Impact of Freeze- and Spray-Drying Microencapsulation Techniques on ß-Glucan Powder Biological Activity: A Comparative Study.

The study compares the impact of freeze- and spray-drying (FD, SD) microencapsulation methods on the content of ß-glucan, total polyphenols (TP), total flavonoids (TF), phenolic acids (PA), and antioxidant activity (AA) in commercially ß-glucan powder (Pleurotus ostreatus) using maltodextrin as a carrier. Morphology (scanning electron microscopy- SEM), yield, moisture content (MC), and water activity (aw) were also evaluated in the samples. Our examinations revealed significant structural differences between powders microencapsulated by the drying methods. As compared to non-encapsulated powder, the SD powder with yield of 44.38 ± 0.55% exhibited more reduced (p < 0.05) values for aw (0.456 ± 0.001) and MC (8.90 ± 0.44%) than the FD one (yield: 27.97 ± 0.33%; aw: 0.506 ± 0.002; MC: 11.30 ± 0.28%). In addition, the highest values for ß-glucan content (72.39 ± 0.38%), TPC (3.40 ± 0.17 mg GAE/g), and TFC (3.07 ± 0.29 mg QE/g) have been detected in the SD powder. Our results allow for the conclusion that the SD microencapsulation method using maltodextrin seems to be more powerful in terms of the ß-glucan powder yield and its contents of ß-glucan, TP, and TF as compared to the FD technique.

GFOGER Peptide Modifies the Protein Content of Extracellular Vesicles and Inhibits Vascular Calcification.

OBJECTIVE: Vascular calcification (VC) is an active process during which vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and release extracellular vesicles (EVs). In turn, the EVs serve as calcification foci via interaction with type 1 collagen (COL1). We recently showed that a specific, six-amino-acid repeat (GFOGER) in the sequence of COL1 was involved in the latter's interaction with integrins expressed on EVs. Our main objective was to test the GFOGER ability to inhibit VC. APPROACH: We synthesized the GFOGER peptide and tested its ability to inhibit the inorganic phosphate (Pi)-induced calcification of VSMCs and aortic rings. Using mass spectrometry, we studied GFOGER's effect on the protein composition of EVs released from Pi-treated VSMCs. RESULTS: Calcification of mouse VSMCs (MOVAS-1 cells), primary human VSMCs, and rat aortic rings was lower in the presence of GFOGER than with Pi alone (with relative decreases of 66, 58, and 91%, respectively; p < 0.001 for all) (no effect was observed with the scramble peptide GOERFG). A comparative proteomic analysis of EVs released from MOVAS-1 cells in the presence or absence of Pi highlighted significant differences in EVs' protein content. Interestingly, the expression of some of the EVs' proteins involved in the calcification process (such as osteogenic markers, TANK-binding kinase 1, and casein kinase II) was diminished in the presence of GFOGER peptide (data are available via ProteomeXchange with identifier PXD018169∗). The decrease of osteogenic marker expression observed in the presence of GFOGER was confirmed by q-RT-PCR analysis. CONCLUSION: GFOGER peptide reduces vascular calcification by modifying the protein content of the subsequently released EVs, in particular by decreasing osteogenicswitching in VSMCs.

The Impact of Plasma Membrane Lipid Composition on Flagellum-Mediated Adhesion of Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC to host tissues is the first step enabling bacterial colonization. Adhesins such as fimbriae and flagella mediate this process. Here, we studied the interaction of the bacterial flagellum with the host cell's plasma membrane using giant unilamellar vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components, including proteins and glycoproteins. In contrast, with GUVs, we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains, or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild-type EHEC but not of a nonflagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagellum-mediated adhesion to plasma membranes has broad implications for host-pathogen interactions.IMPORTANCE Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues, and form biofilm. Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagellum-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagellar adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts, are important in bacterial adhesion. These findings demonstrate that plasma membrane adhesion via bacterial flagella play a significant role for an important human pathogen. This mechanism represents a promising target for the development of novel antiadhesion therapies.

Methylation of Salmonella Typhimurium flagella promotes bacterial adhesion and host cell invasion.

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.

Synthesis and characterization of tethered lipid assemblies for membrane protein reconstitution (Review).

