Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA.     

Epidermal growth factor receptor-dependent control of keratinocyte survival and Bcl-xL expression through a MEK-dependent pathway

Previous work has shown that the epidermal growth factor receptor (EGFR) tyrosine kinase moiety provides protection to normal human keratinocytes against apoptosis. This protection is, at least in part, due to EGFR-dependent expression of the antiapoptotic Bcl-2 family member, Bcl-x(L). Here we focused on intracellular signaling pathways relevant to keratinocyte survival and/or Bcl-x(L) expression. By using pharmacological inhibitors and dominant negative expression constructs, we observed that phosphatidylinositol 3-kinase/AKT and phospholipase C gamma/protein kinase C alpha activation were required for keratinocyte survival independently of EGFR activation or Bcl-x(L) expression. By contrast, MEK activity required EGFR activation and, as shown by use of the MEK inhibitor PD98059 and a dominant negative MEK construct, was necessary for Bcl-x(L) expression and survival. Consistent with an earlier study, blocking SRC kinase activities similarly led to down-regulation of Bcl-x(L) protein expression and impaired keratinocyte survival. In conclusion, our results demonstrate that EGFR-dependent MEK activity contributes to both Bcl-x(L) expression and survival of normal keratinocytes. Other signaling pathways (i.e. phosphatidylinositol 3-kinase/AKT and phospholipase C gamma/protein kinase C alpha) are obligatory to keratinocyte survival but not to Bcl-x(L) expression, and control of these pathways by EGFR activation is not rate-limiting to normal keratinocyte survival.     

Hypnotic accumulation and hangover in elderly inpatients: a controlled double-blind study of temazepam and nitrazepam.

The hypnotic and residual sedative effects of the first and seventh of seven regular night-time doses of nitrazepam 5 mg, temazepam 20 mg, and placebo were studied in 58 elderly inpatients. Plasma temazepam and nitrazepam concentrations rose by about 50% and 113% respectively between the mornings of day 1 and day 7. Patients reported sleeping well more often after the first dose of either hypnotic (p less than 0.05), but there was no difference after the seventh dose. Reaction time was unchanged on the morning after the first dose but was significantly prolonged after the seventh dose of both hypnotics (p less than 0.01). The time taken to eliminate the letter E from a page of prose tended to be prolonged after the first dose of both drugs (temazepam v placebo, p less than 0.05; nitrazepam v placebo, not significant) and was further prolonged on the morning after the seventh dose of nitrazepam (nitrazepam v placebo, p less than 0.05). Thus plasma accumulation of the drug was associated with a deterioration in daytime performance. This change in performance did not correlate with age, cerebral blood flow, or plasma concentration, but patients of low intelligence tended to be more severely affected.     

Accommodating dynamic oceanographic processes and pelagic biodiversity in marine conservation planning

Pelagic ecosystems support a significant and vital component of the ocean's productivity and biodiversity. They are also heavily exploited and, as a result, are the focus of numerous spatial planning initiatives. Over the past decade, there has been increasing enthusiasm for protected areas as a tool for pelagic conservation, however, few have been implemented. Here we demonstrate an approach to plan protected areas that address the physical and biological dynamics typical of the pelagic realm. Specifically, we provide an example of an approach to planning protected areas that integrates pelagic and benthic conservation in the southern Benguela and Agulhas Bank ecosystems off South Africa. Our aim was to represent species of importance to fisheries and species of conservation concern within protected areas. In addition to representation, we ensured that protected areas were designed to consider pelagic dynamics, characterized from time-series data on key oceanographic processes, together with data on the abundance of small pelagic fishes. We found that, to have the highest likelihood of reaching conservation targets, protected area selection should be based on time-specific data rather than data averaged across time. More generally, we argue that innovative methods are needed to conserve ephemeral and dynamic pelagic biodiversity.     

Genome sequence of strain HIMB30, a novel member of the marine Gammaproteobacteria

Strain HIMB30 was isolated from coastal Hawaii seawater by extinction culturing in seawater-based oligotrophic medium. It is a phylogenetically unique member of the class Gammaproteobacteria that is only distantly related to its closest cultured relatives. Here we present the genome sequence of strain HIMB30, including genes for proteorhodopsin-based phototrophy and the Calvin-Benson-Bassham cycle.     

Dexamethasone prevents the growth inhibitory effects of recombinant tumor necrosis factor in a rat hepatoma cell line Reuber-RC-3: an association with the changes in the messenger RNA levels for multidrug resistance gene.

Two days exposure to recombinant tumor necrosis factor (rTNF-alpha) produced a dose-dependent reduction in (methyl-3H) thymidine incorporation in RC-3 cells (ID50 = 25 units/ml). Prolonged treatment with rTNF-alpha further resulted in a significant reduction in colony formation (ID50 = 200 units/ml), which was reversed upon removal of the agent. Interferon levels were undetectable in the supernatants of the rTNF-alpha treated cells. Simultaneous exposure to dexamethasone prevented the growth inhibition in rTNF-alpha-treated RC-3 cells. Significant dose-dependent increase in the steady state levels of the mRNA for multidrug resistance (MDR1) gene was observed after rTNF-alpha treatment while simultaneous exposure to dexamethasone produced a substantial reduction in the mRNA levels for MDR1 gene. These data suggest that growth inhibitory effects of TNF are regulated by dexamethasone and are associated with changes in MDR1 mRNA levels in hepatoma-derived cells.     

Suitability of the biomass crop Miscanthus sinensis as a host for the aphids Rhopalosiphum padi (L.) and Rhopalosiphum maidis (F.), and its susceptibility to the plant luteovirus Barley Yellow Dwarf Virus

1 The reproductive performance of two aphid pest species, Rhopalosiphum padi and Rhopalosiphum maidis, was investigated on two seedling growth stages of Miscanthus sinensis, rhizomatous M. sinensis‘Giganteus’ and barley. Rhopalosiphum padi was unable to complete its development on Miscanthus. Rhopalosiphum maidis was most fecund on rhizomatous plants compared with seedling stages. 2 The ability of R. maidis to transmit the RPV isolate of Barley Yellow Dwarf Virus (BYDV) to M. sinensis seedlings was further investigated. Following successful transmission, host plant symptomology and the effect of infection on the yield of Miscanthus were investigated. Total above soil biomass was reduced by around 23% following infection. 3 The inability of R. padi to utilize Miscanthus is reviewed in light of this species’ origin and inability to utilize C₄ host plants. 4 The potential pest status of R. maidis on Miscanthus is discussed together with the impact that Miscanthus cultivation could have on the ecology of this aphid species and BYDV in the U.K.