The interaction of an antiparasitic peptide active against African Sleeping Sickness with cell membrane models

Zwitterionic peptides with trypanocidal activity are promising lead compounds for the treatment of African Sleeping Sickness, and have motivated research into the design of compounds capable of disrupting the protozoan membrane. In this study, we use the Langmuir monolayer technique to investigate the surface properties of an antiparasitic peptide, namely S-(2,4-dinitrophenyl)glutathione di-2-propyl ester, and its interaction with a model membrane comprising a phospholipid monolayer. The drug formed stable Langmuir monolayers. whose main feature was a phase transition accompanied by a negative surface elasticity. This was attributed to aggregation upon compression due to intermolecular bond associations of the molecules, inferred from surface pressure and surface potential isotherms. Brewster angle microscopy (BAM) images, infrared spectroscopy and dynamic elasticity measurements. When co-spread with dipalmitoyl phosphatidyl choline (DPPC). the drug affected both the surface pressure and the monolayer morphology, even at high surface pressures and with low amounts of the drug. The results were interpreted by assuming a repulsive, cooperative interaction between the drug and DPPC molecules. Such repulsive interaction and the large changes in fluidity arising from drug aggregation may be related to the disruption of the membrane, which is key for the parasite killing property. (C) 2009 Elsevier B.V. All rights reserved.

Interaction of oligonucleotide-based amphiphilic block copolymers with cell membrane models

Oligonucleotides have unique molecular recognition properties, being involved in biological mechanisms such as cell-surface receptor recognition or gene silencing. For their use in human therapy for drug or gene delivery, the cell membrane remains a barrier, but this can be obviated by grafting a hydrophobic tail to the oligonucleotide. Here we demonstrate that two oligonucleotides, one consisting of 12 guanosine units (G(12)), and the other one consisting of five adenosine and seven guanosine (A(5)G(7)) units, when functionalized with poly(butadiene), namely PB-G(12) and PB-A(5)G(7), can be inserted into Langmuir monolayers of dipalmitoyl phosphatidyl choline (DPPC), which served as a cell membrane model. PB-G(12) and PB-A(5)G(7) were found to affect the DPPC monolayer even at high surface pressures. The effects from PB-G(12) were consistently stronger, particularly in reducing the elasticity of the DPPC monolayers, which may have important biological implications. Multilayers of DPPC and nucleotide-based copolymers could be adsorbed onto solid supports, in the form of Y-type LB films, in which the molecular-level interaction led to lower energies in the vibrational spectra of the nucleotide-based copolymers. This successful deposition of solid films opens the way for devices to be produced which exploit the molecular recognition properties of the nucleotides. (C) 2010 Elsevier Inc. All rights reserved.

Comparative study of liponucleosides in Langmuir monolayers as cell membrane models

Liponucleosides may assist the anchoring of nucleic acid nitrogen bases into biological membranes for tailored nanobiotechnological applications. To this end precise knowledge about the biophysical and chemical details at the membrane surface is required. In this paper, we used Langmuir monolayers as simplified cell membrane models and studied the insertion of five lipidated nucleosides. These molecules varied in the type of the covalently attached lipid group, the nucleobase, and the number of hydrophobic moieties attached to the nucleoside. All five lipidated nucleosides were found to be surface-active and capable of forming stable monolayers. They could also be incorporated into dipalmitoylphosphatidylcholine (DPPC) monolayers, four of which induced expansion in the surface pressure isotherm and a decrease in the surface compression modulus of DPPC. In contrast, one nucleoside possessing three alkyl chain modifications formed very condensed monolayers and induced film condensation and an increase in the compression modulus for the DPPC monolayer, thus reflecting the importance of the ability of the nucleoside molecules to be arranged in a closely packed manner. The implications of these results lie on the possibility of tuning nucleic acid pairing by modifying structural characteristics of the liponucleosides. (C) 2010 Elsevier B.V. All rights reserved.

