Distinct membrane mechanical properties of human mesenchymal stem cells determined using laser optical tweezers

The therapeutic efficacy of mesenchymal stem cells (MSCs) in tissue engineering and regenerative medicine is determined by their unique biological, mechanical, and physicochemical characteristics, which are yet to be fully explored. Cell membrane mechanics, for example, has been shown to critically influence MSC differentiation. In this study, we used laser optical tweezers to measure the membrane mechanics of human MSCs and terminally differentiated fibroblasts by extracting tethers from the outer cell membrane. The average tether lengths were 10.6+/-1.1 microm (hMSC) and 3.0+/-0.5 microm (fibroblasts). The tether extraction force did not increase during tether formation, which suggests existence of a membrane reservoir intended to buffer membrane tension fluctuations. Cytoskeleton disruption resulted in a fourfold tether length increase in fibroblasts but had no effect in hMSCs, indicating weak association between the cell membrane and hMSC actin cytoskeleton. Cholesterol depletion, known to decrease lipid bilayer stiffness, caused an increase in the tether length both in fibroblasts and hMSCs, as does the treatment of cells with DMSO. We postulate that whereas fibroblasts use both the membrane rigidity and membrane-cytoskeleton association to regulate their membrane reservoir, hMSC cytoskeleton has only a minor impact on stem cell membrane mechanics.     

Direct observation of membrane tethers formed during neutrophil attachment to platelets or P-selectin under physiological flow

Adhesion and subsequent aggregation between neutrophils and platelets is dependent upon the initial binding of P-selectin on activated platelets to P-selectin glycoprotein ligand 1 (PSGL-1) on the microvilli of neutrophils. High speed, high resolution videomicroscopy of flowing neutrophils interacting with spread platelets demonstrated that thin membrane tethers were pulled from neutrophils in 32 +/- 4% of the interactions. After capture by spread platelets, neutrophil membrane tethers (length of 5.9 +/- 4.1 microm, n = 63) were pulled at an average rate of 6-40 microm/s as the wall shear rate was increased from 100-250 s(-1). The average tether lifetime decreased significantly (P < 0.001) from 630 to 133 ms as the shear rate was increased from 100 s(-1) (F(bond) = 86 pN) to 250 s(-1) (F(bond) = 172 pN), which is consistent with P-selectin/PSGL-1 bond dynamics under stress. Tether formation was blocked by antibodies against P-selectin or PSGL-1, but not by anti-CD18 antibodies. During neutrophil rolling on P-selectin at 150 s(-1), thin membrane tethers were also pulled from the neutrophils. The characteristic jerking motion of the neutrophil coexisted with tether growth (8.9 +/- 8.8 microm long), whereas tether breakage (average lifetime of 3.79 +/- 3.32 s) caused an acute jump in the rolling velocity, proving multiple bonding in the cell surface and the tether surface contact area. Extremely long membrane tethers (>40 microm) were sometimes pulled, which detached in a flow-dependent mechanism of microparticle formation. Membrane tethers were also formed when neutrophils were perfused over platelet monolayers. These results are the first visualization of the often hypothesized tethers that shield the P-selectin/PSGL-1 bond from force loading to regulate neutrophil rolling during inflammation and thrombosis.     

Eukaryotic membrane tethers revisited using magnetic tweezers

Membrane nanotubes, under physiological conditions, typically form en masse. We employed magnetic tweezers (MTW) to extract tethers from human brain tumor cells and compared their biophysical properties with tethers extracted after disruption of the cytoskeleton and from a strongly differing cell type, Chinese hamster ovary cells. In this method, the constant force produced with the MTW is transduced to cells through super-paramagnetic beads attached to the cell membrane. Multiple sudden jumps in bead velocity were manifest in the recorded bead displacement-time profiles. These discrete events were interpreted as successive ruptures of individual tethers. Observation with scanning electron microscopy supported the simultaneous existence of multiple tethers. The physical characteristics, in particular, the number and viscoelastic properties of the extracted tethers were determined from the analytic fit to bead trajectories, provided by a standard model of viscoelasticity. Comparison of tethers formed with MTW and atomic force microscopy (AFM), a technique where the cantilever-force transducer is moved at constant velocity, revealed significant differences in the two methods of tether formation. Our findings imply that extreme care must be used to interpret the outcome of tether pulling experiments performed with single molecular techniques (MTW, AFM, optical tweezers, etc). First, the different methods may be testing distinct membrane structures with distinct properties. Second, as soon as a true cell membrane (as opposed to that of a vesicle) can attach to a substrate, upon pulling on it, multiple nonspecific membrane tethers may be generated. Therefore, under physiological conditions, distinguishing between tethers formed through specific and nonspecific interactions is highly nontrivial if at all possible.     

