Phosphatidylinositol polyphosphate lipids (phosphoinositides) form only a minor pool of membrane phospholipids but are involved in many intracellular signaling processes, including membrane trafficking, cytoskeletal remodeling, and receptor signal transduction. Phosphoinositide properties are largely determined by the characteristics of their headgroup, which at physiological pH is highly charged but also capable of forming hydrogen bonds. Many proteins have developed special binding domains that facilitate specific binding to particular phosphoinositides, while other proteins interact with phosphoinositides via nonspecific electrostatic interactions. Despite its importance, only limited information is available about the ionization properties of phosphoinositides. We have investigated the pH-dependent ionization behavior of all three naturally occurring phosphatidylinositol bisphosphates as well as of phosphatidylinositol 3,4,5-trisphosphate in mixed phosphoinositide/phosphatidylcholine vesicles using magic angle spinning (31)P NMR spectroscopy. For phosphatidylinositol 3,5-bisphosphate, where the two phosphomonoester groups are separated by a hydroxyl group at the 4-position, the pH-dependent chemical shift variation can be fitted with a Henderson-Hasselbalch-type formalism, yielding pK(a)(2) values of 6.96 +/- 0.04 and 6.58 +/- 0.04 for the 3- and 5-phosphates, respectively. In contrast, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] as well as phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] show a biphasic pH-dependent ionization behavior that cannot be explained by a Henderson-Hasselbalch-type formalism. This biphasic behavior can be attributed to the sharing of the last remaining proton between the vicinal phosphomonoester groups. At pH 7.0, the overall charge (including the phosphodiester group charge) is found to be -3.96 +/- 0.10 for PI(3,4)P(2) and -3.99 +/- 0.10 for PI(4,5)P(2). While for PI(3,5)P(2) and PI(4,5)P(2) the charges of the individual phosphate groups in the molecule differ, they are equal for PI(3,4)P(2). Differences in the charges of the phosphomonoester groups can be rationalized on the basis of the ability of the respective phosphomonoester group to form intramolecular hydrogen bonds with adjacent hydroxyl groups. Phosphatidylinositol 3,4,5-trisphosphate shows an extraordinary complex ionization behavior. While at pH 4 the (31)P NMR peak of the 4-phosphate is found downfield from the other two phosphomonoester group peaks, an increase in pH leads to a crossover of the 4-phosphate, which positions this peak eventually upfield from the other two peaks. As a result, the 4-phosphate group shows a significantly lower charge at pH values between 7 and 9.5 than the other two phosphomonoester groups. The charge of the respective phosphomonoester group in PI(3,4,5)P(3) is lower than the corresponding charge of the phosphatidylinositol bisphosphate phosphomonoester groups, leading to an overall charge of PI(3,4,5)P(3) of -5.05 +/- 0.15 at pH 7.0. The charge of all investigated phosphoinos...

In humanitarian emergency settings there is need for low cost and rapidly deployable interventions to protect vulnerable children, in- and out-of-school, from diarrhoeal diseases. Handwashing with soap can greatly reduce diarrhoea but interventions specifically targeting children's handwashing behaviour in humanitarian settings have not been tested. Traditional children's handwashing promotion interventions have been school-focused, resource-intensive and reliant on health-based messaging. However, recent research from non-humanitarian settings and targeting adults suggests that theory-based behaviour change interventions targeting specific motives may be more effective than traditional handwashing interventions. In this proof-of-concept study we test, for the first time, the distribution of a modified soap bar, designed to appeal to the motives of play and curiosity, in a household-level, rapidly deployable, handwashing promotion intervention for older children in a humanitarian setting - an internally displaced persons camp in Iraqi Kurdistan. Out of five total blocks within the camp, one was assigned to intervention and one to control. 40 households from each assigned block were then randomly chosen for inclusion in the study and the practice of handwashing with soap at key times was measured at baseline and four weeks after intervention delivery. Children in intervention households received transparent soaps with embedded toys, delivered within a short, fun, and interactive household session with minimal, non-health-based, messaging. The control group received plain soap delivered in a short standard, health-based, hygiene promotion session. At the 4-week follow-up, children in the intervention group were 4 times more likely to wash their hands with soap after key handwashing occasions than expected in the counterfactual (if there had been no intervention) based on the comparison to children in the control group (adjusted RR = 3.94, 95% CI 1.59-9.79). We show that distributing soaps with toys embedded inside, in a rapidly deployable intervention, can improve child handwashing behaviour in a humanitarian emergency context. Further studies are needed to determine the longer-term behavioural and health impact of such an intervention when delivered at a greater scale in a humanitarian context.

