Résumé
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.
Sujets)
Mâle , Femelle , Animaux , Humains , Suidae , ADN circulaire/génétique , Tumeurs de l'appareil génital féminin , Sperme , ADN , ReproductionRésumé
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.
Sujets)
Humains , Virus de l'hépatite B/génétique , Hépatite B chronique/traitement médicamenteux , Antiviraux/usage thérapeutique , Réplication virale , ADN circulaire/usage thérapeutique , ADN viral/génétique , Hépatite B/traitement médicamenteuxRésumé
A nivel mundial, 300 millones de personas están infectadas por el virus de la hepatitis B (VHB). A pesar de que existe una vacuna que previene la infección y se dispone de tratamiento antiviral que suprime la replicación del virus, no hay cura aún. El principal problema que evita la recuperación total del paciente, incluso para aquel que recibe tratamiento, es la persistencia de dos formas del genoma viral en los hepatocitos: el ADN circular covalentemente cerrado (ADNccc), el cual se encuentra en forma de episoma y tiene la capacidad de replicarse, y las secuencias lineales subge-nómicas que se integran en el genoma humano, con potencial oncogénico. Hasta el momento se dispone de unos pocos biomarcadores para monitorear o predecir la progresión de la enfermedad y la respuesta al tratamiento. Estos biomarcadores se detectan durante la infección, y son la base para la monitorización de la enfermedad y hacer un diagnóstico de la fase clínica de la infección. Recientemente han surgido nuevos biomarcadores como el antígeno relacionado con el core del virus de la hepatitis B (HBcrAg) y la detección del ARN del VHB, que parecen correlacionarse con los niveles transcripcionales del ADNccc, además, durante el tratamiento parecen ayudar a predecir la respuesta y podrían identificar aquellos a quienes se les puede suspender la terapia sin riesgo de recaída. En esta revisión, se describe la utilidad de los principales biomarcadores convencionales en hepatitis B, y se abordan los dos biomarcadores emergentes más estudiados que prometen evaluar el curso de la infección, al igual que determinar la progresión de la enfermedad y la respuesta al tratamiento.
Globally, 300 million people are infected with hepatitis B virus (HBV). Although there is a vaccine that prevents infection and antiviral treatment that suppresses the replication of the virus, there is still no cure. The main problem that prevents the total recovery of the patient, even for those who recei-ve treatment, is the persistence of two forms of the viral genome in hepatocytes: covalently close circular DNA (cccDNA), which is in the form of an episome that has the ability to replicate, and linear subgenomic sequences that are integrated into the human genome, with oncogenic potential. Few biomarkers are currently available to monitor or predict disease progression and response to treatment. These biomarkers are detected during infection and are the basis for monitoring the di-sease and making a diagnosis of the clinical phase of the infection. New biomarkers have recently emerged, such as hepatitis B core-related antigen (HBcrAg) and HBV RNA detection, which seem to correlate with cccDNA transcriptional levels while during treatment seem to help predict response, and could identify those for whom therapy can be discontinued without risk of relapse. In this review, the usefulness of the main conventional biomarkers in hepatitis B is described, and the two most studied emerging biomarkers are mentioned, which promise to evaluate the course of the infection, as well as to determine disease progression and treatment response.
Sujets)
Humains , Marqueurs biologiques , Virus de l'hépatite B , Hépatite , Hépatite B , ADN circulaire , ARN , Risque , Génome , Diagnostic , AntigènesRésumé
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.
Sujets)
Humains , Antiviraux/usage thérapeutique , ADN circulaire/génétique , ADN viral , Période , Hépatite B/traitement médicamenteux , Virus de l'hépatite B/génétique , Hépatite B chronique/traitement médicamenteux , Réplication viraleRésumé
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.
Sujets)
Humains , Antiviraux/usage thérapeutique , Marqueurs biologiques , ADN circulaire , ADN viral , Hépatite B/traitement médicamenteux , Antigènes de surface du virus de l'hépatite B , Virus de l'hépatite B/génétique , Hépatite B chronique , Réplication viraleRésumé
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.
Sujets)
Animaux , Allèles , Systèmes CRISPR-Cas , Réparation de l'ADN par jonction d'extrémités , ADN circulaire/métabolisme , Embryon non mammalien , Édition de gène/méthodes , Techniques de knock-in de gènes , Techniques de knock-out de gènes , Gènes rapporteurs , Locus génétiques , Techniques de génotypage , Protéines à fluorescence verte/métabolisme , Integrases/métabolisme , Protéines luminescentes/métabolisme , Mutagenèse par insertion , Analyse sur cellule unique , Danio zébré/métabolismeRésumé
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.