Molecular structure of membrane tethers

Membrane tethers are nanotubes formed by a lipid bilayer. They play important functional roles in cell biology and provide an experimental window on lipid properties. Tethers have been studied extensively in experiments and described by theoretical models, but their molecular structure remains unknown due to their small diameters and dynamic nature. We used molecular dynamics simulations to obtain molecular-level insight into tether formation. Tethers were pulled from single-component lipid bilayers by application of an external force to a lipid patch along the bilayer normal or by lateral compression of a confined bilayer. Tether development under external force proceeded by viscoelastic protrusion followed by viscous lipid flow. Weak forces below a threshold value produced only a protrusion. Larger forces led to a crossover to tether elongation, which was linear at a constant force. Under lateral compression, tethers formed from undulations of unrestrained bilayer area. We characterized in detail the tether structure and its formation process, and obtained the material properties of the membrane. To our knowledge, these results provide the first molecular view of membrane tethers.     

Multiple membrane tethers probed by atomic force microscopy

Using the atomic force microscope to locally probe the cell membrane, we observed the formation of multiple tethers (thin nanotubes, each requiring a similar pulling force) as reproducible features within force profiles recorded on individual cells. Forces obtained with Chinese hamster ovary cells, a malignant human brain tumor cell line, and human endothelial cells (EA hy926) were found to be 28 +/- 10 pN, 29 +/- 9 pN, and 29 +/- 10 pN, respectively, independent of the nature of attachment to the cantilever. The rather large variation of the tether pulling forces measured at several locations on individual cells points to the existence of heterogeneity in the membrane properties of a morphologically homogeneous cell. Measurement of the summary lengths of the simultaneously extracted tethers provides a measure of the size of the available membrane reservoir through which co-existing tethers are associated. As expected, partial disruption of the actin cytoskeleton and removal of the hyaluronan backbone of the glycocalyx were observed to result in a marked decrease (30-50%) in the magnitude and a significant sharpening of the force distribution indicating reduced heterogeneity of membrane properties. Taken together, our results demonstrate the ability of the plasma membrane to locally produce multiple interdependent tethers-a process that could play an important role in the mechanical association of cells with their environment.     

Modeling the mechanics of tethers pulled from the cochlear outer hair cell membrane

Cell membrane tethers are formed naturally (e.g., in leukocyte rolling) and experimentally to probe membrane properties. In cochlear outer hair cells, the plasma membrane is part of the trilayer lateral wall, where the membrane is attached to the cytoskeleton by a system of radial pillars. The mechanics of these cells is important to the sound amplification and frequency selectivity of the ear. We present a modeling study to simulate the membrane deflection, bending, and interaction with the cytoskeleton in the outer hair cell tether pulling experiment. In our analysis, three regions of the membrane are considered: the body of a cylindrical tether, the area where the membrane is attached and interacts with the cytoskeleton, and the transition region between the two. By using a computational method, we found the shape of the membrane in all three regions over a range of tether lengths and forces observed in experiments. We also analyze the effects of biophysical properties of the membrane, including the bending modulus and the forces of the membrane adhesion to the cytoskeleton. The model's results provide a better understanding of the mechanics of tethers pulled from cell membranes.     

Mechanical properties of neuronal growth cone membranes studied by tether formation with laser optical tweezers.

