Specific and Sensitive Detection of H. pylori in Biological Specimens by Real-Time RT-PCR and In Situ Hybridization

PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens

Fluctuation-Driven Neural Dynamics Reproduce Drosophila Locomotor Patterns.

The neural mechanisms determining the timing of even simple actions, such as when to walk or rest, are largely mysterious. One intriguing, but untested, hypothesis posits a role for ongoing activity fluctuations in neurons of central action selection circuits that drive animal behavior from moment to moment. To examine how fluctuating activity can contribute to action timing, we paired high-resolution measurements of freely walking Drosophila melanogaster with data-driven neural network modeling and dynamical systems analysis. We generated fluctuation-driven network models whose outputs-locomotor bouts-matched those measured from sensory-deprived Drosophila. From these models, we identified those that could also reproduce a second, unrelated dataset: the complex time-course of odor-evoked walking for genetically diverse Drosophila strains. Dynamical models that best reproduced both Drosophila basal and odor-evoked locomotor patterns exhibited specific characteristics. First, ongoing fluctuations were required. In a stochastic resonance-like manner, these fluctuations allowed neural activity to escape stable equilibria and to exceed a threshold for locomotion. Second, odor-induced shifts of equilibria in these models caused a depression in locomotor frequency following olfactory stimulation. Our models predict that activity fluctuations in action selection circuits cause behavioral output to more closely match sensory drive and may therefore enhance navigation in complex sensory environments. Together these data reveal how simple neural dynamics, when coupled with activity fluctuations, can give rise to complex patterns of animal behavior

Age of the Association between Helicobacter pylori and Man

When modern humans left Africa ca. 60,000 years ago (60 kya), they were already infected with Helicobacter pylori, and these bacteria have subsequently diversified in parallel with their human hosts. But how long were humans infected by H. pylori prior to the out-of-Africa event? Did this co-evolution predate the emergence of modern humans, spanning the species divide? To answer these questions, we investigated the diversity of H. pylori in Africa, where both humans and H. pylori originated. Three distinct H. pylori populations are native to Africa: hpNEAfrica in Afro-Asiatic and Nilo-Saharan speakers, hpAfrica1 in Niger-Congo speakers and hpAfrica2 in South Africa. Rather than representing a sustained co-evolution over millions of years, we find that the coalescent for all H. pylori plus its closest relative H. acinonychis dates to 88–116 kya. At that time the phylogeny split into two primary super-lineages, one of which is associated with the former hunter-gatherers in southern Africa known as the San. H. acinonychis, which infects large felines, resulted from a later host jump from the San, 43–56 kya. These dating estimates, together with striking phylogenetic and quantitative human-bacterial similarities show that H. pylori is approximately as old as are anatomically modern humans. They also suggest that H. pylori may have been acquired via a single host jump from an unknown, non-human host. We also find evidence for a second Out of Africa migration in the last 52,000 years, because hpEurope is a hybrid population between hpAsia2 and hpNEAfrica, the latter of which arose in northeast Africa 36–52 kya, after the Out of Africa migrations around 60 kya

Natriuretic peptides increase cAMP production in human thyrocytes via the natriuretic peptide clearance receptor (NPR-C)

The relationship between natriuretic peptides and adenylyl cyclase/cAMP signal transduction has generally been shown to be an inhibitory one, mediated via the NPR-C receptor coupled to adenylyl cyclase by inhibitory G proteins (Gi). In the present studies, we have investigated the modulation of cAMP by natriuretic peptides in a long-term culture of human thyroid cells. Competition of [I-125] rat ANF binding to human thyrocytes (HTU-5) by rat ANF (99-126) and by the NPR-C-specific analog C-ANF (4-23) indicated that greater than 97% of the ANF binding sites on HTU-5 cells are of the NPR-C type. However, rather than inhibiting intracellular cAMP in these cells, ANF increased maximal cAMP to 200-300% of control value. The ANF-induced increase in cAMP was duplicated by C-ANF (4-23). Basal cAMP content was reduced, and the response to ANF was abolished when the cells were grown in low (0.5%) serum without the addition of pituitary and hypothalamic extracts. CNP-22 also increased cAMP above control in HTU-5 cells identically to ANF. Neither ANF nor C-ANF (4-23) had any effect on cAMP in a culture of rat aortic smooth muscle cells. These results provide the first evidence for a positive effect of natriuretic peptides on cAMP mediated through the NPR-C, suggesting the possibility of an alternative mode of signaling by this receptor subtype. (C) 2001 Published by Elsevier Science B.V

Atrial natriuretic factor and C-type natriuretic peptide induce retraction of human thyrocytes in monolayer culture via guanylyl cyclase receptors

