Youngeun Kwon

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.

Electromagnetized gold nanoparticles mediate direct lineage reprogramming into induced dopamine neurons in vivo for Parkinson's disease therapy

Electromagnetic fields (EMF) are physical energy fields generated by electrically charged objects, and specific ranges of EMF can influence numerous biological processes, which include the control of cell fate and plasticity. In this study, we show that electromagnetized gold nanoparticles (AuNPs) in the presence of specific EMF conditions facilitate an efficient direct lineage reprogramming to induced dopamine neurons in vitro and in vivo. Remarkably, electromagnetic stimulation leads to a specific activation of the histone acetyltransferase Brd2, which results in histone H3K27 acetylation and a robust activation of neuron-specific genes. In vivo dopaminergic neuron reprogramming by EMF stimulation of AuNPs efficiently and non-invasively alleviated symptoms in mouse Parkinsons disease models. This study provides a proof of principle for EMF-based in vivo lineage conversion as a potentially viable and safe therapeutic strategy for the treatment of neurodegenerative disorders.

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.

Cross-experimental analysis of microarray gene expression datasets for in silico risk assessment of TiO2 nano-particles

As the production and usage of nanomaterials increase, there are growing concerns on the unidentified detrimental effect of nanoparticles on human health and environmental safety. Systematic assessments of the risks associated with exposure to nanoparticles are needed. DNA microarrays have emerged as a powerful tool for toxicology research. Microarraybased toxicogenomics research provides valuable information for understanding underling mechanisms of toxicological behavior of non-classic contaminants, including ultrafine nanoparticles. In this work, we investigated the effect of nano-titanium oxide exposure on human cells by analyzing the change in transcription levels of cellular DNA. Cross-experimental analysis of heterogeneous gene expression datasets was performed using the RankProd algorithm. Multiple gene expression omnibus series obtained from various experimental conditions were combined and used for risk assessment. Several commonly regulated genes were identified as being unaffected by the laboratory specific conditions. Pathway analysis revealed the genes as being associated with six major pathways: arachidonic acid metabolism, purine metabolism, pentose phosphate pathway, mitogen-activated protein kinase signaling pathway, synthesis and degradation of ketone bodies, and methionine metabolism. The identified differently expressed genes provide a robust set of markers for exposure analysis and risk assessment of titanium oxide nanoparticles.

Antibody-based biosensors for environmental monitoring

Antibody-based biosensors provide sensitive and rapid analytical tools for the detection of a range of pathogens and associated toxins. In this paper, the recent progress in antibody-based biosensors for environmental monitoring is reviewed with particular emphasis on generation and immobilization methods of antibody capture probes. We also describe the current available data on antibody-based detection of pathogens and toxins. Optimal assay design and the strengths and limitations of current sensor technologies for detection of biological agents in environment on site are also discussed.

Intein-mediated protein engineering for biosensor fabrication

Intein-mediated protein engineering has become a valuable tool for biosensing applications. Expressed protein ligation and protein trans-splicing were used to generate proteins with necessary tags or cyclic peptides that can be used to build various biosensing platforms such as biochips or sensor proteins. Especially the use of split-inteins that carry out conditional protein splicing reaction enabled the fabrication of various genetically-encoded biosensors that can monitor various signaling events in vivo including protein- protein interactions and protein translocalization. Biological targets activated split-inteins to generate functioning reporter molecules, such as luciferase and autofluorescent proteins. We here review various sensing platforms that utilize intein-mediated protein engineering technology mainly focusing on cell-based biosensors.

Biomedical applications and safety issues of gold nanoparticles

Recently, nanoparticles (NPs) are introduced to a broad range of biomedical applications including drug delivery, imaging, and sensors. With increased use of NPs and the emergence of consumer products containing NPs, there are growing concerns for the safety of NPs. Among many different types of NPs, gold nanoparticles (GNPs) are popular materials for various biomedical applications. GNPs can be fabricated in various forms using different concentrations of growth-directing surfactant. In this mini-review, we summarize the recent advancements made in the field of safety and biomedical applications of GNPs, and show that GNPs can also be applied in the ablation of thyroid cancer remnants after surgery

Inhibitory Effects of Novel SphK2 Inhibitors on Migration of Cancer Cells

BACKGROUND Cell migration is an essential process for survival and differentiation of mammalian cells. Numerous diseases are induced or influenced by inappropriate regulation of cell migration, which plays a key role in cancer cell metastasis. In fact, very few anti-metastasis drugs are available on the market. SphKs are enzymes that convert sphingosine to sphingosine-1-phosphate (S1P) and are known to control various cellular functions, including migration of cells. In human, SphK2 is known to promote apoptosis, suppresses cell growth, and controls cell migration; in addition, the specific ablation of SphK2 activity was reported to inhibit cancer cell metastasis. OBJECTIVE The previously identified SG12 and SG14 are synthetic analogs of sphingoid and can specifically inhibit the functions of SphK2. We investigated the effects of the SphK2 specific inhibitors on the migratory behavior of cells. METHOD We investigated how SG12 and SG14 affect cell migration by monitoring both cumulative and individual cell migration behavior using HeLa cells. RESULTS SG12 and SG14 mutually showed stronger inhibitory effects with less cytotoxicity compared with a general SphK inhibitor, N,N-dimethylsphingosine (DMS). The mechanistic aspects of specific SphK2 inhibition were studied by examining actin filamentation and the expression levels of motility-related genes. CONCLUSION The data revealed that SG12 and SG14 resemble DMS in decreasing overall cell motility, but differ in that they differentially affect motility parameters and motility-related signal transduction pathways and therefore actin polymerization, which are not altered by DMS. Our findings show that SphK2 inhibitors are putative candidates for anti-metastatic drugs.

Allergen Microarrays for In Vitro Diagnostics of Allergies: Comparison with ImmunoCAP and AdvanSure

Background In vitro detection of the allergen-specific IgE antibody (sIgE) is a useful tool for the diagnosis and treatment of allergies. Although multiple simultaneous allergen tests offer simple and low-cost screening methods, these platforms also have limitations with respect to multiplexibility and analytical performance. As an alternative assay platform, we developed and validated a microarray using allergen extracts that we termed “GOLD” chip. Methods Serum samples of 150 allergic rhinitis patients were used in the study, and the diagnostic performance of the microarray was compared with that of AdvanSure (LG Life Sciences, Daejun, Korea) and ImmunoCAP (Phadia, Uppsala, Sweden). Standard IgE samples were used for the quantitative measurement of sIgEs. Results The microarray-based assay showed excellent performance in the quantitative measurement of sIgEs, demonstrating a linear correlation within the range of sIgE concentrations tested. The limit of detection (LOD) was lower than 0.35 IU/mL, which is the current standard for the LOD cut-off. The assay also provided highly reproducible sets of data. The total agreement percentage of positive and negative calls was 92.2% compared with ImmunoCAP. Moreover, an outstanding correlation was observed between the microarray and the ImmunoCAP results, with Cohens kappa and Pearson correlation coefficient values of 0.80 and 0.79, respectively. Conclusions The microarray-based in vitro diagnostic platform offers a sensitive, reproducible, and highly quantitative method to detect sIgEs. The results showed strong correlations with that of ImmunoCAP. These results suggest that the new allergen microarray can serve as a useful alternative to current screening platforms, ultimately becoming a first-line screening method.