Moo-Yeal Lee

Enzyme activation for nonaqueous media

Highly active enzyme formulations can be prepared for use in nonaqueous media. Considerable progress has been made in the past two years on gaining an improved mechanistic understanding of enzyme function and activation in dehydrated environments. This increased fundamental understanding has led to the development of a broad array of techniques for generating active, stable, and enantioselective and regioselective tailored enzymes for synthetically relevant transformations. This, in turn, is resulting in an exponential increase in the opportunities for enzymatic processes to be developed on a commercial scale.

Removal of heavy metals from aqueous solution by apple residues

Abstract The removal of copper, lead and cadmium by apple residues (AR) was investigated to evaluate cation exchange capacities. The effects of solution pH, ionic strength, ligands and co-ions were studied in batch experiments. Apple residues were modified with phosphorus (V) oxychloride to improve their physico-chemical properties and greatly enhance the capacity of metal removal. Adsorption equilibria were established rapidly initially and decreased markedly after 1 h. Column experiments were carried out in a glass column filled with AR and modified AR to evaluate the metal removal capacity. The influences of the feed concentration, chemical treatment and ligand were also studied. After exhaustion of the residues, the columns were regenerated successfully by a simple elution procedure.

Three-dimensional cell culture microarray for high-throughput studies of stem cell fate.

We have developed a novel three‐dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high‐throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox‐1, whose levels were also measured in situ using a GFP reporter system. In addition, the high‐throughput capacity of the platform was tested using a dual‐slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor‐4 (FGF‐4) on the pluripotency of mouse ES cells. This high‐throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses. Biotechnol. Bioeng. 2010; 106: 106–118.

On-Chip, Cell-Based Microarray Immunofluorescence Assay for High-Throughput Analysis of Target Proteins

We have developed an immunofluorescence-based assay for high-throughput analysis of target proteins on a three-dimensional cellular microarray platform. This process integrates the use of three-dimensional cellular microarrays, which should better mimic the cellular microenvironment, with sensitive immunofluorescence detection and provides quantitative information on cell function. To demonstrate this assay platform, we examined the accumulation of the alpha subunit of the hypoxia-inducible factor (HIF-1alpha) after chemical stimulation of human pancreatic tumor cells encapsulated in 3D alginate spots in volumes as low as 60 nL. We also tested the effect of the known dysregulator of HIF-1alpha, 2-methoxyestradiol (2ME2), on the levels of HIF-1alpha using a dual microarray stamping technique. This chip-based in situ Western immunoassay protocol was able to provide quantitative information on cell function, namely, the cellular response to hypoxia mimicking conditions and the reduction of HIF-1alpha levels after cell treatment with 2ME2. This system is the first to enable high-content screening of cellular protein levels on a 3D human cell microarray platform.

Micro precipitation of lead on the surface of crab shell particles

Crab shell particles were used as a biosorbent to remove lead from aqueous solutions. The equilibrium isotherm showed that crab shell particles took up lead to the extent of 1300 mg Pb g−1 crab shell. The optimum pH range for maximum lead removal was increased to 5·5–11·0 compared to the shell-free control pH of 8·5–11·0. pH values of solutions with crab shell material added were increased spontaneously to about 10 as a result of the CaCO3 present, which formed complexes with lead according to pH. Electron spectroscopy, Fourier transform infrared spectrometry, scanning electron microscopy and X-ray diffraction results confirmed that -NHCOCH3 and CO32 were involved in binding of lead. In addition, the removal of lead occurred mainly through dissolution of CaCO3 followed by precipitation of Pb3(CO3)2(OH)2 and PbCO3 near the surface of crab shell. Micro precipitates formed were then adsorbed to the chitin on the surface of the crab shell particles.

Gene Delivery in Three-Dimensional Cell Cultures by Superparamagnetic Nanoparticles

