Nam-Joon Cho

Quartz crystal microbalance with dissipation monitoring of supported lipid bilayers on various substrates.

Supported lipid bilayers (SLBs) mimic biological membranes and are a versatile platform for a wide range of biophysical research fields including lipid–protein interactions, protein–protein interactions and membrane-based biosensors. The quartz crystal microbalance with dissipation monitoring (QCM-D) has had a pivotal role in understanding SLB formation on various substrates. As shown by its real-time kinetic monitoring of SLB formation, QCM-D can probe the dynamics of biomacromolecular interactions. We present a protocol for constructing zwitterionic SLBs supported on silicon oxide and titanium oxide, and discuss technical issues that need to be considered when working with charged lipid compositions. Furthermore, we explain a recently developed strategy that uses an amphipathic, α-helical (AH) peptide to form SLBs on gold and titanium oxide substrates. The protocols can be completed in less than 3 h.

Quantifying the local tissue volume and composition in individual brains with magnetic resonance imaging

Here, we describe a quantitative neuroimaging method to estimate the macromolecular tissue volume (MTV), a fundamental measure of brain anatomy. By making measurements over a range of field strengths and scan parameters, we tested the key assumptions and the robustness of the method. The measurements confirm that a consistent quantitative estimate of MTV can be obtained across a range of scanners. MTV estimates are sufficiently precise to enable a comparison between data obtained from an individual subject with control population data. We describe two applications. First, we show that MTV estimates can be combined with T1 and diffusion measurements to augment our understanding of the tissue properties. Second, we show that MTV provides a sensitive measure of disease status in individual patients with multiple sclerosis. The MTV maps are obtained using short clinically appropriate scans that can reveal how tissue changes influence behavior and cognition.

Biotechnology Applications of Tethered Lipid Bilayer Membranes

The importance of cell membranes in biological systems has prompted the development of model membrane platforms that recapitulate fundamental aspects of membrane biology, especially the lipid bilayer environment. Tethered lipid bilayers represent one of the most promising classes of model membranes and are based on the immobilization of a planar lipid bilayer on a solid support that enables characterization by a wide range of surface-sensitive analytical techniques. Moreover, as the result of molecular engineering inspired by biology, tethered bilayers are increasingly able to mimic fundamental properties of natural cell membranes, including fluidity, electrical sealing and hosting transmembrane proteins. At the same time, new methods have been employed to improve the durability of tethered bilayers, with shelf-lives now reaching the order of weeks and months. Taken together, the capabilities of tethered lipid bilayers have opened the door to biotechnology applications in healthcare, environmental monitoring and energy storage. In this review, several examples of such applications are presented. Beyond the particulars of each example, the focus of this review is on the emerging design and characterization strategies that made these applications possible. By drawing connections between these strategies and promising research results, future opportunities for tethered lipid bilayers within the biotechnology field are discussed.

pH-Driven Assembly of Various Supported Lipid Platforms: A Comparative Study on Silicon Oxide and Titanium Oxide

Supported lipid platforms are versatile cell membrane mimics whose structural properties can be tailored to suit the application of interest. By identifying parameters that control the self-assembly of these platforms, there is potential to develop advanced biomimetic systems that overcome the surface specificity of lipid vesicle interactions under physiological conditions. In this work, we investigated the adsorption kinetics of vesicles onto silicon and titanium oxides as a function of pH. On each substrate, a planar bilayer and a layer of intact vesicles could be self-assembled in a pH-dependent manner, demonstrating the role of surface charge density in the self-assembly process. Under acidic pH conditions where both zwitterionic lipid vesicles and the oxide films possess near-neutral electric surface charges, vesicle rupture could occur, demonstrating that the process is driven by nonelectrostatic interactions. However, we observed that the initial rupturing process is insufficient for propagating bilayer formation. The role of electrostatic interactions for propagating bilayer formation differs for the two substrates; electrostatic attraction between vesicles and the substrate is necessary for complete bilayer formation on titanium oxide but is not necessary on silicon oxide. Conversely, in the high pH regime, repulsive electrostatic interactions can result in the irreversible adsorption of intact vesicles on silicon oxide and even a reversibly adsorbed vesicle layer on titanium oxide. Together, the results show that pH is an effective tool to modulate vesicle-substrate interactions in order to create various self-assembled lipid platforms on hydrophilic substrates.

