Serum and Soleus Metabolomics Signature of Klf10 Knockout Mice to Identify Potential Biomarkers.

The transcription factor Krüppel-like factor 10 (Klf10), also known as Tieg1 for TGFß (Inducible Early Gene-1) is known to control numerous genes in many cell types that are involved in various key biological processes (differentiation, proliferation, apoptosis, inflammation), including cell metabolism and human disease. In skeletal muscle, particularly in the soleus, deletion of the Klf10 gene (Klf10 KO) resulted in ultrastructure fiber disorganization and mitochondrial metabolism deficiencies, characterized by muscular hypertrophy. To determine the metabolic profile related to loss of Klf10 expression, we analyzed blood and soleus tissue using UHPLC-Mass Spectrometry. Metabolomics analyses on both serum and soleus revealed profound differences between wild-type (WT) and KO animals. Klf10 deficient mice exhibited alterations in metabolites associated with energetic metabolism. Additionally, chemical classes of aromatic and amino-acid compounds were disrupted, together with Krebs cycle intermediates, lipids and phospholipids. From variable importance in projection (VIP) analyses, the Warburg effect, citric acid cycle, gluconeogenesis and transfer of acetyl groups into mitochondria appeared to be possible pathways involved in the metabolic alterations observed in Klf10 KO mice. These studies have revealed essential roles for Klf10 in regulating multiple metabolic pathways whose alterations may underlie the observed skeletal muscle defects as well as other diseases.

Krüppel-like factor 10 regulates the contractile properties of skeletal muscle fibers in mice.

INTRODUCTION/AIMS: Klf10 is a member of the Krüppel-like family of transcription factors, which is implicated in mediating muscle structure (fiber size, organization of the sarcomere), muscle metabolic activity (respiratory chain), and passive force. The aim of this study was to further characterize the roles of Klf10 in the contractile properties of skeletal muscle fibers. METHODS: Fifty-two single fibers were extracted from female wild-type (WT) and Klf10 knockout (KO) oxidative (soleus) and glycolytic (extensor digitorum longus [EDL]) skinned muscles. Each fiber was immersed successively in relaxing (R), washing (W), and activating (A) solutions. Calcium was included in the activating solution to induce a maximum contraction of the fiber. The maximum force (Fmax ) was measured and normalized to the cross-sectional area to obtain the maximum stress (Stressmax ). After a steady state in contraction was reached, a quick stretch-release was performed; the force at the maximum stretch (Fstretch ) was measured and the stiffness was assessed. RESULTS: Deletion of the Klf10 gene induced changes in the contractile parameters (Fmax , Stressmax , Stiffness), which were lower and higher for soleus and EDL fibers compared with littermates, respectively. These measurements also revealed changes in the proportion and resistance of attached cross-bridges. DISCUSSION: Klf10 plays a major role in the homeostasis of the contractile behavior of skeletal muscle fibers in a muscle fiber type-specific manner. These findings further implicate important roles for Klf10 in skeletal muscle function and shed new light on understanding the molecular processes regulating the contractility of skeletal muscle fibers.

First step to the improvement of the blood brain barrier passage of atazanavir encapsulated in sustainable bioorganic vesicles.

The blood - brain barrier (BBB) prevents the majority of therapeutic drugs from reaching the brain following intravenous or oral administration. In this context, polymer nanoparticles are a promising alternative to bypass the BBB and carry drugs to brain cells. Amphiphilic cyclodextrins can form self-assemblies whose nanoparticles have a 100-nm-diameter range and are thus able to encapsulate drugs for controlled release. Our goal is to propose an optimized chemical synthesis of amphiphilic cyclodextrin, which remains a challenging task which commonly leads to only a low-milligram level of the high purity compound. Such cyclodextrin derivatives were used to prepare vesicles and to study their ability to vectorize a drug through the BBB. As a result, we introduced a convergent synthesis for a family of lipophosphoramidyl permethylated ß-CDs (Lip-ß-CDs) with various chain lengths. It was demonstrated that mixed vesicles comprised of phosphatidylcholine (POPC) and LipCDs were able to encapsulate atazanavir (ATV), a well-known protease inhibitor used as an antiretroviral drug against HIV. We highlighted that neo-vesicles promote the penetration of ATV in endothelial cells of the BBB, presumably due to the low fusogenicity of Lip-ß-CDs.

Nitrone-Trolox conjugate as an inhibitor of lipid oxidation: Towards synergistic antioxidant effects.

