Characterisation of ICAM-3 and extracellular vesicles in the clearance of apoptotic cells

Apoptotic cell clearance by phagocytes is a vital part of programmed cell death that prevents dying cells from undergoing necrosis which may lead to inflammatory and autoimmune disorders. Apoptotic cells (AC) are removed by phagocytes, in a process that involves 'find me' and 'eat me' signals that facilitate the synapsing and engulfment of cell corpses. Extracellular vesicles (EV) are shed during apoptosis and promote phagocyte recruitment. Binding of AC is achieved by multiple ligand-receptor interactions. One interesting AC associated ligand is ICAM-3, a highly glycosylated adhesion molecule of the IgSF family, expressed on human leukocytes. On viable cells ICAM-3 participates in initiating immune responses, whereas on AC we show it attracts phagocytes through EV and aids in the binding of AC to the phagocytes. This project aims to characterize the role of ICAM-3 and EV in the clearance of AC and to identify the mechanisms that underlie their function in apoptotic cell clearance. Human B cells induced to apoptosis by UV irradiation were observed during their progression from viable to apoptotic via flow cytometry. The involvement of ICAM-3 in mediating interaction between AC and MØ was assessed. The ability of ICAM3 on EV to mediate chemoattraction was observed using chemotaxis assays. Additionally the anti-inflammatory effect was assessed using LPS-induced TNF-α production that suggested it may have anti-inflammatory effects. Future work in this project will assess the role of ICAM3 on EV from different phases of apoptosis to exert functional effects both in vitro and in vivo

Physical and functional characterisation of apoptotic cell-derived extracellular vesicles

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in the exposure of 'flags' at the dying cell surface and the release of attractive signals to recruit phagocytes. Together these changes ensure efficient phagocytic removal of dying cells and prevention of inflammatory and autoimmune disorders. Extracellular vesicles (EV) are released from a variety of cells (both viable and apoptotic) and they serve as a novel means of intercellular communication. They range in size: 70-100nm ('exosomes') through 100-1000nm ('microparticles') to large vesicles released from dying cells ('apoptotic bodies'). Release of apoptotic cell-derived extracellular vesicles (acdEV) of less than 1000nm is an important mechanism by which phagocytes are attracted to sites of cell death. Using a variety of approaches we characterize the release, physical characteristics and function of acdEV. Using fluorescence microscopy we demonstrate release of ICAM-3 on acdEV from dying leukocytes and, through the use of resistive pulse technology (qNano, IZON Science), we accurately size and quantitate acdEV release. The function of acdEV is revealed through the use of both horizontal chemotaxis assays (Dunn chambers) and vertical transwell migration assays (Cell-IQ, CM Technologies). These assays reveal potent chemoattractive capacity of acdEV and associated ICAM-3. Additionally we demonstrate an additional novel function of acdEV as anti-inflammatory immune-modulators. These data support an integrated approach to the physical and functional analyses of EV

Human aquaporins: regulators of transcellular water flow

Background: Emerging evidence supports the view that (AQP) aquaporin water channels are regulators of transcellular water flow. Consistentwith their expression in most tissues, AQPs are associatedwith diverse physiological and pathophysiological processes. Scope of review: AQP knockout studies suggest that the regulatory role of AQPs, rather than their action as passive channels, is their critical function. Transport through all AQPs occurs by a common passive mechanism, but their regulation and cellular distribution varies significantly depending on cell and tissue type; the role of AQPs in cell volumeregulation (CVR) is particularly notable. This reviewexamines the regulatory role of AQPs in transcellular water flow, especially in CVR.We focus on key systems of the human body, encompassing processes as diverse as urine concentration in the kidney to clearance of brain oedema. Major conclusions: AQPs are crucial for the regulation of water homeostasis, providing selective pores for the rapidmovement ofwater across diverse cellmembranes and playing regulatory roles in CVR. Gatingmechanisms have been proposed for human AQPs, but have only been reported for plant andmicrobial AQPs. Consequently, it is likely that the distribution and abundance of AQPs in a particular membrane is the determinant of membrane water permeability and a regulator of transcellular water flow. General significance: Elucidating the mechanisms that regulate transcellular water flow will improve our understanding of the human body in health and disease. The central role of specific AQPs in regulating water homeostasis will provide routes to a range of novel therapies. This article is part of a Special Issue entitled Aquaporins