Solvent-Assisted Lipid Bilayer Formation on Silicon Dioxide and Gold

Planar lipid bilayers on solid supports mimic the fundamental structure of biological membranes and can be investigated using a wide range of surface-sensitive techniques. Despite these advantages, planar bilayer fabrication is challenging, and there are no simple universal methods to form such bilayers on diverse material substrates. One of the novel methods recently proposed and proven to form a planar bilayer on silicon dioxide involves lipid deposition in organic solvent and solvent exchange to influence the phase of adsorbed lipids. To scrutinize the specifics of this solvent-assisted lipid bilayer (SALB) formation method and clarify the limits of its applicability, we have developed a simplified, continuous solvent-exchange version to form planar bilayers on silicon dioxide, gold, and alkanethiol-coated gold (in the latter case, a lipid monolayer is formed to yield a hybrid bilayer) and varied the type of organic solvent and rate of solvent exchange. By tracking the SALB formation process with simultaneous quartz crystal microbalance-dissipation (QCM-D) and ellipsometry, it was determined that the acoustic, optical, and hydration masses along with the acoustic and optical thicknesses, measured at the end of the process, are comparable to those observed by employing conventional fabrication methods (e.g., vesicle fusion). As shown by QCM-D measurements, the obtained planar bilayers are highly resistant to protein adsorption, and several, but not all, water-miscible organic solvents could be successfully used in the SALB procedure, with isopropanol yielding particularly high-quality bilayers. In addition, fluorescence recovery after photobleaching (FRAP) measurements demonstrated that the coefficient of lateral lipid diffusion in the fabricated bilayers corresponds to that measured earlier in the planar bilayers formed by vesicle fusion. With increasing rate of solvent exchange, it was also observed that the bilayer became incomplete and a phenomenological model was developed in order to explain this feature. The results obtained allowed us to clarify and discriminate likely steps of the SALB formation process as well as determine the corresponding influence of organic solvent type and flow conditions on these steps. Taken together, the findings demonstrate that the SALB formation method can be adapted to a continuous solvent-exchange procedure that is technically minimal, quick, and efficient to form planar bilayers on solid supports.

Identification of a Class of HCV Inhibitors Directed Against the Nonstructural Protein NS4B

An activity identified in hepatitis C virus NS4B paves the way for a distinct new class of antivirals. Hepatitis C is a surreptitious infection leading to inflammation of the liver, chronic liver disease, and ultimately causing cirrhosis. Nearly 150 million infected patients worldwide are in serious need of an alternative to viral suppressants in current use that have significant toxicities and are often ineffective. Cho and colleagues now take a closer look at the molecular virology of hepatitis C and identify a crucial new function for a largely uncharacterized protein, NS4B. They found that a particular region of this protein, which is critically involved in forming small vesicle aggregates that form the hypothesized platform to facilitate viral genome replication, can be manipulated to serve as a readout for high-throughput screens aimed at uncovering small-molecule pharmacological inhibitors of hepatitis C genome replication. Screening through a milieu of possibilities, they demonstrate the utility of two such compounds and tease apart the mechanism and biochemical activities by which these small inhibitors disrupt NS4B function and ultimately viral replication. Whether these new compounds can be used as monotherapies or in amalgamation with current strategies awaits further testing. New classes of drugs are needed to combat hepatitis C virus (HCV), an important worldwide cause of liver disease. We describe an activity of a key domain, an amphipathic helix we termed 4BAH2, within a specific HCV nonstructural protein, NS4B. In addition to its proposed role in viral replication, we validate 4BAH2 as essential for HCV genome replication and identify first-generation small-molecule inhibitors of 4BAH2 that specifically prevent HCV replication within cells. Mechanistic studies reveal that the inhibitors target 4BAH2 function by preventing either 4BAH2 oligomerization or 4BAH2 membrane association. 4BAH2 inhibitors represent an additional class of compounds with potential to effectively treat HCV.

Influence of Osmotic Pressure on Adhesion of Lipid Vesicles to Solid Supports

The adhesion of lipid vesicles to solid supports represents an important step in the molecular self-assembly of model membrane platforms. A wide range of experimental parameters are involved in controlling this process, including substrate material and topology, lipid composition, vesicle size, solution pH, ionic strength, and osmotic pressure. At present, it is not well understood how the magnitude and direction of the osmotic pressure exerted on a vesicle influence the corresponding adsorption kinetics. In this work, using quartz crystal microbalance with dissipation (QCM-D) monitoring, we have experimentally studied the role of osmotic pressure in the adsorption of zwitterionic vesicles onto silicon oxide. The osmotic pressure was induced by changing the ionic strength of the solvent across an appreciably wider range (from 25 to 1000 mM NaCl outside of the vesicle, and 125 mM NaCl inside of the vesicle, unless otherwise noted) compared to that used in earlier works. Our key finding is demonstration that, by changing osmotic pressure, all three generic types of the kinetics of vesicle adsorption and rupture can be observed in one system, including (i) adsorption of intact vesicles, (ii) adsorption and rupture after reaching a critical vesicle coverage, and (iii) rupture just after adsorption. Furthermore, theoretical analysis of pressure-induced deformation of adsorbed vesicles and a DLVO-type analysis of the vesicle-substrate interaction qualitatively support our observations. Taken together, the findings in this work demonstrate that osmotic pressure can either promote or impede the rupture of adsorbed vesicles on silicon oxide, and offer experimental evidence to support adhesion energy-based models that describe the adsorption and spontaneous rupture of vesicles on solid supports.

