Membrane cholesterol strongly influences confined diffusion of prestin.

Prestin is the membrane motor protein that drives outer hair cell (OHC) electromotility, a process that is essential for mammalian hearing. Prestin function is sensitive to membrane cholesterol levels, and numerous studies have suggested that prestin localizes in cholesterol-rich membrane microdomains. Previously, fluorescence recovery after photobleaching experiments were performed in HEK cells expressing prestin-GFP after cholesterol manipulations, and revealed evidence of transient confinement. To further characterize this apparent confined diffusion of prestin, we conjugated prestin to a photostable fluorophore (tetramethylrhodamine) and performed single-molecule fluorescence microscopy. Using single-particle tracking, we determined the microscopic diffusion coefficient from the full time course of the mean-squared deviation. Our results indicate that prestin undergoes diffusion in confinement regions, and that depletion of membrane cholesterol increases confinement size and decreases confinement strength. By interpreting the data in terms of a mathematical model of hop-diffusion, we quantified these cholesterol-induced changes in membrane organization. A complementary analysis of the distribution of squared displacements confirmed that cholesterol depletion reduces prestin confinement. These findings support the hypothesis that prestin function is intimately linked to membrane organization, and further promote a regulatory role for cholesterol in OHC and auditory function.

Unfractionated heparin versus low molecular weight heparin for avoiding heparin-induced thrombocytopenia in postoperative patients.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction presenting as a prothrombotic disorder related to antibody-mediated platelet activation. It is a poorly understood paradoxical immune reaction resulting in thrombin generation in vivo, which leads to a hypercoagulable state and the potential to initiate venous or arterial thrombosis. A number of factors are thought to influence the incidence of HIT including the type and preparation of heparin (unfractionated heparin (UFH) or low molecular weight heparin (LMWH)) and the heparin-exposed patient population, with the postoperative patient population presenting a higher risk.Although LMWH has largely replaced UFH as a front-line therapy, there is evidence supporting a lack of superiority of LMWH compared with UFH regarding prevention of deep vein thrombosis and pulmonary embolism following surgery, and similar frequencies of bleeding have been described with LMWH and UFH. The decision as to which of these two preparations of heparin to use may thus be influenced by adverse reactions such as HIT. We therefore sought to determine the relative impact of UFH and LMWH specifically on HIT in postoperative patients receiving thromboembolism prophylaxis. OBJECTIVES: The objective of this review was to compare the incidence of HIT and HIT complicated by thrombosis in patients exposed to UFH versus LMWH in randomised controlled trials (RCTs) of postoperative heparin therapy. SEARCH METHODS: The Cochrane Peripheral Vascular Diseases Group searched their Specialised Register (March 2012) and CENTRAL (2012, Issue 2). In addition, the authors searched LILACS (March 2012) and additional trials were sought from reference lists of relevant publications. SELECTION CRITERIA: We were interested in comparing the incidence of HIT occurring during exposure to UFH or LMWH after any surgical intervention. Therefore, we studied RCTs in which participants were postoperative patients allocated to receive UFH or LMWH, in a blinded or unblinded fashion. Eligible studies were required to have as an outcome clinically diagnosed HIT, defined as a relative reduction in the platelet count of 50% or greater from the postoperative peak (even if the platelet count at its lowest remained > 150 x 10(9)/L) occurring within five to 14 days after the surgery, with or without a thrombotic event occurring in this timeframe. Additionally, circulating antibodies associated with the syndrome were required to have been investigated through laboratory assays. DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and assessed the risk of bias. Disagreements were resolved by consensus with participation of a third author. MAIN RESULTS: In total two studies involving 923 participants met all the inclusion criteria and were included in the review. Pooled analysis showed a statistically significant reduction in the risk of HIT with LMWH compared with UFH (risk ratio (RR) 0.24, 95% confidence interval (CI) 0.07 to 0.82; P = 0.02). This result suggests that patients treated with LMWH would have a relative risk reduction (RRR) of 76% in the probability of developing HIT compared with patients treated with UFH.Venous thromboembolism (VTE) complicating HIT occurred in 12 of 17 patients who developed HIT. Pooled analysis showed a statistically significant reduction in HIT complicated by VTE with LMWH compared with UFH (RR 0.20, 95% CI 0.04 to 0.90; P = 0.04). This result indicates that patients using LMWH would have a RRR of 80% for developing HIT complicated by VTE compared with patients using UFH. Arterial thrombosis occurred in only one patient who received UFH and there were no amputations or deaths documented. AUTHORS' CONCLUSIONS: There was a lower incidence of HIT and HIT complicated by VTE in postoperative patients undergoing thromboprophylaxis with LMWH compared with UFH. This is consistent with the current clinical use of LMWH over UFH as front-line heparin therapy. However, conclusions are limited by a scarcity of high quality evidence. We did not expect the paucity of RCTs including HIT as an outcome as heparin is one of the most commonly used drugs worldwide and HIT is a life-threatening adverse drug reaction. To address the scarcity of clinically-relevant information on the topic of HIT as a whole, HIT should be included as an outcome in future RCTs of heparin, and HIT as an adverse drug reaction should be considered in clinical recommendations regarding monitoring of the platelet count for HIT.