Sujets)
Humains , Biologie informatique , Méthylation de l'ADN , ADN circulaire/génétique , ADN viral/génétique , Génome humain , Génomique , Génotype , Hepacivirus/génétique , Hépatite C/virologie , Agranulocytes/virologie , Réaction de polymérisation en chaîne , ARN viral/génétique , Alignement de séquencesRésumé
Several studies have reported a good correlation between levels of serum hepatitis B virus surface antigen (HBsAg) and covalently closed circular DNA (cccDNA) before and after antiviral therapy. As a result, the quantification of HBsAg levels has attracted much attention in recent years as an important approach to evaluate viral activity. In this study, mAbs against HBsAg were generated and 9 mAbs (H17, H30, H31, H67, H73, H97, H101, H118, and H128) were investigated for optimization of HBsAg quantitation ELISA. Determination of the best combinations of mAbs for sandwich ELISA identified H17 and H31 mAbs as the ideal capture and horseradish peroxidase (HRP) conjugate mAbs, respectively. A standard curve for the current assay system exhibited linearity up to 40 ng/ml of HBsAg while a detection limit of approximately 1 ng/ml of HBsAg was also estimated, which was comparable to that of the other commercial ELISA kits. The ELISA system established in this study is particularly differentiated from other commercial kits in using mAbs for both capture and HRP conjugate, which provides a solution to inconsistency of quality and ethical issues in polyclonal antibodies production using laboratory animals.
Sujets)
Animaux de laboratoire , Anticorps , Antigènes de surface , ADN circulaire , Test ELISA , Éthique , Antigènes de surface du virus de l'hépatite B , Virus de l'hépatite B , Hépatite B , Hépatite , Horseradish peroxidase , Limite de détectionRésumé
The advent of oral antiviral agents has revolutionized hepatitis B treatment. It has led to decreased incidence and mortality related to hepatocellular carcinoma. However, although nucleos(t)ide analogs (NA) are fast and potent in inhibiting hepatitis B virus (HBV) polymerase and reverse transcriptase activity, complete cure of the virus is not possible. The complete eradication of HBV requires the covalently-closed-circular DNA (cccDNA) to be eliminated. Novel gene editing methods, such as zing finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, designed to target specific DNA sequences has great potential for therapeutic application. Among these, the CRISPR/Cas9 system may be the most feasible approach to eradicate HBV cccDNA. Further studies are needed to develop a more efficient and safer method of delivery using the CRISPR/Cas9 system to achieve complete cure of chronic hepatitis B.
Sujets)
Antiviraux , Séquence nucléotidique , Carcinome hépatocellulaire , Clustered regularly interspaced short palindromic repeats , ADN , ADN circulaire , Doigts , Virus de l'hépatite B , Hépatite B , Hépatite B chronique , Hépatite , Incidence , Méthodes , Mortalité , RNA-directed DNA polymeraseRésumé
<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Sixty 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>
Sujets)
Femelle , Humains , Mâle , Adulte d'âge moyen , ADN circulaire , ADN viral , Hépatite B , Virus de l'hépatite B , Transplantation hépatique , RécidiveRésumé
<p><b>OBJECTIVE</b>To investigate the correlation between intrahepatic eovalently closed circular (ccc)DNA of hepatitis B virus (HBV) and pathogen-and patient-related parameters.</p><p><b>METHODS</b>Ultrasound-guided liver biopsies were obtained from 60 patients with chronic HBV infection. Levels of intrahepatic HBV cccDNA and serum HBV DNA were measured by quantitative fluorescence PCR. Level of serum hepatitis B surface antigen (HBsAg) was measured by chemiluminescence immunoassay. Clinical parameters, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (AKP), albumin, globulin (GLO), white blood cell, platelet, prothrombin-international normalized ratio, were measured by standard assay. Demographic information was recorded.The correlation between intrahepatic HBV cccDNA and pathogen-and patient-related parameters was assessed.</p><p><b>RESULTS</b>Intrahepatic HBV cccDNA level was negatively correlated with age, GLO, ALT and grade of necroinflammation. Patients with age of 30 years or more showed significantly higher level of HBV cccDNA level than patients under 30 years-old (7.44±0.58 and 5.66±1.35; t=7.157, P less than 0.001). Intrahepatic HBV cccDNA level was positively correlated with serum HBV DNA level (r=0.916, P less than 0.001) and serum HBsAg level (r=0.727, P less than 0.001). The median ratio of HBV cccDNA to HBV DNA was 1.18, and of HBV cccDNA to HBsAg was 1.67.</p><p><b>CONCLUSION</b>Intrahepatic HBV cccDNA levels decrease with age, level of ALT, level of GLO and grade of liver necroinflammation, but increase with level of serum HBV DNA and level of serum HBsAg. To a certain extent, serum HBV DNA and serum HBsAg levels may be a sufficient marker of intrahepatic HBV cccDNA levels.</p>
