Hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in Acute lung injury to Reduce Pulmonary dysfunction (HARP-2) trial: study protocol for a randomized controlled trial

Background: Acute lung injury (ALI) is a common devastating clinical syndrome characterized by life-threatening respiratory failure requiring mechanical ventilation and multiple organ failure. There are in vitro, animal studies and pre-clinical data suggesting that statins may be beneficial in ALI. The Hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in Acute lung injury to Reduce Pulmonary dysfunction (HARP-2) trial is a multicenter, prospective, randomized, allocation concealed, double-blind, placebo-controlled clinical trial which aims to test the hypothesis that treatment with simvastatin will improve clinical outcomes in patients with ALI.

Methods/Design: Patients fulfilling the American-European Consensus Conference Definition of ALI will be randomized in a 1: 1 ratio to receive enteral simvastatin 80 mg or placebo once daily for a maximum of 28 days. Allocation to randomized groups will be stratified with respect to hospital of recruitment and vasopressor requirement. Data will be recorded by participating ICUs until hospital discharge, and surviving patients will be followed up by post at 3, 6 and 12 months post randomization. The primary outcome is number of ventilator-free days to day 28. Secondary outcomes are: change in oxygenation index and sequential organ failure assessment score up to day 28, number of non pulmonary organ failure free days to day 28, critical care unit mortality; hospital mortality; 28 day post randomization mortality and 12 month post randomization mortality; health related quality of life at discharge, 3, 6 and 12 months post randomization; length of critical care unit and hospital stay; health service use up to 12 months post-randomization; and safety. A total of 540 patients will be recruited from approximately 35 ICUs in the UK and Ireland. An economic evaluation will be conducted alongside the trial. Plasma and urine samples will be taken up to day 28 to investigate potential mechanisms by which simvastatin might act to improve clinical outcomes.

SMAD inhibition attenuates epithelial to mesenchymal transition by primary keratinocytes in vitro

Epithelial to mesenchymal transition (EMT) is a process whereby epithelial cells undergo transition to a mesenchymal phenotype and contribute directly to fibrotic disease. Recent studies support a role for EMT in cutaneous fibrotic diseases including scleroderma and hypertrophic scarring, though there is limited data on the cytokines and signalling mechanisms regulating cutaneous EMT. We investigated the ability of TGF-β and TNF-α, both over-expressed in cutaneous scleroderma and central mediators of EMT in other epithelial cell types, to induce EMT in primary keratinocytes and studied the signalling mechanisms regulating this process. TGF-β induced EMT in normal human epidermal keratinocytes (NHEK cells) and this process was enhanced by TNF-α. EMT was characterised by changes in morphology, proteome (down-regulation of E-cadherin and Zo-1, and up-regulation of vimentin and fibronectin), MMP secretion and COL1α1 mRNA expression. TGF-β and TNF-α in combination activated SMAD and p38 signalling in NHEK cells. P38 inhibition with SB203580 partially attenuated EMT, whereas SMAD inhibition using SB431542 significantly inhibited EMT and also reversed established EMT. These data highlight the retained plasticity of adult keratinocytes and support further studies of EMT in clinically relevant in vivo models of cutaneous fibrosis, and investigation of SMAD inhibition as a potential therapeutic intervention. This article is protected by copyright. All rights reserved.

The Saccharomyces cerevisiae quinone oxidoreductase Lot6p: stability, inhibition and cooperativity

Lot6p (EC 1.5.1.39; Ylr011wp) is the sole quinone oxidoreductase in the budding yeast, Saccharomyces cerevisiae. Using hexahistidine tagged, recombinant Lot6p, we determined the steady-state enzyme kinetic parameters with both NADH and NADPH as electron donors; no cooperativity was observed with these substrates. The NQO1 inhibitor curcumin, the NQO2 inhibitor resveratrol, the bacterial nitroreductase inhibitor nicotinamide and the phosphate mimic vanadate all stabilise the enzyme towards thermal denaturation as judged by differential scanning fluorimetry. All except vanadate have no observable effect on the chemical cross-linking of the two subunits of the Lot6p dimer. These compounds all inhibit Lot6p's oxidoreductase activity, and all except nicotinamide exhibit negative cooperativity. Molecular modelling suggests that curcumin, resveratrol and nicotinamide all bind over the isoalloxazine ring of the FMN cofactor in Lot6p. Resveratrol was predicted to contact an α-helix that links the two active sites. Mutation of Gly-142 (which forms part of this helix) to serine does not greatly affect the thermal stability of the enzyme. However, this variant shows less cooperativity towards resveratrol than the wild type. This suggests a plausible hypothesis for the transmission of information between the subunits and, thus, the molecular mechanism of negative cooperativity in Lot6p.