A small molecule inhibits HCV replication and alters NS4B's subcellular distribution

Hepatitis C Virus (HCV) is a leading cause of liver disease and represents a significant public health challenge. Treatments for this disease are inadequate and improved antiviral therapies are necessary. Several such antivirals are in development, most of which target the well-characterized NS3 protease or the NS5B polymerase. In contrast, the nonstructural 4B (NS4B) protein, though essential for HCV RNA replication, has been the subject of few pharmacological studies. One of the functions ascribed to this protein is the ability to form intracellular membrane-associated foci (MAF), which are believed to be related to the sites of viral replication. Here, we report the identification of a small molecule that inhibits HCV replication and disrupts the organization of these MAF. Genetic analysis links the compounds mode of action to the NS4B gene product, and transient transfections of NS4B-GFP demonstrate that treatment with this compound can lead to the formation of novel elongated assemblies of NS4B. Furthermore, an in vitro dynamic light scattering assay provides evidence that the second amphipathic helix of NS4B may be the target of the drug. Our results demonstrate that this molecule represents a new potential class of HCV inhibitors and also provides us with a useful tool for studying the HCV life cycle.

Type I Collagen-Functionalized Supported Lipid Bilayer as a Cell Culture Platform

The supported phospholipid bilayer serves as an important biomimetic model for the cell membrane in both basic and applied scientific research. We have constructed a biomimetic platform based on a supported phospholipid bilayer that is functionalized with type I collagen to serve as a substrate for cell culture. To create the type I collagen-functionalized lipid bilayer assembly, a simple chemical approach was employed: lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (DP-NGPE), a carboxylic acid-functionalized phospholipid, were prepared and then fused onto an SiO(2) substrate to form a supported lipid bilayer. Subsequently, type I collagen molecules were introduced to form stable collagen-lipid conjugates via amide linkages with activated DP-NGPE lipids. The binding kinetics of the conjugation process and the resultant changes in film thickness and viscoelasticity were followed using the quartz crystal microbalance with dissipation (QCM-D) monitoring. The morphology of the conjugated collagen adlayer was investigated with atomic force microscopy (AFM). We observed that the adsorbed collagen molecules tended to self-assemble into fibrillar structures. Fluorescence recovery after photobleaching (FRAP) was utilized to estimate lateral lipid mobility, which was reduced by up to 20% after the coupling of type I collagen to the underlying lipid bilayer. As a cell culture platform, the collagen-conjugated supported lipid bilayer showed promising results. Smooth muscle cells (A10) retained normal growth behavior on the collagen-functionalized platform, unlike the bare POPC lipid bilayer and the POPC/DG-NGPE bilayer without collagen. The biomimetic functionalized lipid system presented here is a simple, yet effective approach for constructing a cell culture platform to explore the interactions between extracellular matrix components and cells.

Mechanism of an Amphipathic α-Helical Peptide’s Antiviral Activity Involves Size-Dependent Virus Particle Lysis

The N-terminal region of the hepatitis C virus (HCV) nonstructural protein NS5A contains an amphipathic alpha-helix that is necessary and sufficient for NS5A membrane association. A synthetic peptide (AH) comprising this amphipathic helix is able to lyse lipid vesicles that serve as a model system for virus particles. Based on quartz crystal microbalance-dissipation (QCM-D) experiments, the degree of vesicle rupturing was found to be inversely related to vesicle size, with maximal activity in the size range of several medically important viruses. In order to confirm and further study vesicle rupture, dynamic light scattering (DLS) and atomic force microscopy (AFM) experiments were also performed. The size dependence of vesicle rupturing helps explain the peptides observed effect on the infectivity of a wide range of viruses. Further, in vitro studies demonstrated that AH peptide treatment significantly decreased the infectivity of HCV particles. Thus, the AH peptide might be used to rupture HCV particles extra-corporally (for HCV prevention) and within infected individuals (for HCV therapy).