Optimizing denaturing HPLC as a robust technique for identification of Short Tandem Repeats (STR) in forensic medicine.

INTRODUCTION: Short Tandem Repeats (STRs) are defined as short lengths of 2-7 base pairs spreading through human genome which due to their highly diverse individually distribution are widely applied for identity detection and other forensic medicine purposes. Burdening considerable costs by the conventional methods such as capillary electrophoresis, we aimed to compare concomitant usage of multiplex PCR and denaturing high-performance liquid chromatography (DHPLC) as cheap, fast, highly accurate, and more accessible methods, with capillary electrophoresis (CE) to evaluate their potential for early screening of STRs. MATERIALS AND METHODS: The present study randomly included 20 blood samples from the subjects referred to forensic medicine of Semnan, Iran. According to the size and allele frequency, we selected 8 major STR loci including CSF1PO, VWA, D18S51, TPOX, Amelogenin, FGA, SE33, and Penta D. A quad-STR multiplex PCR was performed for each locus and the PCR products were then analyzed using DHPLC machine and compared with the basic genetic properties obtained by capillary electrophoresis. RESULTS: By optimizing the PCR and DHPLC conditions, our findings suggest this strategy as an effective method for STR detection. The genotypes were determined using size of loci which led to comparable results with capillary electrophoresis confirming an insignificant variation in the detection of TOPX, Amelogenin, CSF1PO, and D18S5 (p = 0.331), but discrepant results for FGA and VWA loci (p = 0.002). CONCLUSION: Our study proposed DHPLC method as an effective screening method to characterize TOPX, Amelogenin, CSF1PO, and D18S51 as frequently used STR loci during identity detection in forensic medicine.

Activity Assay of Glutathione S-Transferase (GSTs) Enzyme as a Diagnostic Biomarker for Liver Hydatid Cyst in Vitro.

BACKGROUND: The aim of this study was to detect the Glutathione S-Transferase(GST) enzyme activity of healthy / cystic liver as a diagnostic biomarker for hydatidosis. In order to compare with liver tissue, the level of the GSTs enzyme activity of parasite was also determined. METHODS: Parasites were collected from sheep liver tissue with hydatid cysts at a local abattoir and washed with PBS buffer. Collected parasites and liver tissues were sonicated or homogenized respectively. Extract solution samples were centrifuged and stored at - 20°C. GST enzyme activities were measured in the extract of parasite and liver tissue samples (healthy and infected livers). Protein amounts and protein bands were detected using Bradford and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods respectively. To determine significant difference between two groups, two-sample t-test was performed. RESULTS: GST specific activities of healthy / infected livers and parasites were estimated 304, 1297 and 146 U/ml/mg respectively. Significant higher GST specific activities in cystic liver than healthy liver was observed (P <0.05). T-test analysis showed GST activity of parasite was lower than healthy liver tissue. SDS-PAGE showed GST protein bands with 24 kDa in parasite samples and25 kDa in liver tissues. CONCLUSION: GST activity incystic liver tissue could be concerned as a biomarker for hydatid cyst diagnosis with other hydatid disease parameters.

Comparative assay of glutathione S-transferase (GSTs) activity of excretory-secretory materials and somatic extract of Fasciola spp parasites.

Fascioliasis is a worldwide parasitic disease in human and domestic animals. The causative agents of fascioliasis are Fasciola hepatica and Fasciola gigantica. In the recent years, fasciola resistance to drugs has been reported in the many of publications. Fasciola spp has detoxification system including GST enzyme which may be responsible for its resistance. Therefore , the aim of the study was to assay of GST enzyme activity in fasciola parasites. Fasciola gigantica and Fasciola hepatica helminths were collected from abattoir as a live and cultured in buffer media for 4 h at 37 °C. Excretory-Secretory products were collected and stored in -80◦C. F. gigantica and Fasciola hepatica were homogenized with homogenizing buffer in a glass homogenizer to prepare of somatic extract. Suspension was then centrifuged and supernatant was stored at -80°C. In order to assay the enzyme activity, excretory-secretory and somatic extracts in the form of cocktails (potassium phosphate buffer, reduced glutathione and 1-chloro-2,4-dinitrobenzene substrates) were prepared and their absorbance recorded for 5 minutes at 340 nm. The total and specific GST activity of F. gigantica somatic and ES products were obtained as 2916.00, 272.01 micromole/minute and 1.33, 1.70 micromole/minute/mg protein, respectively. Fasciola hepatica also showed 2705.00, 276.86 micromole/minute and 1.33, 1.52 micromole/minute/mg protein, respectively. These results are important for analysis of parasite survival / resistance to drugs which use for treatment of fascioliasis.

Association of angiotensin-converting enzyme gene insertion/deletion polymorphism with metabolic syndrome in Iranians with type 2 diabetes mellitus.

BACKGROUND: Angiotensin-converting enzyme insertion/deletion polymorphism has been shown to be associated with diabetes, hypertension, coronary artery diseases, and diabetic nephropathy. The objective of this study was to investigate whether angiotensin-converting enzyme gene insertion/deletion polymorphism is associated with metabolic syndrome in Iranians with type 2 diabetes mellitus. METHODS: A total of 170 patients with type 2 diabetes mellitus and 91 control subjects were studied. The angiotensin-converting enzyme insertion/deletion polymorphism was determined by polymerase chain reaction (PCR) utilizing specific primers. The definition and criteria of metabolic syndrome used in this study matched that proposed in 1998 World Health Organization classification. RESULTS: Of 170 patients studied, 119 (70%) fulfilled the criteria for metabolic syndrome. The prevalence of angiotensin-converting enzyme genotype in the control subjects with DD, ID, and II genotype was 13.2%, 47.3%, and 39.5%, respectively. In patients with metabolic syndrome, the prevalence was 26.9%, 56.3%, and 16.8%, respectively; in patients without metabolic syndrome, it was 21.6%, 62.7%, and 15.7%, respectively. The angiotensin-converting enzyme insertion/deletion polymorphism was not significantly associated with presence of metabolic syndrome in patients with type 2 diabetes (P=0.711). The frequency of DD genotype in the metabolic syndrome group (26.9%) was higher than that (21.6%) in those without metabolic syndrome (P=0.447) and the control group (13.2%, P=0.02). The frequency of D allele in metabolic syndrome patients was 55.1% as compared to those patients without metabolic syndrome (52.9%, P=0.72) and the control subjects (36.8%, P<0.001). CONCLUSION: It seems that the DD genotype and/or D allele of angiotensin-converting enzyme gene may increase the risk for developing type 2 diabetes mellitus, but not metabolic syndrome.