An Investigation Into the Synergism Between Bovine Papillomavirus Type 4 and the Flavonoid Quercetin in the Transformation of Primary Bovine Palate Fibroblasts

Bovine papillomavirus type 4 (BPV 4) infects the upper alimentary canal of cattle causing benign papillomas which can progress to squamous carcinomas in cattle grazing on bracken fern (BF). A single treatment with quercetin, a well characterised and potent mutagen found in BF, can cause full oncogenic transformation of cells partially transformed by BPV-4. Quercetin elevates the activity of the BPV-4 enhancer/promoter element (LCR) by up to four fold but this cannot fully explain the observed effect as the timing of quercetin exposure is critical for full transformation of the cells. We show that quercetin exposure arrests normal PalF cells in the G1 phase of the cell cycle, and this G1 arrest correlates with an increase in p53 protein levels and transcriptional activity. Cells transformed by expression of either BPV 4 E7 and Ha-ras or the BPV4 genome and Ha-ras, fail to arrest in G1 after subsequent quercetin treatments. In these cells which are transformed but non-tumorigenic, p53 protein is elevated and transcriptionally activated in response to quercetin exposure. Yet the lack of cell cycle arrest is probably due to the viral protein E7 inhibiting p2Waf1/Cip1. In the transformed tumorigenic cells the failure to arrest in the G1 phase of the cell cycle is also evident. p53 protein is still present and even its stabilisation in response to quercetin can be observed in some cell lines, however p53 transcriptional activity is inhibited, probably as a result of p53 mutation. Additionally the protein which mediates p53 dependent cell cycle arrest, p2Waf1/Cip1 is also absent from all the tumorigenic cells lending further evidence to the loss of p53 as a transcriptional activator. Here we propose a model in which in normal cells quercetin induces G1 arrest, mediated by p53. Abrogation of this arrest by BPV-4 E7 allows the cell to proliferate allowing the accumulation of inheritable damage, including mutations of the p53 gene at later stages. The net effect of this is full tumorigenic transformation of the cell

Quercetin elevates p27Kip1 and arrests both primary and HPV16 E6/E7 transformed human keratinocytes in G1

Our previous work with primary bovine fibroblasts demonstrated that quercetin, a potent mutagen found in high levels in bracken fern (Pteridium aquilinum), arrested cells in G1 and G2/M, in correlation with p53 activation. The expression of bovine papillomavirus type 4 (BPV-4) E7 overcame this arrest and lead to the development of tumorigenic cells lines (Beniston et al., 2001). Given the possible link between papillomavirus infection, bracken fern in the diet and cancer of the upper gastrointestinal (GI) tract in humans, we investigated whether a similar situation would occur in human cells transformed by human papillomavirus type 16 (HPV-16) oncoproteins. Quercetin arrested primary human foreskin keratinocytes in G1. Arrest was linked to an elevation of the cyclin-dependent kinase inhibitor (cdki) p27Kip1. Expression of the HPV16 E6 and E7 oncoproteins in transformed cells failed to abrogate cell cycle arrest. G1 arrest in the transformed cells was also linked to an increase of p27Kip1 with a concomitant reduction of cyclin E-associated kinase activity. This elevation of p27Kip1 was due not only to increased protein half-life, but also to increased mRNA transcription

Innovation development – an action learning programme for medical scientists and engineers

© 2014, © 2014 Taylor & Francis. There is increasing evidence that action learning is valuable in a higher education setting. This paper goes on to report a personal development programme, based on principles of critical action learning, where the aim is to equip early-career scientists and engineers working in a university setting with the knowledge, skills and confidence to approach the management of innovation. After learning about action learning and critical reflection, the participants, all postdoctorate researchers, completed innovation projects at work, meeting in action learning sets as they proceed. We explain a method of critical thinking before reporting results from an evaluation study based on interviews and focus groups. We consider examples of projects undertaken before considering challenges for students with this approach to learning. Challenges included scepticism about the usefulness of management literature, difficulties in finding ‘problems’ within the constraints of postdoctoral work, and the discomfort and intensiveness of action learning. However, through adaptation by the tutors with students, some significant results were achieved

Metabolic activity in dormant conidia ofAspergillus nigerand developmental changes during conidial outgrowth

The early stages of development of Aspergillus niger conidia during outgrowth were explored by combining genome-wide gene expression analysis (RNAseq), proteomics, Warburg manometry and uptake studies. Resting conidia suspended in water were demonstrated for the first time to be metabolically active as low levels of oxygen uptake and the generation of carbon dioxide were detected, suggesting that low-level respiratory metabolism occurs in conidia for maintenance. Upon triggering of spore germination, generation of CO2 increased dramatically. For a short period, which coincided with mobilisation of the intracellular polyol, trehalose, there was no increase in uptake of O2 indicating that trehalose was metabolised by fermentation. Data from genome-wide mRNA profiling showed the presence of transcripts associated with fermentative and respiratory metabolism in resting conidia. Following triggering of conidial outgrowth, there was a clear switch to respiration after 25 min, confirmed by cyanide inhibition. No effect of SHAM, salicylhydroxamic acid, on respiration suggests electron flow via cytochrome c oxidase. Glucose entry into spores was not detectable before 1 h after triggering germination. The impact of sorbic acid on germination was examined and we showed that it inhibits glucose uptake. O2 uptake was also inhibited, delaying the onset of respiration and extending the period of fermentation. In conclusion, we show that conidia suspended in water are not completely dormant and that conidial outgrowth involves fermentative metabolism that precedes respiration

