The expression of a "Xenopus borealis" cardiac actin gene in normal and transformed frog embryos

The major aim of this project has been to ascertain if the expression of a cloned Xenopus gene, which is normally expressed in a tissue - specific fashion during early development, is regulated in the same way as its chromosomal counterpart in micro- injected Xenopus embryos. A number of clones, which contain genes encoding different actin isoforms, have been isolated from Xenopus borealis genomic libraries. One of these contains an entire cardiac actin gene, on the basis of the isotype - specific sequence of its encoded product. Indeed, in the adult frog, the chromosomal gene is only expressed in the heart. However, in the embryo, transcripts are also detected in the myotomes, which contain skeletal muscle cells. Two transcriptional assays have been developed, so that transcripts from the unmodified, cloned X. borealis cardiac actin gene can be detected separately from endogenous transcripts in micro-injected X. laevis embryos. In such transformed animals, injected linear DNA forms high-molecular-weight extrachromosomal concatenates, which replicate and become relatively evenly distributed throughout all tissues. Properly initiated transcripts from the cloned gene are correctly localised to the myotomes in both neurulae and tadpoles. The temporal regulation of expression also shares strong similarities with that of the endogenous, chromosomal actin gene. In a preliminary investigation of the sequences responsible for this regulation, a fusion construct between the first two actin exons and the last exon of a mouse β-globin gene has been injected. The same wide distribution of DNA, but spatially restricted pattern of expression, as the actin gene is found, whereas transcripts from a histone-globin fusion gene are formed in all tissues. This is the first report of correct spatial control of expression from an injected, cloned gene in Xenopus. I discuss the wider significance of these results for future studies on the early developmental regulation of gene expression

The impact of heat on mortality and morbidity in the Greater Metropolitan Sydney Region: A case crossover analysis

Background: This study examined the association between unusually high temperature and daily mortality (1997-2007) and hospital admissions (1997-2010) in the Sydney Greater Metropolitan Region (GMR) to assist in the development of targeted health program

The gut in the beaker : missing the surfactants

Gastrointestinal drug administration is the preferred route for the majority of drugs however, the natural physiology and physicochemistry of the gastrointestinal tract is critical to absorption but complex and influenced by factors such as diet or disease. The pharmaceutical sciences drive for product consistency has led to the development of in vitro product performance tests whose utility and interpretation is hindered by the complexity, variability and a lack of understanding. This article explores some of these issues with respect to the drug, formulation and the presence of surfactant excipients and how these interact with the natural bile salt surfactants. Interactions start in the mouth and during swallowing but the stomach and small intestine present the major challenges related to drug dissolution, solubility, the impact of surfactants and supersaturation along with precipitation. The behaviour of lipid based formulations and the influence of surfactant excipients is explored along with the difficulties of translating in vitro results to in vivo performance. Possible future research areas are highlighted with the conclusion that, “a great deal of work using modern methods is still required to clarify the situation”

Squalestatin alters the intracellular trafficking of a neurotoxic prion peptide

Neurotoxic peptides derived from the protease-resistant core of the prion protein are used to model the pathogenesis of prion diseases. The current study characterised the ingestion, internalization and intracellular trafficking of a neurotoxic peptide containing amino acids 105-132 of the murine prion protein (MoPrP105-132) in neuroblastoma cells and primary cortical neurons. Fluorescence microscopy and cell fractionation techniques showed that MoPrP105-132 co-localised with lipid raft markers (cholera toxin and caveolin-1) and trafficked intracellularly within lipid rafts. This trafficking followed a non-classical endosomal pathway delivering peptide to the Golgi and ER, avoiding classical endosomal trafficking via early endosomes to lysosomes. Fluorescence resonance energy transfer analysis demonstrated close interactions of MoPrP105-132 with cytoplasmic phospholipase A2 (cPLA2) and cyclo-oxygenase-1 (COX-1), enzymes implicated in the neurotoxicity of prions. Treatment with squalestatin reduced neuronal cholesterol levels and caused the redistribution of MoPrP105-132 out of lipid rafts. In squalestatin-treated cells, MoPrP105-132 was rerouted away from the Golgi/ER into degradative lysosomes. Squalestatin treatment also reduced the association between MoPrP105-132 and cPLA2/COX-1. As the observed shift in peptide trafficking was accompanied by increased cell survival these studies suggest that the neurotoxicity of this PrP peptide is dependent on trafficking to specific organelles where it activates specific signal transduction pathways

Statistical investigation of simulated fed intestinal media composition on the equilibrium solubility of oral drugs

