Detection of hantaviruses in Brazilian rodents by SYBR-Green-based real-time RT-PCR

Current knowledge of the pathogenic hantavirus indicates that wild rodents are its primary natural reservoir. Specific primers to detect the presence of viral genomes were developed using an SYBR-Green-based real-time RT-PCR protocol. One hundred sixty-four rodents native to the Atlantic Forest biome were captured in So Paulo State, Brazil, and their tissues were tested. The presence of hantavirus RNA was detected in sixteen rodents: three specimens of Akodon montensis, three of Akodon cursor, two of Necromys lasiurus, one of Juliomys sp., one of Thaptomys nigrita, five of Oligoryzomys nigripes, and one of Oryzomys sp. This SYBR Green real-time RT-PCR method for detection of hantavirus may be useful for surveying hantaviruses in Brazil.

Arachidonic acid actions on functional integrity and attenuation of the negative effects of palmitic acid in a clonal pancreatic beta-cell line

Chronic exposure of pancreatic beta-cells to saturated non-esterified fatty acids can lead to inhibition of insulin secretion and apoptosis. Several previous studies have demonstrated that saturated fatty acids such as PA (palmitic acid) are detrimental to beta-cell function compared with unsaturated fatty acids. In the present study, we describe the effect of the polyunsaturated AA (arachidonic acid) on the function of the clonal pancreatic beta-cell line BRIN-BD11 and demonstrate AA-dependent attenuation of PA effects. When added to beta-cell incubations at 100 mu M, AA can stimulate cell proliferation and chronic (24 h) basal insulin secretion. Microarray analysis and/or real-time PCR indicated significant AA-dependent up-regulation of genes involved in proliferation and fatty acid metabolism [e.g. Angptl (angiopoietin-like protein 4), Ech1 (peroxisomal Delta(3.5),Delta(2.4)-dienoyl-CoA isomerase), Cox-1 (cyclo-oxygenase-1) and Cox-2, P < 0.05]. Experiments using specific COX and LOX (lipoxygenase) inhibitors demonstrated the importance of COX-1 activity for acute (20 min) stimulation of insulin secretion, suggesting that AA metabolites may be responsible for the insulinotropic effects. Moreover, concomitant incubation of AA with PA dose-dependently attenuated the detrimental effects of the saturated fatty acid, so reducing apoptosis and decreasing parameters of oxidative stress [ROS (reactive oxygen species) and NO levels] while improving the GSH/GSSG ratio. AA decreased the protein expression of iNOS (inducible NO synthase), the p65 subunit of NF-kappa B (nuclear factor kappa B) and the p47 subunit of NADPH oxidase in PA-treated cells. These findings indicate that AA has an important regulatory and protective beta-cell action, which may be beneficial to function and survival in the `lipotoxic` environment commonly associated with Type 2 diabetes mellitus.

Differential gene expression analysis of iodide-treated rat thyroid follicular cell line PCCl3

The inhibitory effect of supraphysiological iodide concentrations on thyroid hormone synthesis (Wolff - Chaikoff effect) and on thyrocyte proliferation is largely known as iodine autoregulation. However, the molecular mechanisms by which iodide modulates thyroid function remain unclear. In this paper, we analyze the transcriptome profile of the rat follicular cell lineage PCCl3 under untreated and treated conditions with 10 (- 3) M sodium iodide (NaI). Serial analysis of gene expression (SAGE) revealed 84 transcripts differentially expressed in response to iodide (p <= 0.001). We also showed that iodide excess inhibits the expression of essential genes for thyroid differentiation: Tshr, Nis, Tg, and Tpo. Relative expression of 14 of 20 transcripts selected by SAGE was confirmed by real-time PCR. Considering the key role of iodide organification in thyroid physiology, we also observed that both the oxidized form of iodide and iodide per se are responsible for gene expression modulation in response to iodide excess. (c) 2008 Elsevier Inc. All rights reserved.

PL spectroscopy of Fermi electrons in regime of the quantum Hall effect

The influence of the interlayer coupling on formation of the quantized Hall phase at the filling factor v = 2 was studied in the multilayer GaAs/AlGaAs heterostructures The disorder broaden Gaussian photoluminescence line due to the localized electrons was found in the quantized Hall phase of the isolated multi-quantum well structure On the other hand. the quantized Hall phase of the weakly-coupled multilayers emitted an asymmetrical line similar to that one observed in the metallic electron systems. We demonstrated that the observed asymmetry indicates a formation of the Fermi Surface in the quantized Hall phase of the multilayer electron system due to the interlayer peicolation. A sharp decrease of the single-particle scattering time associated with the extended states oil the Fermi surface was observed at the filling factor v = 2. (C) 2009 Elsevier B.V All rights reserved

