis B virus (HBV) infection remains a significant global health challenge. Accurate quantification of HBV relaxed circular DNA (rcDNA) is critical for monitoring viral replication and assessing disease progression. However, conventional isothermal amplification methods are often compromised by limited specificity. To address these limitations, we engineered a toehold-triggered switchable DNA three-way junction (3WJ) protective nanoprobe that synergizes RNase H-assisted target recycling amplification (RATRA) with toehold-mediated strand displacement (TMSD). Leveraging the programmability of DNA nanotechnology, we constructed a rigid 3WJ scaffold featuring a "protective" nanoscale cavity at the branching point. This architecture effectively sequesters the ribonucleotides via steric hindrance, preventing non-specific enzymatic cleavage in the absence of the target. Upon specific recognition of the HBV rcDNA gap region, the nanoprobe undergoes a precise conformational switch, initiating a cyclic cleavage and signaling cascade. Governed by a dual-verification mechanism, the assay demonstrates exceptional specificity, enabling the discrimination of single-base mutations. Under optimized conditions, the method achieved a limit of detection (LOD) of 87.28 fM with a broad linear dynamic range. Validation in clinical serum samples revealed strong concordance with gold-standard quantitative real-time PCR (qRT-PCR). Consequently, this work presents a robust, modular, and cost-effective platform for point-of-care viral diagnostics.

mRNA vaccines have emerged as a transformative modality for infectious disease prevention. In response to the emergence of SARS-CoV-2, large-scale in vitro transcription (IVT) of mRNA vaccines was developed. Large-scale IVT currently relies on linearized plasmid DNA (pDNA) as a template for mRNA production. Linearized pDNA production presents several challenges at manufacturing scale, including removal of residual host-cell DNA, protein, endotoxins, and antibiotics. Additionally, pDNA-derived sequences irrelevant to mRNA production must be removed from the final product. Finally, the generation of linear pDNA template is laborious, which reduces mRNA production speed, a renowned advantage of this technology. Enzymatic DNA amplification strategies such as rolling circle amplification (RCA) of a synthetic circular DNA molecule offer a rapid, isothermal reaction as an alternative to pDNA. Therefore, we have developed a fully synthetic, single-vessel mRNA manufacturing platform. Beginning with a chemically synthesized circular DNA template, we amplify via a fit-for-purpose RCA, linearize with a TypeIIS restriction enzyme (RE), and perform IVT in a single vessel. The entire process-from circular template to mRNA-can be completed in as little as two days. This method, termed Unified Sequential Template Amplification and Transcription (USTAT), eliminates bacterial components, large volume pDNA production, and enables rapid, modular mRNA production.

Copy number alterations (CNAs) accumulate non-randomly within cancer genomes reflecting specific DNA damage and repair events. Higher-order patterning of CNAs can illuminate the types and determinants of genome instability (GI), as well as their clinical relevance, highlighting the need to develop analytical frameworks to capture such patterns. To address this issue, we collated a literature-curated compendium of pre-defined CN-based GI scores and extracted de novo CN signatures. Application to 2,763 breast cancer genomes from The Cancer Genome Atlas and METABRIC revealed the complementarity of various GI scores and their differences across immunohistochemical subtypes. Of the eight CN signatures identified, three associated with distinct characteristics of homologous recombination deficiency and showed differential activity between cases with BRCA1 versus BRCA2 loss. Segments assigned to a HER2+ enriched signature strongly overlapped regions of chromothripsis and circular extrachromosomal DNA, suggesting that a common mutational process contributes to these phenotypes. CN "quiet" diploid and tetraploid genomes were apparent, with the latter group capturing a unique subset of whole genome doubled tumors enriched for PIK3CA, MAP3K1, and CDH1 mutations. Finally, combining CN signatures with tumor microenvironment analyses, patients with quiet genomes and low macrophage infiltration showed remarkably better survival outcomes. Collectively, these findings demonstrate the value of deep interrogation of scores and signatures in characterizing the biological processes and clinical implications underlying CN-based GI. The publicly available web portal (https://cnavisualizer.pittlabgenomics.com/home) will facilitate similar analyses across pan-cancer genomes.

Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cell's genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond ∼5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating >5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA "helper" plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large-up to 10 kb DNA-integrations at >20% efficiency in primary human T cells at the TRAC locus and at >60% efficiency in human iPSCs at the AAVS1 locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional in vitro and in vivo . Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.

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.

Extrachromosomal circular DNA (eccDNA) are molecules that originate from chromosomal DNA but exist independently. While large eccDNA (ecDNA) contributes to tumorigenesis, the role of smaller eccDNA (<100,000 base pairs) in cancer remains unclear. Our analysis of 25 colorectal cancer (CRC) tumors and normal adjacent tissues revealed that eccDNA is significantly more abundant in tumor tissues, correlating strongly with chromosomal amplifications. The presence of whole intact genes on 1.29% of eccDNA was nonrandom. We identified 84 genes that recurred across tumors of multiple patients when present on eccDNA, with 19% of genes being cancer-associated. eccDNA-borne genes were often accompanied by increased expression, and their contribution to expression was much larger than that from linear amplifications and the larger ecDNA. The cytokine gene CXCL5 exemplified this phenomenon, showing substantial copy-number increase and upregulation when present on eccDNA. Functional validation in cell lines showed that CXCL5 eccDNA enhanced transcriptional output and immune cell recruitment function. The recurrence and overexpression of CRC-related genes on eccDNA indicate their selection in tumors, suggest that eccDNA can serve as an additional mechanism for dynamically influencing gene expression and is capable of conferring cancer phenotypes on cells. Analysis of chromatin landscapes revealed that eccDNA preferentially forms at sites of open chromatin and active transcription, with architectural boundaries marked by CTCF protein. Clinically, higher eccDNA levels correlated with poorer relapse-free survival in a small patient cohort. These findings suggest that circular DNA elements across the entire size spectrum participate in cancer evolution and warrant further investigation in larger cohorts.

Out of Asia: feline stool-associated circular DNA virus (FeSCV) in Brazilian domestic cats.

I studied medicine to become an endocrinologist but ended up in biochemistry. As a graduate student, I studied tumor mitochondria and discovered the malate-aspartate shuttle (the major route in animal cells for shuttling reducing equivalents into mitochondria). As a postdoc in New York, I switched to the replication of RNA bacteriophages, and on my return to Amsterdam, I started on mitochondrial biogenesis and discovered the circular mitochondrial DNAs (mtDNAs) of animal mitochondria and yeast. I also tackled trypanosomatids, major parasites of humans, in which we characterized their exotic mtDNA networks and discovered the glycosome, an organelle containing most of the glycolytic system. We helped to unravel the mechanism of antigenic variation in African trypanosomes and even discovered a new base, base J, in the DNA of trypanosomatids.After moving to the Netherlands Cancer Institute, I combined the trypanosomatids with a project on mechanisms of multidrug resistance in cancer cells. In particular, studying mice with one or more disrupted ABC transporter genes resulted in interesting findings in drug pharmacokinetics and in the elucidation of the cause of two inborn errors.

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

Extrachromosomal DNA (ecDNA), autonomously replicating circular DNA outside the chromosomes, exists as critical oncogene driver in approximately 20% of all tumors. Massive ecDNA amplification and its asymmetric segregation during mitotic drive high level oncogene amplification and contribute to tumor heterogeneity. Gastric cancer exhibits a high frequency of ecDNA occurrence. KRAS, a key oncogene in multiple cancers, is frequently amplified in gastric cancer; however, its functional implications via ecDNA remain largely understudied. In this study, we performed whole-genome sequencing and single-cell RNA sequencing on a gastric cancer sample to identify genomic amplification and transcription driven by ecDNA. We identified KRAS-ecDNA in gastric cancer, which exhibited significantly elevated KRAS expression and pronounced transcriptional heterogeneity. Functionally, ecDNA_High cells showed enhanced ribosome biogenesis, upregulated DNA repair pathways, differential activation of transcription factors,and reduced MHC-II signaling, indicating potential immune evasion. Drug response predictions suggested that KRAS-ecDNA_High cells are sensitive to MAPK inhibitors and upstream receptor inhibitors, despite showing broad resistance to conventional chemotherapies. Our study uncovers the critical role of KRAS-ecDNA in gastric cancer. These findings provide a rationale for targeting ecDNA-driven oncogenic programs and offer targeted strategies to combat ecDNA-mediated oncogenic evolution.

