Originating but free from chromosomal DNA, extrachromosomal circular DNAs (eccDNAs) are organized in circular form and have long been found in unicellular and multicellular eukaryotes. Their biogenesis and function are poorly understood as they are characterized by sequence homology with linear DNA, for which few detection methods are available. Recent advances in high-throughput sequencing technologies have revealed that eccDNAs play crucial roles in tumor formation, evolution, and drug resistance as well as aging, genomic diversity, and other biological processes, bringing it back to the research hotspot. Several mechanisms of eccDNA formation have been proposed, including the breakage-fusion-bridge (BFB) and translocation-deletion-amplification models. Gynecologic tumors and disorders of embryonic and fetal development are major threats to human reproductive health. The roles of eccDNAs in these pathological processes have been partially elucidated since the first discovery of eccDNA in pig sperm and the double minutes in ovarian cancer ascites. The present review summarized the research history, biogenesis, and currently available detection and analytical methods for eccDNAs and clarified their functions in gynecologic tumors and reproduction. We also proposed the application of eccDNAs as drug targets and liquid biopsy markers for prenatal diagnosis and the early detection, prognosis, and treatment of gynecologic tumors. This review lays theoretical foundations for future investigations into the complex regulatory networks of eccDNAs in vital physiological and pathological processes.
Male ; Female ; Animals ; Humans ; Swine ; DNA, Circular/genetics* ; Genital Neoplasms, Female ; Semen ; DNA ; Reproduction

The resolution of the hepatitis C issue has raised expectations for a chronic hepatitis B cure, driving the industry to expand investment in research and development efforts to strengthen functional cure strategies. These strategies have a wide variety of types, and the published research findings are heterogeneous. The theoretical analysis of these strategies is of great significance for determining prioritized research orientations as well as sensibly allocating research and development resources. However, due to a paucity of necessary conceptual models, current theoretical analysis has not been able to unify various therapeutic strategies into a proper theoretical framework. In view of the fact that the decrease in the quantity of cccDNA is an inevitable core event accompanied by the process of functional cure, this paper intends to analyze several chronic hepatitis B cure strategies using cccDNA dynamics as a framework. Furthermore, there are currently few studies on the dynamics of the cccDNA field, hoping that this article can promote recognition and research in this field.
Humans ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/drug therapy* ; Antiviral Agents/therapeutic use* ; Virus Replication ; DNA, Circular/therapeutic use* ; DNA, Viral/genetics* ; Hepatitis B/drug therapy*

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the template for HBV replication. Currently, there is a lack of therapeutic drugs that directly target cccDNA. Therefore, blocking cccDNA supplements as fast as possible and reducing the existing cccDNA is the key to achieving a complete cure of chronic hepatitis B. Previous studies have suggested that cccDNA had a long half-life, but a recent study showed that it only took a few months to update cycle of cccDNA pool, and its number was much less than previously predicted. In the future, with the advent of new antiviral drugs that can completely inhibit HBV replication, it is expected that the cccDNA pool will be completely cleared due to its supplement complete blockade, so as to achieve virological cure of chronic hepatitis B.
Antiviral Agents/therapeutic use* ; DNA, Circular/genetics* ; DNA, Viral ; Half-Life ; Hepatitis B/drug therapy* ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/drug therapy* ; Humans ; Virus Replication

Hepatitis B virus (HBV) infection remains to be the major cause of chronic liver diseases in China. Since the nucleos(t)ide analogues and pegylated interferon-alpha do not directly target the covalently closed circular DNA (cccDNA) in the nuclei of HBV-infected hepatocytes, those standard-of-care medications cannot efficiently cure the infected hepatocytes and rarely achieve the functional cure of chronic hepatitis B (CHB). Therefore, new antiviral drugs targeting distinct steps of HBV replication and immunotherapeutics reinvigorating antiviral immune responses are urgently needed for the functional cure of CHB. Based on the extensive discussion of the biological and clinical significance of new virologic biomarkers and distinct mechanism of drug candidates currently in clinical development, we propose that the selection of virologic and immunological biomarkers for evaluation of therapeutic efficacy as well as setting the therapeutic endpoints in the clinical trials should be based on the mode of action of investigational drugs. In addition, due to the complexity of CHB pathogenesis, selection of specific subpopulation of CHB patients for the clinical trials of drugs with a specific mode of action should also be considered.
Antiviral Agents/therapeutic use* ; Biomarkers ; DNA, Circular ; DNA, Viral ; Hepatitis B/drug therapy* ; Hepatitis B Surface Antigens ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic ; Humans ; Virus Replication

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.
Alleles ; Animals ; CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA, Circular/metabolism* ; Embryo, Nonmammalian ; Gene Editing/methods* ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genes, Reporter ; Genetic Loci ; Genotyping Techniques ; Green Fluorescent Proteins/metabolism* ; Integrases/metabolism* ; Luminescent Proteins/metabolism* ; Mutagenesis, Insertional ; Single-Cell Analysis ; Zebrafish/metabolism*

