Measuring the composition-curvature coupling in binary lipid membranes by computer simulations.

The coupling between local composition fluctuations in binary lipid membranes and curvature affects the lateral membrane structure. We propose an efficient method to compute the composition-curvature coupling in molecular simulations and apply it to two coarse-grained membrane models-a minimal, implicit-solvent model and the MARTINI model. Both the weak-curvature behavior that is typical for thermal fluctuations of planar bilayer membranes as well as the strong-curvature regime corresponding to narrow cylindrical membrane tubes are studied by molecular dynamics simulation. The simulation results are analyzed by using a phenomenological model of the thermodynamics of curved, mixed bilayer membranes that accounts for the change of the monolayer area upon bending. Additionally the role of thermodynamic characteristics such as the incompatibility between the two lipid species and asymmetry of composition are investigated.

Comparison of ternary bilayer mixtures with asymmetric or symmetric unsaturated phosphatidylcholine lipids by coarse grained molecular dynamics simulations.

We studied the phase behavior of various ternary bilayer mixtures composed of cholesterol, an unsaturated lipid, and a fully saturated lipid, by means of molecular dynamics simulations of the MARTINI coarse grain model. We aimed at comparing lateral organization and local properties of bilayers containing phosphatidylcholine (PC) lipids, either with two unsaturated tails (symmetric), or one unsaturated and one saturated tail (asymmetric), as the low-melting component of the mixture. The number of unsaturations per chain was systematically varied in both classes of unsaturated lipids, to account for its consequences in segregation. In the asymmetric unsaturated PCs, the saturated tail was kept identical to the hydrophobic chains of the fully saturated lipid component. Membranes with a symmetric or an asymmetric unsaturated lipid, with the same kind of unsaturated chain, show different phase behavior. Symmetric polyunsaturated PCs set the separation in two phases. Instead, the asymmetric polyunsaturated lipids induced nonideal mixing of components in single-phase bilayers. A significative drop of temperature, within the accessible temperature range, enhances the segregation in mixtures with the more unsaturated asymmetric PC, but still within a single phase. This different phase behavior between membranes with symmetric and asymmetric unsaturated PCs is also observed for lipids with the same total number of unsaturations. On the other hand, the degree of unsaturation per se enhances the segregation, by increasing the composition fluctuations in single-phase membranes with asymmetric PC lipids, and raising the line tension in the two-phase bilayer mixtures with symmetric polyunsaturated PCs. Dynamic clusters of unsaturated asymmetric lipids can be identified. The clusters show no correlation between leaflets, as observed for the phase domains in mixtures with the symmetric polyunsaturated PCs. Interestingly, we found that asymmetric PC lipids have a preferential orientation such that their saturated tails increase their density toward the periphery of the clusters, facing regions enriched in the fully saturated lipids and cholesterol. The degree of unsaturation increases the cluster size and also enhances the anisotropy of the orientation. The surface density of cholesterol follows a gradient that favors its interaction with the saturated tails. Such gradients in composition lead to gradients in order parameters, such as the conformational order and the area of the tails, which increases away from the unsaturated lipid clusters. We compared, in addition, differences in hydrophobic length mismatch between acyl chains of the low-melting and high-melting components, in mixtures containing either symmetric or asymmetric unsaturated lipids.

Polyunsaturated and saturated phospholipids in mixed bilayers: a study from the molecular scale to the lateral lipid organization.

Polyunsaturated lipids are remarkably flexible molecules, with a great influence on the membrane structure and dynamics, affecting from mechanical properties to domain segregation. In this work, we studied phospholipid mixtures of dipalmitoylphosphatidylcholine (DPPC) and diunsaturated phosphatidylcholine lipids (diunsPC) of different lengths, by means of molecular dynamic simulations of a coarse-grained interaction model. These diunsPC:DPPC binary mixtures show nonideal behavior characterized by one mixed phase with composition fluctuations on a length scale of nanometers. Motivated by this observation, we studied comprehensively the characteristics of molecular structure as a function of the compositional gradient. We analyzed orientational order profiles, density distributions, and pair-pair correlation functions between the molecule residues. We observed that, in diunsPC-enriched regions, DPPC tails become expanded and disordered, especially toward the membrane center. On the other hand, in the more condensed DPPC-enriched patches, diunsaturated acyl chains become displaced toward the interface instead of stretching along the membrane normal. From the comparison of the two diunsPC lipids of different tail length, we measured that the presence of a longer terminal saturated segment induces better mixing with DPPC, and most interestingly eliminates the up-down composition correlation measured with the shorter tail-diunsPC. At molecular level, there is a reduced redistribution of densities and changes in the local order as a function of composition. We interpret these results as indicative that the packing incompatibility between polyunsaturated and saturated lipids rules their mixing behavior.

Micron-scale phase segregation in lipid monolayers induced by myelin basic protein in the presence of a cholesterol analog.

