Sang-Joon Cho

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.

Scanning ion conductance microscopy for imaging biological samples in liquid: a comparative study with atomic force microscopy and scanning electron microscopy.

The present study was designed to show the applicability of scanning ion conductance microscopy (SICM) for imaging different types of biological samples. For this purpose, we first applied SICM to image collagen fibrils and showed the usefulness of the approach-retract scanning (ARS)/hopping mode for such samples with steep slopes. Comparison of SICM images with those obtained by AFM revealed that the ARS/hopping SICM mode can probe the surface topography of collagen fibrils and chromosomes at nanoscale resolution under liquid conditions. In addition, we successfully imaged cultured HeLa cells, with 15 μm in height by ARS/hopping SICM mode. Because SICM can obtain non-contact (or force-free) images, delicate cellular projections were visualized on the surface of the fixed cell. SICM imaging of live HeLa cells further demonstrated its applicability to study the morphological dynamics associated with biological processes on the time scale of minutes under liquid conditions. We further applied SICM for imaging the luminal surface of the trachea and succeeded in visualizing the surface of both ciliated and non-ciliated cells. These SICM images were comparable with those obtained by scanning electron microscopy. Although the dynamic mode of AFM provides better resolution than the ARS/hopping mode of SICM in some samples, only the latter can obtain contact-free images of samples with steep slopes, rendering it an important tool for observing live cells as well as unfixed or fixed soft samples with complicated shapes. Taken together, we demonstrate that SICM imaging, especially using an ARS/hopping mode, is a useful technique with unique capabilities for imaging the three-dimensional topography of a range of biological samples under physiologically relevant aqueous conditions.

Three-dimensional imaging of undercut and sidewall structures by atomic force microscopy

Sidewall surface roughness is an important parameter in electronic device manufacture. At present, no high resolution technique exists to quantitatively characterize this property for undercut structures created by semiconductor processing techniques. We developed a three-dimensional atomic force microscope (3D-AFM) to measure the surface roughness of undercut sidewalls with nanometer precision. Decoupled from the positional scanner, the 3D-AFM probe had a variable tilt up to 40° off the normal. Nonorthogonal scans resolved the sidewall surface roughness, base width, and acute critical angle for undercut structures, including a metal overhang and the transmission line of a photonic device. Compatible with standard cantilevers, the 3D-AFM demonstrates great potential for characterizing the sidewalls of soft materials such as photoresist.

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).

Controlling the Formation of Phospholipid Monolayer, Bilayer, and Intact Vesicle Layer on Graphene

Exciting progress has been made in the use of graphene for bio- and chemical sensing applications. In this regard, interfacing lipid membranes with graphene provides a high-sealing interface that is resistant to nonspecific protein adsorption and suitable for measuring biomembrane-associated interactions. However, a controllable method to form well-defined lipid bilayer coatings remains elusive, and there are varying results in the literature. Herein, we demonstrate how design strategies based on molecular self-assembly and surface chemistry can be employed to coat graphene surface with different classes of lipid membrane architectures. We characterize the self-assembly of lipid membranes on CVD-graphene using quartz crystal microbalance with dissipation, field-effect transistor, and Raman spectroscopy. By employing the solvent-assisted lipid bilayer (SALB) method, a lipid monolayer and bilayer were formed on pristine and oxygen-plasma-treated CVD-graphene, respectively. On these surfaces, vesicle fusion method resulted in formation of a lipid monolayer and intact vesicle layer, respectively. Collectively, these findings provide the basis for improved surface functionalization strategies on graphene toward bioelectronic applications.

A Trachea‐Inspired Bifurcated Microfilter Capturing Viable Circulating Tumor Cells via Altered Biophysical Properties as Measured by Atomic Force Microscopy

Circulating tumor cells (CTCs), though exceedingly rare in the blood, are nonetheless becoming increasingly important in cancer diagnostics. Despite this keen interest and the growing number of potential clinical applications, there has been limited success in developing a CTC isolation platform that simultaneously optimizes recovery rates, purity, and cell compatibility. Herein, a novel tracheal carina-inspired bifurcated (TRAB) microfilter system is reported, which uses an optimal filter gap size satisfying both 100% theoretical recovery rate and purity, as determined by biomechanical analysis and fluid-structure interaction (FSI) simulations. Biomechanical properties are also used to clearly discriminate between cancer cells and leukocytes, whereby cancer cells are selectively bound to melamine microbeads, which increase the size and stiffness of these cells. Nanoindentation experiments are conducted to measure the stiffness of leukocytes as compared to the microbead-conjugated cancer cells, with these parameters then being used in FSI analyses to optimize the filter gap size. The simulation results show that given a flow rate of 100 μL min(-1), an 8 μm filter gap optimizes the recovery rate and purity. MCF-7 breast cancer cells with solid microbeads are spiked into 3 mL of whole blood and, by using this flow rate along with the optimized microfilter dimensions, the cell mixture passes through the TRAB filter, which achieves a recovery rate of 93% and purity of 59%. Regarding cell compatibility, it is verified that the isolation procedure does not adversely affect cell viability, thus also confirming that the re-collected cancer cells can be cultured for up to 8 days. This work demonstrates a CTC isolation technology platform that optimizes high recovery rates and cell purity while also providing a framework for functional cell studies, potentially enabling even more sensitive and specific cancer diagnostics.

