On the Physical Basis of Biological Signaling by Interface Pulses.

Currently, biological signaling is envisaged as a combination of activation and movement, triggered by local molecular interactions and molecular diffusion, respectively. However, here, we suggest that other fundamental physical mechanisms might play an at least equally important role. We have recently shown that lipid interfaces permit the excitation and propagation of sound pulses. Here, we demonstrate that these reversible perturbations can control the activity of membrane-embedded enzymes without a requirement for molecular transport. They can thus facilitate rapid communication between distant biological entities at the speed of sound, which is here on the order of 1 m/s within the membrane. The mechanism described provides a new physical framework for biological signaling that is fundamentally different from the molecular approach that currently dominates the textbooks.

Circulating but not immobilized N-deglycosylated von Willebrand factor increases platelet adhesion under flow conditions.

The role of von Willebrand factor (VWF) as a shear stress activated platelet adhesive has been related to a coiled-elongated shape conformation. The forces dominating this transition have been suggested to be controlled by the proteins polymeric architecture. However, the fact that 20% of VWF molecular weight originates from glycan moieties has so far been neglected in these calculations. In this study, we present a systematic experimental investigation on the role of N-glycosylation for VWF mediated platelet adhesion under flow. A microfluidic flow chamber with a stenotic compartment that allows one to mimic various physiological flow conditions was designed for the efficient analysis of the adhesion spectrum. Surprisingly, we found an increase in platelet adhesion with elevated shear rate, both qualitatively and quantitatively fully conserved when N-deglycosylated VWF (N-deg-VWF) instead of VWF was immobilized in the microfluidic channel. This has been demonstrated consistently over four orders of magnitude in shear rate. In contrast, when N-deg-VWF was added to the supernatant, an increase in adhesion rate by a factor of two was detected compared to the addition of wild-type VWF. It appears that once immobilized, the role of glycans is at least modified if not-as found here for the case of adhesion-negated. These findings strengthen the physical impact of the circulating polymer on shear dependent platelet adhesion events. At present, there is no theoretical explanation for an increase in platelet adhesion to VWF in the absence of its N-glycans. However, our data indicate that the effective solubility of the protein and hence its shape or conformation may be altered by the degree of glycosylation and is therefore a good candidate for modifying the forces required to uncoil this biopolymer.

Propagation of 2D pressure pulses in lipid monolayers and its possible implications for biology.

The existence and propagation of acoustic pressure pulses on lipid monolayers at the air-water interface are directly observed by simple mechanical detection. The pulses are excited by small amounts of solvents added to the monolayer. Controlling the state of the lipid interface, we show that the pulses propagate at velocities c following the lateral compressibility κ. This is manifested by a pronounced minimum in c (∼0.3 m/s) within the transition regime. The role of interface density pulses in biology is discussed, in particular, in the context of communicating localized alterations in protein function (signaling) and nerve pulse propagation.

Chemical and mechanical impact of silica nanoparticles on the phase transition behavior of phospholipid membranes in theory and experiment.

The interaction of nanoparticles (NPs) with lipid membranes is an integral step in the interaction of NPs and living cells. During particle uptake, the membrane has to bend. Due to the nature of their phase diagram, the modulus of compression of these membranes can vary by more than one order of magnitude, and thus both the thermodynamic and mechanical aspects of the membrane have to be considered simultaneously. We demonstrate that silica NPs have at least two independent effects on the phase transition of phospholipid membranes: 1), a chemical effect resulting from the finite instability of the NPs in water; and 2), a mechanical effect that originates from a bending of the lipid membrane around the NPs. Here, we report on recent experiments that allowed us to clearly distinguish both effects, and present a thermodynamic model that includes the elastic energy of the membranes and correctly predicts our findings both quantitatively and qualitatively.

Simultaneously propagating voltage and pressure pulses in lipid monolayers of pork brain and synthetic lipids.