Free radical scavengers like α-phenyl-N-tert-butylnitrone (PBN) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) have been widely used as protective agents in various biomimetic and biological models. A series of three amphiphilic Trolox and PBN derivatives have been designed by adding to those molecules a perfluorinated chain as well as a sugar group in order to render them amphiphilic. In this work, we have studied the interactions between these derivatives and lipid membranes to understand how they influence their ability to prevent membrane lipid oxidation. We showed the derivatives better inhibited the AAPH-induced oxidation of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLiPC) small unilamellar vesicles (SUVs) than the parent compounds. One of the derivatives, bearing both PBN and Trolox moieties on the same fluorinated carrier, exhibited a synergistic antioxidant effect by delaying the oxidation process. We next investigated the ability of the derivatives to interact with DLiPC membranes in order to better understand the differences observed regarding the antioxidant properties. Surface tension and fluorescence spectroscopy experiments revealed the derivatives exhibited the ability to form monolayers at the air/water interface and spontaneously penetrated lipid membranes, underlying pronounced hydrophobic properties in comparison to the parent compounds. We observed a correlation between the hydrophobic properties, the depth of penetration and the antioxidant properties and showed that the location of these derivatives in the membrane is a key parameter to rationalize their antioxidant efficiency. Molecular dynamics (MD) simulations supported the understanding of the mechanism of action, highlighting various key physical-chemical descriptors.

Flax phenolic compounds as inhibitors of lipid oxidation: Elucidation of their mechanisms of action.

Polyunsaturated fatty acids are particularly sensitive to the damages due to reactive oxygen species and lipid oxidation has been reported to be involved in the degradation of food as well as in the early stages of several diseases. Our objective was to study the mechanisms of action of flax (Linum usitatissimum) phenolic compounds to prevent membrane lipid oxidation. To do so, several biophysical techniques (oxidative stress, surface tension, fluorescence spectroscopy and HPLC) were used to investigate the ability of the compounds to prevent lipid oxidation and to interact with membranes. We evidenced a relationship between the structure and the antioxidant efficiency as aglycone compounds were significantly more efficient (p < 0.05) than glucoside compounds. In addition, our results revealed that aglycone lignans spontaneously penetrated the membrane contrary to aglycone hydroxycinnamic acids. To conclude, the comparison of the antioxidant efficiencies revealed that membrane inserted compounds better inhibited lipid oxidation than non-inserted compounds.

Cholesterol-nitrone conjugates as protective agents against lipid oxidation: A model membrane study.

Free radical scavengers such as α-phenyl-N-tert-butylnitrone (PBN) have been widely used as protective agents in several biological models. We recently designed two PBN derivatives by adding a cholesterol moiety to the parent nitrone to increase its lipophilicity. In addition to the cholesterol, a sugar group was also grafted to enhance the hydrophilic properties at the same time. In the present work we report on the synthesis of a third derivative bearing only a cholesterol moiety and the physical chemical and antioxidant characterization of these three derivatives. We demonstrated they were able to form stable monolayers at the air/water interface and with the two derivatives bearing a sugar group, repulsive interactions with 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) were observed. We next investigated the interaction with DLPC on a liposome model. Fluorescence spectroscopy experiments showed the addition of a cholesterol moiety causes an ordering effect whereas the presence of the sugar group led to a disordering effect. The protective effect against lipid oxidation was then investigated using dynamic light scattering and the formation of conjugated dienes was quantified spectrophotometrically. Two oxidizing systems were tested, i.e. the AAPH-thermolysis which generates peroxyl radicals and the Fenton reagent which is responsible of the formation of hydroxyl radicals. Due to their membrane localization, the three cholesteryl-PBN derivatives are able to prevent lipid oxidation with the two types of radical inducers but with a different mode of action.

Synthesis, iron(III) complexation properties, molecular dynamics simulations and P. aeruginosa siderophore-like activity of two pyoverdine analogs.

