Search

Significance Lipid bilayers constitute the matrix of cellular membranes and synthetic vesicles used in drug delivery. This self-assembled structure is only a few nanometers thick but provides an effective barrier between aqueous fluids. The response of lipid bilayers to shear stresses induced by surrounding fluid flows can trigger biophysical processes in cells and influence the efficacy of drug delivery by synthetic vesicles. Here, we use optical tweezers to apply and measure local hydrodynamic shear stresses on both sides of a freestanding lipid bilayer. With this method, we determine the rheological properties of bilayers and capture a previously unreported phenomenon when the intermonolayer friction is so low that the monolayers slip past each other and hydrodynamic shear is not transmitted through the bilayer., Vital biological processes, such as trafficking, sensing, and motility, are facilitated by cellular lipid membranes, which interact mechanically with surrounding fluids. Such lipid membranes are only a few nanometers thick and composed of a liquid crystalline structure known as the lipid bilayer. Here, we introduce an active, noncontact, two-point microrheology technique combining multiple optical tweezers probes with planar freestanding lipid bilayers accessible on both sides. We use the method to quantify both fluid slip close to the bilayer surface and transmission of fluid flow across the structure, and we use numerical simulations to determine the monolayer viscosity and the intermonolayer friction. We find that these physical properties are highly dependent on the molecular structure of the lipids in the bilayer. We compare ordered-phase with liquid disordered-phase lipid bilayers, and we find the ordered-phase bilayers to be 10 to 100 times more viscous but with 100 times less intermonolayer friction. When a local shear is applied by the optical tweezers, the ultralow intermonolayer friction results in full slip of the two leaflets relative to each other and as a consequence, no shear transmission across the membrane. Our study sheds light on the physical principles governing the transfer of shear forces by and through lipid membranes, which underpin cell behavior and homeostasis.

AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ~20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.

AAA+ proteases are degradation machines, which exploit ATP hydrolysis to unfold protein substrates and translocate them through a central pore towards a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. Although cytoplasmic structures are described, the full-length structure of bacterial FtsH is unknown, and the route by which substrates reach the central pore remains unclear. We use electron microscopy to determine the 3D map of the full-lengthAquifex aeolicusFtsH hexamer. Moreover, detergent solubilisation induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. FtsH structures reveal that the cytosolic domain can tilt with respect to the membrane. A flexible linker of ~20 residues between the second transmembrane helix and the cytosolic domain permits the observed large tilting movements, thereby facilitating the entry of substrate proteins towards the central pore for translocation.

We report a simple, cost-effective, and reproducible method to form free-standing lipid bilayer membranes in microdevices made with Norland Optical Adhesive 81 (NOA81). Surface treatment with either alkylsilane or fluoroalkylsilane enables the self-assembly of stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) membranes. Capacitance measurements are used to characterize the lipid bilayer and to follow its formation in real-time. With current recordings, we detect the insertion of single α-hemolysin pores into the bilayer membrane, demonstrating the possibility of using this device for single-channel electrophysiology sensing applications. Optical transparency of the device and vertical position of the lipid bilayer with respect to the microscope focal plane allows easy integration with other single-molecule techniques, such as optical tweezers. Therefore, this method to form long-lived lipid bilayers finds a wide range of applications, from sensing measurements to biophysical studies of lipid bilayers and associated proteins.

International audience; The ability to couple membrane processes and crystallization operations, in order to develop efficient crystallization technologies, is increasingly reported. The main features of membrane crystallization systems are (1) to control and limit the maximum level of supersaturation due to defined mass transfer across the membrane; (2) to act as a support able to activate heterogeneous nucleation; (3) to control solid features such as size, shape, polymorphic form, and purity; and (4) to reduce energy consumption. To achieve these goals, several membrane techniques are used: reverse osmosis, membrane distillation, membrane contactor, and membrane templates. This review describes briefly the principles, applications, and advantages and drawbacks of each technique for crystallization operations.

