Multicentre individual randomised controlled trial of screening and brief alcohol intervention to prevent risky drinking in young people aged 14-15 in a high school setting (SIPS JR-HIGH): study protocol.

INTRODUCTION: Drinking has adverse impacts on health, well-being, education and social outcomes for adolescents. Adolescents in England are among the heaviest drinkers in Europe. Recently, the proportion of adolescents who drink alcohol has fallen, although consumption among those who do drink has actually increased. This trial seeks to investigate how effective and efficient an alcohol brief intervention is with 11-15 years olds to encourage lower alcohol consumption. METHODS AND ANALYSIS: This is an individually randomised two-armed trial incorporating a control arm of usual school-based practice and a leaflet on a healthy lifestyle (excl. alcohol), and an intervention arm that combines usual practice with a 30 min brief intervention delivered by school learning mentors and a leaflet on alcohol. At least 30 schools will be recruited from four regions in England (North East, North West, London, Kent and Medway) to follow-up 235 per arm. The primary outcome is total alcohol consumed in the last 28 days, using the 28 day Timeline Follow Back questionnaire measured at the 12-month follow-up. The analysis of the intervention will consider effectiveness and cost-effectiveness. A qualitative study will explore, via 1:1 in-depth interviews with (n=80) parents, young people and school staff, intervention experience, intervention fidelity and acceptability issues, using thematic narrative synthesis to report qualitative data. ETHICS AND DISSEMINATION: Ethical approval was granted by Teesside University. Dissemination plans include academic publications, conference presentations, disseminating to local and national education departments and the wider public health community, including via Fuse, and engaging with school staff and young people to comment on whether and how the project can be improved. TRIAL REGISTRATION TRIAL: ISRCTN45691494; Pre-results.

Transcriptional changes associated with ethanol tolerance in Saccharomyces cerevisiae.

Saccharomyces spp. are widely used for ethanol production; however, fermentation productivity is negatively affected by the impact of ethanol accumulation on yeast metabolic rate and viability. This study used microarray and statistical two-way ANOVA analysis to compare and evaluate gene expression profiles of two previously generated ethanol-tolerant mutants, CM1 and SM1, with their parent, Saccharomyces cerevisiae W303-1A, in the presence and absence of ethanol stress. Although sharing the same parentage, the mutants were created differently: SM1 by adaptive evolution involving long-term exposure to ethanol stress and CM1 using chemical mutagenesis followed by adaptive evolution-based screening. Compared to the parent, differences in the expression levels of genes associated with a number of gene ontology categories in the mutants suggest that their improved ethanol stress response is a consequence of increased mitochondrial and NADH oxidation activities, stimulating glycolysis and other energy-yielding pathways. This leads to increased activity of energy-demanding processes associated with the production of proteins and plasma membrane components, which are necessary for acclimation to ethanol stress. It is suggested that a key function of the ethanol stress response is restoration of the NAD(+)/NADH redox balance, which increases glyceraldehyde-3-phosphate dehydrogenase activity, and higher glycolytic flux in the ethanol-stressed cell. Both mutants achieved this by a constitutive increase in carbon flux in the glycerol pathway as a means of increasing NADH oxidation.

Retrotransposon expression in ethanol-stressed Saccharomyces cerevisiae.

There are five retrotransposon families in Saccharomyces cerevisiae, three (Ty1, Ty2, and Ty3) of which are known to be transcriptionally active. Early investigations reported yeast retrotransposons to be stress-induced; however, microarray-based studies do not report retrotransposition-related Gene Ontology (GO) categories in the ethanol stress response of S. cerevisiae. In this study, microarray technology was used to investigate the ethanol stress response of S. cerevisiae W303-1A, and the highest stress-induced GO categories, based on z-score, were found to be retrotransposition-related, namely, Retrotransposition Nucleocapsid and Transposition, RNA-Mediated. Further investigation, involving reanalysis of previously published results on the stress response of S. cerevisiae, identified the absence of annotation for retrotransposon genes and associated GO categories and their omission during the printing of spotted arrays as two reasons why these categories in previous gene expression studies on the ethanol stress response of yeast were not reported.

Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae.

Saccharomyces spp. are widely used for ethanologenic fermentations, however yeast metabolic rate and viability decrease as ethanol accumulates during fermentation, compromising ethanol yield. Improving ethanol tolerance in yeast should, therefore, reduce the impact of ethanol toxicity on fermentation performance. The purpose of the current work was to generate and characterise ethanol-tolerant yeast mutants by subjecting mutagenised and non-mutagenised populations of Saccharomyces cerevisiae W303-1A to adaptive evolution using ethanol stress as a selection pressure. Mutants CM1 (chemically mutagenised) and SM1 (spontaneous) had increased acclimation and growth rates when cultivated in sub-lethal ethanol concentrations, and their survivability in lethal ethanol concentrations was considerably improved compared with the parent strain. The mutants utilised glucose at a higher rate than the parent in the presence of ethanol and an initial glucose concentration of 20 g l(-1). At a glucose concentration of 100 g l(-1), SM1 had the highest glucose utilisation rate in the presence or absence of ethanol. The mutants produced substantially more glycerol than the parent and, although acetate was only detectable in ethanol-stressed cultures, both mutants produced more acetate than the parent. It is suggested that the increased ethanol tolerance of the mutants is due to their elevated glycerol production rates and the potential of this to increase the ratio of oxidised and reduced forms of nicotinamide adenine dinucleotide (NAD(+)/NADH) in an ethanol-compromised cell, stimulating glycolytic activity.

