Evaluation of a novel real-time PCR test based on the ssrA gene for the identification of group B streptococci in vaginal swabs

<p>Abstract</p> <p>Background</p> <p>Despite the implementation of prevention guidelines, early-onset group B streptococci (GBS) disease remains a cause of neonatal morbidity and mortality worldwide. Strategies to identify women who are at risk of transmitting GBS to their infant and the administration of intrapartum antibiotics have greatly reduced the incidence of neonatal GBS disease. However, there is a requirement for a rapid diagnostic test for GBS that can be carried out in a labour ward setting especially for women whose GBS colonisation status is unknown at the time of delivery. We report the design and evaluation of a real-time PCR test (<it>RiboSEQ </it>GBS test) for the identification of GBS in vaginal swabs from pregnant women.</p> <p>Methods</p> <p>The qualitative real-time PCR <it>RiboSEQ </it>GBS test was designed based on the bacterial <it>ssrA </it>gene and incorporates a competitive internal standard control. The analytical sensitivity of the test was established using crude lysate extracted from serial dilutions of overnight GBS culture using the IDI Lysis kit. Specificity studies were performed using DNA prepared from a panel of GBS strains, related streptococci and other species found in the genital tract environment. The <it>RiboSEQ </it>GBS test was evaluated on 159 vaginal swabs from pregnant women and compared with the GeneOhm™ StrepB Assay and culture for the identification of GBS.</p> <p>Results</p> <p>The <it>RiboSEQ </it>GBS test is specific and has an analytical sensitivity of 1-10 cell equivalents. The <it>RiboSEQ </it>GBS test was 96.4% sensitive and 95.8% specific compared to "gold standard" culture for the identification of GBS in vaginal swabs from pregnant women. In this study, the <it>RiboSEQ </it>GBS test performed slightly better than the commercial BD GeneOhm™ StrepB Assay which gave a sensitivity of 94.6% and a specificity of 89.6% compared to culture.</p> <p>Conclusion</p> <p>The <it>RiboSEQ </it>GBS test is a valuable method for the rapid, sensitive and specific detection of GBS in pregnant women. This study also validates the <it>ssrA </it>gene as a suitable and versatile target for nucleic acid-based diagnostic tests for bacterial pathogens.</p

Stereotaxical Infusion of Rotenone: A Reliable Rodent Model for Parkinson's Disease

A clinically-related animal model of Parkinson's disease (PD) may enable the elucidation of the etiology of the disease and assist the development of medications. However, none of the current neurotoxin-based models recapitulates the main clinical features of the disease or the pathological hallmarks, such as dopamine (DA) neuron specificity of degeneration and Lewy body formation, which limits the use of these models in PD research. To overcome these limitations, we developed a rat model by stereotaxically (ST) infusing small doses of the mitochondrial complex-I inhibitor, rotenone, into two brain sites: the right ventral tegmental area and the substantia nigra. Four weeks after ST rotenone administration, tyrosine hydroxylase (TH) immunoreactivity in the infusion side decreased by 43.7%, in contrast to a 75.8% decrease observed in rats treated systemically with rotenone (SYS). The rotenone infusion also reduced the DA content, the glutathione and superoxide dismutase activities, and induced alpha-synuclein expression, when compared to the contralateral side. This ST model displays neither peripheral toxicity or mortality and has a high success rate. This rotenone-based ST model thus recapitulates the slow and specific loss of DA neurons and better mimics the clinical features of idiopathic PD, representing a reliable and more clinically-related model for PD research

A Phenotypic Profile of the Candida albicans Regulatory Network

Candida albicans is a normal resident of the gastrointestinal tract and also the most prevalent fungal pathogen of humans. It last shared a common ancestor with the model yeast Saccharomyces cerevisiae over 300 million years ago. We describe a collection of 143 genetically matched strains of C. albicans, each of which has been deleted for a specific transcriptional regulator. This collection represents a large fraction of the non-essential transcription circuitry. A phenotypic profile for each mutant was developed using a screen of 55 growth conditions. The results identify the biological roles of many individual transcriptional regulators; for many, this work represents the first description of their functions. For example, a quarter of the strains showed altered colony formation, a phenotype reflecting transitions among yeast, pseudohyphal, and hyphal cell forms. These transitions, which have been closely linked to pathogenesis, have been extensively studied, yet our work nearly doubles the number of transcriptional regulators known to influence them. As a second example, nearly a quarter of the knockout strains affected sensitivity to commonly used antifungal drugs; although a few transcriptional regulators have previously been implicated in susceptibility to these drugs, our work indicates many additional mechanisms of sensitivity and resistance. Finally, our results inform how transcriptional networks evolve. Comparison with the existing S. cerevisiae data (supplemented by additional S. cerevisiae experiments reported here) allows the first systematic analysis of phenotypic conservation by orthologous transcriptional regulators over a large evolutionary distance. We find that, despite the many specific wiring changes documented between these species, the general phenotypes of orthologous transcriptional regulator knockouts are largely conserved. These observations support the idea that many wiring changes affect the detailed architecture of the circuit, but not its overall output