membranes composed of DOPC/sphingomyelin/cholesterol were prepared on ordered pore-arrays in silicon with different pore diameters by spreading and fusion of giant unilamellar vesicles. To induce vesicle rupture and fusion, the top part of a gold-covered silicon substrate was functionalized with a thiolbearing cholesterol derivative that renders the surface hydrophobic. Confocal laser scanning fluorescence microscopy was used to investigate the phase behavior of the obtained pore-spanning membranes. Coexisting liquid-orderedand liquid-disordered domains were visualized for DOPC/sphingomyelin/ cholesterol (40:40:20) membranes. The same result was obtained for lipid mixtures, in which 5 mol% of sphingomyelin was replaced by 5 mol% of the glycolipid Gb3. Videomicroscopy on these domains demonstrated their lateral mobility on the surface. The size of the lo-phase domains was strongly affected by the underlying pore size of the silicon substrate and could be controlled by temperature, and the cholesterol content in the membrane, which was modulated by the addition of methyl-b-cyclodextrin. Gb3 served as receptor for Shiga toxin B-pentamers, which bind to the membranes and thus considerably modulate the phase behavior of the porespanning membranes. 134-Plat Cholesterol Dependent and Independent Domain Formation in Mixed Phosphatidylinositol/Phosphoinositide Model Membranes

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present in the membrane. We have performed contrast-matching small-angle neutron scattering (SANS) experiments to study the conformational changes of the glycosylated form of A1AT for different concentrations of the osmolyte poly(ethelene glycol) (PEG) and in the presence of two different lipid membranes: POPC and POPS. We also monitor the structural changes of the lipid vesicles in the presence of A1AT by SANS. Guinier fits were used as a first approximation to obtain the radius of gyration (Rg) of A1AT. Bragg peaks were used to study structural changes of lipid vesicles. We observed that the Rg of A1AT changes as a function of PEG concentration in solution and when in the presence of lipid vesicles. The deformations monitored through changed in A1AT Rg in the presence of lipid vesicles are compared to the deformations of the glycoprotein observed under osmotic pressure and to the structural changes observed in the lipid vesicles.[1] Petrusca, et al., JBC 2010.

ObjectiveInvestigate the effect of common inner leaflet plasma membrane lipids such as phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), on phosphoinositide domain behavior in the presence of cholesterol and/or divalent cations.BackgroundPI and phosphoinositides (PIPs) may only comprise a small percentage of the inner leaflet of the plasma membrane, but they have major implications in a variety of signaling events. Previously, we have observed the absence of macroscopically discernible domains in mixtures of PI/PE and PI(4,5)P2/PE. Upon the addition of cholesterol to these mixtures, we observe condensation of the monolayer and domain formation.MethodsWe use epifluorescence microscopy to image domain formation in lipid monolayers at the air/water interfaceResultsWe find that the addition of varying concentrations of Ca2+ to PE/PI(4,5)P2/Cholesterol monolayers result in a decrease in the size of the domains. Our hypothesis is that this is due to a penetration of the Ca2+ ions into the PI(4,5)P2 headgroup region, leading to a disruption of the hydrogen bond network formed by the PI(4,5)P2 headgroup and cholesterol. For both PE/PI(4,5)P2 and PI/PI(4,5)P2, we find homogeneous mixing of the monolayer. Alternatively, when cholesterol is added to PE/PI(4,5)P2 we observe the formation of small domains at low pressures. In addition to cholesterol, we also investigate the effect of divalent cations on these lipid mixtures.

This chapter focuses on the use of cucurbit[n]urils for the functionalisation and controlled assembly of organic and inorganic nanoparticulate components. We begin by exploring the direct electrostatic interaction of the cucurbit[n]uril rims with metallic nanoparticle surfaces and how this can induce the controlled aggregation of nanoparticles. Use of this interaction in nanoparticle formation, surface enhanced Raman spectroscopy (SERS), catalysis and nanowire formation are discussed within. Cucurbit[n]urils can functionalise nanoparticle surfaces indirectly through complexation with surface-bound ligands. Nanoparticles can be assembled through such indirect functionalisation. If stimuli-responsive guests are used, then controllable assembly and disassembly results, and is discussed in depth. The chapter ends by looking at the use of cucurbit[n]urils in the formation of nanoparticles and colloidosomes through host–guest interactions. This wide range of capabilities makes cucurbit[n]uril-based nanosystems of huge interest for use in triggered assembly and delivery with many further areas yet to be investigated.

When phospholipid membranes are exposed to electric fields a variety of phenomena can be observed, such as phase separation, domain movement, electroporation, -deformation, -fusion, and -striction to name but a few. Understanding such responses is of both fundamental interest as well as of practical application. Various thermodynamic susceptibilities of lipid membranes increase strongly in the melting transition, leading to large changes in, for instance, membrane conductivity, compressibility, bending elasticity, relaxation time, and geometry. Another such property (susceptibility) of the membrane is its electrical capacitance. In the phase transition both area and thickness change significantly, but also the dielectric coefficient can increase due changes in membrane composition. This coupling of the membrane's capacitance to its phase state implies that transient currents will appear if the membrane is pushed into the phase transition by changes in e.g. pH, membrane potential, pressure or temperature. On this poster we will show some of these phenomena, and discuss them in the context of the recently proposed soliton model of nerve signal propagation by Heimburg and Jackson, where the coupling between the electrical aspects and the phase state of the system is of central importance.