Yun-Gon Kim

HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software

We present a robust, fully automatable technology platform that includes computer software for the detailed analysis of low femtomoles of N-linked sugars released from glycoproteins. Features include (i) sample immobilization in 96-well plates, glycan release, and fluorescent labeling; (ii) quantitative HPLC analysis, including monosaccharide sequence, linkage, and arm-specific information for charged and neutral glycans; (iii) automatic structural assignment of peaks from HPLC profiles via web-based software that accesses our database (GlycoBase) of more than 350 N-glycan structures, including 117 present in the human serum glycome; and (iv) software (autoGU) that progressively analyzes data from exoglycosidase digestions to produce a refined list of final structures. The N-glycans from a plate of 96 samples can be released and purified in 2 or 3 days and profiled in 2 days. This strategy can be used for (i) identification and screening of disease biomarkers and (ii) monitoring the production of therapeutic glycoproteins, allowing optimization of production conditions. This technology is also suitable for preparing released glycans for other analytical techniques. Here we demonstrate its application to rheumatoid arthritis using 5 microl of patient serum.

Nano/Microfluidics for diagnosis of infectious diseases in developing countries☆

Nano/Microfluidic technologies are emerging as powerful enabling tools for diagnosis and monitoring of infectious diseases in both developed and developing countries. Miniaturized nano/microfluidic platforms that precisely manipulate small fluid volumes can be used to enable medical diagnosis in a more rapid and accurate manner. In particular, these nano/microfluidic diagnostic technologies are potentially applicable to global health applications, since they are disposable, inexpensive, portable, and easy-to-use for detection of infectious diseases. In this paper, we review recent advances in nano/microfluidic technologies for clinical point-of-care applications at resource-limited settings in developing countries.

Quantum dot-based HIV capture and imaging in a microfluidic channel

Globally, over 33.2 million people who mostly live in developing countries with limited access to the appropriate medical care suffer from the human immunodeficiency virus (HIV) infection. We developed an on-chip HIV capture and imaging method using quantum dots (Qdots) from fingerprick volume (10 microl) of unprocessed HIV-infected patient whole blood in anti-gp120 antibody-immobilized microfluidic chip. Two-color Qdots (Qdot525 and Qdot655 streptavidin conjugates) were used to identify the captured HIV by simultaneous labeling the envelope gp120 glycoprotein and its high-mannose glycans. This dual-stain imaging technique using Qdots provides a new and effective tool for accurate identification of HIV particles from patient whole blood without any pre-processing. This on-chip HIV capture and imaging platform creates new avenues for point-of-care diagnostics and monitoring applications of infectious diseases.

Liver Extracellular Matrix Providing Dual Functions of Two-Dimensional Substrate Coating and Three-Dimensional Injectable Hydrogel Platform for Liver Tissue Engineering

Decellularization of tissues or organs can provide an efficient strategy for preparing functional scaffolds for tissue engineering. Microstructures of native extracellular matrices and their biochemical compositions can be retained in the decellularized matrices, providing tissue-specific microenvironments for efficient tissue regeneration. Here, we report the versatility of liver extracellular matrix (LEM) that can be used for two-dimensional (2D) coating and three-dimensional (3D) hydrogel platforms for culture and transplantation of primary hepatocytes. Collagen type I (Col I) has typically been used for hepatocyte culture and transplantation. In this study, LEM was compared with Col I in terms of biophysical and mechanical characteristics and biological performance for enhancing cell viability, differentiation, and hepatic functions. Surface properties of LEM coating and mechanical properties and gelation kinetics of LEM hydrogel could be manipulated by adjusting the LEM concentration. In addition, LEM hydrogel exhibited improved elastic properties, rapid gelation, and volume maintenance compared to Col I hydrogel. LEM coating significantly improved hepatocyte functions such as albumin secretion and urea synthesis. More interestingly, LEM coating upregulated hepatic gene expression of human adipose-derived stem cells, indicating enhanced hepatic differentiation of these stem cells. The viability and hepatic functions of primary hepatocytes were also significantly improved in LEM hydrogel compared to Col I hydrogel both in vitro and in vivo. Albumin and hepatocyte transcription factor expression was upregulated in hepatocytes transplanted in LEM hydrogels. In conclusion, LEM can provide functional biomaterial platforms for diverse applications in liver tissue engineering by promoting survival and maturation of hepatocytes and hepatic commitment of stem cells. This study demonstrates the feasibility of decellularized matrix for both 2D coating and 3D hydrogel in liver tissue engineering.