Many cell phenomena involve major morphological changes, particularly in mitosis and the process of cell migration. For cells or neuronal growth cones to migrate, they must extend the leading edge of the plasma membrane as a lamellipodium or filopodium. During extension of filopodia, membrane must move across the surface creating shear and flow. Intracellular biochemical processes driving extension must work against the membrane mechanical properties, but the forces required to extend growth cones have not been measured. In this paper, laser optical tweezers and a nanometer-level analysis system were used to measure the neuronal growth cone membrane mechanical properties through the extension of filopodia-like tethers with IgG-coated beads. Although the probability of a bead attaching to the membrane was constant irrespective of treatment; the probability of forming a tether with a constant force increased dramatically with cytochalasin B or D and dimethylsulfoxide (DMSO). These are treatments that alter the organization of the actin cytoskeleton. The force required to hold a tether at zero velocity (F0) was greater than forces generated by single molecular motors, kinesin and myosin; and F0 decreased with cytochalasin B or D and DMSO in correlation with the changes in the probability of tether formation. The force of the tether on the bead increased linearly with the velocity of tether elongation. From the dependency of tether force on velocity of tether formation, we calculated a parameter related to membrane viscosity, which decreased with cytochalasin B or D, ATP depletion, nocodazole, and DMSO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deformation and flow of membrane into tethers extracted from neuronal growth cones.

Membrane tethers are extracted at constant velocity from neuronal growth cones using a force generated by a laser tweezers trap. A thermodynamic analysis shows that as the tether is extended, energy is stored in the tether as bending and adhesion energies and in the cell body as "nonlocal" bending. It is postulated that energy is dissipated by three viscous mechanisms including membrane flow, slip between the two monolayers that form the bilayer, and slip between membrane and cytoskeleton. The analysis predicts and the experiments show a linear relation between tether force and tether velocity. Calculations based on the analytical results and the experimental measurements of a tether radius of approximately 0.2 micron and a tether force at zero velocity of approximately 8 pN give a bending modulus for the tether of 2.7 x 10(-19) N.m and an extraordinarily small "apparent surface tension" in the growth cone of 0.003 mN/m, where the apparent surface tension is the sum of the far-field, in-plane tension and the energy of adhesion. Treatments with cytochalasin B and D, ethanol, and nocodazole affect the apparent surface tension but not bending. ATP depletion affects neither, whereas large concentrations of DMSO affect both. Under conditions of flow, data are presented to show that the dominant viscous mechanism comes from the slip that occurs when the membrane flows over the cytoskeleton. ATP depletion and the treatment with DMSO cause a dramatic drop in the effective viscosity. If it is postulated that the slip between membrane and cytoskeleton occurs in a film of water, then this water film has a mean thickness of only approximately 10 A.     

Membrane tether formation from outer hair cells with optical tweezers

Optical tweezers were used to characterize the mechanical properties of the outer hair cell (OHC) plasma membrane by pulling tethers with 4.5-microm polystyrene beads. Tether formation force and tether force were measured in static and dynamic conditions. A greater force was required for tether formations from OHC lateral wall (499 +/- 152 pN) than from OHC basal end (142 +/- 49 pN). The difference in the force required to pull tethers is consistent with an extensive cytoskeletal framework associated with the lateral wall known as the cortical lattice. The apparent plasma membrane stiffness, estimated under the static conditions by measuring tether force at different tether length, was 3.71 pN/microm for OHC lateral wall and 4.57 pN/microm for OHC basal end. The effective membrane viscosity was measured by pulling tethers at different rates while continuously recording the tether force, and estimated in the range of 2.39 to 5.25 pN x s/microm. The viscous force most likely results from the viscous interactions between plasma membrane lipids and the OHC cortical lattice and/or integral membrane proteins. The information these studies provide on the mechanical properties of the OHC lateral wall is important for understanding the mechanism of OHC electromotility.     

Visco-elastic membrane tethers extracted from Escherichia coli by optical tweezers

Tethers were created between a living Escherichia coli bacterium and a bead by unspecifically attaching the bead to the outer membrane and pulling it away using optical tweezers. Upon release, the bead returned to the bacterium, thus showing the existence of an elastic tether between the bead and the bacterium. These tethers can be tens of microns long, several times the bacterial length. Using mutants expressing different parts of the outer membrane structure, we have shown that an intact core lipopolysaccharide is a necessary condition for tether formation, regardless of whether the beads were uncoated polystyrene or beads coated with lectin. A physical characterization of the tethers has been performed yielding visco-elastic tether force-extension relationships: for first pull tethers, a spring constant of 10-12 pN/μm describes the tether visco-elasticity, for subsequent pulls the spring constant decreases to 6-7 pN/μm, and typical relaxation timescales of hundreds of seconds are observed. Studies of tether stability in the presence of proteases, lipases, and amylases lead us to propose that the extracted tether is primarily composed of the asymmetric lipopolysaccharide containing bilayer of the outer membrane. This unspecific tethered attachment mechanism could be important in the initiation of bacterial adhesion.     

Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells

Recognition of the growing role of human mesenchymal stem cells (hMSC) in tissue engineering and regenerative medicine requires a thorough understanding of intracellular biochemical and biophysical processes that may direct the cell's commitment to a particular lineage. In this study, we characterized the distinct biomechanical properties of hMSCs, including the average Young's modulus determined by atomic force microscopy (3.2 +/- 1.4 kPa for hMSC vs. 1.7 +/- 1.0 kPa for fully differentiated osteoblasts), and the average membrane tether length measured with laser optical tweezers (10.6 +/- 1.1 microm for stem cells, and 4.0 +/- 1.1 microm for osteoblasts). These differences in cell elasticity and membrane mechanics result primarily from differential actin cytoskeleton organization in these two cell types, whereas microtubules did not appear to affect the cellular mechanics. The membrane-cytoskeleton linker proteins may contribute to a stronger interaction of the plasma membrane with F-actins and shorter membrane tether length in osteoblasts than in stem cells. Actin depolymerization or ATP depletion caused a two- to threefold increase in the membrane tether length in osteoblasts, but had essentially no effect on the stem-cell membrane tethers. Actin remodeling in the course of a 10-day osteogenic differentiation of hMSC mediates the temporally correlated dynamical changes in cell elasticity and membrane mechanics. For example, after a 10-day culture in osteogenic medium, hMSC mechanical characteristics were comparable to those of mature bone cells. Based on quantitative characterization of the actin cytoskeleton remodeling during osteodifferentiation, we postulate that the actin cytoskeleton plays a pivotal role in determining the hMSC mechanical properties and modulation of cellular mechanics at the early stage of stem-cell osteodifferentiation.     

Membrane tethers formed from blood cells with available area and determination of their adhesion energy

Fundamental to all mammalian cells is the adherence of the lipid bilayer membrane to the underlying membrane associated cytoskeleton. To investigate this adhesion, we physically detach the lipid membrane from the cell by mechanically forming membrane tethers. For the most part these have been tethers formed from either neutrophils or red cells. Here we do a simple thermodynamic analysis of the tether formation process using the entire cell, including tether, as the control volume. For a neutrophil, we show that the total adhesion energy per unit area between lipid membrane and cytoskeleton depends on the square of the tether force. For a flaccid red cell, we show that the total adhesion energy minus the tension in the spectrin cytoskeleton depends also on the square of the tether force. Finally, we discuss briefly the viscous flow of membrane. Using published data we calculate and compare values for the various adhesion energies and viscosities.     

Cell protrusions and tethers: a unified approach

Low pulling forces applied locally to cell surface membranes produce viscoelastic cell surface protrusions. As the force increases, the membrane can locally separate from the cytoskeleton and a tether forms. Tethers can grow to great lengths exceeding the cell diameter. The protrusion-to-tether transition is known as the crossover. Here we propose a unified approach to protrusions and tethers providing, to our knowledge, new insights into their biomechanics. We derive a necessary and sufficient condition for a crossover to occur, a formula for predicting the crossover time, conditions for a tether to establish a dynamic equilibrium (characterized by constant nonzero pulling force and tether extension rate), a general formula for the tether material after crossover, and a general modeling method for tether pulling experiments. We introduce two general protrusion parameters, the spring constant and effective viscosity, valid before and after crossover. Their first estimates for neutrophils are 50 pN μm⁻¹ and 9 pN s μm⁻¹, respectively. The tether elongation after crossover is described as elongation of a viscoelastic-like material with a nonlinearly decaying spring (NLDs-viscoelastic material). Our model correctly describes the results of the published protrusion and tether pulling experiments, suggesting that it is universally applicable to such experiments.     

