Diagnostic application of padlock probes--multiplex detection of plant pathogens using universal microarrays

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology.     

Different modulation by live or killed Helicobacter pylori on cytokine production from peripheral blood mononuclear cells

The mechanisms by which H. pylori colonizes and persists within the gastric mucosa are poorly understood. The induction and maintenance of gastric inflammation appear to depend on the complex interaction between a number of cytokines and chemokines. The gastric immune response observed "in vivo", during H. pylori infection, is characterized by a polarization of Th1 cell type that seems to be responsible for gastric pathology. The purpose of this study was to test the direct effect of H. pylori (live or gentamicin-killed) on human PBMC in order to evaluate the "in vitro" Th1-Th2 balance by monitoring IL-18, IFNgamma and IL-10 production. This study demonstrates for the first time that "in vitro" pretreatment with gentamicin-killed H. pylori of PBMC, followed by infection with live bacteria, downregulates the production of inflammatory cytokines such as IL-18 and IFNgamma Our results provide a possible strategy to restore the immunological disorders determined by H. pylori infection.     

Expression of Caenorhabditis elegans mRNA in n Xenopus oocytes: a model system to study the mechanism of action of avermectins

It has recently been shown that Xenopus oocytes injected with mRNA from the free-living nematode Caenorhabditis elegans express avermectin-sensitive chloride channels. Joseph Arena here reviews what is known about the mechanism of action of avermectin and how these recent results relate to the mechanism in nematodes.     

Spiropiperidine CCR5 antagonists

A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.