Sujets)
Humains , Répartition par âge , Alanine transaminase , Aspartate aminotransferases , Marqueurs biologiques , ADN circulaire , ADN viral , Antigènes de surface du virus de l'hépatite B , Virus de l'hépatite B , Hépatite B chronique , Réaction de polymérisation en chaine en temps réel , Tests sérologiquesRésumé
Antimicrobial screening of several novel 4-thiazolidinones with benzothiazole moiety has been performed. These compounds were evaluated for antimicrobial activity against a panel of bacterial and fungal strains. The strains were treated with these benzothiazole derivatives at varying concentrations, and MIC’s were calculated. Structures of these compounds have been determined by spectroscopic studies viz., FT-IR, 1H NMR, 13C NMR and elemental analysis. Significant antimicrobial activity was observed for some members of the series, and compounds viz. 3-(4-(benzo[d]thiazol-2-yl) phenyl)-2-(4-methoxyphenyl)thiazolidin-4-one and 3-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-hydroxy phenyl)thiazolidin-4-one were found to be the most active against E.coli and C.albicans with MIC values in the range of 15.6–125 μg/ml. Preliminary study of the structure–activity relationship revealed that electron donating groups associated with thiazolidine bearing benzothiazole rings had a great effect on the antimicrobial activity of these compounds and contributes positively for the action. DNA cleavage experiments gave valuable hints with supporting evidence for describing the mechanism of action and hence showed a good correlation between their calculated MIC’s and its lethality.
Sujets)
Antibactériens/synthèse chimique , Antibactériens/composition chimique , Antibactériens/pharmacologie , Antifongiques/synthèse chimique , Antifongiques/composition chimique , Antifongiques/pharmacologie , Benzothiazoles/synthèse chimique , Benzothiazoles/composition chimique , Benzothiazoles/pharmacologie , Candida/effets des médicaments et des substances chimiques , ADN bactérien/effets des médicaments et des substances chimiques , ADN circulaire/effets des médicaments et des substances chimiques , Tests d'agents antimicrobiens par diffusion à partir de disques , Évaluation préclinique de médicament , Électrophorèse sur gel d'agar , Piégeurs de radicaux libres/pharmacologie , Bactéries à Gram négatif/effets des médicaments et des substances chimiques , Bactéries à Gram positif/effets des médicaments et des substances chimiques , Tests de sensibilité microbienne , Structure moléculaire , Thiazolidines/synthèse chimique , Thiazolidines/composition chimique , Thiazolidines/pharmacologieRésumé
Rolling circle amplification (RCA) is a newly developed experimental technique that can specific ally amplify circular DNA. Since 2008, RCA has been extensively used in hepatitis B virus (HBV) research, such as the amplification of the full-length sequence of the HBV genome, and the analysis of the drug-resistant mutations of HBV covalently closed circular DNA (cccDNA), amongst others. To create an easy assay for the analysis of duck hepatitis B virus (DHBV) cccDNA, this study established an RCA-based method. DHBV cccDNA was amplified from the DHBV DNA samples of duck liver with four pairs of sulfur-modified primers, which were designed according to the highly conserved sequence of DHBV using sera DHBV DNA as the negative control. DHBV cccDNA was detected in the obtained RCA products by the sequencing of RCA amplicons that were amplified with primer pairs on both sides of the gap of DH BV relaxed circular DNA, rather than by digesting RCA products with a restriction enzyme. The liver and sera DHBV DNA samples of 39 ducks infected with DHBV were examined with the RCA-based DHBV cccDNA detection method, and the results showed that while DHBV cccDNA was detected from all 39 liver DHBV DNA samples, no DHBV cccDNA was found in any of the sera DHBV DNA samples. These results suggest that the method established in the study is highly specific and sensitive for the detection of DHBV cccDNA. The establishment of this RCA-based DHBV method for cccDNA detection lays the groundwork for using a DHBV model to study the role of cccDNA in the pathogenesis of hepatitis B and to evaluate the effect of anti-virus therapies.