A microfabricated magnetic force transducer-microaspiration system for studying membrane mechanics.

The application of forces to cell membranes is a powerful method for studying membrane mechanics. To apply controlled dynamic forces on the piconewton scale, we designed and characterized a microfabricated magnetic force transducer (MMFT) consisting of current-carrying gold wires patterned on a sapphire substrate. The experimentally measured forces applied to paramagnetic and ferromagnetic beads as a function of applied current agree well with theoretical models. We used this device to pull tethers from microaspirated giant unilamellar vesicles and measure the threshold force for tether formation. In addition, the interlayer drag coefficient of the membrane was determined from the tether-return velocity under magnetic force-free conditions. At high levels of current, vesicles expanded as a result of local temperature changes. A finite element thermal model of the MMFT provided absolute temperature calibration, allowing determination of the thermal expansivity coefficient of stearoyl-oleoyl-phosphatidycholine vesicles (1.7 ± 0.4 × 10(-3) K(-1)) and characterization of the Joule heating associated with current passing through the device. This effect can be used as a sensitive probe of temperature changes on the microscale. These studies establish the MMFT as an effective tool for applying precise forces to membranes at controlled rates and quantitatively studying membrane mechanical and thermo-mechanical properties.

Effects of TGF-beta3 and preculture period of osteogenic cells on the chondrogenic differentiation of rabbit marrow mesenchymal stem cells encapsulated in a bilayered hydrogel composite.

In this work, injectable, biodegradable hydrogel composites of crosslinked oligo(poly(ethylene glycol) fumarate) and gelatin microparticles (MPs) were used to fabricate a bilayered osteochondral construct. Rabbit marrow mesenchymal stem cells (MSCs) were encapsulated with transforming growth factor-beta3 (TGF-beta3)-loaded MPs in the chondrogenic layer and cocultured with cells of different periods of osteogenic preculture (0, 3, 6 and 12 days) in the osteogenic layer to investigate the effects of TGF-beta3 delivery and coculture on the proliferation and differentiation of cells in both layers. The results showed that, in the chondrogenic layer, TGF-beta3 significantly stimulated chondrogenic differentiation of MSCs. In addition, cells of various osteogenic preculture periods in the osteogenic layer, along with TGF-beta3, enhanced gene expression for MSC chondrogenic markers to different extents. In the osteogenic layer, cells maintained their alkaline phosphatase activity during the coculture; however, mineralization was delayed by the presence of TGF-beta3. Overall, this study demonstrated the fabrication of bilayered hydrogel composites which mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers, while illustrating that encapsulated cells of different degrees of osteogenic differentiation can significantly influence the chondrogenic differentiation of cocultured progenitor cells in both the presence and absence of chondrogenic growth factors.

Controlled microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography.

High-pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding and final orientation for sectioning and subsequent transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 microm, the tubing protects small and fragile samples within the thickness constraints of high-pressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.

[Cross-reaction evaluation of PCR-Anaplasma platys positive dogs tested to Anaplasma phagocytophilum antibodies by commercial ELISA]. / Avaliação da ocorrência de reação cruzada em cães PCR-positivos para Anaplasma platys testados em elisa comercial para detecção de anticorpos de Anaplasma phagocytophilum.