It was previously shown that myelin basic protein (MBP) can induce phase segregation in whole myelin monolayers and myelin lipid films, which leads to the accumulation of proteins into a separate phase, segregated from a cholesterol-enriched lipid phase. In this work we investigated some factors regulating the phase segregation induced by MBP using fluorescent microscopy of monolayers formed with binary and ternary lipid mixtures of dihydrocholesterol (a less-oxidable cholesterol analog) and phospholipids. The influence of the addition of salts to the subphase and of varying the lipid composition was analyzed. Our results show that MBP can induce a dihydrocholesterol-dependent segregation of phases that can be further regulated by the electrolyte concentration in the subphase and the composition (type and proportion) of non-sterol lipids. In this way, changes of the lipid composition of the film or the ionic strength in the aqueous media modify the local surface density of MBP and the properties (phase state and composition) of the protein environment.

Biophysics of sphingolipids II. Glycosphingolipids: an assortment of multiple structural information transducers at the membrane surface.

Glycosphingolipids are ubiquitous components of animal cell membranes. They are constituted by the basic structure of ceramide with its hydroxyl group linked to single carbohydrates or oligosaccharide chains of different complexity. The combination of the properties of their hydrocarbon moiety with those derived from the variety and complexity of their hydrophilic polar head groups confers to these lipids an extraordinary capacity for molecular-to-supramolecular transduction across the lateral/transverse planes in biomembranes and beyond. In our opinion, most of the advances made over the last decade on the biophysical behavior of glycosphingolipids can be organized into three related aspects of increasing structural complexity: (1) intrinsic codes: local molecular interactions of glycosphingolipids translated into structural self-organization. (2) Surface topography: projection of molecular shape and miscibility of glycosphingolipids into formation of coexisting membrane domains. (3) Beyond the membrane interface: glycosphingolipid as modulators of structural topology, bilayer recombination and surface biocatalysis.

Protein-mediated surface structuring in biomembranes

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

Protein-mediated surface structuring in biomembranes.

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

The Folch-Lees proteolipid induces phase coexistence and transverse reorganization of lateral domains in myelin monolayers.

Solvent solubilized myelin membranes spread as monomolecular layers at the air-water interface show a heterogeneous pattern at all surface pressures. In order to asses the role of myelin protein and lipid components in the surface structuring we compared the topography, as seen by Brewster angle microscopy (BAM) and epifluorescence microscopy, of monolayers made from mixtures containing all myelin lipids (except gangliosides) and variable proportions of Folch-Lees proteolipid protein (PLP, the major protein component of myelin). The presence of the single PLP, in the absence of the other myelin proteins, can reproduce the surface pattern of the whole myelin extract films in a concentration-dependant manner. Moreover, a threshold mole fraction of PLP is necessary to induce the lipid-protein component reorganization leading to the appearance of a rigid (gray) phase, acting as a surface skeleton, at low surface pressures and of fractal clusters at high surface pressures. The average size of those clusters is also dependent on the PLP content in the monolayer and on the time elapsed from the moment of film spreading, as they apparently result from an irreversible lateral aggregation process. The transverse rearrangement of the monolayer occurring under compression was different in films with the highest and lowest PLP mole fractions tested.

Our results in the treatment of fractures dislocations of Lisfranc's joint.

The authors report their experience in the treatment of traumatic injuries of Lisfranc's joint based on 30 cases treated by surgery between 1984 and 1999. All of the patients were re-evaluated clinically and radiographically. What emerges from the study is the need for surgical stabilization with percutaneous Kirschner wires or by open procedure in cases where there are doubts or where reduction is impossible. The prognosis is worse in injuries of the medial column and in exposed fractures or when mortification of the soft tissues is present.

The sensitivity and specificity of forecasting high-cost users of medical care.

OBJECTIVES: This study compares the ability of 3 risk-assessment models to distinguish high and low expense-risk status within a managed care population. Models are the Global Risk-Assessment Model (GRAM) developed at the Kaiser Permanente Center for Health Research; a logistic version of GRAM; and a prior-expense model. GRAM was originally developed for use in adjusting Medicare payments to health plans. METHODS: Our sample of 98,985 cases was drawn from random samples of memberships of 3 staff/group health plans. Risk factor data were from 1992 and expenses were measured for 1993. Models produced distributions of individual-level annual expense forecasts (or predicted probabilities of high expense-risk status for logistic) for comparison to actual values. Prespecified "high-cost" thresholds were set within each distribution to analyze the models' ability to distinguish high and low expense-risk status. Forecast stability was analyzed through bootstrapping. RESULTS: GRAM discriminates better overall than its comparators (although the models are similar for policy-relevant thresholds). All models forecast the highest-cost cases relatively well. GRAM forecasts high expense-risk status better than its comparators within chronic and serious disease categories that are amenable to early intervention but also generates relatively more false positives within these categories. CONCLUSIONS: This study demonstrates the potential of risk-assessment models to inform care management decisions by efficiently screening managed care populations for high expense-risk. Such models can act as preliminary screens for plans that can refine model forecasts with detailed surveys. Future research should involve multiple-year data sets to explore the temporal stability of forecasts.

Contraction stress testing with nipple stimulation.

The contraction stress test (CST) is used widely as a measure of fetoplacental respiratory reserve. With contractions traditionally induced by intravenous oxytocin, the test has been limited in its use by time, expense and patient discomfort. In a prospective evaluation of the effectiveness of nipple stimulation for the production of uterine contractions, a successful CST was obtained in 94% of the attempts, with a mean total test time of 12 minutes.