Madge - Microplate Array Diagonal Gel Electrophoresis

Microplate array diagonal gel electrophoresis (MADGE) was invented for molecular genetic epidemiological studies. MADGE is a highly flexible, cost effective, microplate compatible solution to high throughput electrophoresis needs. It enables several thousands to million gel lines per day for direct assay of single-base variation in different capacity laboratories. Variants of the standard 96-well MADGE include: 96-well stretch-MADGE, 192-well MADGE, 384-well MADGE, and 768-well MADGE. Melt-MADGE combines the temporal thermal ramp apparatus to achieve similar throughput for de novo mutation scanning. Basic MADGE principles and procedures, preparation of MADGE gels, electrophoresis, visualization and analysis of these gels, as well as modifications of the basic 96-well MADGE will be discussed in detail. For the first time in our country, this revolutionary polyacrylamid electrophoresis was done in 1998. We shortly review our studies which used MADGE for high throughput genotyping of the apolipoprotein E. MADGE and melt-MADGE will have an important role in the future genetic research of complex diseases and especially in pharmacogenomics.5th EFCC Symposium on Proteins - From Electrophoresis to Proteomics, 2009, Belgrade, Serbi

The role of G protein coupled receptor kinases in neurocardiovascular pathophysiology

In coronary artery disease the G protein related kinases (GRKs) play a role in desensitization of beta-adrenoreceptors (AR) after coronary occlusion. Targeted deletion and lowering of cardiac myocyte GRK-2 decreases the risk of post-ischemic heart failure (HF). Studies carried out in humans confirm the role of GRK-2 as a marker for the progression of HF after myocardial infarction (MI). The level of GRK-2 could be an indicator of beta-AR blocker efficacy in patients with acute coronary syndrome. Elevated levels of GRK-2 are an early ubiquitous consequence of myocardial injury. In hypertension an increased level of GRK-2 was reported in both animal models and human studies. The role of GRKs in vagally mediated disorders such as vasovagal syncope and atrial fibrillation remains controversial. The role of GRKs in the pathogenesis of neurocardiological diseases provides an insight into the molecular pathogenesis process, opens potential therapeutic options and suggests new directions for scientific research

The association of ACE I/D gene polymorphism with severe carotid atherosclerosis in patients undergoing carotid endarterectomy

Introduction: The ACE I/D polymorphism was mostly investigated in association with intima-media thickness, rarely with severe atherosclerotic phenotype. Materials and methods: We investigated the association of I/D polymorphism with severe carotid atherosclerosis (CA) (stenosis GT 70%) in asymptomatic and symptomatic patients undergoing carotid endarterectomy. The 504 patients subjected to endarterectomy and 492 healthy controls from a population in Serbia were investigated as a case-control study. Results: The univariate logistic regression analysis revealed ACE DD as a significant risk factor for severe CA (odds ratio [OR] = 1.3, 95% confidence interval [CI] 1.0-1.7, p = 0.04). After adjustment for the common risk factors (age, hypertension, smoking, and HDL) ACE was no longer significant. However, we found a significant independent influence of DD genotype on plaque presence in a normotensive subgroup of patients (OR 1.8, CI 1.2-3.0, p = 0.01, corrected for multiple testing). In symptomatic patients D allele carriers were significantly more frequent compared with asymptomatic patients (OR 1.6 CI 1.0-2.6, p = 0.05). Conclusions: Our data suggests that ACE I/D is not an independent risk factor for severe CA. On the other hand, a significant independent genetic influence of ACE I/D appeared in normotensive and symptomatic patients with severe CA. This should be considered in further research toward resolving the complex genetic background of severe CA phenotype

Gene expression of chemokines CX3CL1 and CXCL16 and their receptors, CX3CR1 and CXCR6, in peripheral blood mononuclear cells of patients with relapsing-remitting multiple sclerosis - a pilot study

Background/Aim. In vitro and in vivo studies show that CX3CL1 and CXCL16 chemokines and their specific receptors, CX3CR1 and CXCR6, respectively, mediate mechanism of neuroinflammation during the pathogenesis of multiple sclerosis (MS). The aim of this study was to investigate relative messenger ribonucleic acid (mRNA) levels of CX3CL1, CXCL16, CX3CR1 and CXCR6 in peripheral blood mononuclear cells, as potential molecular markers of relapsing-remitting (RR) MS. Methods. The study included 43 unrelated RR MS patients, 20 of them with clinically active disease (relapse) and 23 with clinically stable disease (remission), and 28 unrelated healthy subjects as controls. Real-time polymerase chain reactions (PCR) were performed using TaqMan? gene expression assays. Relative expression (mRNA) level of each target gene in each sample of peripheral blood mononuclear cells was calculated as the mean normalized expression. Results. The levels of CX3CR1 mRNA were significantly higher in clinically active RR MS patients compared to controls [fold change = 1.38, p (Mann-Whitney U test) = 0.009], and significantly lower in clinically stable vs active RR MS patients [fold change = - 1.43, p (t-test) = 0.03]. Stable RR MS patients had significantly higher CXCL16 mRNA levels than controls [fold change = 1.33, p (Mann-Whitney U test) = 0.006]. A trend of increased CXCR6 gene expression was found in active RR MS patients compared to controls [fold change = 1.23, p (Mann-Whitney U test) = 0.08]. In either active or stable RR MS patients there were no significant correlations of the clinical parameters with expression levels of the target genes. Conclusion. The current results show that increased CX3CR1 mRNA levels in peripheral blood mononuclear cells could represent a proinflammatory molecular marker of clinically active RR MS