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Animaux , Amorces ADN , Génétique , ADN circulaire , Génétique , ADN viral , Génétique , Canards , Infections à Hepadnaviridae , Virologie , Virus de l'hépatite B du canard , Génétique , Foie , Virologie , Réaction de polymérisation en chaîne , Méthodes , Maladies de la volaille , VirologieRésumé
BACKGROUND/AIMS: Occult HBV infection can persist following HBsAg loss and be transmitted, but the virological features are not well defined. METHODS: Here we investigated 25 Korean patients who lost HBsAg during follow up, either spontaneously or subsequent to therapy. RESULTS: Whereas subtype adr (genotype C) was found in 96% of HBsAg positive patients, 75 % of patients who lost HBsAg spontaneously were seemed to be infected with the ayw subtype with sequence similar to genotype D. Mutations in the major hydrophilic region (MHR) of HBsAg were found in 7 patients who lost HBsAg spontaneously. The mutations include T123S, M125I/N, C139R, D144E, V177A, L192F, and W196L, some of which have not been reported before. Functional analysis via transfection experiments indicate that the C139R and D144E mutations drastically reduced HBsAg antigenicity, while the Y225del mutation found in one interferon-treated patient impaired HBsAg secretion. CONCLUSIONS: Lack of detectable HBsAg in patient serum could be explained by low level of ccc DNA in liver tissue, low antigenicity of the surface protein, or its secretion defect.
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Adulte , Sujet âgé , Femelle , Humains , Mâle , Adulte d'âge moyen , Séquence d'acides aminés , Antiviraux/usage thérapeutique , ADN circulaire/analyse , Génotype , Cellules HepG2 , Hépatite B/traitement médicamenteux , Antigènes de surface du virus de l'hépatite B/sang , Virus de l'hépatite B/génétique , Foie/virologie , Données de séquences moléculaires , Mutation , Rémission spontanée , République de Corée , SérotypieRésumé
No abstract available.
Sujets)
Femelle , Humains , Mâle , ADN circulaire/analyse , Hépatite B/anatomopathologie , Antigènes de surface du virus de l'hépatite B/génétique , Virus de l'hépatite B/métabolismeRésumé
<p><b>OBJECTIVE</b>To generate a mouse model of chronic hepatitis B (CHB) infection by performing in vivo transduction of hepatitis B virus (HBV) covalently closed circular (ccc)DNA.</p><p><b>METHODS</b>Nude mice were injected with HBV cccDNA at doses of 1.5, 1.0 or 0.5 mug/ml. A control group was generated by giving equal injection volumes of physiological saline. The serum levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) on post-injection days 1 and 3, weeks 1-6, 8 and 10 were assayed by reflection immunoassay. At post-injection week 10, all animals were sacrificed and liver tissues were collected. Copies of HBV DNA in serum and liver tissue were detected by real-time PCR. HBV antigens in liver tissue were detected of by immunohistochemistry. Pathological analysis of liver tissue carried out with hematoxylin-eosin staining. Linear correlation of data was determined by statistical analysis.</p><p><b>RESULTS</b>HBsAg and HBeAg were detected in sera from all three groups of cccDNA-injected mice staring at post-injection day 1 and lasting through week 10. The levels of HBsAg over the 10-week period showed two patterns of increase-decrease;the lowest level was detected at week 4 and the highest level was detected at week 8. In contrast, the levels of HBeAg over the 10-week period showed three patterns of increase-decrease; the lower levels were detected at weeks 2 and 4 and the higher levels at weeks 3 and 6. HBV DNA copies in liver tissues showed a cccDNA dose-dependent descending trend over the 10-week study period (1.5 mug/ml:1.14E+07 ± 6.51E+06 copies/g, 1.0 mug/ml:9.81E+06 ± 9.32E+06 copies/g, and 0.5 mug/ml:3.72E+06 ± 2.35E+06 copies/g; Pearson's r =0.979). HBV DNA copies in sera showed the pattern of 1.0 mug/ml cccDNA more than 1.5 mug/ml cccDNA more than 0.5 mug/ml cccDNA, and in general were higher than those detected in the liver tissues. Liver tissues from all cccDNA-injected mice showed positive immunohistochemistry staining for both HBsAg and HBeAg. HE staining showed that the liver tissues of all cccDNA-injected mice had severe fatty and vacuolar degeneration and less obvious structure of liver lobules (compared to the liver tissues from control mice).</p><p><b>CONCLUSION</b>The CHB mouse model successfully established in this study by in vivo transduction of HBV cccDNA may represent a useful tool to study the pathogenic mechanisms and potential antiviral treatments of human CHB.</p>
