Lipid Mono Research Articles

Aqueous Ionic Liquid Mixtures as Minimal Models of Lipid Bilayer Membranes.

We introduce aqueous ionic liquid (IL) mixtures, specifically mixtures of 1-butyl-3-imidazoliumtetrafluoroborate (BMImBF4), with water as a minimal model of lipid bilayer membranes. Imidazolium-based ILs are known to form clustered nanoscale structures in which local inhomogeneities, micellar or lamellar structures, are formed to shield hydrophobic parts of the cation from the polar cosolvent (water). To investigate these nanostructures, dynamic light scattering (DLS) on samples with different mixing ratios of water and BMImBF4 was performed. At mixing ratios of 50% and 45% (v/v), small and homogeneous nanostructures can indeed be detected. To test whether, in particular, these stable nanostructures in aqueous mixtures may mimic the effects of phospholipid bilayer membranes, we further investigated their interaction with myelin basic protein (MBP), a peripheral, intrinsically disordered membrane protein of the myelin sheath. Using dynamic light scattering (DLS), continuous wave (CW) and pulse electron paramagnetic resonance (EPR), and small-angle X-ray scattering (SAXS) on recombinantly produced, "healthy" charge variants rmC1WT and double cysteine variant C1S17CH85C, we find that the size and the shape of the determined nanostructures in an optimum mixture offer model membranes in which the protein exhibits native behavior. SAXS measurements illuminate the size and shape of the nanostructures and indicate IL-rich "beads" clipped together by functional MBP, one of the in vivo roles of the protein in the myelin sheath. All the gathered data combined indicate that the 50% and 45% aqueous IL mixtures can be described as offering minimal models of a lipid mono- or bilayer that allow native processing and potential study of at least peripheral membrane proteins like MBP.

The mechanistic basis of the membrane-permeabilizing activities of the virulence-associated protein A (VapA) from Rhodococcus equi.

Pathogenic Rhodococcus equi release the virulence-associated protein A (VapA) within macrophage phagosomes. VapA permeabilizes phagosome and lysosome membranes and reduces acidification of both compartments. Using biophysical techniques, we found that VapA interacts with model membranes in four steps: (i) binding, change of mechanical properties, (ii) formation of specific membrane domains, (iii) permeabilization within the domains, and (iv) pH-specific transformation of domains. Biosensor data revealed that VapA binds to membranes in one step at pH 6.5 and in two steps at pH 4.5 and decreases membrane fluidity. The integration of VapA into lipid monolayers was only significant at lateral pressures <20 mN m-1 indicating preferential incorporation into membrane regions with reduced integrity. Atomic force microscopy of lipid mono- and bilayers showed that VapA increased the surface heterogeneity of liquid disordered domains. Furthermore, VapA led to the formation of a new microstructured domain type and, at pH 4.5, to the formation of 5 nm high domains. VapA binding, its integration and lipid domain formation depended on lipid composition, pH, protein concentration and lateral membrane pressure. VapA-mediated permeabilization is clearly distinct from that caused by classical microbial pore formers and is a key contribution to the multiplication of Rhodococcus equi in phagosomes.

Drude Polarizable Lipid Force Field with Explicit Treatment of Long-Range Dispersion: Parametrization and Validation for Saturated and Monounsaturated Zwitterionic Lipids.

Accurate empirical force fields of lipid molecules are a critical component of molecular dynamics simulation studies aimed at investigating properties of monolayers, bilayers, micelles, vesicles, and liposomes, as well as heterogeneous systems, such as protein-membrane complexes, bacterial cell walls, and more. While the majority of lipid force field-based simulations have been performed using pairwise-additive nonpolarizable models, advances have been made in the development of the polarizable force field based on the classical Drude oscillator model. In the present study, we undertake further optimization of the Drude lipid force field, termed Drude2023, including improved treatment of the phosphate and glycerol linker region of PC and PE headgroups, additional optimization of the alkene group in monounsaturated lipids, and inclusion of long-range Lennard-Jones interactions using the particle-mesh Ewald method. Initial optimization targeted quantum mechanical (QM) data on small model compounds representative of the linker region. Subsequent optimization targeted QM data on larger model compounds, experimental data, and dihedral potentials of mean force from the CHARMM36 additive lipid force field using a parameter reweighting protocol. The use of both experimental and QM target data during the reweighting protocol is shown to produce physically reasonable parameters that reproduce a collection of experimental observables. Target data for optimization included surface area/lipid for DPPC, DSPC, DMPC, and DLPC bilayers and nuclear magnetic resonance (NMR) order parameters for DPPC bilayers. Validation data include prediction of membrane thickness, scattering form factors, electrostatic potential profiles, compressibility moduli, surface area per lipid, water permeability, NMR T1 relaxation times, diffusion constants, and monolayer surface tensions for a variety of saturated and unsaturated lipid mono- and bilayers. Overall, the agreement with experimental data is quite good, though the results are less satisfactory for the NMR T1 relaxation times for carbons near the ester groups. Notable improvements compared to the additive C36 force field were obtained for membrane dipole potentials, lipid diffusion coefficients, and water permeability with the exception of monounsaturated lipid bilayers. It is anticipated that the optimized polarizable Drude2023 force field will help generate more accurate molecular simulations of pure bilayers and heterogeneous systems containing membranes, advancing our understanding of the role of electronic polarization in these systems.