Biological membranes and their related molecular mechanisms are essential for all living organisms. Membranes host numerous proteins and are responsible for the exchange of molecules and ions, cell signaling, and cell compartmentation. Indeed, the plasma membrane delimits the intracellular compartment from the extracellular environment and intracellular membranes. Biological membranes also play a major role in metabolism regulation and cellular physiology (e.g., mitochondrial membranes). The elaboration of membrane based biomimetic systems allows us to reconstitute and investigate, in controlled conditions, biological events occurring at the membrane interface. A whole variety of model membrane systems have been developed in the last few decades. Among these models, supported membranes were developed on various hydrophilic supports. The use of solid supports enables the direct use of surface sensitive techniques (e.g., surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy) to monitor and quantify events occurring at the membrane surface. Tethered bilayer membranes (tBLMs) could be considered as an achievement of the first solid supported membranes described by the McConnell group. Tethered bilayers on solid supports were designed to delimit an inside compartment from an outside one. They were used for measuring interactions with ligands or incorporating large membrane proteins or complexes without interference with the support. In this context, the authors developed an easy concept of versatile tBLMs assembled on amino coated substrates that are formed upon the vesicle fusion rupture process applicable to protein-free vesicles as well as proteoliposomes. The phospholipid bilayer (natural or synthetic lipids) incorporated 5% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly ethylene glycol-N-hydroxy succinimide to ensure the anchorage of the bilayer to the amino coated surface. The conditions for the formation of tBLMs on amino-coated gold and glass were optimized for protein-free vesicles. This biomimetic membrane delimits an inside "trans" compartment separated from an outside reservoir "cis." Using this tBLM construction, the authors were interested in deciphering two complex molecular mechanisms involving membrane-associated proteins. The first one concerns two mitochondrial proteins, i.e., the porin voltage dependent anion channel (VDAC) embedded in the outer membrane and the nucleotide transporter (adenine nucleotide translocase) that interacts dynamically during mitochondrial pathophysiology. The purified VDAC porin was first reconstituted in proteoliposomes that were subsequently assembled on an amino coated support to form a biomimetic membrane. As a major result, VDAC was reconstituted in this tBLM and calcium channeling was demonstrated across the lipid bilayer. The same two-compartment biomimetic membrane design was further engineered to study the translocation mechanism of a bacterial toxin, the adenylate cyclase toxin, CyaA, from Bordetella pertussis. As a result, the authors developed an elegant in vitro translocation toolkit applicable to potentially a large panel of proteins transported across membranes.

Oligogalacturonic Acid Inhibits Vascular Calcification by Two Mechanisms: Inhibition of Vascular Smooth Muscle Cell Osteogenic Conversion and Interaction With Collagen.

OBJECTIVE: Cardiovascular diseases constitute the leading cause of mortality worldwide. Calcification of the vessel wall is associated with cardiovascular morbidity and mortality in patients having many diseases, including diabetes mellitus, atherosclerosis, and chronic kidney disease. Vascular calcification is actively regulated by inductive and inhibitory mechanisms (including vascular smooth muscle cell adaptation) and results from an active osteogenic process. During the calcification process, extracellular vesicles (also known as matrix vesicles) released by vascular smooth muscle cells interact with type I collagen and then act as nucleating foci for calcium crystallization. Our primary objective was to identify new, natural molecules that inhibit the vascular calcification process. APPROACH AND RESULTS: We have found that oligogalacturonic acids (obtained by the acid hydrolysis of polygalacturonic acid) reduce in vitro inorganic phosphate-induced calcification of vascular smooth muscle cells by 80% and inorganic phosphate-induced calcification of isolated rat aortic rings by 50%. A specific oligogalacturonic acid with a degree of polymerization of 8 (DP8) was found to inhibit the expression of osteogenic markers and, thus, prevent the conversion of vascular smooth muscle cells into osteoblast-like cells. We also evidenced in biochemical and immunofluorescence assays a direct interaction between matrix vesicles and type I collagen via the GFOGER sequence (where single letter amino acid nomenclature is used, O=hydroxyproline) thought to be involved in interactions with several pairs of integrins. CONCLUSIONS: DP8 inhibits vascular calcification development mainly by inhibition of osteogenic marker expression but also partly by masking the GFOGER sequence-thereby, preventing matrix vesicles from binding to type I collagen.

Solid-phase extraction of betanin and isobetanin from beetroot extracts using a dipicolinic acid molecularly imprinted polymer.