Hybrid Mass Spectrometry Methods Reveal Lot-to-Lot Differences and Delineate the Effects of Glycosylation on the Structure of Herceptin®

To consider the measurable variations in biopharmaceuticals we use mass spectrometry and systematically evaluate three lots of Herceptin®, two mAb standards and an intact Fc-hinge fragment. Each mAb is examined in three states; glycan intact, truncated (following endoS2 treatment) and fully deglycosylated. Despite equivalence at the protein level, each lot of Herceptin® gives a distinctive signature in three different mass spectrometry analyses. Ion mobility mass spectrometry (IM-MS) shows that in the API, the attached N-glycans reduce the conformational spread of each mAb by 10.5 – 25 %. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) data supports this, with lower global deuterium uptake in solution when comparing intact to the fully deglycosylated protein. HDX-MS and activated IM-MS map the influence of glycans on the mAb and reveal allosteric effects which extend far beyond the Fc domains into the Fab region. Taken together these findings, and the supplied interactive data sets could be used to provide acceptance criteria with application for MS based characterisation of biosimilars and novel therapeutic mAbs. </p

Hybrid Mass Spectrometry Methods Reveal Lot-to-Lot Differences and Delineate the Effects of Glycosylation on the Structure of Herceptin®

<p>To consider the measurable variations in biopharmaceuticals we use mass spectrometry and systematically evaluate three lots of Herceptin®, two mAb standards and an intact Fc-hinge fragment. Each mAb is examined in three states; glycan intact, truncated (following endoS2 treatment) and fully deglycosylated. Despite equivalence at the protein level, each lot of Herceptin® gives a distinctive signature in three different mass spectrometry analyses. Ion mobility mass spectrometry (IM-MS) shows that in the API, the attached N-glycans reduce the conformational spread of each mAb by 10.5 – 25 %. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) data supports this, with lower global deuterium uptake in solution when comparing intact to the fully deglycosylated protein. HDX-MS and activated IM-MS map the influence of glycans on the mAb and reveal allosteric effects which extend far beyond the Fc domains into the Fab region. Taken together these findings, and the supplied interactive data sets could be used to provide acceptance criteria with application for MS based characterisation of biosimilars and novel therapeutic mAbs. </p

Morphine delays the onset of action of prasugrel in patients with prior history of ST-elevation myocardial infarction

SummaryDelays in the onset of action of prasugrel during primary percutaneous coronary intervention (PPCI) have been reported and could be related to the effects of morphine on gastric emptying and subsequent intestinal absorption. The study objective was to determine whether morphine delays the onset of action of prasugrel in patients with a prior history of ST-elevation myocardial infarction (STEMI) treated with PPCI. This was a crossover study of 11 aspirin-treated patients with prior history of STEMI treated with PPCI, for which prasugrel and morphine had been previously administered. Patients were randomised to receive either morphine (5 mg) or saline intravenously followed by 60 mg prasugrel. Blood samples were collected before randomised treatment and over 24 hours after prasugrel administration. The inhibitory effects of prasugrel on platelets were determined using the VerifyNow P2Y12 assay and light transmission aggregometry. Plasma levels of prasugrel and prasugrel active metabolite were measured. Platelet reactivity determined by VerifyNow PRU, VerifyNow % Inhibition and LTA was significantly higher at 30–120 minutes (min) when morphine had been co-administered compared to when saline had been co-administered. Morphine, compared to saline, significantly delayed adequate platelet inhibition after prasugrel administration (158 vs 68 min; p = 0.006). Patients with delayed onset of platelet inhibition also had evidence of delayed absorption of prasugrel. In conclusion, prior administration of intravenous morphine significantly delays the onset of action of prasugrel. Intravenous drugs may be necessary to reduce the risk of acute stent thrombosis in morphine-treated STEMI patients undergoing PPCI.</jats:p

Economy-wide estimates of the implications of climate change: Sea level rise

The economy-wide implications of sea level rise in 2050 are estimated using a static computable general equilibrium model. This allows for a better estimate of the welfare effects of sea level rise than the common direct cost estimates; and for an estimate of the impact of sea level rise on greenhouse gas emissions. Overall, general equilibrium effects increase the welfare costs of sea level rise, but not necessarily in every sector or region. In the absence of coastal protection, economies that rely most on agriculture are hit hardest. Although energy is substituted for land, overall energy consumption falls with the shrinking economy, hurting energy exporters. With full coastal protection, GDP increases, particularly in regions with substantial dike building, but utility falls, least in regions that protect their coasts and export energy. Energy prices rise and energy consumption falls. The costs of full protection exceed the costs of losing land. The results also show direct costs - the usual method for estimating welfare changes due to sea level rise - are a bad approximation of the general equilibrium welfare effects; previous estimates of the economic impact of sea level rise are therefore biased