Molecular basis of hUHRF1 allosteric activation for synergistic histone modification binding by PI5P.

Chromatin marks are recognized by distinct binding modules, many of which are embedded in multidomain proteins. How the different functionalities of such complex chromatin modulators are regulated is often unclear. Here, we delineated the interplay of the H3 amino terminus– and K9me-binding activities of the multidomain hUHRF1 protein. We show that the phosphoinositide PI5P interacts simultaneously with two distant flexible linker regions connecting distinct domains of hUHRF1. The binding is dependent on both, the polar head group, and the acyl part of the phospholipid and induces a conformational rearrangement juxtaposing the H3 amino terminus and K9me3 recognition modules of the protein. In consequence, the two features of the H3 tail are bound in a multivalent, synergistic manner. Our work highlights a previously unidentified molecular function for PI5P outside of the context of lipid mono- or bilayers and establishes a molecular paradigm for the allosteric regulation of complex, multidomain chromatin modulators by small cellular molecules.

Simple Does Not Mean Trivial: Behavior of Phosphatidic Acid in Lipid Mono- and Bilayers.

Phosphatidic acid (PA) is one of the simplest membrane phospholipids, yet it plays a crucial role in various biologically relevant processes that take place in cells. Since PA generation may be triggered by a variety of factors, very often of antagonistic character, the specific nature of physiological responses driven by PA is not clear. In order to shed more light on these issues, we carried out a systematic characterization of membranes containing one of the three biologically significant PA molecular species. The effect of these molecules on the properties of membranes composed of phosphatidylcholine and/or cholesterol was assessed in a multidisciplinary approach, including molecular dynamic simulations, flicker noise spectroscopy, and Langmuir monolayer isotherms. The first enables the determination of various macroscopic and microscopic parameters such as lateral diffusion, membrane thickness, and defect analysis. The obtained data revealed a strong interaction between unsaturated PA species and phosphatidylcholine. On the other hand, the behavior of saturated PA was greatly influenced by cholesterol. Additionally, a strong effect on mechanical properties was observed in the case of three-component systems, which could not be explained by the simple extrapolation of parameters of the corresponding two-component systems. Our data show that various PA species are not equivalent in terms of their influence on lipid mono- and bilayers and that membrane composition/properties, particularly those related to the presence of cholesterol, may strongly modulate PA behavior.

Surface-active extracts from plants rich in saponins – effect on lipid mono- and bilayers

The aqueous extracts of the seeds of oat (Avena sativa L.), horse chestnut (Aesculus hippocastanum L.), soybean (Glycine max L.), cowherb (Vaccaria hispanica [P. Mill.] Rauschert) and quinoa (Chenopodium quinoa Willd.), and the roots of soapwort (Saponaria officinalis L.) without any preservatives were characterized in terms of their surface tension, surface compression (dilational) rheology, foamability and foam stability. The saponin content in the extracts was determined using UPLC-MS and their interaction with model lipid monolayers consisting of dipalmitoylphosphatidylcholine (DPPC)/cholesterol and Ceramide AP/stearic acid/cholesterol were analyzed by surface pressure relaxation, surface compression elasticity and neutron reflectometry (NR). The lipid composition was chosen to mimic the cell membrane of keratinocytes – major constituents of the human deeper skin layers, and the intercellular lipids (“mortar”) in the “bricks and mortar” model of the outermost layer of the epidermis (stratum corneum). Bilayers of DPPC/cholesterol were additionally characterized using dynamic light scattering (DLS) and NR. The oat and soybean extracts were shown to be much less abundant in saponins as compared to cowherb, horse chestnut, soapwort or quinoa, and showed limited foaming abilities. They did not affect significantly the model lipid mono- and bilayers mimicking the skin outer layers, either. The horse chestnut extract affected both model membranes to the highest extent, yet without solubilizing the lipids.

