Design and Development of a Multiplex Real-Time PCR Assay for Detection of HIV-1 and HCV Using Molecular Beacons

At least 10 million individuals worldwide are co-infected with immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). These two viruses are transmitted most primarily by exposure to infected blood or blood products. Various nucleic acid assays have been developed for diagnostics and therapeutic monitoring of infections. In the present study, a multiplex real-time PCR assay for simultaneous detection of HCV and HIV-1 using molecular beacons were designed and validated. A well-conserved region in the HIV-1 pol gene and 5′NCR of HCV genome were used for primers and molecular beacon design. The analysis of scalar concentrations of the samples indicated that this multiplex procedure detects at least 1,000 copies/ml of HIV-1 and 100 copies/ml of HCV with linear reference curve (R ² > 0.94). The results demonstrate that a specificity of 100 % and sensitivity of 96 % can be achieved. The analytical sensitivity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes only detected HIV-1 and all major variants of HCV. This assay may represent an alternative rapid and relatively inexpensive screening method for detection of HIV-1/HCV co-infection especially in blood screening.     

Ceftriaxone Attenuated Anxiety-Like Behavior and Enhanced Brain Glutamate Transport in Zebrafish Subjected to Alcohol Withdrawal

Neurochemical Research - Chronic and/or excessive consumption of alcohol followed by reduced consumption or abstention can result in Alcohol Withdrawal Syndrome. A number of behavioral changes and...     

Involvement of Syk kinase in TNF-induced nitric oxide production by airway epithelial cells

We have recently found that Syk is widely expressed in lung epithelial cells (EC) and participates in beta1 integrin signaling. In this study, we assessed the role of Syk in regulation of NO production. Stimulation of human bronchial EC line HS-24 by TNF caused an increased expression of inducible nitric oxide synthase (iNOS). Inhibition of Syk using siRNA or piceatannol down-regulated the iNOS expression and reduced NO production. This effect occurred in EC simultaneously stimulated via beta1 integrins, suggesting that TNF and beta1 integrins provide co-stimulatory signals. Inhibition of Syk down-regulated TNF-induced p38 and p44/42 MAPK phosphorylation and nuclear translocation of p65 NF-kappaB. Thus, TNF-induced activation of pro-inflammatory signaling in EC leading to enhanced expression of iNOS and NO production was dependent on Syk. Syk-mediated signaling regulates NO production at least partly via activating the MAPK cascade. Understanding the role of Syk in airway EC may help in developing new therapeutic tools for inflammatory lung disorders.     

Kinetic studies on the inhibition of GABA-T by gamma-vinyl GABA and taurine

Gamma-aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of gamma-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: gamma-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by gamma-vinyl GABA and taurine at concentrations of 51.0 and 78.5 mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (Ki) values for gamma-vinyl GABA and taurine were found to be 26 +/- 3 mM and 68 +/- 7 mM respectively. The transamination process of alpha-ketoglutarate was not affected by the presence of gamma-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when alpha-ketoglutarate was the substrate. The inhibition dissociation constant (Kii) for this system was found to be 96 +/- 10 mM. The Michaelis-Menten constant (Km) in the absence of inhibition, was found to be 0.79 +/- 0.11 mM, and 0.47 +/- 0.10 mM for GABA and alpha-ketoglutarate respectively.     

Ability of alfalfa (Medicago sativa L.) to take up potassium from different micaceous minerals and consequent vermiculitization

Nonexchangeable soil K trapped between interlayers of clay minerals is an important source of K for plants grown on many soils. In this study, the ability of alfalfa, as the most important forage crop worldwide, to take up K from some micaceous minerals and to promote the transformation of micaceous minerals were investigated. Alfalfa was cultivated in pots that consisted of a mixture of Hamadan quartz sand and Amlash biotite, Hamadan muscovite and Hamadan phlogopite. Plants were irrigated with distilled water and complete or K-free nutrient solutions. After 90 days, K uptake by plants was measured by flame photometer. Also, the clay size particles in each pot were analyzed using x-ray diffraction. It was found that alfalfa was able to induce a significant release of interlayer K during cropping. The amount of K taken up by alfalfa was significantly higher for Hamadan phlogopite. X-ray diffractometry showed a strong vermiculitization of Hamadan phlogopite and Amlash biotite, but no X-ray detectable transformation of muscovite was recognized.     

