Evaluation of quantification methods for real-time PCR minor groove binding hybridization probe assays

Real-time PCR data analysis for quantification has been the subject of many studies aimed at the identification of new and improved quantification methods. Several analysis methods have been proposed as superior alternatives to the common variations of the threshold crossing method. Notably, sigmoidal and exponential curve fit methods have been proposed. However, these studies have primarily analyzed real-time PCR with intercalating dyes such as SYBR Green. Clinical real-time PCR assays, in contrast, often employ fluorescent probes whose real-time amplification fluorescence curves differ from those of intercalating dyes. In the current study, we compared four analysis methods related to recent literature: two versions of the threshold crossing method, a second derivative maximum method, and a sigmoidal curve fit method. These methods were applied to a clinically relevant real-time human herpes virus type 6 (HHV6) PCR assay that used a minor groove binding (MGB) Eclipse hybridization probe as well as an Epstein-Barr virus (EBV) PCR assay that used an MGB Pleiades hybridization probe. We found that the crossing threshold method yielded more precise results when analyzing the HHV6 assay, which was characterized by lower signal/noise and less developed amplification curve plateaus. In contrast, the EBV assay, characterized by greater signal/noise and amplification curves with plateau regions similar to those observed with intercalating dyes, gave results with statistically similar precision by all four analysis methods.     

The JNK signal transduction pathway

The c-Jun NH(2)-terminal kinases (JNKs) are an evolutionarily conserved sub-group of mitogen-activated protein (MAP) kinases. Recent studies have improved our understanding of the physiological function of the JNK pathway. Roles of novel molecules that participate in the JNK pathway have been defined and new insight into the role of JNK in survival signaling, cell death, cancer and diabetes has been achieved.     

Translational regulation of neuronal electrical properties

The nervous system has an in-built capability to adjust its responsiveness to excitation according to the history of electrical activity faced by the neurons embedded within its networks. This control over excitability represents a form of homeostasis and is exhibited at multiple stages in the flow of information from the genome to the expression and modification of protein products. Information on the nature of the homeostatic phenomenon at some of these stages is still limited and emerging. This article outlines the various stages at which such neuronal intrinsic plasticity has been observed and draws particular attention to the role of the translation repressor protein, Pumilio, as an important factor in the process. The study of this protein is providing insights into the regulation of neuronal excitability and offers an important research target with benefits to investigators in many areas of neuroscience.     

Cover,not caging,influences chronic physiological stress in a ground‐nesting bird

Predator exclosures (‘nest cages’) around nests are increasingly used to enhance hatching success of declining ground‐nesting birds. However, such exclosures are contentious and have been suggested to have detrimental effects on the species which they aim to protect. This study examines whether exclosures increase physiological stress of incubating birds, a hitherto unrecognised and untested potential drawback of exclosures. Red‐capped plover Charadrius ruficapillus hatching success was radically altered and significantly higher for nests with exclosures (96.2%) compared with those without (6.8%). Chronic physiological stress in parents (as measured by the heterophil/lymphocyte [H/L] ratio in blood) did not vary between nests with and without exclosures, or between the sexes. However the absence of vegetative cover at the nest site was associated with a 62.7% elevation in H/L ratio, indicating that incubating birds which place their nests in the open are subject to increased levels of chronic stress. The results from this study demonstrate the fundamental importance of predation for the nesting success of this species and confirm that chronic stress levels are not a detrimental side effect of exclosure use.     

Co-variation in frequency and severity of cardiovascular and skeletal defects in Sprague-Dawley rats after maternal administration of dimethadione, the N-demethylated metabolite of trimethadione

BACKGROUND: The anticonvulsant trimethadione is a potent inducer of ventricular septation defects, both clinically and in rodents. Teratogenicity requires its N‐demethylation to dimethadione, the proximate teratogen. It was previously demonstrated trimethadione only induced membranous ventricular septation defects in rat (Fleeman et al., 2004 ), and our present goal is to determine whether direct administration of dimethadione increases the incidence and severity of septation defects. METHODS: Pregnant Sprague‐Dawley rats were divided into five groups and administered either distilled water (control) or four different regimens of dimethadione. The core treatment was 300 mg/kg dimethadione b.i.d. on gestation day 9, 10 with additional groups given one additional dose of dimethadione 12 hr earlier, 12 hr later or two additional doses 12 hr earlier and later. Caesarian sections occurred on gestation day 21 and fetuses were examined for standard developmental toxicity endpoints. RESULTS: The broadest dosing regimen yielded the highest incidence and the most severe heart and axioskeletal findings with a decrease in mean fetal body weight. The overall incidence of ventricular septation defects was 74%, of which 68% were membranous and 9% muscular. Outflow tract anomalies (17%) were also observed, as were malformations of the axioskeleton (97%), but not of the long bones, and of particular interest was the high incidence of sternoschesis. CONCLUSIONS: Unlike trimethadione, dimethadione induces more serious muscular septation defects that are believed to be more clinically relevant. This, when taken together with the high incidence of total septation anomalies suggests dimethadione is useful for the study of chemically induced ventricular septation defects. Birth Defects Res (Part B) 92:206–215, 2011. © 2011 Wiley‐Liss, Inc.     

The interchangeability of global positioning system and semiautomated video-based performance data during elite soccer match play

In elite-level soccer, player motion characteristics are commonly generated from match play and training situations using semiautomated video analysis systems and global positioning system (GPS) technology, respectively. Before such data are used collectively to quantify global player load, it is necessary to understand both the level of agreement and direction of bias between the systems so that specific interventions can be made based on the reported results. The aim of this report was to compare data derived from both systems for physical match performances. Six elite-level soccer players were analyzed during a competitive match using semiautomated video analysis (ProZone? [PZ]) and GPS (MinimaxX) simultaneously. Total distances (TDs), high speed running (HSR), very high speed running (VHSR), sprinting distance (SPR), and high-intensity running distance (HIR; >4.0 m·s(-1)) were reported in 15-minute match periods. The GPS reported higher values than PZ did for TD (GPS: 1,755.4 ± 245.4 m; PZ: 1,631.3 ± 239.5 m; p < 0.05); PZ reported higher values for SPR and HIR than GPS did (SPR: PZ, 34.1 ± 24.0 m; GPS: 20.3 ± 15.8 m; HIR: PZ, 368.1 ± 129.8 m; GPS: 317.0 ± 92.5 m; p < 0.05). Caution should be exercised when using match-load (PZ) and training-load (GPS) data interchangeably.     

UniCarb-DB: a database resource for glycomic discovery

Glycosylation is one of the most important post-translational modifications of proteins, known to be involved in pathogen recognition, innate immune response and protection of epithelial membranes. However, when compared to the tools and databases available for the processing of high-throughput proteomic data, the glycomic domain is severely lacking. While tools to assist the analysis of mass spectrometry (MS) and HPLC are continuously improving, there are few resources available to support liquid chromatography (LC)-MS/MS techniques for glycan structure profiling. Here, we present a platform for presenting oligosaccharide structures and fragment data characterized by LC-MS/MS strategies. The database is annotated with high-quality datasets and is designed to extend and reinforce those standards and ontologies developed by existing glycomics databases. AVAILABILITY: http://www.unicarb-db.org