Anaplasma platys, agent of canine cyclic thrombocytopenia parasites exclusively dogs platelets. Its probable vector is the tick Rhipicephalus sanguineus. Among the existent diagnostic methods, the most used include: morulae identification in blood smears; antibody detection by indirect immunofluorescence; or DNA amplification by polymerase chain reaction (PCR). Recently a new commercial ELISA (IDEXX®), capable of detecting A. phagocytophilum antibodies, has been developed. According to the manufacturer it appears to have a serological cross reaction with A. platys which may be used to help in the diagnosis of this agent. The aim of this study was to test serum samples from 16 PCR-positive dogs to A. platys with an ELISA that detects antibodies to A. phagocytophilum, Borrelia burgdorferi and Ehrlichia canis, and antigens of Dirofilaria immitis in order to verify the occurrence of cross reaction between A. platys and A. phagocytophilum. All animals tested negative for A. phagocytophilum, B. burgdorferi, and D. immitis. One animal tested positive for E. canis. Although it's known that A. phagocytophilum presents serological cross reaction with A. platys, the tested animals were negative, once antibodies against A. platys were not detected probably because of the acute phase of the disease.

Electromechanical models of the outer hair cell composite membrane.

The outer hair cell (OHC) is an extremely specialized cell and its proper functioning is essential for normal mammalian hearing. This article reviews recent developments in theoretical modeling that have increased our knowledge of the operation of this fascinating cell. The earliest models aimed at capturing experimental observations on voltage-induced cellular length changes and capacitance were based on isotropic elasticity and a two-state Boltzmann function. Recent advances in modeling based on the thermodynamics of orthotropic electroelastic materials better capture the cell's voltage-dependent stiffness, capacitance, interaction with its environment and ability to generate force at high frequencies. While complete models are crucial, simpler continuum models can be derived that retain fidelity over small changes in transmembrane voltage and strains occurring in vivo. By its function in the cochlea, the OHC behaves like a piezoelectric-like actuator, and the main cellular features can be described by piezoelectric models. However, a finer characterization of the cell's composite wall requires understanding the local mechanical and electrical fields. One of the key questions is the relative contribution of the in-plane and bending modes of electromechanical strains and forces (moments). The latter mode is associated with the flexoelectric effect in curved membranes. New data, including a novel experiment with tethers pulled from the cell membrane, can help in estimating the role of different modes of electromechanical coupling. Despite considerable progress, many problems still confound modelers. Thus, this article will conclude with a discussion of unanswered questions and highlight directions for future research.

Electromechanical effects on tether formation from lipid membranes: a theoretical analysis.

The material properties of biomembranes can be measured by forming a tether, a thin bilayer tube that extends from the membrane surface. Recent experiments have demonstrated that the force required to maintain a tether is sensitive to the transmembrane potential. As a first approach towards understanding this phenomenon, a thermodynamic analysis of the influence of electrical fields on tether formation from an aspirated lipid vesicle is developed. The analysis considers contributions from Maxwell stresses as well as two forms of electromechanical coupling: coupling between the electric field and curvature strain (flexoelectric coupling) and between the electric field and areal strain (piezoelectric coupling). Predictions of equilibrium tether conformations are obtained numerically. For expected values of the dimensionless coupling parameters, flexoelectric coupling alters the force required to form a tether of a given length, while piezoelectric coupling and Maxwell forces do not greatly change the force versus tether length behavior. The results of this analysis indicate that tether experiments have the potential to characterize electromechanical coupling in both synthetic and cellular membranes.

Fractional occurrence of defects in membranes and mechanically driven interleaflet phospholipid transport.

The picture of biological membranes as uniform, homogeneous bileaflet structures has been revised in recent times due to the growing recognition that these structures can undergo significant fluctuations both in local curvature and in thickness. In particular, evidence has been obtained that a temporary, localized disordering of the lipid bilayer structure (defects) may serve as a principal pathway for movement of lipid molecules from one leaflet of the membrane to the other. How frequently these defects occur and how long they remain open are important unresolved questions. In this report, we calculate the rate of molecular transport through a transient defect in the membrane and compare this result to measurements of the net transbilayer flux of lipid molecules measured in an experiment in which the lipid flux is driven by differences between the mechanical stress in the two leaflets of the membrane bilayer. Based on this comparison, we estimate the frequency of defect occurrence in the membrane. The occurrence of defects is rare: the probability of finding a defect in 1.0 microm2 of a lecithin membrane is estimated to be approximately 6.0x10(-6). Based on this fractional occurrence of defects, the free energy of defect formation is estimated to be approximately 1.0x10(-19) J. The calculations provide support for a model in which interleaflet transport in membranes is accelerated by mechanically driven lipid flow.