This paper presents numerical simulations and experimental data on the iodide–iodate reaction in a hollow fiber membrane device. The principle of reactive mixing in a membrane device is the following. Component A flows through the inlet of the lumen side and component B comes from the membrane pores (shell side). Components A and B then mix and react inside the lumen side. Experimental measurements of the segregation index were carried out under various experimental conditions and for two different hollow fiber devices. The numerical and experimental segregation indexes, X S , were found in the range 10 −2 –10 −3 , indicating effective mixing. The numerical simulations showed that I 3 − ions were produced mainly in the first part of the lumen side of the hollow fiber. This confirmed our previous results on precipitation which showed that high supersaturation data and experimental fouling were obtained in this part of the hollow fibers.

International audience; in this second of two papers, we investigated the influence of process parameters on BaSO(4) particle precipitation in a hollow fiber membrane device. The solution of barium chloride was passed tangentially over the membrane surface and reacted with a solution of K(2)SO(4) introduced through the membrane pores. The resulting supersaturation induced nucleation and particle growth on the lumen side of the hollow fibers. A specific technique was developed to measure crystal size directly at the outlet of the hollow fiber device by using EDTA as a neutralizing agent of crystal nucleation and growth. Concentrations of barium chloride and potassium sulphate were shown to influence mainly the CSD, the effect of inlet flow rates on the lumen and shell sides being less pronounced. The chemical conversions measured were between 3.7% and 20.5%. We propose improvements in the design of hollow fiber devices in order to give higher chemical conversions.

We propose a method for calculating directly total body water (TBW) volumes (V (t)) from whole body resistance extrapolated at infinite frequency (R (infinity)) using a XITRON 4200 impedance meter. Mean TBW resistivities for men and women were determined from measurements of R (infinity) and fat-free mass (FFM(d)) measured by DXA in 58 healthy subjects assuming an average hydration coefficient of 73.2%. Mean differences between V (t) measured by our new method and those deduced from DXA data were +0.11 +/- 1.61 L for women and +0.13 +/- 2.16 L for men. For validation, this method was tested with the same resistivities against a 2nd group of 16 volunteers and the mean difference between V (t) from impedance and DXA was -0.80 +/- 1.43 L. Since the resistance at 50 kHz (R (50)) was found to be equal, in average, to 1.230 R (infinity) for men and 1.223 R (infinity) for women, this method can also be applied at 50 kHz with a similar accuracy by estimating R (infinity) from R (50). When our new method was applied to the monitoring of water loss during 28 dialysis runs performed on 13 patients, it predicted a mean water loss equal to 94% of ultrafiltered volume.

Distillation of fermented molasses for alcohol and biofuels production uses large volumes of water. Due to environmental concern, the recycling of condensates in the fermentation medium is encouraged subsequently to a purification step. This step could be performed via reverse osmosis (RO) process, in order to remove in the retentate small organic solutes which act as fermentation inhibitors. Currently, no general rule exists to predict the selectivity of RO membranes according to the nature of organic solutes. Similarly, such insights are missing in nanofiltration processes intended to detoxify biomass hydrolysates prior fermentation. Our objective was to build and validate a molecular model of typical reverse osmosis membranes (exemples: ESPA2 and CPA2 from Hydranautics Membranes, USA), consisting in an active aromatic polyamide layer (APA) with different crosslinking rates. These models will be used in future works to develop general models of transport properties (water swelling, solute partitioning mainly) in RO membranes. The main characteristics of commercial RO membranes were retrieved from isolated APA layers (i.e. separated from the polysulfone-polyester support). Geometrical characteristics such as thickness (about 200 nm), homogeneity and swelling rates were assessed with laser ellipsometry and AFM. The composition and the reticulation rate were inferred from FTIR and XPS measurements. The sorption isotherm of water was sampled with an Intelligent Gravimetric Analyzer. The construction strategy of corresponding atomistic models of dried APA membranes via Molecular Dynamics Simulation (Discover, Accelrys, USA) is presented. The described model gives the theoretical density and neutron scattering spectra of simulated APA. These results could be compared in future works with experimental investigations

