Deborah L. Gater

Two Classes of Cholesterol Binding Sites for the β2AR Revealed by Thermostability and NMR

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

Hydrogen bonding of cholesterol in the lipidic cubic phase.

The addition of cholesterol to the monoolein-based lipidic cubic phase (LCP) has been instrumental in obtaining high-resolution crystal structures of several G protein-coupled receptors. Here, we report the use of high-resolution magic angle spinning NMR spectroscopy to record and assign the isotropic (13)C chemical shifts of cholesterol in lipidic lamellar and cubic phases at different hydration levels with monoolein and chain-deuterated DMPC as host lipids. The hydrogen-bonding patterns of cholesterol in these phases were determined from the NMR data by quantum chemical calculations. The results are consistent with the normal orientation of cholesterol in lipid bilayers and with the cholesterol hydroxyl group located at the hydrophobic/hydrophilic interface. The (13)C chemical shifts of cholesterol are mostly affected by the host lipid identity with little or no dependency on the hydration (20% vs 40%) or the phase identity (lamellar vs LCP). In chain-deuterated DMPC bilayers, the hydroxyl group of cholesterol forms most of its hydrogen bonds with water, while in monoolein bilayers it predominately interacts with monoolein. Such differences in the hydrogen-bonding network of cholesterol may have implications for the design of experiments in monoolein-based LCP.

Connecting spiking neurons to a spiking memristor network changes the memristor dynamics

Memristors have been suggested as neuromorphic computing elements. Spike-time dependent plasticity and the Hodgkin-Huxley model of the neuron have both been modelled effectively by memristor theory. The d.c. response of the memris-tor is a current spike. Based on these three facts we suggest that memristors are well-placed to interface directly with neurons. In this paper we show that connecting a spiking memristor network to spiking neuronal cells causes a change in the memristor network dynamics by: causing a change in current decay rate consistent with a change in memristor state; presenting more-linear I-t dynamics; and increasing the memristor spiking rate, as a consequence of interaction with the spiking neurons. This demonstrates that neurons are capable of communicating directly with memristors, without the need for computer translation.

Formation of the liquid-ordered phase in fully hydrated mixtures of cholesterol and lysopalmitoylphosphatidylcholine

The role of cholesterol (Chol) in promoting lamellar phase formation in mixtures with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PPC) in excess water was investigated using multinuclear solid-state NMR and X-ray scattering. It was found that mixtures containing Chol and Lyso-PPC form a liquid-ordered (Lo) lamellar phase over a range of temperatures and concentrations, as previously observed in mixtures of Chol with various diacylphospholipids. The maximum quadrupolar splitting of the 2H-NMR powder patterns for samples containing per-deuterated Lyso-PPC were 40–50 kHz which is strongly indicative of an Lo phase. This evidence was supported by wide angle X-ray scattering data which showed a characteristic diffuse peak centred at 4.2 A. The Lo phase coexists with an isotropic Lyso-PPC phase at Chol concentrations up to 70 mol% Chol, and with Chol crystals at Chol concentrations above this value. Below 70 mol% Chol, an increase in the concentration of Chol in the system caused a corresponding increase in the proportion of the Lo phase present compared with the amount of isotropic Lyso-PPC. The chemical-shift anisotropy (CSA) of the static 31P-NMR spectra of the Lo phase showed the symmetry of a lamellar phase, but the linewidth, Δσ, was much narrower than CSA powder patterns obtained for diacylphospholipids in similar conditions, being ∼20 ppm as opposed to ∼40 ppm, respectively.

Non-invasive single cell biomechanical analysis using live imaging datasets

ABSTRACT The physiological state of a cell is governed by a multitude of processes and can be described by a combination of mechanical, spatial and temporal properties. Quantifying cell dynamics at multiple scales is essential for comprehensive studies of cellular function, and remains a challenge for traditional end-point assays. We introduce an efficient, non-invasive computational tool that takes time-lapse images as input to automatically detect, segment and analyze unlabeled live cells; the program then outputs kinematic cellular shape and migration parameters, while simultaneously measuring cellular stiffness and viscosity. We demonstrate the capabilities of the program by testing it on human mesenchymal stem cells (huMSCs) induced to differentiate towards the osteoblastic (huOB) lineage, and T-lymphocyte cells (T cells) of naïve and stimulated phenotypes. The program detected relative cellular stiffness differences in huMSCs and huOBs that were comparable to those obtained with studies that utilize atomic force microscopy; it further distinguished naïve from stimulated T cells, based on characteristics necessary to invoke an immune response. In summary, we introduce an integrated tool to decipher spatiotemporal and intracellular dynamics of cells, providing a new and alternative approach for cell characterization. Summary: An automated image-morphing-based method, intricately coupled with automated segmentation and multi-criterion path alignment, for a comprehensive label-free analysis of live-cell biomechanics, morphology and migration.