Micro- and nanomechanics of the cochlear outer hair cell.

Outer hair cell electromotility is crucial for the amplification, sharp frequency selectivity, and nonlinearities of the mammalian cochlea. Current modeling efforts based on morphological, physiological, and biophysical observations reveal transmembrane potential gradients and membrane tension as key independent variables controlling the passive and active mechanics of the cell. The cell's mechanics has been modeled on the microscale using a continuum approach formulated in terms of effective (cellular level) mechanical and electric properties. Another modeling approach is nanostructural and is based on the molecular organization of the cell's membranes and cytoskeleton. It considers interactions between the components of the composite cell wall and the molecular elements within each of its components. The methods and techniques utilized to increase our understanding of the central role outer hair cell mechanics plays in hearing are also relevant to broader research questions in cell mechanics, cell motility, and cell transduction.

A membrane bending model of outer hair cell electromotility.

We propose a new mechanism for outer hair cell electromotility based on electrically induced localized changes in the curvature of the plasma membrane (flexoelectricity). Electromechanical coupling in the cell's lateral wall is modeled in terms of linear constitutive equations for a flexoelectric membrane and then extended to nonlinear coupling based on the Langevin function. The Langevin function, which describes the fraction of dipoles aligned with an applied electric field, is shown to be capable of predicting the electromotility voltage displacement function. We calculate the electrical and mechanical contributions to the force balance and show that the model is consistent with experimentally measured values for electromechanical properties. The model rationalizes several experimental observations associated with outer hair cell electromotility and provides for constant surface area of the plasma membrane. The model accounts for the isometric force generated by the cell and explains the observation that the disruption of spectrin by diamide reduces force generation in the cell. We discuss the relation of this mechanism to other proposed models of outer hair cell electromotility. Our analysis suggests that rotation of membrane dipoles and the accompanying mechanical deformation may be the molecular mechanism of electromotility.

Transverse and lateral mobility in outer hair cell lateral wall membranes.

Cochlear outer hair cell (OHC) electromotility is associated with the cell's lateral wall. The lateral wall contains two distinct membranes: the plasma membrane (PM) and the subsurface cisternae (SSC). We explored biophysical characteristics of these lipid structures using membrane-specific fluorescent dyes. We have previously demonstrated that di-8-ANEPPS stains the PM while NBD-C6-ceramide partitions to the SSC. In this report we show that NBD-cholesterol also partitions to the SSC. Transmigration of the SSC dyes across the PM was visualized under confocal microscopy, after separating the two membranes using the micropipette aspiration technique. The transverse mobility of NBD-cholesterol was faster than that of NBD-C6-ceramide. We then measured the lateral mobility of the dyes within both the PM and the SSC using fluorescence recovery after photobleaching (FRAP). The diffusion coefficients at 12 37 degrees C and the activation energies for diffusion were found to be similar to those of other biological membranes. These data indicate that both the PM and the SSC are membranes in the fluid phase, with no evidence of temperature-dependent phase transitions. Our observations are consistent with a fluid-mosaic model of the lateral wall membranes.

The effects of gramicidin on electroporation of lipid bilayers.

The effects of the channel-forming peptide gramicidin D (gD) on the conductance and electroporation thresholds of planar bilayer lipid membranes, made of the synthetic lipid 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 200-900 mV) rectangular voltage pulses of 15 ms duration were used to perturb the bilayers and monitor the transmembrane conductance. Electroporation voltage thresholds were found, and conductance was recorded before and after electroporation. Gramicidin was added to the system in peptide/lipid ratios of 1:10, 000, 1:500, and 1:15. The addition of gD in a ratio of 1:10,000 had no effect on electroporation, but ratios of 1:500 and 1:15 significantly increased the thresholds by 16% (p < 0.0001) and 40% (p < 0.0001), respectively. Membrane conductance before electroporation was measurable only after the addition of gD and increased monotonically as the peptide/lipid ratio increased. The effect of gD on the membrane area expansivity modulus (K) was tested using giant unilamellar vesicles (GUVs). When gD was incorporated into the vesicles in a 1:15 ratio, K increased by 110%, consistent with the increase in thresholds predicted by an electromechanical model. These findings suggest that the presence of membrane proteins may affect the electroporation of lipid bilayers by changing their mechanical properties.

Changes in electroporation thresholds of lipid membranes by surfactants and peptides.