The lipid composition of a cell membrane modulates the interaction of an antiparasitic peptide at the air-water interface

The antiparasitic property of peptides is believed to be associated with their interactions with the protozoan membrane, which calls for research on the identification of membrane sites capable of peptide binding. In this study we investigated the interaction of a lipophilicglutathioine peptide known to be effective against the African Sleeping Sickness (ASS - African Trypanosomiasis) and cell membrane models represented by Langmuir monolayers. It is shown that even small amounts of the peptide affect the monolayers of some phospholipids and other lipids, which points to a significant interaction. The latter did not depend on the electrical charge of the monolayer-forming molecules but the peptide action was particularly distinctive for cholesterol + sphingomyelin monolayers that roughly resemble rafts on a cell membrane. Using in situ polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), we found that the orientation of the peptide is affected by the phospholipids and dioctadecyldimethylammonium bromide (DODAB), but not in monolayers comprising cholesterol + sphingomyelin. In this mixed monolayer resembling rafts, the peptide still interacts and has some induced order, probably because the peptide molecules are fitted together into a compact monolayer. Therefore, the lipid composition of the monolayer modulates the interaction with the lipophilic glutathioine peptide, and this may have important implications in understanding how the peptide acts on specific sites of the protozoan membrane. (C) 2011 Elsevier B.V. All rights reserved.

Chitosan as a removing agent of beta-lactoglobulin from membrane models

Many chitosan biological activities depend on the interaction with biomembranes, but so far it has not been possible to obtain molecular-level evidence of chitosan action. In this article, we employ Langmuir phospholipid monolayers as cell membrane models and show that chitosan is able to remove beta-lactoglobulin (BLG) from negatively charged dimyristoyl phosphatidic acid (DMPA) and dipalmitoyl phosphatidyl glycerol (DPPG). This was shown with surface pressure isotherms and elasticity and PM-IRRAS measurements in the Langmuir monolayers, in addition to quartz crystal microbalance and fluorescence spectroscopy measurements for Langmuir-Blodgett (LB) films transferred onto solid substrates. Some specificity was noted in the removal action because chitosan was unable to remove BLG incorporated into neutral dipalmitoyl phosphatidyl choline (DPPC) and cholesterol monolayers and had no effect on horseradish peroxidase and urease interacting with DMPA. An obvious biological implication of these findings is to offer reasons that chitosan can remove BLG from lipophilic environments, as reported in the recent literature.

The lipid composition of a cell membrane modulates the interaction of an antiparasitic peptide at the air-water interface

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Formation of the three-dimensional geometry of the red blood cell membrane

Red blood cells (RBCs) are nonnucleated liquid capsules, enclosed in deformable viscoelastic membranes with complex three dimensional geometrical structures. Generally, RBC membranes are highly incompressible and resistant to areal changes. However, RBC membranes show a planar shear deformation and out of plane bending deformation. The behaviour of RBCs in blood vessels is investigated using numerical models. All the characteristics of RBC membranes should be addressed to develop a more accurate and stable model. This article presents an effective methodology to model the three dimensional geometry of the RBC membrane with the aid of commercial software COMSOL Multiphysics 4.2a and Fortran programming. Initially, a mesh is generated for a sphere using the COMSOL Multiphysics software to represent the RBC membrane. The elastic energy of the membrane is considered to determine a stable membrane shape. Then, the actual biconcave shape of the membrane is obtained based on the principle of virtual work, when the total energy is minimised. The geometry of the RBC membrane could be used with meshfree particle methods to simulate motion and deformation of RBCs in micro-capillaries

Arenavirus budding resulting from viral-protein-associated cell membrane curvature