Hydrated interfaces are ubiquitous in biology and appear on all length scales from ions and individual molecules to membranes and cellular networks. In vivo, they comprise a high degree of self-organization and complex entanglement, which limits their experimental accessibility by smearing out the individual phenomenology. The Langmuir technique, however, allows the examination of defined interfaces, the controllable thermodynamic state of which enables one to explore the proper state diagrams. Here we demonstrate that voltage and pressure pulses simultaneously propagate along monolayers comprised of either native pork brain or synthetic lipids. The excitation of pulses is conducted by the application of small droplets of acetic acid and monitored subsequently employing time-resolved Wilhelmy plate and Kelvin probe measurements. The isothermal state diagrams of the monolayers for both lateral pressure and surface potential are experimentally recorded, enabling us to predict dynamic voltage pulse amplitudes of 0.1-3 mV based on the assumption of static mechanoelectrical coupling. We show that the underlying physics for such propagating pulses is the same for synthetic and natural extracted (pork brain) lipids and that the measured propagation velocities and pulse amplitudes depend on the compressibility of the interface. Given the ubiquitous presence of hydrated interfaces in biology, our experimental findings seem to support a fundamentally new mechanism for the propagation of signals and communication pathways in biology (signaling), which is based neither on protein-protein or receptor-ligand interaction nor diffusion.

Thermomechanic-electrical coupling in phospholipid monolayers near the critical point.

Lipid monolayers have been shown to represent a powerful tool in studying mechanical and thermodynamic properties of lipid membranes as well as their interaction with proteins. Using Einstein's theory of fluctuations we here demonstrate that an experimentally derived linear relationship both between transition entropy S and area A as well as between transition entropy and charge q implies a linear relationships between compressibility κT, heat capacity cπ, thermal expansion coefficient αT, and electric capacity CT. We demonstrate that these couplings have strong predictive power as they allow calculating electrical and thermal properties from mechanical measurements. The precision of the prediction increases as the critical point TC is approached.

Acoustic driven flow and lattice Boltzmann simulations to study cell adhesion in biofunctionalized mu-fluidic channels with complex geometry.

Accurately mimicking the complexity of microvascular systems calls for a technology which can accommodate particularly small sample volumes while retaining a large degree of freedom in channel geometry and keeping the price considerably low to allow for high throughput experiments. Here, we demonstrate that the use of surface acoustic wave driven microfluidics systems successfully allows the study of the interrelation between melanoma cell adhesion, the matrix protein collagen type I, the blood clotting factor von Willebrand factor (vWF), and microfluidic channel geometry. The versatility of the tool presented enables us to examine cell adhesion under flow in straight and bifurcated microfluidic channels in the presence of different protein coatings. We show that the addition of vWF tremendously increases (up to tenfold) the adhesion of melanoma cells even under fairly low shear flow conditions. This effect is altered in the presence of bifurcated channels demonstrating the importance of an elaborate hydrodynamic analysis to differentiate between physical and biological effects. Therefore, computer simulations have been performed along with the experiments to reveal the entire flow profile in the channel. We conclude that a combination of theory and experiment will lead to a consistent explanation of cell adhesion, and will optimize the potential of microfluidic experiments to further unravel the relation between blood clotting factors, cell adhesion molecules, cancer cell spreading, and the hydrodynamic conditions in our microcirculatory system.

Transport, separation, and accumulation of proteins on supported lipid bilayers.

Transport, separation, and accumulation of proteins in their natural environment are central goals in protein biotechnology. Miniaturized assays of supported lipid bilayers (SLBs) have been proposed as promising candidates to realize such technology on a chip, but a modular system for the controlled transport of membrane proteins does not exist. In this letter, we demonstrate that standing surface acoustic waves drive the in-plane redistribution of proteins on planar SLBs over macroscopic distances (3.5 mm). Accumulation of proteins in periodic patterns of about 10-fold protein concentration difference is accomplished and shown to relax into the homogeneous state by diffusion. Different proteins separate in individual fractions from a homogeneous distribution and are transported and accumulated into clusters using beats. The modular planar setup has the potential of integrating other lab-on-a-chip tools, for monitoring the membrane-protein integrity or adding microfluidic features for blood screening or DNA analysis.

