TaqMan Real-time Polymerase Chain Research Articles

A study of changing trends of prevalence and genotypic distribution of hepatitis C virus among high risk groups in North India

Purpose: Hepatitis C virus (HCV) has emerged as a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. There is a great variability in HCV’s geographical presence, transmission routes, genotypic distribution etc., in studied populations. We undertook this study in a North Indian hospital on patients of chronic liver disease to observe any emerging trend in risk groups, transmission patterns, genotypic distribution of the virus in this geographical region and its correlation with viral load. Materials and Methods: There were 54 anti-HCV positive patients including 31 HCV Ribonucleic acid (RNA) positive patients were included in the study. HCV genotyping was carried out by restriction fragment length polymorphism (RFLP) followed by direct sequencing of the core region. Viral load estimation was carried out by Taqman real time polymerase chain reaction system. Results: In 45/54 (83.3%) anti-HCV positive patients, iatrogenic procedures were responsible for transmission with blood transfusion alone responsible in 36/54 (67%). Genotype 3 was observed to be the commonest type found in all risk groups followed by type 1 and 2. Subtype 3b (35.5%) was found more prevalent than subtype 3a. A higher frequency of subtype 1b (19.4%) was also seen. Genotype 1 was associated with a significantly higher viral load compared to genotypes 3 and 2. No significant difference was observed in the biochemical profile among the three genotypes except for the levels of the enzyme, aspartate aminotransferase (AST). Conclusions: Iatrogenic procedures, especially contaminated blood transfusion etc., still contributes significantly to the pool of HCV infection. Genotype 3 is the predominant genotype in North India, though the subtype distribution within genotype 3 may be changing. The association of severe liver disease is significantly more with genotype 1 as evidenced by higher viral load and deranged AST levels.

DNA methylation of the p66Shc promoter is decreased in placental tissue from women delivering intrauterine growth restricted neonates

The adaptor protein p66Shc generates mitochondrial reactive oxygen species and translates oxidative signals into apoptosis. We aimed to analyze potential alterations in total methylation and in p66Shc activation in placental tissues from women delivering intrauterine growth restricted neonates (IUGR) versus appropriate for gestational age (AGA) and small for gestational age (SGA) neonates. DNA methylation of the p66Shc promoter and of long interspersed nuclear elements (LINE-1), as a marker for total methylation, was quantified by automatic pyrosequencing in 15 IUGR, 25 AGA and 15 SGA placentas. Placental gene expression of p66Shc was determined by TaqMan real-time polymerase chain reaction. No significant difference was found for LINE-1 methylation between IUGR, AGA and SGA newborns. DNA methylation of the p66Shc promoter was significantly decreased in the IUGR compared with the AGA group (p < 0.0001) and the SGA group (p < 0.0001). However, analysis of placental p66Shc gene expression did not show a significant difference between the three groups. It remains speculative if the decreased p66Shc promoter methylation might play a role in the pathophysiology of endothelial dysfunction and cardiovascular disease after IUGR.

Notch Signaling Modulates MUC16 Biosynthesis in an In Vitro Model of Human Corneal and Conjunctival Epithelial Cell Differentiation

Notch proteins are a family of transmembrane receptors that coordinate binary cell fate decisions and differentiation in wet-surfaced epithelia. We sought to determine whether Notch signaling contributes to maintaining mucosal homeostasis by modulating the biosynthesis of cell surface-associated mucins in an in vitro model of human corneal (HCLE) and conjunctival (HCjE) epithelial cell differentiation. HCLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction. Here we demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4. These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface.

