Design and characterization of a SYBR Green I-based melting curve method for investigation of HER2I655V polymorphism in breast cancer.

BACKGROUND: Breast cancer is a disease in which cell grows rapidly forming a mass in the breast. HER2 polymorphisms Ile655Val have been studied as biomarkers for breast cancer and may comprise a risk factor of cardiac toxicity for breast cancer-consuming trastuzumab. AIM OF WORK: In this study, we developed a simple, low cost, and rapid test to detect polymorphism at HER2 gene using SYBR Green I-based melting curve method. SUBJECTS AND METHODS: In this report, we performed allelic discrimination with real-time temperature melting (Tm) Shift SYBR Green I-based melting curve method. The melting profiles of amplified DNA HER-2 Ile655Val and its characteristics were analyzed. RESULT: Tm value of HER2 GG and AA alleles were 85 ± 0.14 °C and 82.5 ± 0.23 °C, respectively, while cycle threshold (Ct) value of GG, AG, and AA alleles were 19.6 ± 0.27, 22.5 ± 0.23, 18.6 ± 0.22 correspondingly; furthermore, no template control has shown consisting Ct value at 31.18 ± 0.27. The developed methods' characteristics were optimum annealing at 62 °C and Kappa coefficient value 1 with the mean almost consistent with PCR-sequencing. The coefficient of variability for intra-assay of GG, AG, and AA was in the range of 0.2-1%, while the coefficient of variability for inter-assay for each were in the range 0.7-1%. Further, based on PCR, shelf-life assay has shown stability for 3 months of storage observation. CONCLUSION: This approach may be considered as simple, rapid, and low cost supporting the rapid study of HER2 epidemiology. Furthermore, the developed methods potentially facilitate clinicians in dealing with breast cancer patients, especially in considering about the cardiotoxicity effect of trastuzumab.

The neuroprotective effect of ethanolic extract Ocimum sanctum Linn. in the regulation of neuronal density in hippocampus areas as a central autobiography memory on the rat model of Alzheimer's disease.

The aim of this study was to identify the effects of Ocimum sanctum Linn. ethanolic extract (OSE) on the neurons of the CA1, CA3, and DG hippocampal areas with the use of in vivo and in vitro models of Alzheimer's diseases (AD). Twenty-one two-month-old male rats were divided into three groups: untreated (Group A, n = 3), AD rats model pretreated with OSE followed by induction for Trimethyltin (TMT) on day 7 (group B, n = 9), and AD rats model treated with OSE both as pre-TMT introduction for 7 days and post-TMT induction for 21 days (group C, n = 9). AD rats were sacrificed on days 7, 14, and 21, and brain samples were collected and analyzed for neuronal density and neuropeptide Y (NPY) immunoreactivity. To corroborate the in vivo observations, HEK-293 cells were treated with TMT and used as an in vitro model of AD. The results were then analyzed using FITC Annexin V and flow cytometry. Nuclear fragmentation was observed in cells stained with Hoechst 33342 by confocal microscopy. The results showed a significant increase in the number of neurons and NPY expression in the AD rats that were pre- and post-treated with OSE (p < 0.05). Indeed, OSE was able to retain and promote neuronal density in the rat model of AD. Further studies of an in vitro model of neurodegeneration with Ocimum sanctum Linn. ethanolic extract inhibited apoptosis in TMT-induced HEK-293 cells. Moreover, OSE prevented nuclear fragmentation, which was confirmed by staining the nuclei of HEK-293 cells. Taken together, there findings suggest that OSE has the potential as a neuroprotective agent (retaining the autobiographical memory),and the neuroproliferation of neurons in the CA1, CA3, and DG hippocampal areas in the rats¡ model of neurodegeneration was mediated by activation of NPY expression.

Exploration of Adhesion Molecule Expression in Cardiac Muscle of Early Atherosclerosis Dyslipidemic Sprague Dawley Rats.

OBJECTIVE: This study is aimed to examine the expression of ICAM-1 and VCAM-1 in cardiac tissue of dyslipidemic Sprague Dawley rats. METHODS: Eight Sprague Dawley strain rats, with 150-200 gram body weight, were divided into two groups. The control group was fed a standard diet, the positive control group was fed a high-fat diet as our previous study for 8 weeks. The pattern of distribution of ICAM-1 and VCAM-1 in cardiac muscle cell was examined by immunofluorescence and observed with a confocal laser scanning microscope. Lipid profile was also examined at the end of the study. RESULT: Independent t-test showed no differences in ICAM-1 and VCAM-1 expression in cardiac muscle of hypercholesterol-diet-fed Sprague Dawley rat compared to control. CONCLUSION: The expression of ICAM-1 and VCAM-1 in cardiac muscle did not change after the onset of atherosclerosis.

Reduction in Vasa Vasorum Angiogenesis by Lp-PLA2 Selective Inhibitor Through The HIF-1α and VEGF Expression Under Dyslipidemic Conditions in Atherosclerosis Pathogenesis.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease which may lead to major cardiovascular events. The primary cause of atherosclerosis is Dyslipidemia. The increased level of lipid profile triggers endothelial dysfunction. This results in inflammation with the recruitment of monocyte, macrophage, T lymphocyte, and Mast cells secreted by an Lp-PLA2 enzyme which causes binding between macrophage and oxidized LDL. This binding results in the formation of foam cells and also the migration of smooth muscle cells. Following that, an Lp-PLA2 receptor hydrolizes OxPC which results in LysoPC and OxNEFA, bioactive compounds which stimulate the progression of atherosclerosis plaques. This process leads to cell hypoxia, which may result in the increase of HIF-1α and VEGF expressions and induction of vasa vasorum angiogenesis. Employing darapladib as an agent of Lp-PLA2 selective inhibitors, this study aimed to find out the effect of darapladib as an Lp- PLA2 selective inhibitor agent on the formation of vasa vasorum angiogenesis and the decrease of HIF-1α and VEGF expression in aortic tissue of rats with dyslipidemia. METHOD: A true laboratory experiment with a randomized post-test control group design used 30 male spraque dowley rats as animal models which were divided into 6 groups: Normal 8 weeks, Normal 16 weeks, Dyslipidemia (DL) 8 weeks, Dyslipidemia (DL) 16 weeks, Dyslipidemia with darapladib treatment (DLDP) 8 weeks and Dyslipidemia with darapladib treatment (DLDP) 16 weeks. The data measured in this study were the lipid profile (total cholesterol, HDL, and LDL). Using EnzyChrom TM kit, hematoxylin eosin, and double-labelling immunofluorescene, the levels of lipid profile, vasa vasorum, HIF-1α and VEGF were measured. RESULTS: The study results which were analyzed using NOVA test showed that with darapladib administration, there was a significant decrease in vasa vasorum angiogenesis (p=0.000), HIF-1α (p=0.005) and VEGF (p=0.009) expression in each time series. This result proves that Lp-PLA2 inhibitor reduces inflammatory process. CONCLUSION: Darapladib injection as an Lp-PLA2 selective inhibitor correlates with the decreasing vasa vasorum angiogenesis through alteration in HIF-1α and VEGF expressions in the aorta of high fat diet rats. We recommend further experiments to determine the effectiveness of darapladib with earlier time series in the atherosclerosis process.