BIOPHYSICAL APPLICATIONS OF SCANNING ION CONDUCTANCE MICROSCOPY (SICM)

Scanning probe microscopy (SPM) techniques represent one of the most promising approaches to probe the physical and chemical properties of nanoscale materials. The growing convergence of physics and biology has demanded nanotechnology tools to understand the fundamental physics of biological systems. Despite the advantages of SPM techniques, there have been challenges with its application to characterization of biological specimens. In recent times, the development of one class of SPM technique, scanning ion conductance microscopy (SICM), has overcome these limitations and enabled noninvasive, nanoscale investigation of live cells. In this review article, we present the theory behind the SICM operating principles and data modeling. Based on this framework, we discuss recent research advances where the SICM technique has proven technically superior. SICM applications discussed herein include imaging of cell topography, monitoring of live cell dynamics, mechanical stimulation of live cells, and surface patterning. Additional findings on the combination of SICM with other SPM techniques as well as patch clamp electrophysiology are presented in the context of building integrated knowledge on the structure and function of live cells. In summary, SICM bridges physics and biology to enable a range of important biomedical applications.

Nanoscale fluctuations on epithelial cell surfaces investigated by scanning ion conductance microscopy

Nanoscale fluctuations on the apical surfaces of epithelial cells connected to neighboring cells were investigated by scanning ion conductance microscopy. Mapping the ion current as a function of the tip–surface distance revealed that in untreated cells, the apparent fluctuation amplitude increased towards the cell center. We found that the spatial dependence was less correlated with the heterogeneities of cell stiffness but was significantly reduced when actin filaments were disrupted. The results indicate that apical surface fluctuations are highly constrained at the cell–cell interface, in the vertical direction to the surface and by the underlying actin filaments.

High-throughput automatic defect review for 300mm blank wafers with atomic force microscope

While feature size in lithography process continuously becomes smaller, defect sizes on blank wafers become more comparable to device sizes. Defects with nm-scale characteristic size could be misclassified by automated optical inspection (AOI) and require post-processing for proper classification. Atomic force microscope (AFM) is known to provide high lateral and the highest vertical resolution by mechanical probing among all techniques. However, its low throughput and tip life in addition to the laborious efforts for finding the defects have been the major limitations of this technique. In this paper we introduce automatic defect review (ADR) AFM as a post-inspection metrology tool for defect study and classification for 300 mm blank wafers and to overcome the limitations stated above. The ADR AFM provides high throughput, high resolution, and non-destructive means for obtaining 3D information for nm-scale defect review and classification.

Quantitative Evaluation of Viral Protein Binding to Phosphoinositide Receptors and Pharmacological Inhibition

There is significant interest in developing analytical methods to characterize molecular recognition events between proteins and phosphoinositides, which are a medically important class of carbohydrate-functionalized lipids. Within this scope, one area of high priority involves quantitatively evaluating drug candidates that pharmacologically inhibit protein–phosphoinositide interactions. As full-length proteins are often difficult to produce, establishing methods to study these interactions with shorter, bioactive peptides would be advantageous. Herein, we report an atomic force microscopy (AFM)-based force spectroscopic approach to detect the specific interaction between an amphipathic, α-helical (AH) peptide derived from the hepatitis C virus NS5A protein and its biological target, the phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] phosphoinositide receptor. After optimization of the peptide tethering strategy and measurement parameters, the binding specificity of AH peptide for PI(4,5)P2 receptors was comparatively evaluated across a panel of phosphoinositides and the influence of ionic strength on AH–PI(4,5)P2 binding strength was tested. Importantly, these capabilities were translated into the development of a novel experimental methodology to determine the inhibitory activity of a small-molecule drug candidate acting against the AH–PI(4,5)P2 interaction, and extracted kinetic parameters agree well with literature values obtained by conventional biochemical methods. Taken together, our findings provide a nanomechanical basis for explaining the high binding specificity of the NS5A AH to PI(4,5)P2 receptors, in turn establishing an analytical framework to study phosphoinositide-binding viral peptides and proteins as well as a broadly applicable approach to evaluate candidate inhibitors of protein–phosphoinositide interactions.