Spectroscopic evidence of extended states in quantized Hall phase of weakly coupled GaAs/AlGaAs multi-layers

The influence of the interlayer coupling on formation of the quantized Hall conductor phase at the filling factor v = 2 was studied in the multi-layer GaAs/AlGaAs heterostructures. The disorder broadened Gaussian photoluminescence line due to the localized electrons was found in the quantized Hall phase of the isolated multi-quantum well structure. On the other hand, the quantized Hall phase of the weakly coupled multi-layers emitted an unexpected asymmetrical line similar to that one observed in the metallic electron systems. We demonstrated that the observed asymmetry is caused by a partial population of the extended electron states formed in the quantized Hall conductor phase due to the interlayer percolation. A sharp decrease of the single-particle scattering time associated with these extended states was observed at the filling factor v = 2. (c) 2007 Elsevier B.V. All rights reserved.

Quantitative Detection of Enterotoxigenic Bacteroides fragilis Subtypes Isolated from Children with and without Diarrhea

A rapid real-time PCR ( RT-PCR) approach was developed to detect the bft gene subtypes in Bacteroides fragilis isolated from fecal samples. DNA obtained from diarrhea (110) and nondiarrhea (150) samples was evaluated. Subtype 1 was observed in 9 (8.2%) diarrhea and 7 (4.7%) nondiarrhea samples. Subtype 2 was not detected in any DNA samples, and subtype 3 was observed in only 1 diarrhea sample. The presence of the bft-1 gene did not show any statistically significant differences between the groups of children. This technique could be used to evaluate a possible correlation between disease and the presence of B. fragilis enterotoxin.

Quantitative detection of periodontopathic bacteria in atherosclerotic plaques from coronary arteries

Oral pathogens, including periodontopathic bacteria, are thought to be aetiological factors in the development of cardiovascular disease. In this study, the presence of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum-periodonticum-simiae group, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Tannerella forsythia in atheromatous plaques from coronary arteries was determined by real-time PCR. Forty-four patients displaying cardiovascular disease were submitted to periodontal examination and endarterectomy of coronary arteries. Approximately 60-100 mg atherosclerotic tissue was removed surgically and DNA was obtained. Quantitative detection of periodontopathic bacteria was performed using universal and species-specific TaqMan probe/primer sets. Total bacterial and periodontopathic bacterial DNA were found in 94.9 and 92.3 %, respectively, of the atheromatous plaques from periodontitis patients, and in 80.0 and 20.0%, respectively, of atherosclerotic tissues from periodontally healthy subjects. All periodontal bacteria except for the F. nucleatum-periodonticum-simiae group were detected, and their DNA represented 47.3 % of the total bacterial DNA obtained from periodontitis; patients. Porphyromonas gingivalis, A. actinomycetemcomitans and Prevotella intermedia were detected most often. The presence of two or more periodontal species could be observed in 64.1 % of the samples. In addition, even in samples in which a single periodontal species was detected, additional unidentified microbial DNA could be observed. The significant number of periodontopathic bacterial DNA species in atherosclerotic tissue samples from patients with periodontitis suggests that the presence of these micro-organisms in coronary lesions is not coincidental and that they may in fact contribute to the development of vascular diseases.

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis

Aim: To develop a TaqMan probe-based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. Methods and Results: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay`s specificity, detection limit, intra- and inter-assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100-fold more sensitive than the cPCR. No cross-reactivity with nontarget pig mycoplasmas was observed. An average of 1.62 x 10(11) and 2.75 x 10(8) target copies ml(-1) of blood were detected in the acutely and chronically infected pigs, respectively. Three (7.5%) pigs and 32 (80.0%) sows were positive while all peccaries were negative for Myc. suis. Conclusion: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. Significance and Impact of the Study: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.

A non-parametric vessel detection method for complex vascular structures

Modern medical imaging techniques enable the acquisition of in vivo high resolution images of the vascular system. Most common methods for the detection of vessels in these images, such as multiscale Hessian-based operators and matched filters, rely on the assumption that at each voxel there is a single cylinder. Such an assumption is clearly violated at the multitude of branching points that are easily observed in all, but the Most focused vascular image studies. In this paper, we propose a novel method for detecting vessels in medical images that relaxes this single cylinder assumption. We directly exploit local neighborhood intensities and extract characteristics of the local intensity profile (in a spherical polar coordinate system) which we term as the polar neighborhood intensity profile. We present a new method to capture the common properties shared by polar neighborhood intensity profiles for all the types of vascular points belonging to the vascular system. The new method enables us to detect vessels even near complex extreme points, including branching points. Our method demonstrates improved performance over standard methods on both 2D synthetic images and 3D animal and clinical vascular images, particularly close to vessel branching regions. (C) 2008 Elsevier B.V. All rights reserved.