Betanin is a natural pigment with significant antioxidant and biological activities currently used as food colorant. The isolation of betanin is problematic due to its instability. In this work, we developed a fast and economic procedure based on molecularly imprinted solid-phase extraction (MISPE) for the selective clean-up of betanin and its stereoisomer isobetanin from beetroot extracts. Dipicolinic acid was used as template for the MIP preparation because of its structural similarity with the chromophore group of betanin. The MISPE procedures were fully optimized allowing the almost complete removal of matrix components such as sugars and proteins, resulting in high extraction recovery of betanin/isobetanin in a single step. Moreover, the whole extraction procedure was performed in environmentally friendly solvents with either ethanol or water. Our MISPE method is very promising for the future development of well-formulated beetroot extract with specified betanin/isobetanin content, ready for food or medicinal use.

Toward a Universal Method for Preparing Molecularly Imprinted Polymer Nanoparticles with Antibody-like Affinity for Proteins.

We describe a potentially universal, simple and cheap method to prepare water-compatible molecularly imprinted polymer nanoparticles (MIP-NPs) as synthetic antibodies against proteins. The strategy is based on a solid phase synthesis approach where glass beads (GBs) are functionalized with a metal chelate, acting as a general affinity ligand to attract surface-bound histidines present on proteins. This configuration enables an oriented immobilization of the proteins, upon which thermoresponsive MIP-NPs are synthesized. The GBs play the role of both a reactor and a separation column since, after synthesis, the MIP-NPs are released from the support by a simple temperature change, resulting in protein-free polymers. The resulting MIP-NPs are endowed with improved binding site homogeneity, since the binding sites have the same orientation. Moreover, they are stable (no aggregation) in a buffer solution for prolonged storage time and exhibit apparent dissociation constants in the nanomolar range, with little or no cross-reactivity toward other proteins.

Betanin-Enriched Red Beetroot (Beta vulgaris L.) Extract Induces Apoptosis and Autophagic Cell Death in MCF-7 Cells.

Recent studies have pointed out the preventive role of beetroot extracts against cancers and their cytotoxic activity on cancer cells. Among many different natural compounds, these extracts contained betanin and its stereoisomer isobetanin, which belongs to the betalain group of highly bioavailable antioxidants. However, a precise identification of the molecules responsible for this tumor-inhibitory effect was still required. We isolated a betanin/isobetanin concentrate from fresh beetroots, corresponding to the highest purified betanin extract used for studying anticancer activities of these molecules. The cytotoxicity of this betanin-enriched extract was then characterized on cancer and normal cells and we highlighted the death signalling pathways involved. Betanin/isobetanin concentrate significantly decreased cancer cell proliferation and viability. Particularly in MCF-7-treated cells, the expressions of apoptosis-related proteins (Bad, TRAILR4, FAS, p53) were strongly increased and the mitochondrial membrane potential was altered, demonstrating the involvement of both intrinsic and extrinsic apoptotic pathways. Autophagosome vesicles in MCF-7-treated cells were observed, also suggesting autophagic cell death upon betanin/isobetanin treatment. Importantly, the betanin-enriched extract had no obvious effect towards normal cell lines. Our data bring new insight to consider the betanin/isobetanin mix as therapeutic anticancer compound, alone or in combination with classical chemotherapeutic drugs, especially in functional p53 tumors.

EGFR Inhibition by Curcumin in Cancer Cells: A Dual Mode of Action.

Epidermal Growth Factor Receptor (EGFR) is an important target of anticancer therapy. Nowadays, the search for new molecules inhibiting this receptor is turning toward natural substances. One of the most promising natural compounds that have shown an anti-EGFR activity is curcumin, a polyphenol found in turmeric. Its effect on the receptor kinase activity and on the receptor autophosphorylation has been already described, but the mechanism of how curcumin interacts with EGFR is not fully elucidated. We demonstrate that the mode of action of curcumin is dual. This polyphenol is able to inhibit directly but partially the enzymatic activity of the EGFR intracellular domain. The present work shows that curcumin also influences the cell membrane environment of EGFR. Using biomimetic membrane models, we show that curcumin insertion into the lipid bilayer leads to its rigidification. Single particle tracking analyses performed in the membrane of A431 cancer cells confirmed that this effect of curcumin on the membrane slows down the receptor diffusion. This is likely to affect the receptor dimerization and, in turn, its activation.

Synergism of antioxidant action of vitamins E, C and quercetin is related to formation of molecular associations in biomembranes.