Quantification of sterol-specific response in human macrophages using automated imaged-based analysis

BackgroundThe transformation of normal macrophage cells into lipid-laden foam cells is an important step in the progression of atherosclerosis. One major contributor to foam cell formation in vivo is the intracellular accumulation of cholesterol.MethodsHere, we report the effects of various combinations of low-density lipoprotein, sterols, lipids and other factors on human macrophages, using an automated image analysis program to quantitatively compare single cell properties, such as cell size and lipid content, in different conditions.ResultsWe observed that the addition of cholesterol caused an increase in average cell lipid content across a range of conditions. All of the sterol-lipid mixtures examined were capable of inducing increases in average cell lipid content, with variations in the distribution of the response, in cytotoxicity and in how the sterol-lipid combination interacted with other activating factors. For example, cholesterol and lipopolysaccharide acted synergistically to increase cell lipid content while also increasing cell survival compared with the addition of lipopolysaccharide alone. Additionally, ergosterol and cholesteryl hemisuccinate caused similar increases in lipid content but also exhibited considerably greater cytotoxicity than cholesterol.ConclusionsThe use of automated image analysis enables us to assess not only changes in average cell size and content, but also to rapidly and automatically compare population distributions based on simple fluorescence images. Our observations add to increasing understanding of the complex and multifactorial nature of foam-cell formation and provide a novel approach to assessing the heterogeneity of macrophage response to a variety of factors.

Development of transdermal vitamin D3 (VD3) delivery system using combinations of PLGA nanoparticles and microneedles

Although vitamin D3 (VD3), which is the main form of vitamin D, can be produced in human skin under the sunlight, vitamin D deficiency emerged as a major public health problem worldwide. Mainly, oral supplements or vitamin D-fortified foods are distributed to help supplementation of vitamin D. However, those oral methods are limitedly supplied in the Middle East countries, and oral absorption has low efficiency due to many barriers and various changes of conditions along the route. Then, it is recommended to take them every day in order to maintain the adequate serum level of vitamin D. Alternatively, transdermal delivery system could provide a convenient way to get sustained supplement of vitamin D by its advantages like avoiding first-pass effect of the liver and providing release for long periods of time. In this study, we introduced transdermal delivery system for sustained vitamin D release using coating microneedles that easily pierce the skin layer with enough mechanical strength and allow the localization of drugs within the dermal region. According to the experimental results, poly (lactic-co-glycolic acid) (PLGA) successfully encapsulated VD3 as a nanoparticle form with appropriate properties for transdermal delivery such as size distribution, skin compatibility, and effective release of encapsulated compound. Finally, PVD3 layers coated on solid microneedles were completely dissolved into intradermal region in porcine skin model and revealed better performance for VD3 release into plasma compared to ointment base transdermal method.

Neural net to neuronal network memristor interconnects

Hardware-based Artificial Intelligence (A.I.) has many potential applications in biomedical technology; for example, connecting expert systems to biosensors for real-time physiological monitoring of a range of biomarkers, or interfacing with the brain.We suggest that memristors are well-placed to interface directly with neurons to interconnect between computer hardware and the brain for 3 reasons: memristors are widely-touted as neuromorphic computing elements; memristor theory has been successfully used to model spike-time dependent plasticity and the Hodgkin-Huxley model of the neuron; and, the d.c. response of the memristor is a current spike. In this chapter we show that connecting a spiking memristor network to electrically active neuronal cells causes a change in the memristor network dynamics by: removing the memristor spikes, which we show is due to the effects of connection to aqueous medium; causing a change in current decay rate consistent with a change in memristor state; presenting more-linear I − t dynamics; and increasing the memristor spiking rate, as a consequence of interaction with the active cells. This demonstrates that such cells are capable of communicating directly with memristors, without the need for computer translation.