Viral replication occurs within cells, with release (and onward infection) primarily achieved through two alternative mechanisms: lysis, in which virions emerge as the infected cell dies and bursts open; or budding, in which virions emerge gradually from a still living cell by appropriating a small part of the cell membrane. Virus budding is a poorly understood process that challenges current models of vesicle formation. Here, a plausible mechanism for arenavirus budding is presented, building on recent evidence that viral proteins embed in the inner lipid layer of the cell membrane. Experimental results confirm that viral protein is associated with increased membrane curvature, whereas a mathematical model is used to show that localized increases in curvature alone are sufficient to generate viral buds. The magnitude of the protein-induced curvature is calculated from the size of the amphipathic region hypothetically removed from the inner membrane as a result of translation, with a change in membrane stiffness estimated from observed differences in virion deformation as a result of protein depletion. Numerical results are based on experimental data and estimates for three arenaviruses, but the mechanisms described are more broadly applicable. The hypothesized mechanism is shown to be sufficient to generate spontaneous budding that matches well both qualitatively and quantitatively with experimental observations.

Copper and lactational hormones influence the CTR1 copper transporter in PMC42-LA mammary epithelial cell culture models

Adequate amounts of copper in milk are critical for normal neonatal development, however the mechanisms regulating copper supply to milk have not been clearly defined. PMC42-LA cell cultures representative of resting, lactating and suckled mammary epithelia were used to investigate the regulation of the copper uptake protein, CTR1. Both the degree of mammary epithelial differentiation (functionality) and extracellular copper concentration greatly impacted upon CTR1 expression and its plasma membrane association. In all three models (resting, lactating and suckling) there was an inverse correlation between extracellular copper concentration and the level of CTR1. Cell surface biotinylation studies demonstrated that as extracellular copper concentration increased membrane associated CTR1 was reduced. There was a significant increase in CTR1 expression (total and membrane associated) in the suckled gland model in comparison to the resting gland model, across all copper concentrations investigated (0-50 μM). Regulation of CTR1 expression was entirely post-translational, as quantitative real-time PCR analyses showed no change to CTR1 mRNA between all models and culture conditions. X-ray fluorescence microscopy on the differentiated PMC42-LA models revealed that organoid structures distinctively accumulated copper. Furthermore, as PMC42-LA cell cultures became progressively more specialised, successively more copper accumulated in organoids (resting

The third dimension: new developments in cell culture models for colorectal research

Cellular models are important tools in various research areas related to colorectal biology and associated diseases. Herein, we review the most widely used cell lines and the different techniques to grow them, either as cell monolayer, polarized two-dimensional epithelia on membrane filters, or as three-dimensional spheres in scaffoldfree or matrix-supported culture conditions. Moreover, recent developments, such as gut-on-chip devices or the ex vivo growth of biopsy-derived organoids, are also discussed. We provide an overview on the potential applications but also on the limitations for each of these techniques, while evaluating their contribution to provide more reliable cellular models for research, diagnostic testing, or pharmacological validation related to colon physiology and pathophysiology.

Ceruloplasmin is regulated by copper and lactational hormones in PMC42-LA mammary epithelial cell culture models

Ceruloplasmin (Cp) is a multicopper ferroxidase that is considered to be an important source of copper in milk for normal neonatal development. We investigated the expression, subcellular localization and secretion of Cp in PMC42-LA cell culture models representative of resting, lactating and suckled human mammary epithelia. Both secreted Cp (sCp) and plasma membrane associated glycosylphosphatidylinositol-linked Cp (GPI-Cp) were expressed in PMC42-LA cells. In all three epithelial models (resting, lactating and suckled), the expression and secretion of copper-bound, ferroxidase active, Cp (holo-Cp) was dependent on media copper concentration. In low copper (bathocuproinedisulphonic acid/d-penicillamine treated models) there was greater than a 2-fold decrease in holo-Cp expression and secretion, which was mirrored by a 2-fold increase in the expression and secretion of copper-free Cp protein (apo-Cp). Cell surface biotinylation studies revealed that the state of PMC42-LA cell differentiation (functionality), and the level of extracellular copper, had no significant effect on the level of plasma membrane bound GPI-Cp. Quantitative real time PCR analyses determined that there was no significant (P > 0.05) difference in Cp mRNA levels across all copper conditions investigated (0, 5, 50 μM). However, there was a significant (P < 0.05) increase (∼2-fold) in Cp mRNA in both the lactating and suckled models in comparison to the resting model. Furthermore, the Cp mRNA increase in response to PMC42-LA differentiation corresponded with more secreted Cp protein, both apo and holo forms, indicating a link between function and Cp requirement. Our results provide significant insight on the regulation of Cp expression and secretion in lactation and copper incorporation into milk.