Vitamins E, C and polyphenols (flavonoids and non-flavonoids) are major natural antioxidants capable of preventing damage generated by oxidative stress. Here we show the capacity of these antioxidants to form non-covalent association within lipid bilayers close to the membrane/cytosol interface. Antioxidant regeneration is significantly enhanced in these complexes.

Real-time QCM-D monitoring of cancer cell death early events in a dynamic context.

Since a few years, the acoustic sensing of whole cell is the focus of increasing interest for monitoring the cytoskeletal cellular response to morphological modulators. We aimed at illustrating the potentialities of the quartz crystal microbalance with dissipation (QCM-D) technique for the real-time detection of the earliest morphological changes that occur at the cell-substrate interface during programmed cell death. Human breast cancer cells (MCF-7) grown on serum protein-coated gold sensors were placed in dynamic conditions under a continuous medium flow. The mass and viscoelasticity changes of the cells were tracked by monitoring the frequency and dissipation shifts during the first 4h of cell exposure to staurosporine, a well-known apoptosis inducer. We have identified a QCM-D signature characteristic of morphological modifications and cell detachment from the sensing surface that are related to the pro-apoptotic treatment. In particular, for low staurosporine doses below 1 µM, we showed that recording the dissipation shift allows to detect an early cell response which is undetectable after the same duration by the classical analytical techniques in cell biology. Furthermore, this sensing method allows quantifying the efficiency of the drug effect in less than 4h without requiring labeling and without interfering in the system, thus preventing any loss of information. In the actual context of targeted cancer therapy development, we believe that these results bring new insights in favor of the use of the non invasive QCM-D technique for quickly probing the cancer cell sensitivity to death inducer drugs.

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer.

Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study's in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions.

Enhanced cellular adhesion on titanium by silk functionalized with titanium binding and RGD peptides.

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. A quartz crystal microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived arginine-glycine-aspartic acid (RGD) peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by scanning electron microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required.

Quartz crystal microbalance immunosensor for the quantification of immunoglobulin G in bovine milk.

The development of precise and sensitive methods for milk analysis remains a challenging task in the milk quality control field. A piezoelectric immunosensor was developed for the real-time quantification of immunoglobulin G (IgG) in bovine milk and colostrum. The sensing surface was designed with rabbit antibovine IgG as the detecting molecule, coupled onto a carboxymethyl dextran-coated gold crystal. Total binding and non-specific binding were measured using a quartz crystal microbalance with dissipation (QCM-D). Conditions of analysis, including ligand immobilization, dilution ratio of milk, salinity, and pH of the dilution buffer were optimized by Doehlert experimental design in order to enhance the detection specificity. The performances of the optimized immunosensor were evaluated. The standard curve was established from QCM-D responses and was linear until an IgG concentration of 2500 ng/mL, with a detection limit of 46 ng/mL. The total assay time is 5 min per sample, including the regeneration step. The intra- and inter-assay variation coefficients were equal to or below 4.7 and 6.1%, respectively. The sensing surface was stable for 100 analyses. This technique was successfully applied to the detection of colostrum addition in milk, with a minimum threshold of 0.1%. This new IgG quantification method is particularly interesting as a cost-effective and time-saving alternative for the dairy analytical laboratories when compared with the existing quantification methods.

Autophosphorylation activation and inhibition by curcumin of the epidermal growth factor receptor reconstituted in liposomes.

The epidermal growth factor receptor (EGFR) is a 170-kDa transmembrane protein with intrinsic protein kinase activity. It is involved in the regulation of essential cellular processes such as proliferation, differentiation, survival, and migration. An increase in EGFR activity has been correlated to malignant evolution of the cells. We have used proteoliposomes as a platform to study the mechanism of activation and inhibition of EGFR. We have been able to reconstitute functional EGFR in liposomes through detergent removal by Bio-Beads and have measured the receptor dimerization and its autophosphorylation resulting from its inherent tyrosine kinase activity. In particular, we have studied the activation of autophosphorylation by the natural ligand epidermal growth factor and its inhibition by curcumin, a polyphenol from Curcuma longa. This artificial membrane model provides a convenient tool to both qualitatively and quantitatively elucidate the mechanism of activation and inhibition of EGFR. It allows studying the isolated receptor under well-defined conditions, which enables one to use a number of biochemical and physico-chemical techniques that are difficult to put into practice with living cells. We believe that this platform can be used as a systematic screening tool for membrane receptor modulators, which are potential drug candidates.