Lipid-Dependent Interaction of Human N-BAR Domain Proteins with Sarcolemma Mono- and Bilayers.

The N-BAR domain of the human Bin1 protein is indispensable for T-tubule biogenesis in skeletal muscles. It binds to lipid mono- and bilayers that mimic the sarcolemma membrane composition, and it transforms vesicles into uniform tubules by generating a decorating protein scaffold. We found that Δ(1-33)BAR, lacking the N-terminal amphipathic helix (H0), and H0 alone bind to sarcolemma monolayers, although both proteins are not able to tubulate sarcolemma vesicles. By variation of the lipid composition, we elucidated the role of PI(4,5)P2, cholesterol, and an asymmetric sarcolemma composition for Bin1-N-BAR binding and sarcolemma tubulation. Our results indicate that Bin1-N-BAR binding is low in the absence of PI(4,5)P2 and it is affected by additional changes in the negative headgroup charge and lipid acyl chain composition. However, it is not dependent on the cholesterol content. The results from Langmuir monolayer experiments are complementary to lipid bilayer studies using electron microscopy that provides information on membrane curvature generation.

Modifying Membrane Morphology and Interactions with DNA Origami Clathrin-Mimic Networks.

We describe the triggered assembly of a bioinspired DNA origami meshwork on a lipid membrane. DNA triskelia, three-armed DNA origami nanostructures inspired by the membrane-modifying protein clathrin, are bound to lipid mono- and bilayers using cholesterol anchors. Polymerization of triskelia, triggered by the addition of DNA staples, links triskelion arms to form a mesh. Using transmission electron microscopy, we observe nanoscale local deformation of a lipid monolayer induced by triskelion polymerization that is reminiscent of the formation of clathrin-coated pits. We also show that the polymerization of triskelia bound to lipid bilayers modifies interactions between them, inhibiting the formation of a synapse between giant unilamellar vesicles and a supported lipid bilayer.

Interaction of DNA with likely-charged lipid monolayers: An experimental study.

Anionic lipids are increasingly being used in lipoplexes for synthetic gene vectors as an alternative to cationic lipids. This is primarily due to their lower toxicity, which makes them biocompatible and adaptable to be tissue specific. However, anionic lipoplexes require the presence of multivalent cations to promote the electrostatic attraction between DNA and anionic lipid mono- and bilayers. In this work we provide for the first time experimental results of the adsorption of linear DNA onto anionic/zwitterionic lipid monolayers without any addition of cations. This is demonstrated experimentally by means of Langmuir monolayers of DOPE/DOPG (1:1) lipids spread on a water subphase that contains calf thymus DNA. The adsorption of DNA onto anionic/zwitterionic lipid monolayers is discussed in terms of the surface pressure-molecular area isotherms recorded in the absence and in the presence of different electrolytes. Measurements of the surface potential provide additional evidence of the different interaction of DNA anionic/zwitterionic lipid monolayers depending on the presence and nature of electrolyte. These experimental results are further analysed in terms of the overall dipole moment normal to the monolayers providing new insight into the behaviour of anionic lipoplexes and the role of zwitterionic lipids.

Interaction of membrane palmitoylated protein-1 with model lipid membranes.

Membrane palmitoylated protein-1 (MPP1) plays an important role in the formation of raft domains in erythroid membranes. We have shown recently that MPP1 interacts with membrane lipids composed of dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM) and cholesterol. Here we further extend this investigation. Our results obtained from FRET assays revealed that MPP1 binds liposomes with high affinity (KD ~ 135 ± 15 nM). Preincubation of MPP1 with cholesterol before its addition to the Langmuir subphase resultedin a dramatic reduction in the membrane insertion/binding of MPP1, indicating the role of direct MPP1/cholesterol complexes in the interaction of MPP1 with membrane lipids. The generalized polarization values of liposomes as well as the constant surface area experiments on monolayers composed of DOPC/SM/Chol indicated a change in the lipid mono- and bilayer properties upon the addition of MPP1. Furthermore, the presence of flotillins did not affect the binding of MPP1 to membrane lipids. Also, MPP1 containing palmitoylation-mimicking mutation (C242F) bound DOPC/SM/Chol mono- and bilayer with an affinity very similar to that obtained for wild-type MPP1. In conclusion, our results suggest that the direct binding of MPP1 with membrane lipids could be involved in the mechanism of membrane association of MPP1 in erythroid cells.