G-quadruplex DNA has become an important target for tumor therapy and anti-tumor development. Modern pharmacology has proved that Macleaya cordata has anti-inflammatory, antibacterial, anti-tumor and other pharmacological effects. Affinity ultrafiltration method can screen active ingredients from compounds rapidly, but G-quadruplex DNA ligands are difficult to dissociate, which is a key step in conventional ultrafiltration method. In this paper, the filtrates after ultrafiltration were determined by HPLC-MS in substitution. The peaks with 20% reduction of MS response from the incubation vs control were considered to be ligand components to G-quadruplex. Two of the peaks with the relative abundance above 30% were identified as sanguinarine(SAN) and chelerine(CHE). Their circular dichroism conformations further proved that SAN and CHE are active ligands of HT4. In addition, another two gradients with high relative abundance were identified as protopine(PRO) and allpcryprotopine(ALL). The binding rate of SAN, CHE, PRO and ALL was calculated according to the HPLC-MS results, and the results showed a consistency with that of the molecular docking method. The proposed method can be used to screen active components from mixture.
Chromatography, High Pressure Liquid ; Chromatography, Liquid ; G-Quadruplexes ; Ligands ; Mass Spectrometry ; Molecular Docking Simulation ; Ultrafiltration

Avian malaria is one of the most important general blood parasites of poultry in Southeast Asia. Plasmodium (P.) juxtanucleare causes avian malaria in wild and domestic fowl. This study aimed to identify and characterize the Plasmodium species infecting in Thai native fowl. Blood samples were collected for microscopic examination, followed by detection of the Plasmodium cox I gene by using PCR. Five of the 10 sampled fowl had the desired 588 base pair amplicons. Sequence analysis of the five amplicons indicated that the nucleotide and amino acid sequences were homologous to each other and were closely related (100% identity) to a P. juxtanucleare strain isolated in Japan (AB250415). Furthermore, the phylogenetic tree of the cox I gene showed that the P. juxtanucleare in this study were grouped together and clustered with the Japan strain. The presence of P. juxtanucleare described in this study is the first report of P. juxtanucleare in the Thai native fowl of Thailand.
Amino Acid Sequence ; Animals ; Asia, Southeastern ; Asian Continental Ancestry Group ; Base Pairing ; Cytochromes c ; Cytochromes ; Electron Transport Complex IV ; Humans ; Japan ; Malaria, Avian ; Parasites ; Plasmodium ; Polymerase Chain Reaction ; Poultry ; Sequence Analysis ; Thailand ; Trees

Rolling circle amplification is a rapid, sensitive and isothermal single-stranded DNA amplification technique that can be used with staining or probes to amplify the detection signal. This technology has been widely used in biological detection and other aspects. The present paper introduces how to design rolling circle amplification, summarize its application in the detection of pathogens, nucleic acid tumor markers, proteins, biological small biomolecules, and viruses in recent years and prospects for future development.
DNA, Single-Stranded ; Nucleic Acid Amplification Techniques

The chinstrap (Pygoscelis antarcticus) and gentoo (P. papua) penguins are distributed throughout Antarctica and the sub-Antarctic islands. In this study, high-quality de novo assemblies of blood transcriptomes from these penguins were generated using the Illumina MiSeq platform. A total of 22.2 and 21.8 raw reads were obtained from chinstrap and gentoo penguins, respectively. These reads were assembled using the Oases assembly platform and resulted in 26,036 and 21,854 contigs with N50 values of 929 and 933 base pairs, respectively. Functional gene annotations through pathway analyses of the Gene Ontology, EuKaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were performed for each blood transcriptome, resulting in a similar compositional order between the two transcriptomes. Ortholog comparisons with previously published transcriptomes from the Adélie (P. adeliae) and emperor (Aptenodytes forsteri) penguins revealed that a high proportion of the four penguins’ transcriptomes had significant sequence homology. Because blood and tissues of penguins have been used to monitor pollution in Antarctica, immune parameters in blood could be important indicators for understanding the health status of penguins and other Antarctic animals. In the blood transcriptomes, KEGG analyses detected many essential genes involved in the major innate immunity pathways, which are key metabolic pathways for maintaining homeostasis against exogenous infections or toxins. Blood transcriptome studies such as this may be useful for checking the immune and health status of penguins without sacrifice.
Animals ; Base Pairing ; Gene Ontology ; Genes, Essential ; Genome ; Homeostasis ; Immunity, Innate ; Islands ; Metabolic Networks and Pathways ; Molecular Sequence Annotation ; Sequence Homology ; Spheniscidae ; Transcriptome

OBJECTIVE: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. METHODS: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. RESULTS: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. CONCLUSIONS The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.
Computational Biology ; DNA Methylation ; DNA, Circular/genetics* ; DNA, Viral/genetics* ; Genome, Human ; Genomics ; Genotype ; Hepacivirus/genetics* ; Hepatitis C/virology* ; Humans ; Leukocytes, Mononuclear/virology* ; Polymerase Chain Reaction ; RNA, Viral/genetics* ; Sequence Alignment