Solubilization of planar bilayers with detergent

AbstractThe interaction of the nonionic detergent Triton X-100 with supported phosphatidylcholine planar lipid bilayers has been investigated by optically monitoring changes in the bilayer, using the technique of optical waveguide lightmode spectroscopy (OWLS). This technique has several advantages over the methods applied to the problem hitherto, including: high sensitivity; measurement in situ with good time resolution; the fact that the free detergent concentration is well-defined, and the lipid concentration in solution is zero; ease of studying the reversibility of the interaction; and the readiness with which absolute rather than effective amounts of detergent incorporated into the lipid can be determined. The main finding is that as the free Triton concentration increases, the detergent is first incorporated reversibly into the bilayer, then partly but never completely removes lipid, and finally (at or above the cmc) completely solubilizes the bilayer. The behaviour of the planar supported lipid bilayers is thus similar to that previously reported for lipid vesicles

Ligand-Specific Targeting of Microspheres to Phagocytes by Surface Modification with Poly(L-Lysine)-Grafted Poly(Ethylene Glycol) Conjugate

Purpose. The purpose of this study was to demonstrate specific receptor-mediated targeting of phagocytes by functional surface coatings of microparticles, shielding from nonspecific phagocytosis and allowing ligand-specific interactions via molecular recognition. Methods. Coatings of the comb polymer poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) were investigated for potential to inhibit 1) nonspecific spreading of human blood-derived macrophages (MOs) and dendritic cells (DCs) on glass and 2) nonspecific phagocytosis of PLL-g-PEG-coated, carboxylated polystyrene (PS) or biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres. Coating was performed by adsorption of positively charged PLL-g-PEG on negatively charged microparticles or plasma-cleaned glass through electrostatic interaction. The feasibility of ligand-specific interactions was tested with a model ligand, RGD, conjugated to PEG chains of PLL-g-PEG to form PLL-g-PEG-RGD and compared with inactive ligand conjugate, PLL-g-PEG-RDG. Results. Coatings with PLL-g-PEG largely impaired the adherence and spreading of MOs and DCs on glass. The repellent character of PLL-g-PEG coatings drastically reduced phagocytosis of coated PS and PLGA microparticles to 10% in presence of serum. With both MOs and DCs, we observed ligand-specific interactions with PLL-g-PEG-RGD coatings on glass and PS and PLGA microspheres. Ligand specificity was abolished when using inactive ligand conjugate PLL-g-PEG-RDG, whereas repellency of coating was maintained. Conclusions. Coatings of PLL-g-PEG-ligand conjugates provide a novel technology for ligand specific targeting of microspheres to MOs and DCs while reducing nonspecific phagocytosi

Ligand-specific targeting of microspheres to phagocytes by surface modification with poly(L-lysine)-grafted poly(ethylene glycol) conjugate

ISSN:0724-8741ISSN:1573-904

Simultaneous analysis of large-scale RNAi screens for pathogen entry

Large-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in human host cells for eight pathogens using image-based kinome-wide siRNA screens with siRNAs from three vendors. We propose a Parallel Mixed Model (PMM) approach that simultaneously analyzes several non-identical screens performed with the same RNAi libraries.; We show that PMM gains statistical power for hit detection due to parallel screening. PMM allows incorporating siRNA weights that can be assigned according to available information on RNAi quality. Moreover, PMM is able to estimate a sharedness score that can be used to focus follow-up efforts on generic or specific gene regulators. By fitting a PMM model to our data, we found several novel hit genes for most of the pathogens studied.; Our results show parallel RNAi screening can improve the results of individual screens. This is currently particularly interesting when large-scale parallel datasets are becoming more and more publicly available. Our comprehensive siRNA dataset provides a public, freely available resource for further statistical and biological analyses in the high-content, high-throughput siRNA screening field