P. aeruginosa ranks among the top five organisms causing nosocomial infections. Among the many novel strategies for developing new therapeutics against infection, targeting iron uptake mechanism seems promising as P. aeruginosa needs iron for its growth and survival. To scavenge iron, the bacterium produces siderophores possessing a very high affinity towards Fe(III) ions such as pyoverdines. In this work, we decided to study two pyoverdine analogs, aPvd2 and aPvd3, structurally close to the endogen pyoverdine. The pFe constants calculated with the values of formation showed a high affinity of aPvd3 towards Fe(III). Molecular dynamics calculations demonstrated that aPvd3-Fe forms with Fe(III) stable 1:1 complexes in water, whereas aPvd2 does not. Only aPvd3 is able to increase the bacterial growth and represents thus an alternative to pyoverdine for iron acquisition by the bacterium. The aPvd2-3 interaction studies with a lipid membrane indicated that they were unable to interact and to cross the plasma membrane of bacteria by passive diffusion. Consequently, the penetration of aPvd3 is ruled by a transport membrane protein. These results showed that aPvd3 may be used to inhibit pyoverdine uptake or to promote the accumulation and release of antibiotics into the cell following a Trojan horse strategy.

Antioxidant and Membrane Binding Properties of Serotonin Protect Lipids from Oxidation.

Serotonin (5-hydroxytryptamine, 5-HT) is a well-known neurotransmitter that is involved in a growing number of functions in peripheral tissues. Recent studies have shown nonpharmacological functions of 5-HT linked to its chemical properties. Indeed, it was reported that 5-HT may, on the one hand, bind lipid membranes and, on the other hand, protect red blood cells through a mechanism independent of its specific receptors. To better understand these underevaluated properties of 5-HT, we combined biochemical, biophysical, and molecular dynamics simulations approaches to characterize, at the molecular level, the antioxidant capacity of 5-HT and its interaction with lipid membranes. To do so, 5-HT was added to red blood cells and lipid membranes bearing different degrees of unsaturation. Our results demonstrate that 5-HT acts as a potent antioxidant and binds with a superior affinity to lipids with unsaturation on both alkyl chains. We show that 5-HT locates at the hydrophobic-hydrophilic interface, below the glycerol group. This interfacial location is stabilized by hydrogen bonds between the 5-HT hydroxyl group and lipid headgroups and allows 5-HT to intercept reactive oxygen species, preventing membrane oxidation. Experimental and molecular dynamics simulations using membrane enriched with oxidized lipids converge to further reveal that 5-HT contributes to the termination of lipid peroxidation by direct interaction with active groups of these lipids and could also contribute to limit the production of new radicals. Taken together, our results identify 5-HT as a potent inhibitor of lipid peroxidation and offer a different perspective on the role of this pleiotropic molecule.

Hematin loses its membranotropic activity upon oligomerization into malaria pigment.

Malaria is an infectious disease caused by Plasmodium type parasites transmitted by the bites of infected female anopheles mosquitoes. The malaria parasite multiplies in red blood cells where it degrades hemoglobin. This degradation of hemoglobin proteins releases hematin, an iron-containing porphyrin, which provokes membrane disruption and lysis. The malaria parasite blocks hematin-induced lysis by biocrystallization, a process that converts hematin into insoluble and chemically inert crystals. Hematin molecules are especially prone to self-assembly as dimers, oligomers and aggregates depending on environmental conditions (pH, solvent, temperature, concentration, ionic strength). Considering the different forms of hematin-based assemblies, it is still unclear which are the ones able to interact with membranes. We have prepared hematin under different conditions to form hematin-based assemblies and to measure their ability to interact and to disorganize membranes. Our results show that different forms of hematin molecules are able to penetrate lipid membranes. Interestingly, this membrane activity is spontaneously inhibited at acidic pH and it can be restored under neutral pH. By contrast, the oligomers of ß-hematin were found to be completely harmless toward lipid membranes. Finally, the AFM visualization of hematin interaction with supported lipid bilayers showed for the first time its preferential interaction with defaults in membranes, at the boundaries between two distinct lipid phases. The superficial adsorption of aggregates on membranes and the absence of effect due to oligomers were also confirmed with AFM.

PBN derived amphiphilic spin-traps. II/Study of their antioxidant properties in biomimetic membranes.