Gastrointestinal fluid is a complex milieu and it is recognised that gut drug solubility is different to that observed in simple aqueous buffers. Simulated gastrointestinal media have been developed covering fasted and fed states to facilitate in vitro prediction of gut solubility and product dissolution. However, the combination of bile salts, phospholipids, fatty acids and proteins in an aqueous buffered system creates multiple phases and drug solubility is therefore a complex interaction between these components, which may create unique environments for each API. The impact on solubility can be assessed through a statistical design of experiment (DoE) approach, to determine the influence and relationships between factors. In this paper DoE has been applied to fed simulated gastrointestinal media consisting of eight components (pH, bile salt, lecithin, sodium oleate, monoglyceride, buffer, salt and pancreatin) using a two level D-optimal design with forty-four duplicate measurements and four centre points. The equilibrium solubility of a range of poorly soluble acidic (indomethacin, ibuprofen, phenytoin, valsartan, zafirlukast), basic (aprepitant, carvedilol, tadalafil, bromocriptine) and neutral (fenofibrate, felodipine, probucol, itraconazole) drugs was investigated. Results indicate that the DoE provides equilibrium solubility values that are comparable to literature results for other simulated fed gastrointestinal media systems or human intestinal fluid samples. For acidic drugs the influence of pH predominates but other significant factors related to oleate and bile salt or interactions between them are present. For basic drugs pH, oleate and bile salt have equal significance along with interactions between pH and oleate and lecithin and oleate. Neutral drugs show diverse effects of the media components particularly with regard to oleate, bile salt, pH and lecithin but the presence of monoglyceride, pancreatin and buffer have significant but smaller effects on solubility. There are fourteen significant interactions between factors mainly related to the surfactant components and pH, indicating that the solubility of neutral drugs in fed simulated media is complex. The results also indicate that the equilibrium solubility of each drug can exhibit individualistic behaviour associated with the drug’s chemical structure, physicochemical properties and interaction with media components. The utility of DoE for fed simulated media has been demonstrated providing equilibrium solubility values comparable with similar in vitro systems whilst also providing greater information on the influence of media factors and their interactions. The determination of a drug’s gastrointestinal solubility envelope provides useful limits that can potentially be applied to in silico modelling and in vivo experiments

Statistical investigation of the full concentration range of fasted and fed simulated intestinal fluid on the equilibrium solubility of oral drugs

Upon oral administration the solubility of a drug in intestinal fluid is a key property influencing bioavailability. It is also recognised that simple aqueous solubility does not reflect intestinal solubility and to optimise in vitro investigations simulated intestinal media systems have been developed. Simulated intestinal media which can mimic either the fasted or fed state consists of multiple components each of which either singly or in combination may influence drug solubility, a property that can be investigated by a statistical design of experiment technique. In this study a design of experiment covering the full range from the lower limit of fasted to the upper limit of fed parameters and using a small number of experiments has been performed. The measured equilibrium solubility values are comparable with literature values for simulated fasted and fed intestinal fluids as well as human fasted and fed intestinal fluids. The equilibrium solubility data range is statistically equivalent to a combination of published fasted and fed design of experiment data in six (indomethacin, phenytoin, zafirlukast, carvedilol, fenofibrate and probucol) drugs with three (aprepitant, tadalafil and felodipine) drugs not equivalent. In addition the measured equilibrium solubility data sets were not normally distributed. Further studies will be required to determine the reasons for these results however it implies that a single solubility measurement without knowledge of the solubility distribution will be of limited value. The statistically significant media factors which promote equilibrium solubility (pH, sodium oleate and bile salt) were in agreement with published results but the number of determined significant factors and factor interactions was fewer in this study, lecithin for example did not influence solubility. This may be due to the reduction in statistical sensitivity from the lower number of experimental data points or the fact that using the full range will examine media parameters ratios that are not biorelevant. Overall the approach will provide an estimate of the solubility range and the most important media factors but will not be equivalent to larger scale focussed studies. Further investigations will be required to determine why some drugs do not produce equivalent DoE solubility distributions, for example combined fasted and fed DoE, but this simply may be due to the complexity and individuality of the interactions between a drug and the media components

Implementing the contextual safeguarding approach: a study in one local authority

Purpose This study aims to explore how local authority child and family practitioners understood and implement the contextual safeguarding approach focusing, in particular, on what practitioners felt supported and hindered implementation. Design/methodology/approach This qualitative study drew upon semi-structured interviews and focus groups to explore the perspectives of 18 frontline workers, team managers and senior managers in a London authority. The transcribed accounts were analysed using thematic analysis. Findings Participants reported that the strain on services because of sustained budget cuts was overwhelming, even without the additional challenge of implementing this new approach. Further challenges in relation to implementation included parental-capacity focused legislation and conflicting perspectives between stakeholders. This study recommends that proper funding must be committed to safeguarding partnerships if contextual safeguarding is to be successfully implemented. Additionally, child protection practitioners should aim to develop a collaborative and child-welfare focused network of community agencies and organisations if young people are to be safeguarded in their communities. Research limitations/implications As the sample required specialist knowledge to participate in this study, this study cannot claim that the findings are generalisable to all social workers. Originality/value To the best of the authors’ knowledge, this is the first external evaluation of the implementation of the contextual safeguarding approach in a local authority independent of the contextual safeguarding team at the University of Bedfordshire since the evaluation of Hackney