Gene Annotation and Drug Target Discovery in Candida albicans with a Tagged Transposon Mutant Collection

Candida albicans is the most common human fungal pathogen, causing infections that can be lethal in immunocompromised patients. Although Saccharomyces cerevisiae has been used as a model for C. albicans, it lacks C. albicans' diverse morphogenic forms and is primarily non-pathogenic. Comprehensive genetic analyses that have been instrumental for determining gene function in S. cerevisiae are hampered in C. albicans, due in part to limited resources to systematically assay phenotypes of loss-of-function alleles. Here, we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants, using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency. We identified 269 genes that were haploinsufficient in four growth conditions, the majority of which were condition-specific. These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes. We also screened 57 chemically diverse compounds that more potently inhibited growth of C. albicans versus S. cerevisiae. For four of these compounds, we examined the genetic basis of this differential inhibition. Notably, Sec7p was identified as the target of brefeldin A in C. albicans screens, while S. cerevisiae screens with this compound failed to identify this target. We also uncovered a new C. albicans-specific target, Tfp1p, for the synthetic compound 0136-0228. These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification

Brain Derived Neurotrophic Factor (BDNF) Expression Is Regulated by MicroRNAs miR-26a and miR-26b Allele-Specific Binding

Brain-derived neurotrophic factor (BDNF) is a neurotrophin that plays an essential role in neuronal development and plasticity. MicroRNA (miRNAs) are small non-coding RNAs of about 22-nucleotides in length regulating gene expression at post-transcriptional level. In this study we explore the role of miRNAs as post-transcriptional inhibitors of BDNF and the effect of 3′UTR sequence variations on miRNAs binding capacity. Using an in silico approach we identified a group of miRNAs putatively regulating BDNF expression and binding to BDNF 3′UTR polymorphic sequences. Luciferase assays demonstrated that these miRNAs (miR-26a1/2 and miR-26b) downregulates BDNF expression and that the presence of the variant alleles of two single nucleotide polymorphisms (rs11030100 and rs11030099) mapping in BDNF 3′UTR specifically abrogates miRNAs targeting. Furthermore we found a high linkage disequilibrium rate between rs11030100, rs11030099 and the non-synonymous coding variant rs6265 (Val66Met), which modulates BDNF mRNA localization and protein intracellular trafficking. Such observation led to hypothesize that miR-26s mediated regulation could extend to rs6265 leading to an allelic imbalance with potentially functional effects, such as peptide's localization and activity-dependent secretion. Since rs6265 has been previously implicated in various neuropsychiatric disorders, we evaluated the distribution of rs11030100, rs11030099 and rs6265 both in a control and schizophrenic group, but no significant difference in allele frequencies emerged. In conclusion, in the present study we identified two novel miRNAs regulating BDNF expression and the first BDNF 3′UTR functional variants altering miRNAs-BDNF binding

Paleotemperature Proxies from Leaf Fossils Reinterpreted in Light of Evolutionary History

Present-day correlations between leaf physiognomic traits (shape and size) and climate are widely used to estimate paleoclimate using fossil floras. For example, leaf-margin analysis estimates paleotemperature using the modern relation of mean annual temperature (MAT) and the site-proportion of untoothed-leaf species (NT). This uniformitarian approach should provide accurate paleoclimate reconstructions under the core assumption that leaf-trait variation principally results from adaptive environmental convergence, and because variation is thus largely independent of phylogeny it should be constant through geologic time. Although much research acknowledges and investigates possible pitfalls in paleoclimate estimation based on leaf physiognomy, the core assumption has never been explicitly tested in a phylogenetic comparative framework. Combining an extant dataset of 21 leaf traits and temperature with a phylogenetic hypothesis for 569 species-site pairs at 17 sites, we found varying amounts of non-random phylogenetic signal in all traits. Phylogenetic vs. standard regressions generally support prevailing ideas that leaf-traits are adaptively responding to temperature, but wider confidence intervals, and shifts in slope and intercept, indicate an overall reduced ability to predict climate precisely due to the non-random phylogenetic signal. Notably, the modern-day relation of proportion of untoothed taxa with mean annual temperature (NT-MAT), central in paleotemperature inference, was greatly modified and reduced, indicating that the modern correlation primarily results from biogeographic history. Importantly, some tooth traits, such as number of teeth, had similar or steeper slopes after taking phylogeny into account, suggesting that leaf teeth display a pattern of exaptive evolution in higher latitudes. This study shows that the assumption of convergence required for precise, quantitative temperature estimates using present-day leaf traits is not supported by empirical evidence, and thus we have very low confidence in previously published, numerical paleotemperature estimates. However, interpreting qualitative changes in paleotemperature remains warranted, given certain conditions such as stratigraphically closely-spaced samples with floristic continuity