The natriuretic peptides signal through three receptor subtypes, of which two (NPR-A and NPR-B) are membrane-bound guanylyl cyclases for which the principal ligands are respectively atrial natriuretic factor (ANF) and C-type natriuretic peptide (CNP). In the human thyroid cell, a third receptor, NPR-C, has been implicated in the regulation of thyroglobulin, but functional roles for NPR-A and NPR-B have not yet been defined. In the present study we used RT-PCR to identify transcripts of all three receptor subtypes, both in human thyroid and in HTU-5 cells, a long-term culture of thyroid-derived cells. Both ANF and CNP induced a twofold increase in intracellular cGMP content in HTU-5 cells. Morphologic changes (a significant increase in cells of the retracted phenotype) were observed in ANF- and CNP-treated cells within 3 and 5 h of treatment respectively. Significant increases in retracted cell number were induced by ANF and CNP, but not the NPR-C-specific ring-deleted ANF analog, C-ANF(4-23), during a 15-day treatment. All three natriuretic peptides, however, induced a small (15-20%) but significant (P<0.001) increase in DNA content per well. The stable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), also increased the number of retracted HTU-5 cells, and was equipotent with the cAMP analog, 8-BrcAMP, in this effect. The cGMP-dependent protein kinase inhibitor, KT5823, however, had no significant effect on the ANF-induced increase in numbers of retracted cells. These results suggest that the actions of NPR-A and NPR-B, functional receptors in the human thyroid cell, may in part be mediated by cGMP-induced alterations in the cytoskeleton

Intracellular and interstitial expression of Helicobacter pylori virulence genes in gastric precancerous intestinal metaplasia and adenocarcinoma

Gastric intestinal metaplasia (IM) and gastric cancer are associated with Helicobacter pylori, but the bacterium often is undetectable in these lesions. To unravel this apparent paradox, IM, H. pylori presence, and the expression of H. pylori virulence genes were quantified concurrently using histologic testing, in situ hybridization, and immunohistochemistry. H. pylori was detected inside metaplastic, dysplastic, and neoplastic epithelial cells, and cagA and babA2 expression was colocalized. Importantly, expression of cagA was significantly higher in patients with IM and adenocarcinoma than in control subjects. The preneoplastic "acidic" MUC2 mucin was detected only in the presence of H. pylori, and MUC2 expression was higher in patients with IM, dysplasia, and cancer. These novel findings are compatible with the hypothesis that all stages of gastric carcinogenesis are fostered by persistent intracellular expression of H. pylori virulence genes, especially cagA inside MUC2-producing precancerous gastric cells and pleomorphic cancer cells

Association of the PPP3CA c.249G&gt;A variant with clinical outcomes of tacrolimus-based therapy in kidney transplant recipients

Patricia C Salgado,1 Fabiana DV Genvigir,1 Claudia R Felipe,2 Helio Tedesco-Silva Jr,2 Jose O Medina-Pestana,2 Sonia Q Doi,3 Mario H Hirata,1 Rosario DC Hirata1 1Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of Sao Paulo, 2Division of Nephrology, Hospital do Rim, Federal University of Sao Paulo, Sao Paulo, Brazil; 3School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, USA Background: The effects of genetic variants related to the pharmacodynamic mechanisms of immunosuppressive drugs on their therapeutic efficacy and safety have been poorly explored. This study was performed to investigate the influence of the PPP3CA c.249G&gt;A variant on the clinical outcomes of kidney transplant recipients. Patients and methods: A total of 148 Brazilian patients received tacrolimus (TAC)-based immunosuppressive therapy for 90 days post-kidney transplantation. The PPP3CA rs3730251 (c.249G&gt;A) polymorphism was determined by real-time polymerase chain reaction. Single-nucleotide polymorphism (SNP) data for CYP3A5 rs776746 (CYP3A5*3C; g.6986A&gt;G) were used to eliminate the confounding effects of this variant. Results: The PPP3CA c.249G&gt;A SNP did not influence early TAC exposure, renal function, or other laboratory parameters, including levels of urea, creatinine, glucose, and lipids, and blood counts. This variant also did not account for the cumulative incidence of biopsy-confirmed acute rejection or delayed graft function. Regarding adverse events, PPP3CA c.249A allele carriers initially had a 3.05-fold increased probability of treatment-induced blood and lymphatic system disorders compared with c.249GG genotype individuals (95% confidence interval: 1.10&ndash;8.48, p=0.032). However, this result was not maintained after adjusting for body weight and CYP3A5*3C SNP status (p=0.086). Conclusion: The PPP3CA c.249G&gt;A variant does not influence the clinical outcomes of Brazilian patients in the early phase of TAC-based immunosuppressive regimen. Keywords: kidney transplantation, polymorphism, PPP3CA, tacrolimus, adverse events, Brazilian patient