Kyoungmi Min

Cross experimental analysis of microarray gene expression data from volatile organic compounds treated targets

DNA microarrays have revolutionized environmental research by enabling the discovery of genomic markers that reflect the toxic effect of various chemicals and by providing information on the underlying mechanisms. Microarray-based toxicogenomics approaches have become a popular tool to investigate potential risks of exposure to various environmental contaminants at the DNA level. Especially, the analysis of microarray data that are generated under various experimental conditions is critically important for validation of biomarkers and, thus, diagnosis and treatment of environmental targets. Presently, we identified commonly regulated genes whose expression level varied upon exposure to volatile organic compounds (VOCs) by performing cross-experimental analysis of public gene expression datasets using the RankProd algorithm. VOCs are chemical contaminants that often exhibit long-term adverse effects upon chronic exposure. Since VOCs are often used in household items and residential buildings, it is important to understand their effect on human health in a more systematic way. This cross-experiment resulted in a valid set of commonly regulated genes. The functional analysis of these differentially expressed genes (DEGs) generated several significantly over-represented Gene Ontology terms and identified metabolic pathways tightly-associated with cancer development. The functional analysis of identified up-regulated genes (RPL27, RPS6, RPS11, RPS27A, AURKA, FNTA, HSP90AB1) revealed concordance with genes related to various respiratory symptoms such as non-small cell lung cancer. The selected commonly regulated diseaserelated genes were also compared with the DEGs identified in previous analysis performed individually for validation of biomarkers.

Optimized magnetic bead-based immunoassay for automated detection of protein toxins

Rapid, accurate, and autonomous analysis of bioagents in the environment is critical in protecting human health from natural and intentional environmental contamination with biological toxicants. We previously developed and tested an immunoassay protocol that can be utilized for automated and simultaneous detection of selected biological agents and toxins. We adopted an antibody-based approach for the detection of pathogens and/or toxins. The fluorescent eTags™ were used as reporter molecules and the immunoassay was modified for automated field-deployed detection of pathogens and/or toxins. The present study improved the limit of detection of this system to be suitable for the detection of environmental toxins. We tested different settings to optimize the assay protocol and successfully detected 10 ng/mL (or 100 fg) of an toxin analog, ovalbumin. The developed assay represents a notable improvement from currently available assays in terms of reduced time, increased sensitivity, and automation potential. Additionally, this assay can be easily modified, with the appropriate antibodies, to detect a wide range of proteins and infectious agents.

Site-specific and effective immobilization of proteins by Npu DnaE split-intein mediated protein trans-splicing reaction

Immobilized proteins on solid supports provide a useful platform for various biological assays including proteomics research, protein-protein interaction studies, and diagnosis. In fabricating protein chips, proteins should be immobilized to maintain activity and specificity towards their targets. Here, we describe an approach to attach a protein of interest onto a solid support through a covalent bond as well as in situ monitoring of protein immobilization and subsequent binding assays. Our system utilized self-assembled monolayers of alkanethiolates on gold as biologically inert solid substrates, and Nostoc punctiforme DnaE splitinteins were used to mediate biospecific interactions and covalently conjugate proteins on the solid support. Use of a gold substrate enabled in situ monitoring of the protein-protein interactions using surface plasmon resonance spectroscopy. This approach provides a flexible method for further protein immobilization applications.

Efficient and wash-free labeling of membrane proteins using engineered Npu DnaE split-inteins: Intein Mediated Labeling of Membrane Proteins

An efficient and wash‐free method to conjugate a fluorescent tag to a target membrane protein is developed, using engineered Npu DnaE split‐inteins. This approach allowed fast labeling while avoiding the strenuous synthesis of a long polypeptide. Two different modes of labeling, namely specific binding and covalent conjugation, are observed. The covalent labeling was monitored within 5 min, without background staining.