Wave propagation in lipid monolayers.

Sound waves are excited on lipid monolayers using a set of planar electrodes aligned in parallel with the excitable medium. By measuring the frequency-dependent change in the lateral pressure, we are able to extract the sound velocity for the entire monolayer phase diagram. We demonstrate that this velocity can also be directly derived from the lipid monolayer compressibility, and consequently displays a minimum in the phase transition regime. This minimum decreases from v(0) = 170 m/s for one-component lipid monolayers down to v(m) = 50 m/s for lipid mixtures. No significant attenuation can be detected confirming an adiabatic phenomenon. Finally, our data propose a relative lateral density oscillation of Deltarho/rho approximately 2%, implying a change in all area-dependent physical properties. Order-of-magnitude estimates from static couplings therefore predict propagating changes in surface potential of 1-50 mV, 1 unit in pH (electrochemical potential), and 0.01 K in temperature, and fall within the same order of magnitude as physical changes measured during nerve pulse propagation. These results therefore strongly support the idea of propagating adiabatic sound waves along nerves as first thoroughly described by Kaufmann in 1989 and recently by Heimburg and Jackson, but already claimed by Wilke in 1912.

Phase-state dependent current fluctuations in pure lipid membranes.

Current fluctuations in pure lipid membranes have been shown to occur under the influence of transmembrane electric fields (electroporation) as well as a result from structural rearrangements of the lipid bilayer during phase transition (soft perforation). We demonstrate that the ion permeability during lipid phase transition exhibits the same qualitative temperature dependence as the macroscopic heat capacity of a D15PC/DOPC vesicle suspension. Microscopic current fluctuations show distinct characteristics for each individual phase state. Although current fluctuations in the fluid phase show spikelike behavior of short timescales (approximately 2 ms) with a narrow amplitude distribution, the current fluctuations during lipid phase transition appear in distinct steps with timescales of approximately 20 ms. We propose a theoretical explanation for the origin of timescales and permeability based on a linear relationship between lipid membrane susceptibilities and relaxation times near the phase transition.

Phase transition induced fission in lipid vesicles.

In this work we demonstrate how the first order phase transition in giant unilamellar vesicles (GUVs) can function as a trigger for membrane fission. When driven through their gel-fluid phase transition GUVs exhibit budding or pearl formation. These buds remain connected to the mother vesicle presumably by a small neck. Cooling these vesicles from the fluid phase (T>T(m)) through the phase transition into the gel state (T

Thermodynamic relaxation drives expulsion in giant unilamellar vesicles.

We investigated the thermodynamic relaxation of giant unilamellar vesicles (GUVs) which contained small vesicles within their interior. Quenching these vesicles from their fluid phase (T > T(m)) through the phase transition in the gel state (T < T(m)) drives the inner vesicles to be expelled from the larger mother vesicle via the accompanying decrease in the vesicle area by approximately 25% which forces a pore to open in the mother vesicle. We demonstrate that the proceeding time evolution of the resulting efflux follows the relaxation of the membrane area and describes the entire relaxation process using an Onsager-like non-equilibrium thermodynamics ansatz. As a consequence of the volume efflux, internal vesicles are expelled from the mother vesicle. Although complete sealing of the pore may occur during the expulsion, the global relaxation dynamics is conserved. Finally, comparison of these results to morphological relaxation phenomena found in earlier studies reveals a universal relaxation behaviour in GUVs. When quenched from the fluid to gel phase the typical time scale of relaxation shows little variation and ranges between 4 and 5 s.

Relaxation of ultralarge VWF bundles in a microfluidic-AFM hybrid reactor.

