Chloe Thomas

Mutations in the sterol-sensing domain of Patched suggest a role for vesicular trafficking in Smoothened regulation.

The tumor suppressor gene patched (ptc) encodes an approximately 140 kDa polytopic transmembrane protein [1-3] [corrected] that binds members of the Hedgehog (Hh) family of signaling proteins [4-6] [corrected] and regulates the activity of Smoothened (Smo), a G protein-coupled receptor-like protein essential for Hh signal transduction [7-9] [corrected]. Ptc contains a sterol-sensing domain (SSD) [10, 11] [corrected], a motif found in proteins implicated in the intracellular trafficking of cholesterol [12] [corrected], and/or other cargoes [13-15] [corrected]. Cholesterol plays a critical role in Hedgehog (Hh) signaling by facilitating the regulated secretion and sequestration of the Hh protein [16] [corrected], to which it is covalently coupled. In addition, cholesterol synthesis inhibitors block the ability of cells to respond to Hh [18, 19] [corrected], and this finding points to an additional requirement for the lipid in regulating downstream components of the Hh signaling pathway. Although the SSD of Ptc has been linked to both the sequestration of, and the cellular response to Hh [16, 20, 21] [corrected], definitive evidence for its function has so far been lacking. Here we describe the identification and characterization of two missense mutations in the SSD of Drosophila Ptc; strikingly, while both mutations abolish Smo repression, neither affects the ability of Ptc to interact with Hh. We speculate that Ptc may control Smo activity by regulating an intracellular trafficking process dependent upon the integrity of the SSD.

Planar polarity specification through asymmetric subcellular localization of Fat and Dachsous.

Summary Two pathways regulate planar polarity: the core proteins and the Fat-Dachsous-Four-jointed (Ft-Ds-Fj) system. Morphogens specify complementary expression patterns of Ds and Fj that potentially act as polarizing cues. It has been suggested that Ft-Ds-Fj-mediated cues are weak and that the core proteins amplify them [1, 2]. Another view is that the two pathways act independently to generate and propagate polarity [3, 4]: if correct, this raises the question of how gradients of Ft and Ds expression or activity might be interpreted to provide strong cellular polarizing cues and how such cues are propagated from cell to cell. Here, we demonstrate that the complementary expression of Ds and Fj results in biased Ft and Ds protein distribution across cells, with Ft and Ds accumulating on opposite edges. Furthermore, boundaries of Ft and Ds expression result in subcellular asymmetries in protein distribution that are transmitted to neighboring cells, and asymmetric Ds localization results in a corresponding asymmetric distribution of the myosin Dachs. We show that the generation of subcellular asymmetries of Ft and Ds and the core proteins is largely independent in the wing disc and additionally that ommatidial polarity in the eye can be determined without input from the Ft-Ds-Fj system, consistent with the two pathways acting in parallel.

The roles of the cadherins Fat and Dachsous in planar polarity specification in Drosophila

Planar polarity is generated through the activity of two groups of proteins, the “core” system and the Fat (Ft)/Dachsous (Ds) system. Although both are conserved from insects to mammals, vertebrate studies into planar polarity have primarily focussed on core planar polarity proteins and have only recently branched into the study of the Ft/Ds system. In Drosophila, however, years of detailed analysis have started to elucidate some of the mechanisms by which Ft/Ds signalling might set up polarity across a tissue, and how this may impact upon core protein‐mediated planar polarity. In this review, we discuss the major findings, models, and controversies that have emerged from Drosophila research into the Ft/Ds system, and indicate some areas for further investigation. Developmental Dynamics 241:27–39, 2012.

Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila.

The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. Here, we identify domains essential for Smo activity and investigate the sub-cellular distribution of the wild type protein in vivo. We find that deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins we show that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. We further demonstrate that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction.

JNK signalling influences intracellular trafficking during Drosophila morphogenesis through regulation of the novel target gene Rab30

JNK-mediated closure of the Drosophila dorsal epidermis during embryogenesis is a well-characterised model for morphogenesis. However, little is known about how JNK signalling modifies particular cellular behaviours such as intracellular transport. Here we demonstrate that the gene encoding the small GTPase Rab30 is a new JNK transcriptional target whose function is required during embryonic and adult morphogenesis including JNK-dependent dorsal closure, embryonic head involution and thorax closure. Using immuno-fluorescence and live imaging, we show that EGFP-Rab30 localises to trans-Golgi in addition to small unidentified vesicles, and moves in a microtubule-dependent, polarised dorso-ventral manner in the leading edge during dorsal closure. We propose that JNK activity upregulates genes involved in intracellular transport in order to provide an increased level of trafficking activity in cells undergoing complex morphogenetic arrangements such as dorsal closure.

Rabaptin-5 and Rabex-5 are neoplastic tumour suppressor genes that interact to modulate Rab5 dynamics in Drosophila melanogaster

Endocytosis plays an important role in the regulation of tumour growth and metastasis. In Drosophila, a number of endocytic neoplastic tumour suppressor genes have been identified that when mutated cause epithelial disruption and over-proliferation. Here we characterise the Drosophila homologue of the Rab5 effector Rabaptin-5, and show that it is a novel neoplastic tumour suppressor. Its ability to bind Rab5 and modulate early endosomal dynamics is conserved in Drosophila, as is its interaction with the Rab5 GEF Rabex5, for which we also demonstrate neoplastic tumour suppressor characteristics. Surprisingly, we do not observe disruption of apico-basal polarity in Rabaptin-5 and Rabex-5 mutant tissues; instead the tumour phenotype is associated with upregulation of Jun N-terminal Kinase (JNK) and Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signalling.