Control of lipid domain organization by a biomimetic contractile actomyosin cortex.

The cell membrane is a heterogeneously organized composite with lipid-protein micro-domains. The contractile actin cortex may govern the lateral organization of these domains in the cell membrane, yet the underlying mechanisms are not known. We recently reconstituted minimal actin cortices (MACs) (Vogel et al., 2013b) and here advanced our assay to investigate effects of rearranging actin filaments on the lateral membrane organization by introducing various phase-separated lipid mono- and bilayers to the MACs. The addition of actin filaments reorganized membrane domains. We found that the process reached a steady state where line tension and lateral crowding balanced. Moreover, the phase boundary allowed myosin driven actin filament rearrangements to actively move individual lipid domains, often accompanied by their shape change, fusion or splitting. Our findings illustrate how actin cortex remodeling in cells may control dynamic rearrangements of lipids and other molecules inside domains without directly binding to actin filaments.

X-ray Reflectivity Study of the Interaction of an Imidazolium-Based Ionic Liquid with a Soft Supported Lipid Membrane.

Ionic liquids (ILs) are important for their antimicrobial activity and are found to be toxic to some microorganisms. To shed light on the mechanism of their activities, the interaction of an imidazolium-based IL 1-butyl-3-methylimidazolium tetrfluoroborate ([BMIM][BF4]) with E. coli bacteria and cell-membrane-mimicking lipid mono- and bilayers has been studied. The survival of the bacteria and corresponding growth inhibition are observed to be functions of the concentration of the IL. The IL alters the pressure-area isotherm of the monolayer formed at an air-water interface by the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid. The in-plane elasticity of the lipid layer is reduced as a consequence of the insertion of this IL. The X-ray reflectivity study from a polymer-supported lipid bilayer shows strong perturbation in the self-assembled structure of the bilayer due to the interaction. As a consequence, there is a considerable decrease in bilayer thickness and a corresponding increase in electron density. These results, however, depend on the chain configurations of the lipid molecules.

Efficient Cellular Knockdown Mediated by siRNA Nanovectors of Gemini Cationic Lipids Having Delocalizable Headgroups and Oligo-Oxyethylene Spacers.

The use of small interfering RNAs (siRNAs) to silence specific genes is one of the most promising approaches in gene therapy, but it requires efficient nanovectors for successful cellular delivery. Recently, we reported liposomal gene carriers derived from a gemini cationic lipid (GCL) of the 1,2-bis(hexadecyl dimethyl imidazolium) oligo-oxyethylene series ((C16Im)2(C2H4O)nC2H4 with n = 1, 2, or 3) and 1,2-dioleyol phosphatidylethanolamine as highly efficient cytofectins for pDNA. On the basis of the satisfactory outcomes of the previous study, the present work focuses on the utility of coliposomes of these gemini lipids with the biocompatible neutral lipid mono oleoyl glycerol (MOG) as highly potent vectors for siRNA cellular transport in the presence of serum. The (C16Im)2(C2H4O)nC2H4/MOG-siRNA lipoplexes were characterized through (i) a physicochemical study (zeta potential, cryo-transmission electron microscopy, small-angle X-ray scattering, and fluorescence anisotropy) to establish the relationship between size, structure, fluidity, and the interaction between siRNA and the GCL/MOG gene vectors and (ii) a biological analysis (flow cytometry, fluorescence microscopy, and cell viability) to report the anti-GFP siRNA transfections in HEK 293T, HeLa, and H1299 cancer cell lines. The in vitro biological analysis confirms the cellular uptake and indicates that a short spacer, a very low molar fraction of GCL in the mixed lipid, and a moderate effective charge ratio of the lipoplex yielded maximum silencing efficacy. At these experimental conditions, the siRNA used in this work is compacted by the GCL/MOG nanovectors by forming two cubic structures (Ia3d and Pm3n) that are correlated with excellent silencing activity. These liposomal nanocarriers possess high silencing activity with a negligible cytotoxicity, which strongly supports their practical use for in vivo knockdown studies.