The crucial role of the biopolymer "Von Willebrand factor" (VWF) in blood platelet binding is tightly regulated by the shear forces to which the protein is exposed in the blood flow. Under high-shear conditions, VWFs ability to immobilize blood platelets is strongly increased due to a change in conformation which at sufficient concentration is accompanied by the formation of ultra large VWF bundles (ULVWF). However, little is known about the dynamic and mechanical properties of such bundles. Combining a surface acoustic wave (SAW) based microfluidic reactor with an atomic force microscope (AFM) we were able to study the relaxation of stretched VWF bundles formed by hydrodynamic stress. We found that the dynamical response of the network is well characterized by stretched exponentials, indicating that the relaxation process proceeds through hopping events between a multitude of minima. This finding is in accordance with current ideas of VWF self-association. The longest relaxation time does not show a clear dependence on the length of the bundle, and is dominated by the internal conformations and effective friction within the bundle.

Shear-induced unfolding triggers adhesion of von Willebrand factor fibers.

von Willebrand factor (VWF), a protein present in our circulatory system, is necessary to stop bleeding under high shear-stress conditions as found in small blood vessels. The results presented here help unravel how an increase in hydrodynamic shear stress activates VWF's adhesion potential, leading to the counterintuitive phenomena of enhanced adsorption rate under strong shear conditions. Using a microfluidic device, we were able to mimic a wide range of bloodflow conditions and directly visualize the conformational dynamics of this protein under shear flow. In particular, we find that VWF displays a reversible globule-stretch transition at a critical shear rate gamma(crit) in the absence of any adsorbing surface. Computer simulations reproduce this sharp transition and identify the large size of VWF's repeating units as one of the keys for this unique hydrodynamic activation. In the presence of an adsorbing collagen substrate, we find a large increase in the protein adsorption at the same critical shear rate, suggesting that the globule unfolding in bulk triggers the surface adsorption in the case of a collagen substrate, which provides a sufficient density of binding sites. Monitoring the adsorption process of multiple VWF fibers, we were able to follow the formation of an immobilized network that constitutes a "sticky" grid necessary for blood platelet adhesion under high shear flow. Because areas of high shear stress coincide with a higher chance for vessel wall damage by mechanical forces, we identified the shear-induced increase in the binding probability of VWF as an effective self-regulating repair mechanism of our microvascular system.

Shear-flow-induced unfolding of polymeric globules.

The behavior of a single collapsed polymer under shear flow is examined using hydrodynamic simulations and scaling arguments. Below a threshold shear rate gamma[.]{*}, the chain remains collapsed and only deforms slightly, while above gamma[.]{*} the globule exhibits unfolding/refolding cycles. Hydrodynamics are crucial: In the free draining case, gamma[.]{*} scales with the globule radius R as gamma[.]{*} approximately R{-1}, while in the presence of hydrodynamic interactions gamma[.]{*} approximately R. Experiments on the globular von Willebrand protein confirm the presence of an unfolding transition at a well-defined critical shear rate.

Homocysteinemia in psychiatric disorders: association with dementia and depression, but not schizophrenia in female patients.

Homocysteinemia has been reported to be a risk factor for dementia, depression and also schizophrenia, the latter in a gender-specific manner. We have determined homocysteine in female inpatients suffering from various psychiatric diseases to further investigate a possible association between homocysteinemia and psychiatric disorders. Homocysteine was not elevated in schizophrenic females (mean, 11.6+/-5.8 micromol/l); in accordance with previous studies, homocysteinemia could be found frequently in dementia of different aetiology (mean, 17.2+/-6.7 micromol/l), but also to a slighter extent in depressive disorders (mean, 12.9+/-3.8 micromol/l), especially in elderly subjects. We thus suggest that homocysteinemia, at least in females, is an unspecific risk factor for organic brain disorders, but not endogenous psychoses.

Neuropathology and binding studies in anti-amphiphysin-associated stiff-person syndrome.