CXCL16 in Vascular Pathology Research: from Macro Effects to microRNAs

Chemokines and their receptors have become significant factors in atherosclerosis research. CXCL16 is a multifunctional agent located on a separate locus to all other known chemokines and binds only to its unique receptor named CXCR6. As a scavenger receptor, adhesion molecule, and chemokine, it quickly became an interesting target in atherosclerosis research as all its functions have a role in vascular pathology. The investigation of the role of CXCL16 in atherosclerosis, although shown in in vitro studies, animal knockout models, and CXCL16 gene polymorphisms, haplotypes, and circulating levels, still shows puzzling results. Genetic and epigenetic studies have just scratched the surface of research necessary for a better assessment of the significance and perspective of this marker in plaque development and progression. In this review, we will summarize current knowledge about CXCL16 in atherosclerosis. Additionally, we will point out the importance of bioinformatics tools for the detection of potentially new CXCL16 regulatory networks through microRNA activity. This review aims to provide a better understanding of the underlying mechanisms, define more specific biomarkers, and discover new therapeutic targets

Polymorphism rs10757278 in 9p21 region is associated with severe carotid atherosclerosis in sex specific manner: Preliminary results

Genome wide association studies have recognized the 9p21 rs10757278 polymorphism as a significant independent genetic prognostic marker for coronary artery disease. The aim of this study was to explore possible association of the rs10757278 polymorphism with advanced carotid atherosclerosis (CA) in the population of Serbia. The study group included 147 controls and 428 patients consecutively admitted for carotid endarterectomy. 9p21 rs10757278 polymorphism was genotyped using TaqMan technology on 7500 ABI Real Time PCR. There was no significant association of this polymorphism and CA, either in study group overall or in males. The GG genotype, according to recessive model of inheritance (AA+AG vs. GG), was significantly associated with advanced CA in females only (OR=2.15, 95% CI 1.07- 4.29, p=0.03). Preliminary results in this study suggest that rs10757278 GG genotype might be a significant predictive sex-specific marker for advanced CA in the population of Serbia.5th Congress of the Serbian Genetic Society : Book of abstracts; September 28 - October 2, Kladovo, 2014

Angiotensin II receptor type 2 (AT2R) -1332 A/G gene polymorphism as a risk factor for multiple sclerosis

Multiple sclerosis (MS) is a complex inflammatory,demyelinating disease of central nervous system (CNS). All the essen-tial components of the renin-angiotensin system (RAS) are presentedin the mammalian brain. The angiotensin II (Ang II), biologicallyactive octapeptide is not only a vasoconstrictor, but also a pro-inflam-matory factor. Many of the classical and of the hypothetical functionsof brain Ang II are mediated by stimulation of AT1 receptors (AT1R).Brain AT2 receptors (AT2R) are highly expressed during development.In the adults, AT2R are restricted to areas predominantly involved inthe process of sensory information. The AT2R1332 A/G polymorph-ism was proposed to influence AT2R protein expression, and is themost studied polymorphism in this gene, in other diseases. Recently,the striking appearance of the RAS in MS brain was described.However, the role of AT2R remains to be clarified. Thus, the aim ofour study was to establish if there is an association between AT2R1332 A/G gene polymorphism and predisposition of MS Methods:Subjected group consisted of 122 female and 70 malepatients with MS and 75 female and 50 male controls from populationof Serbia. Genotyping was done by PCR and restriction digestion withEcoRI enzyme.Results:The genotype and allele frequencies for AT2R1332A/Ggene polymorphism are analyzed separately in females and males,since this gene is located on X chromosome. We detected significantoverrepresentation of1332A/G AA genotype (OR 1.6, 95% CI:1.0–2.7, p<0.05) in female patients with MS compared to female controls.In hemizygous males we didn’t found any difference between patientsand controls.Conclusion:The role of RAS genes in MS was neglected untilrecently. Than, it was shown that the role of RAS in the CNS isbeyond the regulation of cardiovascular function. Until now AT2R(1332A/G) gene polymorphism was widely studied and associatedwith hypertension and other vascular disease. Until now, there wereno studies concerning role of Ang II receptor polymorphisms in MS.This study suggest possible role of AT2R in MS. Further studies areneeded to elucidate this result.ECTRIMS : The 26th Congress of the European Committee for Treatment and Research in Multiple Sclerosis : Book of abstract