This article reviews recent work from our laboratory that explores how chemical additives may alter the threshold of electroporation of synthetic lipid bilayers. The addition of the nonionic block copolymeric surfactant, poloxamer 188 (P188), at a concentration of 1 mM increased the electroporation thresholds of planar lipid bilayer membranes made of azolectin. For a 10-microsecond rectangular pulse, P188-treated membranes were found to have a statistically higher threshold voltage, longer latency time to rupture, and lower postpulse conductance. Addition of the nonionic surfactant, octaethyleneglycol-mono-n-dodecyl-ether (C12E8), decreased the electroporation threshold of bilayer membranes made of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) by 10-40% for 10-microsecond- to 10-s-duration pulses, in a concentration-dependent manner over concentrations ranging from 0.1 to 10 mM. Postpulse membrane conductance also increased. The opposite effects of the two surfactants on electroporation thresholds may result from their very different structures, which would encourage different modes of surfactant-lipid interactions. To examine protein-lipid interactions and their effects on the electroporation threshold, the effects of a channel-forming polypeptide, gramicidin D (gD), was studied on membrane conductance and electroporation threshold. Electroporation thresholds for 15-ms pulses were unaffected by addition of gramicidin to POPC at a peptide:lipid concentration estimated to be 1:10,000, but increased significantly at ratios of 1:500 and 1:15, while membrane conductance increased monotonically with peptide concentration. A micropipette aspiration technique was applied to giant unilamellar POPC vesicles to measure changes in the membrane physical properties. When gD was added to give an estimated peptide:lipid ratio of 1:15, the membrane area expansivity modulus increased, indicating that the increase in electroporation threshold is correlated with a change in membrane stiffness. Thus, these findings demonstrate that surfactants or peptides can mediate the electroporation threshold of lipid bilayers.

Theoretical analysis of the effect of the transbilayer movement of phospholipid molecules on the dynamic behavior of a microtube pulled out of an aspirated vesicle.

Observations over extended times of a lipid microtube (tether) formed from a lecithin vesicle have shown that under constant external loads the tether exhibits a continuous slow growth. It is considered that this growth is a consequence of the net transbilayer movement of phospholipid molecules in a direction which relieves the membrane strain resulting from the elastic deformation of the vesicle. The elastic deformation mode responsible for this effect is identified as the relative expansion of the two membrane layers reflecting the non-local contribution to membrane bending. An equation for the consequent rate of transbilayer movement of phospholipid molecules is derived. The dynamic behavior of the system is modeled by including frictional contributions due to interlayer slip and Stokes drag on the glass bead used to form the tether. The general numerical solution reveals a complex dependence of the tether growth rate on the system parameters and a continuous increase in the rate of tether growth at long times. Closed form expressions approximating the system behavior are derived and the conditions under which they can be applied are specified. Modeling the mechanically-driven lipid transport as a simple, stochastic, thermal process, allows the rate of lipid translocation to be related to the equilibrium transbilayer exchange rate of phospholipid molecules. Consideration of experimental results shows that the time constant for mechanically-driven translocation is shorter than the time for diffusion-driven translocation by approximately two orders of magnitude, indicating that lipid translocation is not a simple diffusive process.

Accelerated interleaflet transport of phosphatidylcholine molecules in membranes under deformation.

Biological membranes are lamellar structures composed of two leaflets capable of supporting different mechanical stresses. Stress differences between leaflets were generated during micromechanical experiments in which long thin tubes of lipid (tethers) were formed from the surfaces of giant phospholipid vesicles. A recent dynamic analysis of this experiment predicts the relaxation of local differences in leaflet stress by lateral slip between the leaflets. Differential stress may also relax by interleaflet transport of lipid molecules ("flip-flop"). In this report, we extend the former analysis to include interleaflet lipid transport. We show that transmembrane lipid flux will evidence itself as a linear increase in tether length with time after a step reduction in membrane tension. Multiple measurements were performed on 24 different vesicles composed of stearoyl-oleoyl-phosphatidylcholine plus 3% dinitrophenol-linked di-oleoyl-phosphatidylethanolamine. These tethers all exhibited a linear phase of growth with a mean value of the rate of interlayer permeation, cp = 0.009 s-1. This corresponds to a half-time of approximately 8 min for mechanically driven interleaflet transport. This value is found to be consistent with longer times obtained for chemically driven transport if the lipids cross the membrane via transient, localized defects in the bilayer.