Understanding the yeast host cell response to recombinant membrane protein production

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes

The SAMI Galaxy Survey: a statistical approach to an optimal classification of stellar kinematics in galaxy surveys

Large galaxy samples from multi-object IFS surveys now allow for a statistical analysis of the z~0 galaxy population using resolved kinematics. However, the improvement in number statistics comes at a cost, with multi-object IFS survey more severely impacted by the effect of seeing and lower S/N. We present an analysis of ~1800 galaxies from the SAMI Galaxy Survey and investigate the spread and overlap in the kinematic distributions of the spin parameter proxy $\lambda_{Re}$ as a function of stellar mass and ellipticity. For SAMI data, the distributions of galaxies identified as regular and non-regular rotators with \textsc{kinemetry} show considerable overlap in the $\lambda_{Re}$-$\varepsilon_e$ diagram. In contrast, visually classified galaxies (obvious and non-obvious rotators) are better separated in $\lambda_{Re}$ space, with less overlap of both distributions. Then, we use a Bayesian mixture model to analyse the observed $\lambda_{Re}$-$\log(M_*/M_{\odot})$ distribution. Below $\log(M_{\star}/M_{\odot})\sim10.5$, a single beta distribution is sufficient to fit the complete $\lambda_{Re}$ distribution, whereas a second beta distribution is required above $\log(M_{\star}/M_{\odot})\sim10.5$ to account for a population of low-$\lambda_{Re}$ galaxies. While the Bayesian mixture model presents the cleanest separation of the two kinematic populations, we find the unique information provided by visual classification of kinematic maps should not be disregarded in future studies. Applied to mock-observations from different cosmological simulations, the mixture model also predicts bimodal $\lambda_{Re}$ distributions, albeit with different positions of the $\lambda_{Re}$ peaks. Our analysis validates the conclusions from previous smaller IFS surveys, but also demonstrates the importance of using kinematic selection criteria that are dictated by the quality of the observed or simulated data.Comment: 30 pages and 17 figures, accepted for publication in MNRAS. Abstract abridged for Arxiv. The key figures of the paper are: 3, 7, 8, and 1

STEPWISE - STructured lifestyle Education for People WIth SchizophrEnia : a study protocol for a randomised controlled trial

BACKGROUND: People with schizophrenia are two to three times more likely to be overweight than the general population. The UK National Institute of Health and Care Excellence (NICE) recommends an annual physical health review with signposting to, or provision of, a lifestyle programme to address weight concerns and obesity. The purpose of this randomised controlled trial is to assess whether a group-based structured education programme can help people with schizophrenia to lose weight. METHODS: Design: a randomised controlled trial of a group-based structured education programme. SETTING: 10 UK community mental health trusts. PARTICIPANTS: 396 adults with schizophrenia, schizoaffective, or first-episode psychosis who are prescribed antipsychotic medication will be recruited. Participants will be overweight, obese or be concerned about their weight. INTERVENTION: participants will be randomised to either the intervention or treatment as usual (TAU). The intervention arm will receive TAU plus four 2.5-h weekly sessions of theory-based lifestyle structured group education, with maintenance contact every 2 weeks and 'booster' sessions every 3 months. All participants will receive standardised written information about healthy eating, physical activity, alcohol and smoking. OUTCOMES: the primary outcome is weight (kg) change at 1 year post randomisation. Secondary outcomes, which will be assessed at 3 and 12 months, include: the proportion of participants who maintained or reduced their weight; waist circumference; body mass index; objectively measured physical activity (wrist accelerometer); self-reported diet; blood pressure; fasting plasma glucose, lipid profile and HbA1c (baseline and 1 year only); health-related quality of life (EQ-5D-5L and RAND SF-36); (adapted) brief illness perception questionnaire; the Brief Psychiatric Rating Scale; the Client Service Receipt Inventory; medication use; smoking status; adverse events; depression symptoms (Patient Health Questionnaire-9); use of weight-loss programmes; and session feedback (intervention only). Outcome assessors will be blind to trial group allocation. Qualitative interviews with a subsample of facilitators and invention-arm participants will provide data on intervention feasibility and acceptability. Assessment of intervention fidelity will also be performed. DISCUSSION: The STEPWISE trial will provide evidence for the clinical and cost-effectiveness of a tailored intervention, which, if successful, could be implemented rapidly in the NHS. TRIAL REGISTRATION: ISRCTN19447796 , registered on 20 March 2014