The authors report a 71-year-old woman with amphiphysin-associated paraneoplastic stiff-person syndrome, opsoclonus, and encephalopathy. The patient's symptoms temporarily responded to plasmapheresis in parallel with a decline of serum anti-amphiphysin antibody titers. Later, the encephalopathy progressed rapidly and the patient died. Binding studies and the detection of autoantibodies in the patient's CNS as well as the treatment response suggest a pathogenic role of the anti-amphiphysin antibodies.

Neuroleptic malignant syndrome in Kufs' disease.

A patient with adult neuronal ceroid lipofuscinosis (ANCL; Kufs' disease) is described in whom neuroleptic malignant syndrome occurred, initially presenting as catatonic syndrome. Comprehensive neuroimaging studies were conducted including FDG-PET, IBZM-SPECT, and beta-CIT-SPECT, electrophysiological examinations and an ex vivo contracture test exposing muscle biopsy specimens to neuroleptics. Collectively the results argued for an involvement of the muscle in neuroleptic malignant syndrome at least in ANCL.

Delayed dedifferentiation and retention of properties in dissociated adult skeletal muscle fibers in vitro.

Adult skeletal muscle fibers can be isolated and cultured but tend to dedifferentiate and sprout with time in culture. We examined isolated adult mouse flexor digitorum brevis muscle fibers under various culture conditions by monitoring maintenance of the same fibers at 2-d intervals using survival analysis. Fibers plated on laminin and cultured in serum-free media did not show sprouting and exhibited significantly (P < 0.0001) longer survival (median survival time, T(50) = 10.2 d) than fibers in serum-containing media (T(50) = 3.3 d). Cell proliferation was markedly suppressed in serum-free cultures. Multiple or delayed Ca(2+) transients in response to brief field stimulation were often observed in dedifferentiated fibers after several d in serum-containing media but were not observed in fibers in serum-free media. The addition of cytosine arabinoside to serum-containing cultures did not prolong fiber survival (P = 0.39) and did not eliminate sprouting but did greatly suppress proliferation of nonmuscle cells. Fibers cultured in agarose gel with serum exhibited small, bud-like extensions but no sprouts and did not survive as long (T(50) = 6.2 d) as fibers plated on laminin and cultured in serum-free media (T(50) = 10.2 d) did. These results demonstrate that both morphological and physiological properties of fibers become modified in serum-containing media but can be retained by culturing without serum.

Type 1 and type 3 ryanodine receptors generate different Ca(2+) release event activity in both intact and permeabilized myotubes.

In this investigation we use a "dyspedic" myogenic cell line, which does not express any ryanodine receptor (RyR) isoform, to examine the local Ca(2+) release behavior of RyR3 and RyR1 in a homologous cellular system. Expression of RyR3 restored caffeine-sensitive, global Ca(2+) release and causes the appearance of relatively frequent, spontaneous, spatially localized elevations of [Ca(2+)], as well as occasional spontaneous, propagating Ca(2+) release, in both intact and saponin-permeabilized myotubes. Intact myotubes expressing RyR3 did not, however, respond to K(+) depolarization. Expression of RyR1 restored depolarization-induced global Ca(2+) release in intact myotubes and caffeine-induced global release in both intact and permeabilized myotubes. Both intact and permeabilized RyR1-expressing myotubes exhibited relatively infrequent spontaneous Ca(2+) release events. In intact myotubes, the frequency of occurrence and properties of these RyR1-induced events were not altered by partial K(+) depolarization or by application of nifedipine, suggesting that these RyR1 events are independent of the voltage sensor. The events seen in RyR1-expressing myotubes were spatially more extensive than those seen in RyR3-expressing myotubes; however, when analysis was limited to spatially restricted "Ca(2+) spark"-like events, events in RyR3-expressing myotubes were larger in amplitude and duration compared with those in RyR1. Thus, in this skeletal muscle context, differences exist in the spatiotemporal properties and frequency of occurrence of spontaneous release events generated by RyR1 and RyR3. These differences underscore functional differences between the Ca(2+) release behavior of RyR1 and RyR3 in this homologous expression system.