Defective IL10 Signaling Defining a Subgroup of Patients With Inflammatory Bowel Disease

Early onset inflammatory bowel diseases (EO-IBD) developing during the first year of life are likely to reflect inherited defects in key mechanism(s) controlling intestinal homeostasis, as recently suggested for interleukin 10 (IL10). Thus, we aimed to further elaborate the hypothesis of defective anti-inflammatory responses in patients with IBD. The capacities of transforming growth factor β (TGFβ) and IL10 to inhibit proinflammatory cytokine production by monocyte-derived dendritic cells (MoDC) or peripheral blood cells (PBMC) was analyzed in 75 children with IBD, including 13 infants with EO-IBD (in whom autoimmune diseases or classical immunodeficiencies were ruled out). IL10 receptor-A/-B expression, STAT3 activation in response to IL6, IL10, IL21, IL22 were analyzed by FACS and western blotting. IL10RA and B genes were sequenced. The response to IL22 was tested in ileal/colonic tissue cultures. Tissue gene expression was analyzed by Taqman real-time polymerase chain reaction. Production of IL10 in response to bacterial motifs was normal in all IBD patients. In contrast to our original hypothesis, no defect of the anti-inflammatory potential of TGFβ and IL10 was observed in children with IBD or EO-IBD except two infants who presented with granuloma-positive colitis at 3 months of life: no response to IL10 was observed secondary to mutations in the α (p.R262C) or β (p.E141X) chain of IL10R, respectively, although a fully functional Jak-STAT3 pathway was present in both patients. When analyzing the regulation of intestinal bacterial clearance, we detected a defect in the patient with absent IL10 RB to upregulate protective transcripts in response to IL22, whereas all other EO-IBD patients, including the patient with an abnormal α chain, responded normally. Impaired IL10 signaling characterizes a subgroup of IBD patients, whereas the majority of children with severe IBD including EO forms normally produces and responds to IL10. Defective IL22 signaling may additionally impair intestinal epithelial clearance. Our data point out the complexity of IBD, which represent a group of distinct diseases with several pathogenetic abnormalities.

Development of a rapid triplex TaqMan real-time PCR assay for the detection of toxigenic Vibrio cholerae

Objective To develop a rapid triplex TaqMan real-time quantitative polymerase chain reaction (PCR) assay for the discrimination of toxigenic Vibrio cholerae O1 and Vibrio cholerae O139 simultaneously. Methods Primers and Taqman probes were designed for multiplex PCR reaction using rfb-O1, rfb-O139 specific O antigen biosynthetic gene and cholera toxin gene ctxA as the target regions for amplification. Smart cycler system was used to evaluate the sensitivity of this method, and a total of 19 other common enteropathogenic bacteria or frequent isolates causing nosocomial infection were also used to measure the specificity of constructed method. Results Constructed assay method allowed the detection as few as 102 copies per reaction for the testing of ctxA, rfb-O1 and rfb-O139 genes using mixed plasmids as the templates, and the linearity range of assay varied from 102 to 109 copies per reaction. Using the Vibrio cholerae O1 and O139 genome DNAs as the starting materials for detection, constructed assay method could detect 1.0×10-1 pg and 1.0×100 pg per reaction,respectively. Nan-specific amplifications were not presented when testing 19 other common enteropathogenic bacteria or frequent isolates causing nosocomial infection. In addition, the assay could get valid results within only 2 hours. Conclusion The triplex TaqMan real-time PCR assay described in this study was specific, sensitive,rapid and suitable not only for the discrimination of toxigenic strains,O1 and O139 serogroup of Vibrio cholerae, but also for the detection of their virulent abilities simultaneously. Key words: Vibrio cholerae; Polymerase chain reaction; Taq polymerase; Cholera toxin