Comparative analysis of new peptide conjugates of antitubercular drug candidates—Model membrane and in vitro studies

Novel peptide conjugates of two antitubercular drug candidates were synthesised and characterised using new tuftsin peptide derivative (OT14) as carrier moiety. As antitubercular drug candidates two pyridopyrimidine derivatives, TB803 (2-allylamino-4-oxopyrido[1,2-a]pyrimidine-3-carbaldehyde) and TB820 (4-oxo-2-(pyrrolidin-1-yl)-pyrido[1,2-a]pyrimidin-3-carbaldehyde) inhibiting vital enzyme of Mycobacterium tuberculosis were applied. Membrane affinity of the compounds TB803 and TB820 and their peptide conjugates was evaluated using experimental lipid mono- and bilayer models. Penetration ability was assessed tensiometrically from Langmuir monolayer study and applying quartz crystal microbalance for the supported lipid bilayer (SLB) system. Minimal inhibitory concentration (MIC) values remained in a similar micromolar range for both of the conjugates while their cellular uptake rate was improved significantly compared to the drug candidates. A correlation was found between membrane affinity properties and results of in vitro biological investigations. Analysis of physical/structural properties of SLB in contact with bioactive components and visualization of the structural change by atomic-force microscopy (AFM) provided information on the type and route of molecular interaction of drug construction with lipid layers. The possible role of electrostatic interactions between lipid layer and drug candidates was tested in Langmuir-balance experiments using negatively charged lipid mixture (DPPC+DPPG). Especially the peptide conjugates presented increased membrane affinity due to cationic character of the peptide sequence selected for the conjugate formation. That is supposed to be one reason for the enhanced cellular uptake observed in vitro on MonoMac6 cell line. The conjugation of antitubercular agents to a peptidic carrier is a promising approach to enhance membrane affinity, cellular uptake rate and in vitro selectivity.

CHARMM-GUI Input Generator for NAMD, Gromacs, Amber, Openmm, and CHARMM/OpenMM Simulations using the CHARMM36 Additive Force Field

Proper treatment of nonbonded interactions is essential for the accuracy of molecular dynamics (MD) simulations, especially in studies of lipid mono- and bilayers. The use of the CHARMM36 force field (C36 FF) in different MD simulation programs can result in disagreements with published simulations performed with CHARMM due to differences in the protocols used to treat the long-range and 1-4 nonbonded interactions. In this study, we systematically test the use of the C36 lipid FF in NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM. A wide range of Lennard-Jones (LJ) cutoff schemes and integrator algorithms were tested to find the optimal simulation protocol to best match lipid properties of six bilayers with varying acyl chain saturation and head groups. MD simulations of 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) bilayers were used to obtain optimal protocols for each program. MD simulations with all programs were found to reasonably match the DPPC bilayer membrane properties, such as surface area per lipid, chain order parameters, and area compressibility modulus, obtained using the standard protocol used in CHARMM as well as from experiments. The optimal simulation protocol was then applied to the other five lipid simulations and resulted in excellent agreement between results from most simulation programs as well as with experimental data. AMBER compared least favorably with the expected membrane properties, which appears to due to its use of the hard-truncation of the LJ potential versus a force-based switching function used to smooth the LJ potential as it approaches the cutoff distance. The optimal simulation protocol for each program has been implemented in CHARMM-GUI. We expect this protocol to be applicable to the remainder of the additive C36 FF including the proteins, nucleic acids, carbohydrates, and small molecules.

New Insights into Lipid Monolayers from Coarse-Grained Simulation Techniques

New Insights into Lipid Monolayers from Coarse-Grained Simulation Techniques

Interaction of mixed-ligand monolayer-protected Au₁₄₄ clusters with biomimetic membranes as a function of the transmembrane potential.

Understanding the interaction of nanoparticles with cell membranes is a high-priority research area for possible biomedical applications. We describe our findings concerning the interaction of Au144 monolayer-protected clusters (MPCs) with biomimetic membranes and their permeabilizing effect as a function of the transmembrane potential. We synthesized Au144(SCH2CH2Ph)60 and modified the capping monolayer with 8-mercaptooctanoic acid (Au144OctA) or thiolated trichogin (Au144TCG), a channel-forming peptide. The interactions of these MPCs with mercury-supported lipid mono- and bilayers were studied with a combination of electrochemical techniques specifically sensitive to changes in the properties of biomimetic membranes and/or charge-transfer phenomena. Permeabilization effects were evaluated through the influence of MPC uptake on the reduction of cadmium(II) ions. The nature and properties of the Au144 capping molecules play a crucial role in controlling how MPCs interact with membranes. The native MPC causes a small effect, whereas both Au144OctA and Au144TCG interact significantly with the lipid monolayer and show electroactivity. Whereas Au144OctA penetrates the membrane, Au144TCG pierces the membrane with its peptide appendage while remaining outside of it. Both clusters promote Cd(2+) reduction but with apparently different mechanisms. Because of the different way that they interact with the membrane, Au144OctA is more effective in Cd(2+) reduction when interacting with the lipid bilayer and Au144TCG performs particularly well when piercing the lipid monolayer.

