Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eukaryotic cells. They have long been recognized as the generators of energy for the cell and also have been known to associate with several metabolic pathways that are crucial for cellular function. Mitochondria have their own genome, mitochondrial DNA (mtDNA), that is completely separated and independent from the much larger nuclear genome, and even have their own system for making proteins from the genes in this mtDNA genome. The human mtDNA is a small (~16.5 kb) circular DNA and defects in this genome can cause a wide range of inherited human diseases. Despite of the significant advances in discovering the mtDNA defects, however, there are currently no effective therapies for these clinically devastating diseases due to the lack of technology for introducing specific modifications into the mitochondrial genomes and for generating accurate mtDNA disease models. The ability to engineer the mitochondrial genomes would provide a powerful tool to create mutants with which many crucial experiments can be performed in the basic mammalian mitochondrial genetic studies as well as in the treatment of human mtDNA diseases. In this review we summarize the current approaches associated with the correction of mtDNA mutations in cells and describe our own efforts for introducing engineered mtDNA constructs into the mitochondria of living cells through bacterial conjugation.
Conjugation, Genetic ; Cytoplasm ; DNA ; DNA, Circular ; DNA, Mitochondrial ; Eukaryotic Cells ; Genome ; Genome, Mitochondrial ; Humans ; Membranes ; Metabolic Networks and Pathways ; Mitochondria ; Organelles ; Proteins

DNA, Circular ; DNA, Viral ; Hepatitis B virus ; genetics

DNA, Circular ; isolation & purification ; DNA, Viral ; isolation & purification ; Hepatitis B ; diagnosis ; Hepatitis B virus ; genetics ; Humans

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

Animals ; DNA, Circular ; analysis ; Hepatitis B ; virology ; Hepatitis B virus ; genetics ; Humans

OBJECTIVETo explore clinical value of circular DNA in acute myocardial infarction.

METHODSVenous blood (2 ml/head) of 40 healthy control and 40 patients with acute myocardial infarction within 48h of onset of illness and convalescent period was collected. The level of plasma circular DNA was detected by duplex real-time polymerase chain reaction assay. The levels of myocardial enzyme spectrum and cardiac troponin T (cTnT) were detected by biochemistry method.

RESULTSThe level of circular DNA in control group and group of acute myocardial infarction before treatment was (21.5 +/- 10.7) ng/ml and (253.6 +/- 45.7) ng/ml, respectively (P = 0.000). The levels of serum myocardial enzyme spectrum and cTnT before treatment in patients with acute myocardial infarction were significantly higher than those of control group (P < 0.05). The level of circular DNA after treatment in patients with acute myocardial infarction was significantly decreased compared with that before treatment (P = 0.000), the levels of myocardial enzyme spectrum and cTnT were also significantly reduced (P < 0.05). There was significant correlation between the level of circular DNA and those of CK-MB and cTnT, r = 0.613, 0.654, P = 0.032, 0.021.

CONCLUSIONCircular DNA can be used as a marker of sensitively reflecting myocardial cell injury.

Aged ; Case-Control Studies ; DNA, Circular ; blood ; Female ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; blood ; diagnosis

OBJECTIVETo investigate the levels of hepatitis B virus (HBV) total DNA and covalently closed circular (ccc) DNA in liver transplant recipients due to HBV-associated liver diseases and detemaine their clinical significance.

METHODSSixty patients undergoing liver transplantation (LT) due to HBV-associated liver diseases were enrolled for the study. Levels of HBV total and ccc DNA in plasma, liver and PBMCs were measured by RT-PCR.

RESULTSThe ratio of male:female participants was 48:12. The mean age was 52.98+/-9.40 years old, and the median duration post-LT was 72 (25-128) months. 59 of the patients had no detectable HBV DNA in plasma.Four patients had detectable levels of total HBV DNA in PBMCs, but no detectable ccc DNA. Five patients had detectable levels of total HBV DNA in liver, and two of those also had detectable levels of ccc DNA. One patients who had detectable HBV DNA in PBMCs suffered HBV recurrence.

CONCLUSIONThe liver transplant recipients with detectable levels of HBV total and ccc DNA in PBMCs and liver should be considered high risk for HBV recurrence following LT.

DNA, Circular ; DNA, Viral ; Female ; Hepatitis B ; Hepatitis B virus ; Humans ; Liver Transplantation ; Male ; Middle Aged ; Recurrence

BACKGROUNDSuccessful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).

METHODSBased on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard.

RESULTSThe nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0 × 10(2) copies/ml to 3.9 × 10(8) copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative.

CONCLUSIONThe nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.

Cells, Cultured ; DNA, Circular ; genetics ; DNA, Viral ; genetics ; Hepatitis B virus ; genetics ; Humans ; Real-Time Polymerase Chain Reaction ; methods

DNA binding compounds were previously shown to bind to the right-handed DNA forms and hybrid B-Z forms in a highly cooperative manner and indicate that structural specificity plays a key role in a ligand binding to DNA. In this study, the modes of binding and structural specificity of agents to unusual DNA are examined by a variety of fluorescence techniques (intensity, polarization and quenching, etc.) to explore a reliable method to detect the association environment of ligands to deoxyoligonucleotides initially containing a B-Z junction between the left-handed Z-DNA and right-handed B-DNA. The results of fluorescence energy transfer measurement demonstrated that the ligand molecules bind to the allosterically converted DNA structures by intercalation. In the absence of high-resolution structural data, this fluorescence energy transfer measurement allowed reliable measures and infer the binding environment of ligands to the allosteric DNA structures.
Allosteric Regulation ; Circular Dichroism ; DNA/*chemistry/*metabolism ; Energy Transfer ; Ethidium/*metabolism ; Exodeoxyribonucleases/metabolism ; Ligands ; Motion ; Nucleic Acid Conformation ; Oligodeoxyribonucleotides/*metabolism

BACKGROUND: The TT virus (TTV) is a recently discovered, single-stranded circular DNA virus in the serum of the patients with post-transfusion hepatitis and it is thought to be one of the causative agents of cryptogenic hepatitis. In this study, we evaluated the prevalence and clinical significance of TTV viremia in general populations and patients on hemodialysis. METHODS: Sera of 115 general populations and 69 patients on hemodialysis were examined for TTV viremia by semi-nested polymerase chain reaction using primers deduced from the N22 region. RESULTS: The TTV was detected in 26.1% (18 of 69) of the patients on hemodialysis and was not different from the 20.9% (24 of 115) prevalence in general populations. Two patients on hemodialysis that revealed biochemical evidence of acute hepatitis were the sole TTV infection without other hepatitis virus infection but the mean alanine aminotransferase level was not significantly different according to the TTV viremia. The TTV was persistently detected in the sera of eight of thirteen patients (61.5%) 12 month later without any evidence of hepatitis. CONCLUSIONS: TTV is widespread in general populations and shows similar prevalence in patients on hemodialysis. Viral persistence and nonparenteral transmission may be possible. The relationship between the TTV viremia and hepatitis was not proved.
Alanine Transaminase ; DNA, Circular ; Hepatitis ; Hepatitis Viruses ; Humans ; Polymerase Chain Reaction ; Prevalence* ; Renal Dialysis* ; Torque teno virus* ; Viremia*