Three-dimensional (3D) cellular assays closely mimic the in vivo milieu, providing a rapid, inexpensive system for screening drug candidates for toxicity or efficacy in the early stages of drug discovery. However, 3D culture systems may suffer from mass transfer limitations, particularly in delivery of large polypeptide or nucleic acid compounds. Nucleic acids (e.g., genes, silencing RNA) are of particular interest both as potential therapeutics and due to a desire to modulate the gene-expression patterns of cells exposed to small-molecule pharmacological agents. In the present study, polyethylenimine (PEI)-coated superparamagnetic nanoparticles (SPMNs) were designed to deliver interfering RNA and green fluorescent protein (GFP) plasmids through a collagen-gel matrix into 3D cell cultures driven by an external magnetic field. The highest transfection efficiency achieved was 64% for siRNA and 77% for GFP plasmids. Delivery of an shRNA plasmid against GFP by PEI-coated SPMNs silenced the GFP expression with 82% efficiency. We further demonstrated that this delivery approach could be used for screening interfering RNA constructs for therapeutic or toxic effects for cells grown in 3D cultures. Four known toxic shRNA plasmids were delivered by PEI-coated SPMNs into 3D cell cultures, and significant toxicities (41-51% cell death) were obtained.

High-Throughput Screening (HTS) of Anticancer Drug Efficacy on a Micropillar/Microwell Chip Platform

Contemporary cancer therapy refers to treatment based on genetic abnormalities found in patients tumor. However, this approach is faced with numerous challenges, including tumor heterogeneity and molecular evolution, insufficient tumor samples available along with genetic information linking to clinical outcomes, lack of therapeutic drugs containing pharmacogenomic information, and technical limitations of rapid drug efficacy tests with insufficient quantities of primary cancer cells from patients. To address these problems and improve clinical outcomes of current personalized gene-targeted cancer therapy, we have developed a micropillar/microwell chip platform, which is ideally suited for encapsulating primary cancer cells in nanoscale spots of hydrogels on the chip, generating efficacy data with various drugs, eventually allowing for a comparison of the in vitro data obtained from the chip with clinical data as well as gene expression data. As a proof of concept in this study, we have encapsulated a U251 brain cancer cell line and three primary brain cancer cells from patients (448T, 464T, and 775T) in 30 nL droplets of alginate and then tested the therapeutic efficacy of 24 anticancer drugs by measuring their dose responses. As a result, the IC50 values of 24 anticancer drugs obtained from the brain cancer cells clearly showed patient cell-specific efficacy, some of which were well-correlated with their oncogene overexpression (c-Met and FGFR1) as well as the in vivo previous results of the mouse xenograft model with the three primary brain cancer cells.

Effects of N-acetylation degree on N-acetylated chitosan hydrolysis with commercially available and modified pectinases

Three types of N-acetylated chitosans (NACs) with different degrees of acetylation (DA) were prepared and used as a substrate for enzymatic hydrolysis with a commercially available pectinase and a modified one. Pectinase modification was conducted using polyalkyleneoxide-maleic anhydride copolymer (PEO-MA copolymer). The effects of DA on enzymatic reaction with native and modified pectinases were investigated experimentally. Initial hydrolysis rate and Michaelis-Menten kinetic parameters were measured by analysis of reducing sugars. DA of NAC strongly affected the hydrolytic characteristics of native and modified pectinases. N-acetylation of chitosan increased the initial hydrolysis rate and the enzyme-substrate affinity with respect to both pectinases: NACs with DA over 0.3 showed high initial hydrolysis rate and strong affinity between enzyme and substrate. Especially, when NAC with DA over 0.3 was treated with modified pectinase, the affinity became much stronger than the native pectinase.

High-throughput and combinatorial gene expression on a chip for metabolism-induced toxicology screening

Differential expression of various drug-metabolizing enzymes in the human liver may cause deviations of pharmacokinetic profiles, resulting in inter-individual variability of drug toxicity and/or efficacy. Here we present the “Transfected Enzyme and Metabolism Chip” (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip. The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2E1, and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray. Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner.

Removal of Lead in a Fixed-Bed Column Packed with Activated Carbon and Crab Shell

ABSTRACT Crab shell particles (Protunus trituberculatus) and activated carbon (Norit 0,8 SUPRA) were used as packing materials in a fixed-bed column. When 1 g crab shell was added in a column packed with 10 g activated carbon, breakthrough occurred at 1500 bed volumes as compared to 380 bed volumes for 10 g activated carbon only. The addition of crab shell particles into an activated carbon column resulted in an increased uptake of lead. The dramatic improvement might be attributed to an increase in and OH− available for binding lead. From the results of SEM, XRD, and FT-IR analyses, the major mechanism of lead removal was based on dissolution of CaCO3 in the crab shell followed by precipitation of Pb3(CO3)2(OH)2(s) on the surface of activated carbon. The lead uptake increased twofold when the influent lead concentration was increased from 10 to 50 mg/L.