BackgroundDuring the course of a bacterial infection, the rapid identification of the causative agent(s) is necessary for the determination of effective treatment options. We have developed a method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level. The broad-range PCR primer mixture was designed using conserved regions of the bacterial topoisomerase genes gyrB and parE. The primer design allowed the use of a novel DNA amplification method, which produced labeled, single-stranded DNA suitable for microarray hybridization. The probes on the microarray were designed from the alignments of species- or genus-specific variable regions of the gyrB and parE genes flanked by the primers. We included mecA-specific primers and probes in the same assay to indicate the presence of methicillin resistance in the bacterial species. The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples.ResultsComparison of our results with those of a conventional culture-based method revealed a sensitivity of 96% (initial sensitivity of 82%) and specificity of 98%. Furthermore, only one cross-reaction was observed upon investigating 102 culture isolates from 70 untargeted bacteria. The total assay time was only three hours, including the time required for the DNA extraction, PCR and microarray steps in sequence.ConclusionThe assay rapidly provides reliable data, which can guide optimal antimicrobial treatment decisions in a timely manner.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. An unstable expansion of a dodecamer repeat in the CSTB promoter accounts for the majority of EPM1 disease alleles worldwide. We here describe a novel PCR protocol for detection of the dodecamer repeat expansion. We describe two novel EPM1-associated mutations, c.149G4A leading to the p.G50E missense change and an intronic 18-bp deletion (c.168 þ 1_18del), which affects splicing of CSTB. The p.G50E mutation that affects the conserved QVVAG amino acid sequence critical for cathepsin binding fails to associate with lysosomes. This further supports the previously implicated physiological importance of the CSTB-lysosome association. Expression of CSTB mRNA and protein was markedly reduced in lymphoblastoid cells of the patients irrespective of the mutation type. Patients homozygous for the dodecamer expansion mutation showed 5-10% expression compared to controls. By combining database searches with RT-PCR we identified several alternatively spliced CSTB isoforms. One of these, CSTB2, was also present in mouse and was analyzed in more detail. In real-time PCR quantification, CSTB2 expression was less than 5% of total CSTB expression in all human adult and fetal tissues analyzed. In patients homozygous for the minisatellite mutation, the level of CSTB2 was reduced similarly to that of CSTB implicating regulation from the same promoter. The physiological significance of CSTB2 remains to be determined.

Benzodiazepines are in wide clinical use for their sedative and tranquilizing actions, the former being mediated via a1-containing GABA A receptors. The signal transduction pathways elicited beyond the receptor are only poorly understood. Changes of transcript levels in cerebral cortex induced by acute diazepam administration were therefore compared by microarray analysis between wild-type and point mutated a1(H101R) mice, in which the a1 GABA A receptor subunit had been rendered insensitive to diazepam. In wild-type animals, diazepam reduced the expression levels of the a subunit of the calcium/calmodulin-dependent protein kinase II, as well as brain-derived neurotrophic factor, MAP kinase phosphatase, transcription factor GIF, c-fos and nerve growth factor induced gene-A. None of these transcripts was changed in the a1(H101R) mice after treatment with diazepam. Thus, the sedative action of diazepam is correlated with a selective down-regulation of transcripts involved in the regulation of neuronal plasticity and neurotrophic responses. Most transcript changes were transient except for the decrease of the CaMKIIa transcript which persisted even 40 h after the single dose of diazepam. This long-term alteration is likely to contribute to the resetting of the neuronal responsiveness, which may be involved in rebound phenomena and, under chronic treatment, in the development of tolerance and dependence.

Phosphomannose isomerase (PMI40) catalyzes the conversion between fructose 6-phosphate and mannose 6-phosphate and thus connects glycolysis, i.e. energy production and GDP-mannose biosynthesis or cell wall synthesis in Saccharomyces cerevisiae. After PMI40 deletion (pmi ؊ ) the cells were viable only if fed with extracellular mannose and glucose. In an attempt to force the GDP-mannose synthesis in the pmi ؊ strain by increasing the extracellular mannose concentrations, the cells showed significantly reduced growth rates without any alterations in the intracellular GDPmannose levels. To reveal the mechanisms resulting in reduced growth rates, we measured genome-wide gene expression levels, several metabolite concentrations, and selected in vitro enzyme activities in central metabolic pathways. The increasing of the initial mannose concentration led to an increase in the mannose 6-phosphate concentration, which inhibited the activity of the second enzyme in glycolysis, i.e. phosphoglucose isomerase converting glucose 6-phosphate to fructose 6-phosphate. As a result of this limitation, the flux through glycolysis was decreased as was the median expression of the genes involved in glycolysis. The expression levels of RAP1, a transcription factor involved in the regulation of the mRNA levels of several enzymes in glycolysis, as well as those of cell cycle regulators CDC28 and CLN3, decreased concomitantly with the growth rates and expression of many genes encoding for enzymes in glycolysis.Phosphomannose isomerase enzyme (PMIe) 1 catalyzes the interconversion of fructose 6-phosphate (Fru-6-P) in glycolysis to mannose 6-phosphate (Man-6-P) through a mannose pathway. In the eukaryotic model organism, yeast Saccharomyces cerevisiae, phosphomannose isomerase is encoded by the PMI40 gene (1). In a PMI40 deletion strain (pmi Ϫ ), the synthesis of Man-6-P from Fru-6-P is not possible, disabling the growth of such a strain on medium without mannose. The inability to grow, caused by defective glycosylation of a temperature-sensitive pmi40 mutant of S. cerevisiae, and repairing the defects by addition of mannose to the growth medium have been described previously (2). In humans PMIe deficiency is the cause of carbohydrate-deficient glycoprotein syndrome type Ib, but the condition can be successfully treated by mannose administration (3). Man-6-P produced either from Fru-6-P or mannose serves as a precursor for the de novo biosynthesis of GDP-mannose. Man-6-P is converted to mannose 1-phosphate (Man-1-P) by phosphomannomutase encoded by SEC53. Subsequently, Man-1-P is ligated with the guanosine 5-triphosphate molecule (GTP) to form GDP-mannose by Man-1-P guanylyltransferase encoded by PSA1 (4). The de novo formation of the purine ring of GTP, required for the biosynthesis of GDPmannose, starts from ribose 5-phosphate in the pentose phosphate pathway and requires also 3-phosphoglycerate in the glycolysis as a precursor. Taken together, the biosynthesis of GTP is more complex than the mannose pathway (4). GDPmannose is needed in ...