Cost-effectiveness analyses for mirtazapine and sertraline in dementia: randomised controlled trial

BACKGROUND Depression is a common and costly comorbidity in dementia. There are very few data on the cost-effectiveness of antidepressants for depression in dementia and their effects on carer outcomes. AIMS To evaluate the cost-effectiveness of sertraline and mirtazapine compared with placebo for depression in dementia. METHOD A pragmatic, multicentre, randomised placebo-controlled trial with a parallel cost-effectiveness analysis (trial registration: ISRCTN88882979 and EudraCT 2006-000105-38). The primary cost-effectiveness analysis compared differences in treatment costs for patients receiving sertraline, mirtazapine or placebo with differences in effectiveness measured by the primary outcome, total Cornell Scale for Depression in Dementia (CSDD) score, over two time periods: 0-13 weeks and 0-39 weeks. The secondary evaluation was a cost-utility analysis using quality-adjusted life years (QALYs) computed from the Euro-Qual (EQ-5D) and societal weights over those same periods. RESULTS There were 339 participants randomised and 326 with costs data (111 placebo, 107 sertraline, 108 mirtazapine). For the primary outcome, decrease in depression, mirtazapine and sertraline were not cost-effective compared with placebo. However, examining secondary outcomes, the time spent by unpaid carers caring for participants in the mirtazapine group was almost half that for patients receiving placebo (6.74 v. 12.27 hours per week) or sertraline (6.74 v. 12.32 hours per week). Informal care costs over 39 weeks were £1510 and £1522 less for the mirtazapine group compared with placebo and sertraline respectively. CONCLUSIONS In terms of reducing depression, mirtazapine and sertraline were not cost-effective for treating depression in dementia. However, mirtazapine does appear likely to have been cost-effective if costing includes the impact on unpaid carers and with quality of life included in the outcome. Unpaid (family) carer costs were lower with mirtazapine than sertraline or placebo. This may have been mediated via the putative ability of mirtazapine to ameliorate sleep disturbances and anxiety. Given the priority and the potential value of supporting family carers of people with dementia, further research is warranted to investigate the potential of mirtazapine to help with behavioural and psychological symptoms in dementia and in supporting carers

Fine-Tuning of PI3K/AKT Signalling by the Tumour Suppressor PTEN Is Required for Maintenance of Flight Muscle Function and Mitochondrial Integrity in Ageing Adult Drosophila melanogaster

Insulin/insulin-like growth factor signalling (IIS), acting primarily through the PI3-kinase (PI3K)/AKT kinase signalling cassette, plays key evolutionarily conserved regulatory roles in nutrient homeostasis, growth, ageing and longevity. The dysfunction of this pathway has been linked to several age-related human diseases including cancer, Type 2 diabetes and neurodegenerative disorders. However, it remains unclear whether minor defects in IIS can independently induce the age-dependent functional decline in cells that accompany some of these diseases or whether IIS alters the sensitivity to other aberrant signalling. We identified a novel hypomorphic allele of PI3K’s direct antagonist, Phosphatase and tensin homologue on chromosome 10 (Pten), in the fruit fly, Drosophila melanogaster. Adults carrying combinations of this allele, Pten5, combined with strong loss-of-function Pten mutations exhibit subtle or no increase in mass, but are highly susceptible to a wide range of stresses. They also exhibit dramatic upregulation of the oxidative stress response gene, GstD1, and a progressive loss of motor function that ultimately leads to defects in climbing and flight ability. The latter phenotype is associated with mitochondrial disruption in indirect flight muscles, although overall muscle structure appears to be maintained. We show that the phenotype is partially rescued by muscle-specific expression of the Bcl-2 homologue Buffy, which in flies, maintains mitochondrial integrity, modulates energy homeostasis and suppresses cell death. The flightless phenotype is also suppressed by mutations in downstream IIS signalling components, including those in the mechanistic Target of Rapamycin Complex 1 (mTORC1) pathway, suggesting that elevated IIS is responsible for functional decline in flight muscle. Our data demonstrate that IIS levels must be precisely regulated by Pten in adults to maintain the function of the highly metabolically active indirect flight muscles, offering a new system to study the in vivo roles of IIS in the maintenance of mitochondrial integrity and adult ageing