In hepatitis B surface antigen (HBsAg)-negative recipients with antibody to hepatitis B core antigen (anti-HBc)-positive liver grafts, hepatitis B virus (HBV) can be reactivated under post-transplant immunosuppression. We recently reported a median intrahepatic covalently closed circular DNA (cccDNA) level of 238 copies/μg in HBsAg-positive patients. Meanwhile, the potential levels of intrahepatic cccDNA in anti-HBc-positive grafts have never been focused on as a factor for HBV recurrence. Among 168 patients who underwent living donor liver transplantation (LDLT) between 2018 and 2022, 4 HBsAg-negative recipients with anti-HBc-positive grafts were consecutively enrolled in this prospective study. Intrahepatic cccDNA levels in donor grafts were measured based on an intraoperative liver biopsy and digital droplet polymerase chain reaction. In all four donor grafts, intrahepatic cccDNA levels ranged from 5.2 to 408 copies/μg, and all were negative for the high-sensitivity hepatitis B core-related antigen. Three of four recipients experienced HBV reactivation 14-24 months after LDLT. Two patients developed hepatitis with high HBV DNA and HBsAg levels, escape mutations (G145R ± K141R) with genotype B or C were detected. Entecavir therapy achieved serological and virological clearance within 2-9 months. The fourth patient remained recurrence-free during 36 months of follow-up. Even among patients with resolved HBV infection, there are cases possessing intrahepatic cccDNA levels as high as or even higher than those in HBsAg-positive patients. To resolve this critical pitfall in immunosuppressed patients, the development of serum markers that precisely reflect intrahepatic cccDNA levels will be essential for improving HBV prophylaxis strategies.

Circular DNA molecules, including mitochondrial DNA (mtDNA) and extrachromosomal circular DNA (ecDNA), are critical genetic elements in human cells. Pathological alterations in their length—through deletions, duplications, or complex rearrangements—are directly implicated in a spectrum of diseases, from inherited mitochondrial disorders to cancer progression and therapy resistance. This review provides a comprehensive analysis of the methodologies available for detecting these specific structural changes. We systematically evaluate classical techniques, such as long-range PCR, Southern blotting, and electron microscopy, which offer direct visualization and validation. Furthermore, we examine modern high-throughput approaches, including short- and long-read sequencing technologies that enable genome-wide discovery and precise breakpoint mapping at single-nucleotide resolution. Special emphasis is placed on quantitative methods like digital droplet PCR (ddPCR) for ultra-sensitive detection and monitoring in clinical settings. A comparative framework is presented to guide the selection of appropriate methods based on resolution, throughput, quantifiability, and cost. We conclude that an integrated, multi-method strategy is often essential for robust analysis, from initial discovery to clinical validation. The accurate detection of circular DNA length alterations is rapidly evolving from a research tool into a cornerstone of precision diagnostics, offering significant potential for advancing our understanding and management of complex human diseases.

Extrachromosomal circular DNA is an emerging regulatory element implicated in genomic stability and gene regulation, yet its role in preimplantation development remains elusive. Here, we report the widespread presence of extrachromosomal circular DNA in preimplantation embryos, characterized by homologous junction sequences and originating from genomic regions enriched for active histone marks and RNA Polymerase II occupancy. Functional perturbations demonstrate that RNA Polymerase II inhibition suppresses extrachromosomal circular DNA production, whereas disruption of the Fanconi anemia pathway elevates it, suggesting that transcription-replication conflicts affect its biogenesis. Notably, extrachromosomal circular DNA levels surge during major zygotic genome activation. Synthetic extrachromosomal circular DNAs carrying putative enhancers for the zygotic genome activation genes Mycn and Egfl7, and the developmental gene Emx1, significantly upregulate the expression of their respective genes upon transfection into fibroblasts and zygotes. Collectively, this study unveils the extrachromosomal circular DNA landscape in preimplantation embryos, elucidates a transcription-replication conflict mechanism underlying its generation, and establishes its regulatory potential during mammalian preimplantation development.