A tethered bilayer assembled on top of immobilized calmodulin to mimic cellular compartmentalization.

BACKGROUND: Biomimetic membrane models tethered on solid supports are important tools for membrane protein biochemistry and biotechnology. The supported membrane systems described up to now are composed of a lipid bilayer tethered or not to a surface separating two compartments: a "trans" side, one to a few nanometer thick, located between the supporting surface and the membrane; and a "cis" side, above the synthetic membrane, exposed to the bulk medium. We describe here a novel biomimetic design composed of a tethered bilayer membrane that is assembled over a surface derivatized with a specific intracellular protein marker. This multilayered biomimetic assembly exhibits the fundamental characteristics of an authentic biological membrane in creating a continuous yet fluid phospholipidic barrier between two distinct compartments: a "cis" side corresponding to the extracellular milieu and a "trans" side marked by a key cytosolic signaling protein, calmodulin. METHODOLOGY/PRINCIPAL FINDINGS: We established and validated the experimental conditions to construct a multilayered structure consisting in a planar tethered bilayer assembled over a surface derivatized with calmodulin. We demonstrated the following: (i) the grafted calmodulin molecules (in trans side) were fully functional in binding and activating a calmodulin-dependent enzyme, the adenylate cyclase from Bordetella pertussis; and (ii) the assembled bilayer formed a continuous, protein-impermeable boundary that fully separated the underlying calmodulin (trans side) from the above medium (cis side). CONCLUSIONS: The simplicity and robustness of the tethered bilayer structure described here should facilitate the elaboration of biomimetic membrane models incorporating membrane embedded proteins and key cytoplasmic constituents. Such biomimetic structures will also be an attractive tool to study translocation across biological membranes of proteins or other macromolecules.

Quantification of immunoglobulin g in bovine and caprine milk using a surface plasmon resonance-based immunosensor.

Detection of colostrum in bovine and caprine milks is essential for dairy industries to avoid negative economical and technological consequences. One of the best markers of the presence of colostrum is immunoglobulin G (IgG). Two quantification methods have been evaluated for IgG in bovine or caprine milk, based on the real-time immunodetection of IgG by surface plasmon resonance (SPR) spectroscopy. Calibration curves were established by extracting affinity data from the sensorgrams, either using the residual bound IgG level after the association and dissociation phases or using the IgG binding rate during the association phase. The binding rate method allows for substantially reduced analysis times of below 4 min, which make it compatible with the milking time of small ruminants. Moreover, the binding rate method showed a better analytical performance, with lower detection limit and higher precision and accuracy than the residual binding method.

Exploring the membrane mechanism of the bioactive peptaibol ampullosporin a using lipid monolayers and supported biomimetic membranes.

Ampullosporin A is an antimicrobial, neuroleptic peptaibol, the behavior of which was investigated in different membrane mimetic environments made of egg yolk L-α-phosphatidylcholine. In monolayers, the peptaibol adopted a mixed α/3(10)-helical structure with an in-plane orientation. The binding step was followed by the peptide insertion into the lipid monolayer core. The relevance of the inner lipid leaflet nature was studied by comparing ampullosporin binding on a hybrid bilayer, in which this leaflet was a rigid alkane layer, and on supported fluid lipid bilayers. The membrane binding was examined by surface plasmon resonance spectroscopy and the effect on lipid dynamics was explored using fluorescence recovery after photobleaching. In the absence of voltage and at low concentration, ampullosporin A substantially adsorbed onto lipid surfaces and its interaction with biomimetic models was strongly modified depending on the inner leaflet structure. At high concentration, ampullosporin A addition led to the lipid bilayers disruption.

Formation of tethered bilayer lipid membranes probed by various surface sensitive techniques.

Tethered bilayer lipid membranes are promising biomimetic architectures. Their formation has been investigated using four different surface sensitive techniques, including optical, acoustic, and electrical methods. The lipid bilayers are built in a two-step procedure; the proximal layer is formed by self-assembly and is then completed to a bilayer by fusion with small vesicles. The different technical approaches revealed specific aspects of the layer formation processes, namely, first a fast adsorption process followed by a longer rearrangement period. Similar phenomena have been observed for the vesicle fusion process. The results allow for a more controlled assembly protocol for the preparation of highly insulating lipid membranes.