Sujets)
Animaux , Mâle , Souris , ADN circulaire , ADN viral , Modèles animaux de maladie humaine , Antigènes de surface du virus de l'hépatite B , Sang , Antigènes e du virus de l'hépatite virale B , Sang , Virus de l'hépatite B , Génétique , Physiologie , Hépatite B chronique , Virologie , Souris nude , Transduction génétique , Réplication viraleRésumé
<p><b>OBJECTIVE</b>To explore clinical value of circular DNA in acute myocardial infarction.</p><p><b>METHODS</b>Venous 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Circular DNA can be used as a marker of sensitively reflecting myocardial cell injury.</p>
Sujets)
Sujet âgé , Femelle , Humains , Mâle , Adulte d'âge moyen , Études cas-témoins , ADN circulaire , Sang , Infarctus du myocarde , Sang , DiagnosticRésumé
To verify that the circular forms of bocavirus genome exist in their host, bocavirus episomes were detected in fecal samples of healthy piglets using a semi-nested PCR method. Two species of porcine bocaviruses (PBoVG2-episome and PBoVG3-episome) were identified for the first time. The relevant terminal sequences of the noncoding region (405 and 511 nt, respectively) were also obtained. Sequence analyses and secondary structure prediction indicated that the PBoVG2-episome was more similar to that of human bocavirus 3 (HBoV3) but the PBoVG3-episome was quite different from that of other members of the genus Bocavirus. Discovery of episomal forms of porcine bocaviruses (PBoV) suggested that PBoV, like HBoV, used a different replication mechanism from other parvoviruses. The sequencing of episome Inverted Terminal Repeats (ITRs) also contributes to a possible alternative strategy for constructing infectious molecular clones of bocavirus in a future study.
Sujets)
Animaux , Séquence nucléotidique , Bocavirus , Génétique , Physiologie , ADN circulaire , Génétique , ADN viral , Génétique , Génome viral , Génétique , Données de séquences moléculaires , Réaction de polymérisation en chaîne , Suidae , VirologieRésumé
OBJECTIVE@#To evaluate the antioxidant potential of different extract/fractions of Anthocephalus cadamba (A. cadamba) (Roxb.) Miq. (Rubiaceae) and study the tentative identification of their active constituents.@*METHODS@#The extract/fractions were screened for antioxidant activity using various in vitro assays viz. DPPH assay, ABTS assay, superoxide anion radical scavenging assay, reducing power assay and plasmid DNA nicking assay. Total phenolic content of extract/fractions was determined by colorimetric method. An ultra-performance LC-electrospray-quadrupole-time of flight mass spectrometry method was used to analyse the active constituents of extract/fractions of A. cadamba.@*RESULTS@#The ethyl acetate fraction was found to be most active fraction in all the assays as compared to other extract/fractions. The IC(50) value of ethyl acetate fraction (ETAC fraction) was 21.24 μg/mL, 1.12 μg/mL, 9.68 μg/mL and 57.81 μg/mL in DPPH assay, ABTS assay, reducing power assay and superoxide scavenging assay respectively. All the extract/fractions also showed the potential to protect the plasmid DNA (pBR322) against the attack of hydroxyl radicals generated by Fentońs reagent. The bioactive compounds were identified by UPLC-ESI-QTOF-MS, by comparing the mass and λ(max) with literature values.@*CONCLUSIONS@#The potential of the extract/fractions to scavenge different free radicals in different systems indicated that they may be useful therapeutic agents for treating radical-related pathologic damage.
Sujets)
Acétates , Chimie , Antioxydants , Chimie , Pharmacologie , Dérivés du biphényle , Chimie , Chromatographie en phase liquide à haute performance , Méthodes , Colorimétrie , Altération de l'ADN , ADN circulaire , Chimie , Oxydoréduction , Phénols , Chimie , Pharmacologie , Picrates , Chimie , Extraits de plantes , Chimie , Pharmacologie , Feuilles de plante , Chimie , Plasmides , Génétique , Analyse de régression , Rubiaceae , Chimie , Spectrométrie de masse ESI , MéthodesRésumé
<p><b>BACKGROUND</b>Successful 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).</p><p><b>METHODS</b>Based 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.</p>