Inhibition of ribosome biogenesis induces apoptosis in p53-/- cells

The nucleolus is an important region of the nucleus that acts as the main site of rRNA transcription by RNA polymerase I (Pol-I), and one of the most prominent morphological markers of proliferative and invasive cancers is increased nucleolar size. Increases in Pol-I transcription leads to increased levels of ribosome biogenesis that are able to fuel rapid cell growth and proliferation due to increased ribosome numbers. Therefore Pol-I transcription seems a viable target for the development of anticancer therapeutics as abrogation of Pol-I transcription leads to cessation of cell growth and eventual cell death. We have confirmed that ellipticine compound, 9-Hydroxyellipticine (9HE), is an efficient inhibitor of Pol-I transcription in vitro and in p53+/+ and -/- cell lines in vivo. Short-term treatments (≤24 h) with micromolar concentrations of 9HE leads to decreased cell viability and proliferation, and leads to activation of caspases 3, 8 and 9, indicating that both intrinsic and extrinsic cell death mechanisms are activated upon Pol-I inhibition. Reactive oxygen species levels were also studied following short and long term treatments with 9HE and there was a 2/3-fold increase in cellular ROS levels. Long-term 9HE treatment (≥24 h) leads to cellular senescence as indicated by increased cellular morphology and senescence associated β-galactosidase staining, and this senescence is not accompanied by induction of autophagy. Following 24 h treatment there is also accumulation of cells in the G0/G1 phase of the cell cycle and qPCR analysis of cell cycle regulators shows down-regulation of G1/S transition associated cyclins. These data show that Pol-I transcription is a viable target for the development of novel chemotherapeutics, although further delineation of the cell death pathways remains. To further elucidate the mechanism, the role of mitochondrial death signals will be investigated by determination of cytochrome c release and regulation of Bcl2 family member proteins.

A simple inhibition coefficient for quantifying potency of biocontrol agents against plant-pathogenic fungi

Microbial interactions depend on a range of biotic and environmental variables, and are both dynamic and unpredictable. For some purposes, and under defined conditions, it is nevertheless imperative to evaluate the inhibitory efficacy of microbes, such as those with potential as biocontrol agents. We selected six, phylogenetically diverse microbes to determine their ability to inhibit the ascomycete Fusarium
coeruleum, a soil-dwelling pathogen of potato tubers that causes the storage disease dry rot. Interaction assays, where colony development was quantified (for both fungal pathogen and potential control agents), were therefore carried out on solid media. The key parameters that contributed to, and were indicative of, inhibitory efficacy were identified as: fungal growth-rates (i) prior to contact with the biocontrol
agent and (ii) if/once contact with the biocontrol agent was established (i.e. in the zone of mixed
culture), and (iii) the ultimate distance traveled by the fungal mycelium. It was clear that there was no correlation between zones of fungal inhibition and the overall reduction in the extent of fungal colony development. An inhibition coefficient was devised which incorporated the potential contributions of distal inhibition of fungal growth-rate; prevention of mycelium development in the vicinity of the biocontrol
agent; and ability to inhibit plant-pathogen growth-rate in the zone of mixed culture (in a ratio of 2:2:1). The values derived were 84.2 for Bacillus subtilis (QST 713), 74.0 for Bacillus sp. (JC12GB42), 30.7 for Pichia anomala (J121), 19.3 for Pantoea agglomerans (JC12GB34), 13.9 for Pantoea sp. (S09:T:12), and
21.9 (indicating a promotion of fungal growth) for bacterial strain (JC12GB54). This inhibition coefficient, with a theoretical maximum of 100, was consistent with the extent of F. coeruleum-colony development (i.e. area, in cm2) and assays of these biocontrol agents carried out previously against Fusarium
spp., and other fungi. These findings are discussed in relation to the dynamics and inherent complexity of natural ecosystems, and the need to adapt models for use under specific sets of conditions.