Trehalose promotes the survival of Saccharomyces cerevisiae during lethal ethanol stress, but does not influence growth under sublethal ethanol stress.

Trehalose is known to protect cells from various environmental assaults; however, its role in the ethanol tolerance of Saccharomyces cerevisiae remains controversial. Many previous studies report correlations between trehalose levels and ethanol tolerance across a variety of strains, yet variations in genetic background make it difficult to separate the impact of trehalose from other stress response factors. In the current study, investigations were conducted on the ethanol tolerance of S. cerevisiae BY4742 and BY4742 deletion strains, tsl1Delta and nth1Delta, across a range of ethanol concentrations. It was found that trehalose does play a role in ethanol tolerance at lethal ethanol concentrations, but not at sublethal ethanol concentrations; differences of 20-40% in the intracellular trehalose concentration did not provide any growth advantage for cells incubated in the presence of sublethal ethanol concentrations. It was speculated that the ethanol concentration-dependent nature of the trehalose effect supports a mechanism for trehalose in protecting cellular proteins from the damaging effects of ethanol.

CT findings and mistopathology of intratumoral steatosis in focal nodular hyperplasia: case report and review of the literature.

Focal nodular hyperplasia (FNH) is a benign hepatic lesion characterized by homogeneous enhancement following intravenous contrast. The authors report a patient with a markedly heterogeneous FNH due to extensive intratumoral fat.

Evaluation of a recombinant Klebsiella oxytoca strain for ethanol production from cellulose by simultaneous saccharification and fermentation: comparison with native cellobiose-utilising yeast strains and performance in co-culture with thermotolerant yeast and Zymomonas mobilis.

In the simultaneous saccharification and fermentation to ethanol of 100 g l(-1) microcrystalline cellulose, the cellobiose-fermenting recombinant Klebsiella oxytoca P2 outperformed a range of cellobiose-fermenting yeasts used in earlier work, despite producing less ethanol than reported earlier for this organism under similar conditions. The time taken by K. oxytoca P2 to produce up to about 33 g l(-1) ethanol was much less than for any other organism investigated, including ethanol-tolerant strains of Saccharomyces pastorianus, Kluyveromyces marxianus and Zymomonas mobilis. Ultimately, it produced slightly less ethanol (maximum 36 g l(-1)) than these organisms, reflecting its lower ethanol tolerance. Significant advantages were obtained by co-culturing K. oxytoca P2 with S. pastorianus, K. marxianus or Z. mobilis, either isothermally, or in conjunction with temperature-profiling to raise the cellulase activity. Co-cultures produced significantly more ethanol, more rapidly, than either of the constituent strains in pure culture at the same inoculum density. K. oxytoca P2 dominated the early stages of the co-cultures, with ethanol production in the later stages due principally to the more ethanol tolerant strain. The usefulness of K. oxytoca P2 in cellulose simultaneous saccharification and fermentation should be improved by mutation of the strain to increase its ethanol tolerance.

Product formation and phosphoglucomutase activities in Lactococcus lactis: cloning and characterization of a novel phosphoglucomutase gene.

Maltose metabolism in Lactococcus lactis involves the conversion of beta-glucose 1-phosphate to glucose 6-phosphate, a reaction which is reversibly catalysed by a maltose-inducible and glucose-repressible beta-phosphoglucomutase (beta-PGM). The gene encoding beta-PGM (pgmB) was cloned from a genomic library of L. lactis using antibodies. The nucleotide sequence of a 5695 bp fragment was determined and six ORFs, including the pgmB gene, were found. The gene expressed a polypeptide with a calculated molecular mass of 24210 Da, which is in agreement with the molecular mass of the purified beta-PGM (25 kDa). A short sequence at the N-terminus was found to be similar to known metal-binding domains. The expression of beta-PGM in L lactis was found to be induced also by trehalose and sucrose, and repressed by lactose in the growth medium. This indicates that beta-PGM does not serve solely to degrade maltose, but that it is also involved in the metabolism of other carbohydrates. The specific activity of beta-PGM during fermentation was dependent on the maltose concentration in the medium. The maximum specific activity of beta-PGM increased by a factor of 4.6, and the specific growth rate by a factor of 7, when the maltose concentration was raised from 0.8 to 11.0 g l-1. Furthermore, a higher amount of lactate produced relative to formate, acetate and ethanol was observed when the initial maltose concentration in the medium was increased. The specific activity of alpha-PGM responded similarly to beta-PGM, but the magnitude of the response was lower. Preferential sugar utilization and alpha- and beta-PGM suppression was observed when L. lactis was grown on the substrate combinations glucose and maltose, or lactose and maltose; maltose was the least-preferred sugar. In contrast, galactose and maltose were utilized concurrently and both PGM activities were high throughout the fermentation.