Formation and maintenance of tubular membrane projections require mechanical force, but their elongation and shortening do not require additional force

Living cells develop their own characteristic shapes depending on their physiological functions, and their morphologies are based on the mechanical characteristics of the cytoskeleton and of membranes. To investigate the role of lipid membranes in morphogenesis, we constructed a simple system that can manipulate liposomes and measure the forces required to transform their shapes. Two polystyrene beads (1 microm in diameter) were encapsulated in giant liposomes and were manipulated using double-beam laser tweezers. Without any specific interaction between the lipid membrane and beads, mechanical forces could be applied to the liposome membrane from the inside. Spherical liposomes transformed into a lemon shape with increasing tension, and tubular membrane projections were subsequently generated in the tips at either end. This process is similar to the liposomal transformation caused by elongation of encapsulated cytoskeletons. In the elongation stage of lemon-shaped liposomes, the force required for the transformation became larger as the end-to-end length increased. Just before the tubular membrane was generated, the force reached the maximum strength (approximately 11 pN). However, immediately after the tubular membrane developed, the force suddenly decreased and was maintained at a constant strength (approximately 4 pN) that was independent of further tube elongation or shortening, even though there was no excess membrane reservoir as occurs in living cells. When the tube length was shortened to approximately 2 microm, the liposome reversed to a lemon shape and the force temporarily increased (to approximately 7 pN). These results indicate that the simple application of mechanical force is sufficient to form a protrusion in a membrane, that a critical force and length is needed to form and to maintain the protrusion, and suggest that the lipid bilayer itself has the ability to buffer the membrane tension.     

Effects of chlorpromazine on mechanical properties of the outer hair cell plasma membrane

An optical tweezers system was used to characterize the effects of chlorpromazine (CPZ) on the mechanical properties of the mammalian outer hair cell (OHC) through the formation of plasma membrane tethers. Such tethers exhibited force relaxation when held at a constant length for several minutes. We used a second-order generalized Kelvin body to model tether-force behavior from which several mechanical parameters were then calculated including stiffness, viscosity-associated measures, and force relaxation time constants. The results of the analysis portray a two-part relaxation process characterized by significantly different rates of force decay, which we propose is due to the local reorganization of lipids within the tether and the flow of external lipid into the tether. We found that CPZ's effect was limited to the latter phenomenon since only the second phase of relaxation was significantly affected by the drug. This finding coupled with an observed large reduction in overall tether forces implies a common basis for the drug's effects, the plasma membrane-cytoskeleton interaction. The CPZ-induced changes in tether viscoelastic behavior suggest that alterations in the mechanical properties of the OHC lateral wall could play a role in the modulation of OHC electromotility by CPZ.     

Extensional flow of erythrocyte membrane from cell body to elastic tether. II. Experiment.

This is the second of two papers on an analytical and experimental study of the flow of erythrocyte membrane. In the experiments discussed here, preswollen human erythrocytes are sphered by aspirating a portion of the cell membrane into a small micropipette; and long, thin, membrane filaments or tethers are steadily withdrawn from the cell at a point diametrically opposite to the point of aspiration. The aspirated portion of the membrane furnishes a reservoir of material that replaces the membrane as it flows as a liquid from the nearly spherical cell body to the cylindrical tether. The application of the principle of conservation of mass permits the tether radius Rt to be measured with the light microscope as the tether is formed and extended at a constant rate. The tether behaves as an elastic solid such that the tether radius decreases as the force or axial tension acting on the tether is increased. For the range of values for Rt is these experiments (100 A less than or equal to Rt less than or equal to 200 A), the slope of the tether-force, tether-radius line is -1.32 dyn/cm. The surface viscosity of the membrane as it flows from cell body to tether is 3 x 10(-3) dyn.s/cm. This viscosity is essentially constant for characteristic rates of deformation between 10 and 200 s-1.     

Tether extrusion from red blood cells: integral proteins unbinding from cytoskeleton