Brucella: A fastidious bacteria but a virulent pathogen

To investigate the presence of brucellosis in graved human and animal buried in the same graves around 3 500 years ago.Two ancient graves in AH2XX1 trench in Gohar Tepe (Behshahr, North of Iran) in which human and animal were buried in single grave were selected for this study. After DNA extraction from human and animal tooth samples, TaqMan real time polymerase chain reaction method was utilized to check and measure the presence of brucellae bacteria.Brucellosis was found in human with 3.9×105 copy/0.05 gr and in animal with 6.5×103 copy/0.05 gr in grave one. In grave two, 2.6×105 copy/0.05 gr in human and 7.7×103 copy/0.05 gr in animal were found.This study demonstrated the presence of brucellosis infections in this region in 3 500 years ago. Also, it showed that the transmission of brucellosis in Gohar Tepe might be due to the frequent use of milk and meat as the main energy source by the residents. Although previous research claimed that brucellosis was limited to the European continent in the Middle Bronze Age, the current research showed the presence of brucellosis in Middle East region. An advantage of this study over previous research is the use of real time polymerase chain reaction which is more rigorous for studying the presence of brucellae in ancient objects compared to Paleopathological method.

DCC is a potential methylation marker in human cancers

15054 Background: DCC (deleted in colorectal cancer) is a tumor suppressor gene, and known to be inactivated by mutation in human cancers. In this study, we revealed DCC gene promoter methylation in primary human esophageal and breast cancers and refer to its excellence as a cancer-prone marker. Methods: Quantititative TaqMan real time polymerase chain reaction; 20 esophageal squamous cell carcinoma (ESCC) and the corresponding non-cancerous esophageal mucosal tissues, 58 breast cancer tissues, and ESCC cell lines. Results: (1) In ESCC tissues, DCC hypermethylation is detected in 18/20 cases (sensitivity=90%), while it is detected in only 2/20 normal cases (specificity=90%). (2) In breast cancer tissues, DCC hypermethylation is detected in 29/58 cases (50%), and patients with higher methylation of DCC showed size larger than 6 cm (100%) as compared with those less than 6 cm (45.3%)(p&lt;0.025). (3) DCC is silenced in cancer cell lines, and found to be reactivated by demethylating agent in several cell lines, while still silenced in other cell lines. Conclusions: In ESCC, DCC hypermethylation could be an excellent potential marker. DCC inactivation is partially explained by methylation, however other mechanism could be involved like p53. The accurate mechanism of DCC requires further elucidation, but its potential as a tumor marker in SCC is considered excellent to apply for cancer detection using human body fluid. No significant financial relationships to disclose.

Increased plasma levels of LDL cholesterol in rabbits after adenoviral overexpression of human scavenger receptor class B type I

Scavenger receptor class B type I (SR-BI), a CD36 family member, plays a key role in high-density lipoprotein (HDL) metabolism, reverse cholesterol transport, and whole body cholesterol homeostasis, and is shown to be involved in the development of atherosclerosis in mice. In this report, we describe the effects of the adenoviral overexpression of human SR-BI (hSR-BI) in New Zealand White (NZW) rabbits, a wild-type animal model that expresses cholesteryl ester transfer protein (CETP) in plasma, displays a manlike lipoprotein profile, and is susceptible to atherosclerosis. A total of 1x10(12) adenoviral particles containing either hSR-BI or lacZ complementary deoxyribonucleic acid (control) were infused into the ear vein of NZW rabbits. Transgene expression was ascertained by TaqMan Real Time polymerase chain reaction measurements. Rabbits infected with Ad/hSR-BI (adenoviral plasmids containing hSR-BI) showed a faster clearance of administered [3H]HDL cholesterol and significantly decreased apolipoprotein (apo) A-I levels when compared to control rabbits, respectively. Interestingly, we found markedly increased levels of low-density lipoprotein (LDL) cholesterol exclusively in SR-BI-overexpressing rabbits. These changes were not accompanied by alterations in LDL receptor expression but by increased levels of CE transfer in these animals. By lowering HDL cholesterol and increasing plasma apoB-containing lipoprotein levels, the overexpression of SR-BI leads to a lipoprotein pattern, which is believed to enhance the development of atherosclerosis. The role of SR-BI in lipoprotein metabolism and atherogenesis in rabbits--a CETP-expressing animal model displaying a manlike lipoprotein profile--may therefore be different from the one found in rodents.

