Development of a cellular assay system to study the genome replication of high- and low-risk mucosal and cutaneous human papillomaviruses

We found that recircularized high-risk (type 16 and 18) and low-risk mucosal (type 6b and 11) and cutaneous (type 5 and 8) human papillomavirus (HPV) genomes replicate readily when delivered into U2OS cells by electroporation. The replication efficiency is dependent on the amount of input HPV DNA and can be followed for more than 3 weeks in proliferating cell culture without selection. Cotransfection of recircularized HPV genomes with a linear G418 resistance marker plasmid has allowed subcloning of cell lines, which, in a majority of cases, carry multicopy episomal HPV DNA. Analysis of the HPV DNA status in these established cell lines showed that HPV genomes exist in these cells as stable extrachromosomal oligomers. When the cell lines were cultivated as confluent cultures, a 3- to 10-fold amplification of the HPV genomes per cell was induced. Two-dimensional (2D) agarose gel electrophoresis confirmed amplification of mono- and oligomeric HPV genomes in these confluent cell cultures. Amplification occurred as a result of the initiation of semiconservative two-dimensional replication from one active origin in the HPV oligomer. Our data suggest that the system described here might be a valuable, cost-effective, and efficient tool for use in HPV DNA replication studies, as well as for the design of cell-based assays to identify potential inhibitors of all stages of HPV genome replication.     

1,4‐Dihydropyridine derivatives without Ca2+‐antagonist activity up‐regulate Psma6 mRNA expression in kidneys of intact and diabetic rats

Impaired degradation of proteins by the ubiquitin–proteasome system (UPS) is observed in numerous pathologies including diabetes mellitus (DM) and its complications. Dysregulation of proteasomal degradation might be because of altered expression of genes and proteins involved in the UPS. The search for novel compounds able to normalize expression of the UPS appears to be a topical problem. A novel group of 1,4‐dihydropyridine (1,4‐DHP) derivatives lacking Ca2+‐antagonists activities, but capable to produce antidiabetic, antioxidant and DNA repair enhancing effects, were tested for ability to modify Psma6 mRNA expression levels in rat kidneys and blood in healthy animals and in rats with streptozotocin (STZ) induced DM. Psma6 gene was chosen for the study, as polymorphisms of its human analogue are associated with DM and cardiovascular diseases. 1,4‐DHP derivatives (metcarbatone, etcarbatone, glutapyrone, J‐9‐125 and AV‐153‐Na) were administered per os for three days (0.05 mg/kg and/or 0.5 mg/kg). Psma6 gene expression levels were evaluated by quantitative PCR. Psma6 expression was higher in kidneys compared to blood. Induction of diabetes caused increase of Psma6 expression in kidneys, although it was not changed in blood. Several 1,4‐DHP derivatives increased expression of the gene both in kidneys and blood of control and model animals, but greater impact was observed in kidneys. The observed effect might reflect coupling of antioxidant and proteolysis‐promoting activities of the compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitative approach using multiple single parameters versus visual assessment in dobutamine stress echocardiography

ABSTRACT: BACKGROUND: A number of myocardial Doppler-derived velocity, strain myocardial imaging parameters (DMI) and speckle tracking imaging (STI) have been proposed for the quantification of myocardial ischemia during stress echocardiography. The purpose of the study was to identify the best single ultrasound quantitative parameter for prediction of significant coronary stenosis and compare it with visual assessment during dobutamine stress echocardiography (DSE). METHODS: Prospective analysis included data of 151 patients (age 61.8 +/- 9.2) who underwent dobutamine stress echocardiography for known (n=35) or suspected CAD (n=36) or symptomatic chest pain (n=80), excluding patients with previous myocardial infarction. Systolic, post-systolic and diastolic velocities, strain and strain rate parameters were obtained at rest and at peak dobutamine challenge. Derivative markers as E'/A' ratio, post-systolic index and changes from rest to stress were calculated (98 parameters overall, predominantly longitudinal). Coronary angiography was chosen as reference method considering at least one stenosis greater than or equal to 70% per patient as significant CAD. The predictive value of quantitative parameters and wall motion score index (WMSI) was obtained using logistic regression and ROC analysis. RESULTS: The value of single parameters discriminated as independent predictors of CAD appeared to be modest (area under the curve [AUC] ranged from 0.63 to 0.72 for 16 PW-DMI, 12 CC-DMI and 12 STI markers), comparing to AUC of WMSI 0.88. Sensitivity, specificity and accuracy of visual DSE evaluation was 82.4% (95%CI 77.4%; 85.2%), 92.6% (95%CI 83.4%; 97.5%) and 86.0% (95%CI 79.5%; 89.6%), respectively, Youden index 0.75. Sensitivity, specificity and accuracy of single predictors ranged from 40.0% to 93.3% (95% CI 22.7%; 99.2%), from 34.2% to 88.7% (95% CI 25.6%; 94.1%) and from 45.8% to 80.0% (95% CI 37.5%; 87.2%) respectively, Youden index ranged from 0.20 to 0.52. CONCLUSIONS: Multiple single quantitative parameters showed limited predictive ability to identify significant coronary artery stenosis. Visual assessment of DSE appears to be more accurate than single velocity and strain/strain rate markers in the diagnosis of CAD.     