The work reported herein deals with the evaluation of the antioxidant properties of bitailed amphiphilic α-phenyl-N-tert-butylnitrone derivatives (BPBNs) towards oxidation of an unsaturated lipid, the 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC). Oxidation was induced either by UV light irradiation or radical initiators, i.e. the water soluble AAPH and the Fenton reaction, and the antioxidant evaluation was carried out using two biomimetic systems, namely Langmuir monolayers and large unilamellar vesicles. Measurement of the molecular area and the membrane fluidity of pure nitrone monolayers before and after UV-irradiation demonstrated the better stability and antioxidant properties of B17PBN, the derivative with two C17H35 alkyl chains, compared to its analogue B11PBN with two C11H23 alkyl chains. At only 5% molar ratio of nitrone in mixed DLoPC/nitrone monolayers, a complete inhibition of the molecular area decrease was observed for B17PBN whereas B11PBN showed lower protection. The oxidation of mixed DLoPC/nitrones large unilamellar vesicles in the presence of free radicals arising from AAPH decomposition or Fenton reaction was assessed by measuring lipid conjugated dienes and thiobarbituric acid reactive substances on the whole series of nitrone, i.e. C11-, C13-, C15- and C17-based compounds. Compared to the saturated 1,2-dimyristoyl-sn-glycero-3-phosphocholine, all bitailed amphiphilic nitrones were able to decrease conjugated dienes and TBARS in both oxidative paradigms, demonstrating therefore antioxidant property. The inhibition of phospholipids oxidation was increased when increasing the concentration of nitrone with the two B11PBN and B13PBN derivatives exhibiting higher potency. This study underlines the importance in the choice of a model membrane system when evaluating the potency of antioxidants against lipid oxidation.

Topological effects and binding modes operating with multivalent iminosugar-based glycoclusters and mannosidases.

Multivalent iminosugars have been recently explored for glycosidase inhibition. Affinity enhancements due to multivalency have been reported for specific targets, which are particularly appealing when a gain in enzyme selectivity is achieved but raise the question of the binding mode operating with this new class of inhibitors. Here we describe the development of a set of tetra- and octavalent iminosugar probes with specific topologies and an assessment of their binding affinities toward a panel of glycosidases including the Jack Bean α-mannosidase (JBαMan) and the biologically relevant class II α-mannosidases from Drosophila melanogaster belonging to glycohydrolase family 38, namely Golgi α-mannosidase ManIIb (GM) and lysosomal α-mannosidase LManII (LM). Very different inhibitory profiles were observed for compounds with identical valencies, indicating that the spatial distribution of the iminosugars is critical to fine-tune the enzymatic inhibitory activity. Compared to the monovalent reference, the best multivalent compound showed a dramatic 800-fold improvement in the inhibitory potency for JBαMan, which is outstanding for just a tetravalent ligand. The compound was also shown to increase both the inhibitory activity and the selectivity for GM over LM. This suggests that multivalency could be an alternative strategy in developing therapeutic GM inhibitors not affecting the lysosomal mannosidases. Dynamic light scattering experiments and atomic force microscopy performed with coincubated solutions of the compounds with JBαMan shed light on the multivalent binding mode. The multivalent compounds were shown to promote the formation of JBαMan aggregates with different sizes and shapes. The dimeric nature of the JBαMan allows such intermolecular cross-linking mechanisms to occur.

Atomic force microscopy of model lipid membranes.

Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir-Blodgett or Langmuir-Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

Probing the nature of the cluster effect observed with synthetic multivalent galactosides and peanut agglutinin lectin.

We designed a set of multi-galactosides with valencies ranging from one to seven and different spacer-arm lengths. The compounds display a high structural homology for a strict assessment of multivalent phenomena. The multimers were first evaluated by an enzyme-linked lectin assay (ELLA) toward the peanut agglutinin (PNA). The binding affinity was shown to be dependent on the spacer-arm length, and cluster effects were observed for the galactosides bearing the shortest and the longest linkers. The latter compounds were shown to be much more potent PNA cross-linkers in a "sandwich assay". Dynamic light scattering (DLS) experiments also revealed the formation of soluble aggregates between heptavalent derivatives with medium or long linkers and the labeled PNA. ELLA experiments performed with valency-controlled clusters and labeled lectins are therefore not always devoid from aggregative processes. The precise nature of the multivalent interaction observed by ELLA for the compounds bearing the shortest linkers, which are unable to form PNA aggregates, was further investigated by atomic force microscopy (AFM). The galactosides were grafted onto the tip of a cantilever and the PNA lectin onto a gold surface. Similar unbinding forces were registered when the valency of the ligands was increased, thus showing that the multimers cannot interact more strongly with PNA. Multiple binding events to the PNA were also never observed, thus confirming that a chelate binding mode does not operate with the multivalent galactosides, probably because the linkers are too short. Altogether, these results suggest that the cluster effect that operates in ELLA with the multimers is not related to additional PNA stabilizations and can be ascribed to local concentration effects that favor a dynamic turnover of the tethered galactosides in the PNA binding sites.

Titanium dioxide nanoparticles disturb the fibronectin-mediated adhesion and spreading of pre-osteoblastic cells.