Gene expression in peripheral blood mononuclear cells from patients with chronic fatigue syndrome

Background: Chronic fatigue syndrome (CFS) is a multisystem disease, the pathogenesis of which remains undetermined. Aims: To test the hypothesis that there are reproducible abnormalities of gene expression in patients...

Comparison of aerosol therapy with different perfluorocarbons in surfactant-depleted animals

The study investigates the effectiveness of aerosol treatment on gas exchange and pulmonary inflammatory reaction using perfluorocarbons with different molecular structure and vapor pressure. Experimental, prospective, randomized, controlled study. Experimental laboratory at a university hospital. Twenty anesthetized neonatal piglets assigned to four groups. After establishment of lung injury by bronchoalveolar lavage, piglets either received aerosolized FC77 (n = 5), perfluorooctylbromide (n = 5), or FC43 (n = 5, 10 mL x kg(-1) x hr(-1) for 2 hrs) or intermittent mandatory ventilation (control, n = 5). Thereafter, animals were supported for another 6 hrs. Pao2 significantly improved in the perfluorocarbon groups compared with control (p < .01). Final Pao2 (mean +/- SEM) was FC77, 406 +/- 27 mm Hg; perfluorooctylbromide, 332 +/- 32 mm Hg; FC43, 406 +/- 19 mm Hg; control, 68 +/- 8 mm Hg. Paco2 and mean pulmonary arterial pressure were lower in all perfluorocarbon groups compared with control. The ratio of terminal dynamic compliance to total compliance was significantly higher in the FC77 than in the FC43, perfluorooctylbromide, and control groups. Relative gene expression of interleukin-1beta, interleukin-8, P-selectin, E-selectin, and intercellular adhesion molecule-1 in lung tissue was determined by TaqMan real time polymerase chain reaction normalized to hypoxanthineguanine-phosphoribosyl-transferase and was shown to be reduced by all perfluorocarbons. Aerosol treatment with all the perfluorocarbons investigated improved gas exchange and reduced pulmonary inflammatory reaction independently from molecular structure and vapor pressure of the perfluorocarbons. Although differences in vapor pressure and molecular structure may account for varying optimal dosing strategies, several different perfluorocarbons were shown to be principally suitable for aerosol treatment.

MRNA Quantification by Real Time TaqMan Polymerase Chain Reaction: Validation and Comparison with RNase Protection

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common inborn long-chain fatty acid oxidation (FAO) disorder. VLCAD deficiency is characterized by distinct phenotypes. The severe phenotypes are potentially life-threatening and affect the heart or liver, with a comparatively milder phenotype characterized by myopathic symptoms. There is an unmet clinical need for effective treatment options for the myopathic phenotype. The molecular mechanisms driving the gradual decrease in mitochondrial function and associated alterations of muscle fibers are unclear.The peroxisome proliferator-activated receptor (PPAR) pan-agonist bezafibrate is a potent modulator of FAO and multiple other mitochondrial functions and has been proposed as a potential medication for myopathic cases of long-chain FAO disorders. In vitro experiments have demonstrated the ability of bezafibrate to increase VLCAD expression and activity. However, the outcome of small-scale clinical trials has been controversial.We found VLCAD deficient patient fibroblasts to have an increased oxidative stress burden and deranged mitochondrial bioenergetic capacity, compared to controls. Applying heat stress under fasting conditions to bezafibrate pretreated patient cells, caused a marked further increase of mitochondrial superoxide levels. Patient cells failed to maintain levels of the essential thiol peptide antioxidant glutathione and experienced a decrease in cellular viability. Our findings indicate that chronic PPAR activation is a plausible initiator of long-term pathogenesis in VLCAD deficiency. Our findings further implicate disruption of redox homeostasis as a key pathogenic mechanism in VLCAD deficiency and support the notion that a deranged thiol metabolism might be an important pathogenic factor in VLCAD deficiency.