We investigate the mechanical strength of adhesion and the dynamics of detachment of the membrane from the cytoskeleton of red blood cells (RBCs). Using hydrodynamical flows, we extract membrane tethers from RBCs locally attached to the tip of a microneedle. We monitor their extrusion and retraction dynamics versus flow velocity (i.e., extrusion force) over successive extrusion-retraction cycles. Membrane tether extrusion is carried out on healthy RBCs and ATP-depleted or -inhibited RBCs. For healthy RBCs, extrusion is slow, constant in velocity, and reproducible through several extrusion-retraction cycles. For ATP-depleted or -inhibited cells, extrusion dynamics exhibit an aging phenomenon through extrusion-retraction cycles: because the extruded membrane is not able to retract properly onto the cell body, each subsequent extrusion exhibits a loss of resistance to tether growth over the tether length extruded at the previous cycle. In contrast, the additionally extruded tether length follows healthy dynamics. The extrusion velocity L depends on the extrusion force f according to a nonlinear fashion. We interpret this result with a model that includes the dynamical feature of membrane-cytoskeleton association. Tether extrusion leads to a radial membrane flow from the cell body toward the tether. In a distal permeation regime, the flow passes through the integral proteins bound to the cytoskeleton without affecting their binding dynamics. In a proximal sliding regime, where membrane radial velocity is higher, integral proteins can be torn out, leading to the sliding of the membrane over the cytoskeleton. Extrusion dynamics are governed by the more dissipative permeation regime: this leads to an increase of the membrane tension and a narrowing of the tether, which explains the power law behavior of L(f). Our main result is that ATP is necessary for the extruded membrane to retract onto the cell body. Under ATP depletion or inhibition conditions, the aging of the RBC after extrusion is interpreted as a perturbation of membrane-cytoskeleton linkage dynamics.     

The viscoelastic properties of microvilli are dependent upon the cell-surface molecule

We studied at nanometer resolution the viscoelastic properties of microvilli and tethers pulled from myelogenous cells via P-selectin glycoprotein ligand 1 (PSGL-1) and found that in contrast to pure membrane tethers, the viscoelastic properties of microvillus deformations are dependent upon the cell-surface molecule through which load is applied. A laser trap and polymer bead coated with anti-PSGL-1 (KPL-1) were used to apply step loads to microvilli. The lengthening of the microvillus in response to the induced step loads was fitted with a viscoelastic model. The quasi-steady state force on the microvillus at any given length was approximately fourfold lower in cells treated with cytochalasin D or when pulled with concanavalin A-coated rather than KPL-1-coated beads. These data suggest that associations between PSGL-1 and the underlying actin cytoskeleton significantly affect the early stages of leukocyte deformation under flow.     

Cell Membrane Tethers Generate Mechanical Force in Response to Electrical Stimulation

Living cells maintain a huge transmembrane electric field across their membranes. This electric field exerts a force on the membrane because the membrane surfaces are highly charged. We have measured electromechanical force generation by cell membranes using optically trapped beads to detach the plasma membrane from the cytoskeleton and form long thin cylinders (tethers). Hyperpolarizing potentials increased and depolarizing potentials decreased the force required to pull a tether. The membrane tether force in response to sinusoidal voltage signals was a function of holding potential, tether diameter, and tether length. Membrane electromechanical force production can occur at speeds exceeding those of ATP-based protein motors. By harnessing the energy in the transmembrane electric field, cell membranes may contribute to processes as diverse as outer hair cell electromotility, ion channel gating, and transport.     

Determination of bilayer membrane bending stiffness by tether formation from giant, thin-walled vesicles.

The curvature elastic modulus (bending stiffness) of stearoyloleoyl phosphatidylcholine (SOPC) bilayer membrane is determined from membrane tether formation experiments. R. E. Waugh and R. M. Hochmuth 1987. Biophys. J. 52:391-400) have shown that the radius of a bilayer cylinder (tether) is inversely related to the force supported along its axis. The coefficient that relates the axial force on the tether to the tether radius is the membrane bending stiffness. Thus, the bending stiffness can be calculated directly from measurements of the tether radius as a function of force. Giant (10-50-microns diam) thin-walled vesicles were aspirated into a micropipette and a tether was pulled out of the surface by gravitational forces on small glass beads that had adhered to the vesicle surface. Because the vesicle keeps constant surface area and volume, formation of the tether requires displacement of material from the projection of the vesicle in the pipette. Tethers can be made to grow longer or shorter or to maintain equilibrium by adjusting the aspiration pressure in the micropipette at constant tether force. The ratio of the change in the length of the tether to the change in the projection length is proportional to the ratio of the pipette radius to the tether radius. Thus, knowing the density and diameter of the glass beads and measuring the displacement of the projection as a function of tether length, independent determinations of the force on the tether and the tether radius were obtained. The bending stiffness for an SOPC bilayer obtained from these data is approximately 2.0 x 10(-12) dyn cm, for tether radii in the range of 20-100 nm.(ABSTRACT TRUNCATED AT 250 WORDS)