The cost-effectiveness of a theory-based online health behaviour intervention for new university students: an economic evaluation

BackgroundToo many young people engage in unhealthy behaviours such as eating unhealthily, being physically inactive, binge drinking and smoking. This study aimed to estimate the short-term and long-term cost-effectiveness of a theory-based online health behaviour intervention (“U@Uni”) in comparison with control in young people starting university.MethodsA costing analysis was conducted to estimate the full cost of U@Uni and the cost of U@Uni roll-out. The short-term cost-effectiveness of U@Uni was estimated using statistical analysis of 6-month cost and health-related quality of life data from the U@Uni randomised controlled trial. An economic modelling analysis combined evidence from the trial with published evidence of the effect of health behaviours on mortality risk and general population data on health behaviours, to estimate the lifetime cost-effectiveness of U@Uni in terms of incremental cost per QALY. Costs and effects were discounted at 1.5% per annum. A full probabilistic sensitivity analysis was conducted to account for uncertainty in model inputs and provide an estimate of the value of information for groups of important parameters.ResultsTo implement U@Uni for the randomised controlled trial was estimated to cost £292 per participant, whereas roll-out to another university was estimated to cost £19.71, both giving a QALY gain of 0.0128 per participant. The short-term (6-month) analysis suggested that U@Uni would not be cost-effective at a willingness-to-pay threshold of £20,000 per QALY (incremental cost per QALY gained = £243,926). When a lifetime horizon was adopted the results suggest that the full implementation of U@Uni is unlikely to be cost-effective, whereas the roll-out of U@Uni to another university is extremely likely to be cost-effective. The value of information analysis suggests that the most important drivers of decision uncertainty are uncertainties in the effect of U@Uni on health behaviours.ConclusionsThe study provides the first estimate of the costs and cost-effectiveness of an online health behaviour intervention targeted at new university students. The results suggest that the roll-out, but not the full implementation, of U@Uni would be a cost-effective decision for the UK Department of Health, given a lifetime perspective and a willingness-to pay threshold of £20,000 per QALY.Trial registrationCurrent Controlled Trials ISRCTN67684181.

The impact of Type 2 diabetes prevention programmes based on risk-identification and lifestyle intervention intensity strategies : a cost-effectiveness analysis

The following article, published online on 17 November 2015 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the journal Editor in Chief, Richard Holt and John Wiley & Sons Ltd. The retraction has been agreed due the fact the authors have made Wiley aware that there was an error in the way the diabetes prevention intervention effect was implemented in this version of the model. The underlying model is not affected by this error. However, the magnitude of the cost savings and health benefits are substantially lower than stated.

Cost-effectiveness of population-based, community, workplace and individual policies for diabetes prevention in the UK

To analyse the cost‐effectiveness of different interventions for Type 2 diabetes prevention within a common framework.

Assessing the potential return on investment of the proposed UK NHS diabetes prevention programme in different population subgroups: an economic evaluation

Objectives To evaluate potential return on investment of the National Health Service Diabetes Prevention Programme (NHS DPP) in England and estimate which population subgroups are likely to benefit most in terms of cost-effectiveness, cost-savings and health benefits. Design Economic analysis using the School for Public Health Research Diabetes Prevention Model. Setting England 2015–2016. Population Adults aged ≥16 with high risk of type 2 diabetes (HbA1c 6%–6.4%). Population subgroups defined by age, sex, ethnicity, socioeconomic deprivation, baseline body mass index, baseline HbA1c and working status. Interventions The proposed NHS DPP: an intensive lifestyle intervention focusing on dietary advice, physical activity and weight loss. Comparator: no diabetes prevention intervention. Main outcome measures Incremental costs, savings and return on investment, quality-adjusted life-years (QALYs), diabetes cases, cardiovascular cases and net monetary benefit from an NHS perspective. Results Intervention costs will be recouped through NHS savings within 12 years, with net NHS saving of £1.28 over 20 years for each £1 invested. Per 100 000 DPP interventions given, 3552 QALYs are gained. The DPP is most cost-effective and cost-saving in obese individuals, those with baseline HbA1c 6.2%–6.4% and those aged 40–74. QALY gains are lower in minority ethnic and low socioeconomic status subgroups. Probabilistic sensitivity analysis suggests that there is 97% probability that the DPP will be cost-effective within 20 years. NHS savings are highly sensitive to intervention cost, effectiveness and duration of effect. Conclusions The DPP is likely to be cost-effective and cost-saving under current assumptions. Prioritising obese individuals could create the most value for money and obtain the greatest health benefits per individual targeted. Low socioeconomic status or ethnic minority groups may gain fewer QALYs per intervention, so targeting strategies should ensure the DPP does not contribute to widening health inequalities. Further evidence is needed around the differential responsiveness of population subgroups to the DPP.