In the context of rapid development of nanoparticles (NPs) for industrial applications, the question of their toxicity and biological effects must be considered. In this work, we have assessed the influence of titanium dioxide NPs on the adhesion and spreading of MC-3T3 pre-osteoblasts by using a cell subclone that does not produce its own extracellular matrix. Petri dishes were coated with the important adhesion protein fibronectin (Fn). By incubating these Fn-coated surfaces with different amounts of TiO(2) NPs, we have shown that the adhesion of pre-osteoblasts is disturbed, with an important decrease in the number of adherent cells (from 40 to 75% depending upon the concentration and type of NPs). Petri-dish surfaces were analyzed with environmental scanning electron microscropy (ESEM), with images showing that TiO(2) NP aggregates are bound to the layer of adsorbed Fn molecules. The cells cultured on these Fn/NP surfaces adopted an irregular shape and an aberrant organization of actin cytoskeleton, as revealed by fluorescence microscopy. Most importantly, these results, taken together, have revealed that the actin cytoskeleton forms abnormal aggregates, even on top of the nucleus, that coincide with the presence of large aggregates of NPs on top of cells. On the basis of these observations, we propose that some Fn molecules are able to desorb from the Petri dish surface to coat TiO(2) NPs. Fn/NP complexes are not attached firmly enough on the surface to allow for normal cell adhesion/spreading and the development of tense actin fibers. These results stress the paramount need for the assessment of the toxicology of NPs, with special attention to their interactions with biomolecules.

Preosteoblasts and fibroblasts respond differently to anatase titanium dioxide nanoparticles: a cytotoxicity and inflammation study.

There is a bundle of proofs suggesting that some industrial nanoparticles (NPs) can provoke diseases and pollute the environment durably. However, these issues still remain controversial. In the biomedical field, TiO(2) NPs were recently proposed to serve as fillers in polymeric materials to improve bone prostheses and scaffolds. Submicrometer TiO(2) particles could also result from wear debris of prostheses. Thus, it appears to be of the highest importance to elucidate the effects of well-characterized TiO(2) NPs on the behaviour of osteoblasts. In this work, we have measured the toxicity of anatase TiO(2) NPs with two different cell types, on L929 fibroblasts and for the first time on MC-3T3 pre-osteoblasts, with the aim to determine the level of cellular toxicity and inflammation. Our results clearly show that these NPs provoke different dose-response effects, with the pre-osteoblasts being much more sensitive than fibroblasts. Furthermore, we observed that anatase TiO(2) NPs had no effect on cell adhesion. By contrast, both cell types had their morphology and LDH release modified in the presence of NPs. Their DNA was also found to be fragmented as analyzed by quantifying the sub-G1 cell population with flow cytometry. By measuring the production of IL-6 and TNF-α proinflammatory cytokines, we have shown that TNF-α was never produced and that MC-3T3 cells were secreting IL-6. Most importantly, our results highlight the necessity of evaluating the toxicity of prostheses wear debris, and of NP coatings of medical implants, to determine if they can possibly provoke inflammation and inhibit bone reconstruction.

The natural antioxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidation in situ.

Exogenous molecules from dietary sources such as polyphenols are very efficient in preventing the alteration of lipid membranes by oxidative stress. Among the polyphenols, we have chosen to study rosmarinic acid (RA). We investigated the efficiency of RA in preventing lipid peroxidation and in interacting with lipids. We used liposomes of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) to show that RA was an efficient antioxidant. By HPLC, we determined that the maximum amount of RA associated with the lipids was ~1 mol%. Moreover, by using Langmuir monolayers, we evidenced that cholesterol decreases the penetration of RA. The investigation of transferred lipid/RA monolayers by atomic force microscopy revealed that 1 mol% of RA in the membrane was not sufficient to alter the membrane structure at the nanoscale. By fluorescence, we observed no significant modification of membrane permeability and fluidity caused by the interaction with RA. We also deduced that RA molecules were mainly located among the polar headgroups of the lipids. Finally, we prepared DLPC/RA vesicles to evidence for the first time that up to 1 mol% of RA inserts spontaneously in the membrane, which is high enough to fully prevent lipid peroxidation without any noticeable alteration of the membrane structure due to RA insertion.

The potent antimalarial peptide cyclosporin A induces the aggregation and permeabilization of sphingomyelin-rich membranes.

