Non-invasive single-cell biomechanical analysis using live-imaging datasets.

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.

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

BACKGROUND: The 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. METHODS: Here, 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. RESULTS: We 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. CONCLUSIONS: The 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.

Two classes of cholesterol binding sites for the ß2AR 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 (ß2AR) 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 ß2AR. By analyzing the ß2AR 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 ß2AR, 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.

Phase Heterogeneity in Cholesterol-Containing Ternary Phospholipid Lamellar Phases.

Pseudo-ternary mixtures of lamellar phase phospholipids (DPPC and brain sphingomyelin with cholesterol) were studied below T m while comparing the influence of cholesterol content, temperature, and the presence of small quantities of vitamin D binding protein (DBP) or vitamin D receptor (VDR). The measurements, conducted by X-ray diffraction (XRD) and nuclear magnetic resonance (NMR), cover a range of cholesterol concentrations (20% mol. wt to 40% mol. wt.) and physiologically relevant temperature range (294-314 K). In addition to rich intraphase behavior, data and modeling are used to approximate the lipids' headgroup location variations under the abovementioned experimental conditions.

Effect of cholecalciferol on unsaturated model membranes.

Vitamin D plays an important role in many physiological processes, particularly calcium and phosphorous homeostasis. The biochemistry of vitamin D is also complex, encompassing a range of active molecules that may be either endogenous or dietary in origin. The role of lipids and fats in the production, processing and use of vitamin D is an interesting one, with a relative paucity of model studies into the interactions of vitamin D with lipidic systems such as micelles and vesicles. Here, we have studied the effect of vitamin D3 in simple unsaturated phospholipid systems. We used NMR and FTIR spectroscopy to investigate the effect of increasing vitamin D concentration on the structure and dynamics of the lipid chains and interfacial region. In order to link these model studies with more complex biomimetic environments, we compare results in the presence of buffer and vitamin D binding protein. We have also used DLS to determine that vitamin D3-DOPC vesicles can retain their size distribution for varying amounts of time in different conditions. We find that the acyl chain region of vitamin D3-DOPC membranes are generally disordered, and that the addition of buffer and/or protein alters the properties of the interfacial region.

Therapeutic vitamin delivery: Chemical and physical methods with future directions.

Vitamins are a diverse group of "life nourishing" molecules that are essential for proper childhood development, and for maintaining health throughout adulthood into old age. Vitamin supplementation is an important strategy for reducing the severe and chronic effects of malnutrition in subsets of the population of the developing world. Additionally, the precise role of many vitamins in certain conditions, including cancer and cardiovascular disease, remains an area of active research, although guidelines for vitamin supplementation in otherwise adequately nourished populations remain controversial. This review describes vitamin delivery methods and techniques, focusing on the most recent advances and novel approaches. Specific attention has been given to physical methods and novel formulations for delivery with an emphasis on reporting pros and cons of each technique and highlighting future directions. Of particular interest is the potential for transdermal delivery of certain vitamins, which is an approach that may provide advantages in some populations (e.g. for vitamin D), but that still requires considerable additional research and clinical validation.

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

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 Å. 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.

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.