Recombination-Dependent Oligomerization of Human Papillomavirus Genomes upon Transient DNA Replication

We describe the extensive and progressive oligomerization of human papillomavirus (HPV) genomes after transfection into the U2OS cell line. The HPV genomic oligomers are extrachromosomal concatemeric molecules containing the viral genome in a head-to-tail orientation. The process of oligomerization does not depend on the topology of the input DNA, and it does not require any other viral factors besides replication proteins E1 and E2. We provide evidence that oligomerization of the HPV18 and HPV11 genomes involves homologous recombination. We also demonstrate oligomerization of the HPV18 and HPV11 genomes in SiHa, HeLa, and C-33 A cell lines and provide examples of oligomeric HPV genomes in clinical samples obtained from HPV-infected patients.     

Episomal maintenance of plasmids with hybrid origins in mouse cells

Bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and human herpesvirus 8 genomes are stably maintained as episomes in dividing host cells during latent infection. The mitotic segregation/partitioning function of these episomes is dependent on single viral protein with specific DNA-binding activity and its multimeric binding sites in the viral genome. In this study we show that, in the presence of all essential viral trans factors, the segregation/partitioning elements from both BPV1 and EBV can provide the stable maintenance function to the mouse polyomavirus (PyV) core origin plasmids but fail to do so in the case of complete PyV origin. Our study is the first which follows BPV1 E2- and minichromosome maintenance element (MME)-dependent stable maintenance function with heterologous replication origins. In mouse fibroblast cell lines expressing PyV large T antigen (LT) and either BPV1 E2 or EBV EBNA1, the long-term episomal replication of plasmids carrying the PyV minimal origin together with the MME or family of repeats (FR) element can be monitored easily for 1 month under nonselective conditions. Our data demonstrate clearly that the PyV LT-dependent replication function and the segregation/partitioning function of the BPV1 or EBV are compatible in certain, but not all, configurations. The quantitative analysis indicates a loss rate of 6% per cell, doubling in the case of MME-dependent plasmids, and 13% in the case of FR-dependent plasmids in nonselective conditions. Our data clearly indicate that maintenance functions from different viruses are principally interexchangeable and can provide a segregation/partitioning function to different heterologous origins in a variety of cells.     

Correction of glycaemia and GLUT1 level by mildronate in rat streptozotocin diabetes mellitus model

Anti‐ischaemic drug mildronate suppresses fatty acid metabolism and increases glucose utilization in myocardium. It was proposed that it could produce a favourable effect on metabolic parameters and glucose transport in diabetic animals. Rats with streptozotocin diabetes mellitus were treated with mildronate (100 mg/kg daily, per os, 6 weeks). Therapeutic effect of mildronate was monitored by measuring animal weight, concentrations of blood glucose, insulin, blood triglycerides, free fatty acids, blood ketone bodies and cholesterol, glycated haemoglobin per cent (HbA1c%) and glucose tolerance. GLUT1 mRNA and protein expression in kidneys, heart, liver and muscles were studied by means of real time RT‐PCR and immunohistochemistry correspondingly. In the streptozotocin + mildronate group, mildronate treatment caused a significant decrease in mean blood glucose, cholesterol, free fatty acid and HbA1c concentrations and improved glucose tolerance. Induction of streptozotocin diabetes mellitus provoked increase of both GLUT1 gene and protein expression in kidneys, heart and muscle, mildronate treatment produced normalization of the GLUT1 expression levels. In the liver a similar effect was observed for GLUT1 protein expression, while GLUT1 gene expression was increased by mildronate. Mildronate produces therapeutic effect in streptozotocin diabetes model. Mildronate normalizes the GLUT1 expression up‐regulated by streptozotocin diabetes mellitus in kidneys, heart, muscle and liver. Copyright © 2011 John Wiley & Sons, Ltd.