International audience; Particle formation by precipitation is an important unit operation for the production of fine solids such as catalysts, pigments, and pharmaceuticals. It is the decisive step for determining properties such as particle size, shape, and purity, which in turn determine the quality of the end product. in this study, we investigated the precipitation of BaSO(4) particles in a. hollow fiber membrane device. A solution of BaCl(2), was passed tangentially over a membrane surface and reacted with a solution of K(2)SO(4) introduced through the membrane pores. The resulting supersaturation induced nucleation and particles grew on the lumen side of the hollow fibers. Particulate fouling was observed using X-ray images with BaSO(4) as a radiocontrast agent. It was shown that this fouling was attributed primarily to fiber blockage by newly formed crystals rather than to pore blockage. Moreover, a larger inner diameter of hollow fiber was shown to slow down considerably the fouling of the device. A numerical simulation was developed to calculate the supersaturation inside a hollow fiber, and the results of the simulation were compared with experimental data.

International audience; Liquid-liquid phase membrane contactors are increasingly being used for mixing and reaction. The principle is the following: component A flows through the membrane device inlet to mix/react with component B which comes from the membrane pores. This study presents a numerical simulation using computational fluid dynamics (CFD) of momentum and mass transfer in a tubular membrane contactor for laminar flow conditions. The velocity and concentration profiles of components A-C are obtained by resolution of the Navier-Stokes and convection-diffusion equations. The numerical simulations show that mixing between A and B is obtained by diffusion along the streamlines separating both components. The mixing/reaction zone width is within the region of a few hundred of microns, and depends on the diffusion coefficients of A and B. Hollow fiber membrane devices are found to be of particular interest because their inner diameter is close to the mixing zone width.

The regulation of protein trafficking across cellular membranes, done by transmembrane proteins, is a vital process known as translocation. However, protein translocation is not completely understood and traditional electrophysiology techniques cannot give an insight into the mechanical properties of translocation. On the other hand, optical tweezers has been shown to be a suitable candidate to study the translocation of soluble proteins but becomes a challenge with transmembrane proteins. Hence there is a need to develop a biophysical tool that allows the study of transmembrane proteins into their membrane environment with optical tweezers.There is an increasing effort to miniaturize black lipid membranes (BLM), and several approaches have now been developed to study the translocation of proteins in miniaturized systems. In this work, we develop a microfluidic system suitable for the study of transmembrane proteins into artificial membranes. The advantages of our approach include: real-time control over the charge gradient across the membrane, dynamic exchange of buffers, and capability to combine with force-spectroscopy techniques.We demonstrate the formation of a free-standing BLM on a glass micro-device by measures of capacitance and electron flux detection through toxin pores. This microfluidic device is combined with a high-resolution optical tweezers for the study of protein translocation across membranes and the evaluation of current translocation models.

Objectives The first objective was to compare the accuracy of a foot-to-foot impedance meter with a multifrequency bioimpedance for measurements of fat-free mass (FFM) and fat mass (FM) using dual energy X-ray absorptiometry (DXA) as reference. The second objective was to validate measurements of extracellular water resistance and volume by the foot-to-foot impedance meter, using multifrequency bioimpedance as reference. Methods This investigation was carried out in 60 volunteers 18 to 71 y of age. Impedance meters were a Tefal Bodymaster Vision (foot-to-foot) that featured a square wave signal and a Xitron Hydra 4200 (5 to 1000 kHz) by using the bioimpedance spectroscopic method. Results Bland-Altman tests showed that FFM differences between Tefal and DXA data were 1.98 ± 3.09 kg in men and −0.08 ± 2.98 kg in women. Total body water was measured by the Xitron, and FFM as measured with the Xitron was calculated as total body water divided by 0.732. Mean differences between Xitron-measured and DXA-measured FFM were 2.37 ± 3.03 kg for men and 2.84 ± 2.40 kg for women, indicating a systematic underestimation by the Xitron of intracellular volume. Extracellular water resistances measured by Tefal were in good agreement with those measured by Xitron with electrodes pasted under the subject’s feet (mean difference 8.5 ± 31 Ω). Extracellular water volumes were calculated from Tefal-measured extracellular water resistances by using a modified bioimpedance spectroscopic method and differed from those measured with Xitron by −0.03 ± 0.66 L. Conclusion Limits of agreement with DXA-measured FFM produced by the foot-to-foot impedance meter tested are too large for clinical measurements in individuals, but they are sufficient to assess FFM in groups of subjects and for home use. Our prototype was also capable of estimating extracellular water volume with a similar accuracy as multifrequency bioimpedance in normal subjects.