Arf1 Induced Membrane Remodeling and Morphological Changes Studied by Cryo-Em, Confocal Microscopy and Langmuir Film Balance

The small GTPase Arf1 of the RAS superfamily plays an important role in vesicular trafficing. On the Golgi membrane, the formation and fission of coat protein I (COPI) transport vesicles proceeds via local deformation of the lipid bilayer by a curvature generating COPI protein coat. The assembly of this complex is initiated by the GTPase Arf1 in a nucleotide-dependent manner. After GDP/GTP exchange, soluble Arf1 becomes membrane bound by insertion of its myristoylated N-terminal amphipathic helix (myrAH) into the proximal leaflet of the Golgi membrane. The subsequent liberation of transport vesicles requires the full COPI complex and has been observed in vivo and in vitro.As the role of Arf1 in the process of curvature induction has not been fully elucidated, we have studied binding and incorporation of recombinant S. cerevisiae Arf1p into lipid mono- and bilayers using binding assays with a Langmuir film balance setup and artificial, unilamellar liposomes. We observe myristoylation-dependent binding to membranes and an increase in membrane surface area upon addition of Arf1p. Confocal laser scanning microscopy and cryo electron microscopy reveal highly curved membrane structures upon incorporation of myristoylated Arf1p. Our results support a mechanism of curvature induction based on the bilayer couple theory.

Abstract 18: The Effect of Lipid Modification on Peripheral Arterial Disease after Endovascular Intervention Trial (ELIMIT)

BACKGROUND The Effect of Lipid Modification on Peripheral Arterial Disease after Endovascular Intervention Trial (ELIMIT), a prospective double-blind randomized study, was designed to determine over 24-months, the effects of triple drug lipid modification therapy versus mono therapy on the progression of atherosclerotic lesions in the distal superficial femoral artery (SFA), as assessed by 3.0T magnetic resonance imaging (MRI). METHODS A total of 95 patients were randomized to either mono therapy with simvastatin (40mg) or triple therapy with simvastatin (40mg), extended-release niacin (1.5g), and ezetimibe (10mg). MR imaging was performed at baseline, 6-, 12-, and 24-months. SFA wall, lumen, and total vessel volumes were quantified using inter-visit co-registered proton-density-weighted turbo spin echo MRI sequences. MRI derived SFA parameters and lipids were analyzed with multi-level models and non-parametric tests, respectively. RESULTS Baseline characteristics did not differ between mono and triple therapy groups, except for ethnicity (Table). There was no difference in adverse cardiovascular events between groups (p=0.99). SFA wall, lumen, and vessel volumes increased non-significantly for both groups between baseline and 24-months. Non-high-density lipoprotein cholesterol showed a larger reduction in triple therapy compared with mono therapy at 12-months (p=0.01). CONCLUSION Despite improved atherogenic lipid profiles, there was no significant difference between mono therapy with simvastatin and triple therapy with simvastatin, extended-release niacin, and ezetimibe for the 24-month changes in total SFA wall, lumen, and vessel volumes.

Phase Separation in a DMPC/DCHOL Mixed Langmuir Film: A Combined Brewster Angle, Fluorescence and Light Scattering Microscopy Study

Fluorescence microscopy [FM] is one of the most direct imaging techniques for in situ observation of morphology and phase-separation at the macroscopic scale [1] in lipid mono- or bi-layers. However, the presence of fluorescent dye-molecules can affect the system. In Brewster Angle Microscopy (BAM), one can image monomolecular Langmuir films without probes. Here, using a composite set-up of BAM, FM and Light Scattering Microscopy (LSM), we present a comparative study of the three techniques on a binary lipid mixture in the presence of two different probes. In most cases, all three techniques show precisely the same domains. However, depending on conditions, some domain types were more evident in one technique than the others. This established, we can directly test the influence of probe on the domain structure. The appearance of collapsed cholesterol-rich crystallites with film compression, and the influence of probe on this collapse, is also discussed.