Cyclosporin A (CsA) is a hydrophobic peptide drug produced by the fungus Tolypocladium inflatum. CsA is commonly used as an immunosuppressive drug, but it also has antimalarial activity. The immunosuppressive activity of CsA is clearly due to its association with specific proteins of immune cells such as cyclophilins. By contrast, the antimalarial properties of this peptide are completely independent of the association with a parasite's cyclophilins. Because of its hydrophobicity, CsA may interact with biological membranes, which may participate in its therapeutic effect. Recently, we have shown a marked preference of CsA for insertion into sphingomyelin (SM) monolayers. In this article, we measure for the first time the ability of CsA to induce permeabilization and aggregation and to change the lipid order, especially in the presence of SM. Calcein-release experiments permitted us to show that CsA causes the leakage of the fluorescent probe from SM-rich liposomes by 40% and PC liposomes by 11%, suggesting a lipid-selective effect. Electron microscopy and dynamic light scattering experiments confirmed the different interaction of CsA with SM and PC vesicles: it formed much larger aggregates with SM than with PC. Our results taken together suggest that CsA could specifically weaken and aggregate SM-rich membranes, which could in turn explain why CsA is efficient in the treatment of malaria. Indeed, CsA could inhibit the development of Plasmodium by permeabilizing and aggregating the SM-rich membrane network formed by the parasite during its intraerythrocytic growth cycle.

Cytochrome c provokes the weakening of zwitterionic membranes as measured by force spectroscopy.

Cytochrome c (cyt c) is a small soluble protein from the intermembrane space of mitochondria. This protein is essential because it transfers electrons between two membrane complexes of the respiratory chain. In fact, during this transfer, the positively charged amino-acid residues surrounding the heme in the protein structure allow the cyt c to interact properly with the anionic part of other molecules: mainly the cardiolipin-rich membrane of mitochondria and respiratory complexes. We have previously shown that besides its interaction with anionic lipids, the cyt c is also able to cross neutral lipid membranes. In this work, with the help of AFM and punch-through experiments, we have measured the force required to penetrate the membrane in the fluid and in the gel phases with or without cyt c molecules. In the presence of cyt c molecules, the structures generated by the interaction with the protein were considerably weakened, which led to the desorption of the fluid bilayer and to a considerable loss of cohesion of the gel phase. These results show the usefulness of punch-through experiments in determining the changes of membrane properties in the presence of external agents.

Preparation of an electrochemical biosensor based on lipid membranes in nanoporous alumina.

Model lipid bilayers are versatile tools to investigate the molecular processes occurring at the membrane level. Among the model membranes, substrate supported bilayers have attracted much interest because they are robust and they can be investigated by powerful surface sensitive techniques such as electrochemical measurements. In a biosensor, lipid films can be used not only as a support for the biological sensing elements but also as sensing elements themselves to detect molecules that are able to alter the structure and the properties of biomembranes. In this work, we have prepared a tethered lipid membrane-based biosensor able to detect the alterations of membrane structure and fluidity. This tethered lipid membrane was prepared in a nanoporous aluminium oxide that provides a high surface area and a protective environment against dewetting. The membrane contained PEG-PE lipids as hydrating, protective and tethering agents and ubiquinone which is a redox lipophilic mediator embedded within the acyl chains of the lipid bilayer. The lipid membrane was prepared inside the pores of the nanoporous support by a PEG-triggered fusion of liposomes. This sensing system was efficient to detect the alterations of lipid membranes that are induced by the addition of a commonly used non-ionic detergent: Triton X-100.

The potent antimalarial drug cyclosporin A preferentially destabilizes sphingomyelin-rich membranes.

Cyclosporin A (CsA) is a hydrophobic cyclic peptide produced by a fungus. CsA is widely used as an immunosuppressive agent to inhibit the rejection of transplanted organs. CsA also exhibits an antiparasitic activity against Plasmodium, the microorganism responsible for malaria disease. This antimalarial activity is not completely understood yet. In this study, we have used Langmuir monolayers and atomic force microscopy to investigate the interaction of CsA with different lipids: phosphatidylcholines with different molecular packing, cholesterol, and sphingomyelin. We have shown that CsA inserts in all kinds of lipid monolayers but it has a marked preference for sphingomyelin monolayers. This preferential insertion of CsA within sphingomyelin-enriched membranes could explain the antimalarial activity of CsA. Indeed, the parasites need to produce a membrane network inside the erythrocytes, which allows for their proper development/multiplication by exchanging nutrients with the external medium. This membrane network is particularly enriched in sphingomyelin, so the preferential insertion of CsA in these bilayers may destabilize them, thereby inhibiting the development of the parasite.