The MS risk allele of CD40 is associated with reduced cell-membrane bound expression in antigen presenting cells: Implications for gene function

Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS. © 2015 Field et al.

Voltage and Temperature Scalable Standard Cell Leakage Models Based on Stacks for Statistical Leakage Characterization

With extensive use of dynamic voltage scaling (DVS) there is increasing need for voltage scalable models. Similarly, leakage being very sensitive to temperature motivates the need for a temperature scalable model as well. We characterize standard cell libraries for statistical leakage analysis based on models for transistor stacks. Modeling stacks has the advantage of using a single model across many gates there by reducing the number of models that need to be characterized. Our experiments on 15 different gates show that we needed only 23 models to predict the leakage across 126 input vector combinations. We investigate the use of neural networks for the combined PVT model, for the stacks, which can capture the effect of inter die, intra gate variations, supply voltage(0.6-1.2 V) and temperature (0 - 100degC) on leakage. Results show that neural network based stack models can predict the PDF of leakage current across supply voltage and temperature accurately with the average error in mean being less than 2% and that in standard deviation being less than 5% across a range of voltage, temperature.

NANOLOCALIZED SINGLE CELL MEMBRANE NANOELECTROPORATION

Today single cell research is a great interest to analyze cell to cell or cell to environment behavior with their intracellular compounds, where bulk measurement can provide average value. To deliver biomolecules precise and localized way into single living cell with high transfection rate and high cell viability is a challenging and promisible task for biological and therapeutic research. In this report, we present a nano-localized single cell nano-electroporation technique, where electroporation take place in a very precise and localized area on a single cell membrane to achieve high efficient delivery with high cell viability. We fabricated 60nm gap with 40 nm triangular Indium Tin Oxide (ITO) based nano-eletcrode tip, which can intense electric field in a nano-localized area of a single cell to permeabilize cell membrane and deliver exogenous biomolecules from outside to inside of the cell. This device successfully deliver dyes, proteins into single cell with high cell viability (98%). The process not only control precise delivery mechanism into single cell with membrane reversibility, but also it can provide special, temporal and qualitative dosage control, which might be beneficial for therapeutic and biological cell studies.

Cell-Membrane-Mimicking Lipid-Coated Nanoparticles Confer Raman Enhancement to Membrane Proteins and Reveal Membrane-Attached Amyloid-beta Conformation

Identifying the structures of membrane bound proteins is critical to understanding their function in healthy and diseased states. We introduce a surface enhanced Raman spectroscopy technique which can determine the conformation of membrane-bound proteins, at low micromolar concentrations, and also in the presence of a substantial membrane-free fraction. Unlike conventional surface enhanced Raman spectroscopy, our approach does not require immobilization of molecules, as it uses spontaneous binding of proteins to lipid bilayer-encapsulated Ag nanoparticles. We apply this technique to probe membrane-attached oligomers of Amyloid-beta(40) (A beta(40)), whose conformation is keenly sought in the context of Alzheimer's disease. Isotope-shifts in the Raman spectra help us obtain secondary structure information at the level of individual residues. Our results show the presence of a beta-turn, flanked by two beta-sheet regions. We use solid-state NMR data to confirm the presence of the beta-sheets in these regions. In the membrane-attached oligomer, we find a strongly contrasting and near-orthogonal orientation of the backbone H-bonds compared to what is found in the mature, less-toxic A beta fibrils. Significantly, this allows a ``porin'' like beta-barrel structure, providing a structural basis for proposed mechanisms of A beta oligomer toxicity.