Load transfer along the bone–dental implant interface

In this paper the variation of normal and shear stresses along a path defined on the bone–dental implant interface is investigated. In particular, the effects of implant diameter, collar length and slope, body length, and the effects of four different types of external threads on the interfacial stress distribution are studied. The geometry of the bone is digitized from a CT scan of a mandibular incisor and the surrounding bone. The bone and the implant are assumed to be perfectly bonded. The finite element method with 2D plane strain assumption is used to compute interfacial stresses. Highest continuous interfacial stresses are encountered in the region where the implant collar engages the cortical region, and near the apex of the implant in the subcortical region. Stress concentrations in the interfacial stresses occur near the geometric discontinuities on the implant contour, and jumps in stress values occur where the elastic modulus of the bone transitions between the cortical and trabecular bone values. Among the six contour parameters, the slope and the length of the implant collar, and the implant diameter influence the interfacial stress levels the most, and the effects of changing these parameters are significantly noticed only in the cortical bone (alveolar ridge) area. External threads cause significant stress concentrations in interfacial stresses in otherwise smoothly varying regions. This work shows that the presence of external threads could cause significant variations in both normal and shear stresses along the bone–implant interface, but not reduction in shear stress as previously thought.     

Calpain 1 binding capacities of the N1-line region of titin are significantly enhanced by physiological concentrations of calcium

Calpain 1, an ubiquitous well-known calcium-dependent intracellular protease, was recently shown to bind tightly to the proximal end of the I-band titin segment in a calcium-dependent manner [Raynaud et al. (2005) FEBS J. 272, 2578-2590]. In the present work we identified the titin Ig-domain of concern by this interaction and the role of calcium in this interaction using a recombinant fragment of titin spanning the I2-I6 region and its subfragments. The heterodimeric form of calpain 1 binds to this titin fragment with a very high affinity ( K d = 5.1 +/- 0.2 x 10 (-7) M) at much lower calcium levels than those saturating the high-affinity binding sites of the peptidase ( K d = 25 microM). Investigation of this interaction with I2-I6 subfragments clearly showed that the dimeric form of calpain 1 binds exclusively to the Ig-domain I4 of titin with an affinity similar to that of the whole I2-I6 segment. As for the I2-I6 fragment, this interaction is calcium regulated. Calcium was shown to bind tightly to titin ( K d = 1.9 x 10 (-7) M), causing an oligomerization of the titin segment. At physiological calcium concentration (10 (-6) to 10 (-8) M), the prevailing form of the titin fragment is a trimer, suggesting that calpain 1 binds to this titin structure. From the present findings, it was concluded that calcium binding to titin increased the amount of bound calpain 1 (up to 40% of the total calpain 1) and that this bound calpain 1 might constitute a reservoir for this peptidase. In this context, we proposed a schematic diagram of this series of calcium-dependent events with the inherent unanswered questions. These events are probably under a complex regulation involving undoubtedly different yet unidentified proteins.     

Effect of Pedoclimatic Conditions on the Chemical Composition of the Sigoise Olive Cultivar

The present work focused on the quality and the chemical composition of monovarietal virgin olive oil from the Sigoise variety grown in two different locations in Tunisia, viz., a sub‐humid zone (Béjaoua, Tunis) and an arid zone (Boughrara, Sfax). In addition to the quality characteristics (acidity, peroxide value, and the spectrophotometric indices K232 and K270) and the chemical composition (content of fatty acids, antioxidants, and volatile compounds) of the oil, the fruit characteristics of the olives were studied. Except for the content of the majority of the fatty acids, there were significant differences observed in the oil composition of olives that were cultivated in different locations. The content of total phenols and lipoxygenase (LOX) oxidation products was higher for olives grown at the higher altitude, whereas that of α‐tocopherol, carotenes, and chlorophylls was higher for olives from the Boughrara region (lower altitude). Moreover, olives produced at the higher altitude showed a higher ripeness index and oil content than those cultivated at the lower altitude.     

AdipoR1 and AdipoR2 gene expression are regulated by thyroid hormones in adipose tissue

The aim of this study was to examine whether the relative gene expression of AdipoR1 and AdipoR2 in rat adipose tissue is altered by thyroid hormones, and whether this might relate to their circulating thyroid hormones and adiponectin levels. Hyper- and hypothyroidism were induced by daily oral administration of levothyroxine and methimazole in rats, respectively, over a 42 days period. Real-time PCR analysis was performed to evaluate the changes in AdipoR1 and AdipoR2 mRNA levels in the adipose tissue on days 15, 28, 42, and also 2 weeks after the cessation of treatment. In response to treatment with methimazole, mRNA levels of AdipoR1 and AdipoR2 decreased in the white adipose tissue compared to the euthyroid rats (p < 0.05). This decline was reversible 2 weeks after treatment cessation. The mRNA levels of AdipoR1 and AdipoR2 were increased in the hyperthyroid group of animals compared to euthyroid control (p < 0.05), and its changes were reversible 2 weeks after treatment cessation (P < 0.05). Adiponectin receptors gene expression levels in the adipose tissue of treated animals have positive correlations with thyroid hormones concentrations. Our results suggest that AdipoR1 and AdipoR2 gene expression is regulated by thyroid hormones in hypo- and hyperthyroidism.