Design of experiments to study the impact of process parameters on droplet size and development of non-invasive imaging techniques in tablet coating

Atomisation of an aqueous solution for tablet film coating is a complex process with multiple factors determining droplet formation and properties. The importance of droplet size for an efficient process and a high quality final product has been noted in the literature, with smaller droplets reported to produce smoother, more homogenous coatings whilst simultaneously avoiding the risk of damage through over-wetting of the tablet core. In this work the effect of droplet size on tablet film coat characteristics was investigated using X-ray microcomputed tomography (XμCT) and confocal laser scanning microscopy (CLSM). A quality by design approach utilising design of experiments (DOE) was used to optimise the conditions necessary for production of droplets at a small (20 μm) and large (70 μm) droplet size. Droplet size distribution was measured using real-time laser diffraction and the volume median diameter taken as a response. DOE yielded information on the relationship three critical process parameters: pump rate, atomisation pressure and coating-polymer concentration, had upon droplet size. The model generated was robust, scoring highly for model fit (R2 = 0.977), predictability (Q2 = 0.837), validity and reproducibility. Modelling confirmed that all parameters had either a linear or quadratic effect on droplet size and revealed an interaction between pump rate and atomisation pressure. Fluidised bed coating of tablet cores was performed with either small or large droplets followed by CLSM and XμCT imaging. Addition of commonly used contrast materials to the coating solution improved visualisation of the coating by XμCT, showing the coat as a discrete section of the overall tablet. Imaging provided qualitative and quantitative evidence revealing that smaller droplets formed thinner, more uniform and less porous film coats

Human aquaporins: regulators of transcellular water flow

Background: Emerging evidence supports the view that (AQP) aquaporin water channels are regulators of transcellular water flow. Consistentwith their expression in most tissues, AQPs are associatedwith diverse physiological and pathophysiological processes. Scope of review: AQP knockout studies suggest that the regulatory role of AQPs, rather than their action as passive channels, is their critical function. Transport through all AQPs occurs by a common passive mechanism, but their regulation and cellular distribution varies significantly depending on cell and tissue type; the role of AQPs in cell volumeregulation (CVR) is particularly notable. This reviewexamines the regulatory role of AQPs in transcellular water flow, especially in CVR.We focus on key systems of the human body, encompassing processes as diverse as urine concentration in the kidney to clearance of brain oedema. Major conclusions: AQPs are crucial for the regulation of water homeostasis, providing selective pores for the rapidmovement ofwater across diverse cellmembranes and playing regulatory roles in CVR. Gatingmechanisms have been proposed for human AQPs, but have only been reported for plant andmicrobial AQPs. Consequently, it is likely that the distribution and abundance of AQPs in a particular membrane is the determinant of membrane water permeability and a regulator of transcellular water flow. General significance: Elucidating the mechanisms that regulate transcellular water flow will improve our understanding of the human body in health and disease. The central role of specific AQPs in regulating water homeostasis will provide routes to a range of novel therapies. This article is part of a Special Issue entitled Aquaporins