Cell lipid membranes are the site of vital biological processes, such as motility, trafficking, and sensing, many of which involve mechanical forces. Elucidating the interplay between such bioprocesses and mechanical forces requires the use of tools that apply and measure piconewton-level forces, e.g., optical tweezers. Here, we introduce the combination of optical tweezers with free-standing lipid bilayers, which are fully accessible on both sides of the membrane. In the vicinity of the lipid bilayer, optical trapping would normally be impossible due to optical distortions caused by pockets of the solvent trapped within the membrane. We solve this by drastically reducing the size of these pockets via tuning of the solvent and flow cell material. In the resulting flow cells, lipid nanotubes are straightforwardly pushed or pulled and reach lengths above half a millimeter. Moreover, the controlled pushing of a lipid nanotube with an optically trapped bead provides an accurate and direct measurement of important mechanical properties. In particular, we measure the membrane tension of a free-standing membrane composed of a mixture of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) to be 4.6 × 10-6 N/m. We demonstrate the potential of the platform for biophysical studies by inserting the cell-penetrating trans-activator of transcription (TAT) peptide in the lipid membrane. The interactions between the TAT peptide and the membrane are found to decrease the value of the membrane tension to 2.1 × 10-6 N/m. This method is also fully compatible with electrophysiological measurements and presents new possibilities for the study of membrane mechanics and the creation of artificial lipid tube networks of great importance in intra- and intercellular communication.

Although the main application of reverse osmosis (RO) filtration remains the separation or concentration of electrolytes from aqueous solvents (e.g. desalination), new promising applications including the recycling or purification of organic-rich effluents used in fermentation processes (bio-ethanol and other biofuel productions). Currently, no model exists to predict a priori the selectivity of given RO (mostly in aromatic polyamide) membrane to non-electrolyte organic compounds, such as non-dissociated acids, aldehydes, esters or aromatic compounds. The complication arises due to the mutual diffusion of water and small solutes within membrane, whose swelling is controlled by the stiffness of polymer segments and their cross-linking rates. The general objective of this study is to build atomistic-scale models of typical commercial aromatic polyamide (APA) membranes in order to analyze the contribution of their polymer chemical structure on swelling rate to water and on mutual diffusion mechanisms in the bulk. As APA membranes are polymerized in-situ as approximately 200 nm thick layer, our computational effort was combined with experimental isolation and deformulation of two commercial APA membranes (references: ESPA2 and CPA2, Hydranautics Membranes, USA) to provide both i) initial assumptions to build the atomistic model (monomers composition, average cross-linking rate, approximate swelling rate) and ii) independent reference data to validate the generated model at different relative humidity (X-ray structure factors).The active APA layer was separated from support by removing iteratively each support layer with N,N-dimethylformamide (DMF). Macroscopic properties such as swelling rates, sorption isotherms were assessed by ellipsometry and Intelligent Gravimetric Analyser respectively. A maximum swelling rate of 38% was determined. A cross-linking rate of 65 % was inferred from chemical composition analyses in X-ray photoelectron spectroscopy (XPS) and attenuated-total reflection mode Fourier transform IR (ATR-FTIR). A first atomistic model including the number of desired monomers and water content was built by a conventional compression box technique and classical molecular dynamics code (Discover, Accelrys, San-Diego). Cross-linking was subsequently performed by applying a specific reactive force-field within our own software. The degree of freedoms were i) the initial ratios in monomers (benzene-1,3,5-tricarbonyle chloride with a connectivity of 3, benzene-1,3-diamine with a connectivity of 2), ii) the initial length of block-oligomers (ranging from 5 to 62). Each system was finally equilibrated during long-term isobaric and isothermal molecular dynamics simulations at 600 K and 298 K. Final structure factors were in good agreement with RX scattering spectra.