A relative and absolute quantification of neutral N-linked oligosaccharides using modification with carboxymethyl trimethylammonium hydrazide and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Quantification of oligosaccharides is of great importance to investigate variations or changes in the glycans of glycoconjugates. Mass spectrometry (MS) has been widely applied to identification and structural analysis of complex oligosaccharides. However, quantification using MS alone is still quite challenging due to heterogeneous charge states and different ionization efficiency of various types of oligosaccharides. To overcome such shortcomings, derivatization with carboxymethyl trimethylammonium hydrazide (Girards reagent T [GT]) was introduced to generate a permanent cationic charge at the reducing end of neutral oligosaccharides, resulting in mainly [M](+) ion using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), so that the ambiguities caused by metal adduct peaks such as [M+K](+) and [M+Na](+) were avoided. To verify our method, the relative and absolute quantification of neutral glycans from human immunoglobulin G (IgG) and ovalbumin with internal standards of dextran ladders using MALDI-TOF MS were compared with those performed by conventional normal-phase high-performance liquid chromatography (NP-HPLC) profiling. The quantification using GT derivatization and MALDI-TOF MS agreed well with the HPLC profiling data and showed excellent reliability and reproducibility with better resolution and sensitivity. This method was further applied to quantify the enzymatically desialylated N-glycans from miniature pig kidney membrane proteins. The results showed that the low-abundance structures that could not be resolved by NP-HPLC were quantified with high sensitivity. Thus, this novel method of using modification of neutral sugars with GT is quite powerful for neutral glycan analysis, especially to quantify minute glycan samples with undetectable levels using HPLC.

In situ monitoring of antibiotic susceptibility of bacterial biofilms in a microfluidic device

Antibiotic resistance of biofilms is a growing public health concern due to overuse and improper use of antibiotics. Thus, determining an effective minimal concentration of antibiotics to eradicate bacterial biofilms is crucial. Here we present a simple, novel one-pot assay for the analysis of antibiotic susceptibility of bacterial biofilms using a microfluidics system where continuous concentration gradients of antibiotics are generated. The results of minimal biofilm eradication concentration (MBEC) clearly confirm that the concentration required to eradicate biofilm-grown Pseudomonas aeruginosa is higher than the minimal inhibitory concentration (MIC) that has been widely used to determine the lowest concentration of antibiotics against planktonically grown bacteria.

Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

Drop-on-Demand Single Cell Isolation and Total RNA Analysis

Technologies that rapidly isolate viable single cells from heterogeneous solutions have significantly contributed to the field of medical genomics. Challenges remain both to enable efficient extraction, isolation and patterning of single cells from heterogeneous solutions as well as to keep them alive during the process due to a limited degree of control over single cell manipulation. Here, we present a microdroplet based method to isolate and pattern single cells from heterogeneous cell suspensions (10% target cell mixture), preserve viability of the extracted cells (97.0±0.8%), and obtain genomic information from isolated cells compared to the non-patterned controls. The cell encapsulation process is both experimentally and theoretically analyzed. Using the isolated cells, we identified 11 stem cell markers among 1000 genes and compare to the controls. This automated platform enabling high-throughput cell manipulation for subsequent genomic analysis employs fewer handling steps compared to existing methods.

High-Throughput Quantitative Analysis of Total N-Glycans by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Accurate and reproducible quantification of glycans from protein drugs has become an important issue for quality control of therapeutic proteins in biopharmaceutical and biotechnology industries. Mass spectrometry is a promising tool for both qualitative and quantitative analysis of glycans owing to mass accuracy, efficiency, and reproducibility, but it has been of limited success in quantitative analysis for sialylated glycans in a high-throughput manner. Here, we present a solid-phase permethylation-based total N-glycan quantitative method that includes N-glycan releasing, purification, and derivatization on a 96-well plate platform. The solid-phase neutralization enabled us to perform reliable absolute quantification of the acidic N-glycans as well as neutral N-glycans from model glycoproteins (i.e., chicken ovalbumin and porcine thyroglobulin) by only using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, low-abundance sialylated N-glycans from human serum prostate specific antigen (PSA), an extremely valuable prostate cancer marker, were initially quantified, and their chemical compositions were proposed. Taken together, these results demonstrate that our all-inclusive glycan preparation method based on a 96-well plate platform may contribute to the precise and reliable qualitative and quantitative analysis of glycans.

Structural analysis of α‐Gal and new non‐Gal carbohydrate epitopes from specific pathogen‐free miniature pig kidney

The major barrier in transplantation of pig organs into humans is the presence of surface carbohydrate antigens (e.g., the Galα1‐3Galβ1‐4GlcNAc‐R (α‐Gal) epitope) expressed on pig endothelial cells. In this study, total N‐glycans from membrane glycoproteins derived from specific pathogen‐free miniature pig kidney are identified by MALDI‐TOF, negative ion ESI MS/MS and normal‐phase HPLC (NP‐HPLC) combined with exoglycosidase digestion. Over 100 N‐glycans, including sialylated and neutral types, were identified. As well as the known α‐Gal antigens, some of these glycans contained novel non‐Gal carbohydrate antigens such as (Neu5Gc‐Gal‐GlcNAc) and Galα1‐3Lewisx (Gal‐Gal‐(Fuc)GlcNAc) which have not been reported before in N‐glycans from pig organs. The ability of MALDI, ESI, and HPLC to measure the relative proportions of the glycans was evaluated. The HPLC resolution was insufficient for accurate work and some minor differences were noted in the ionization efficiencies of different glycan groups when measured by the two mass spectrometric techniques. However, the results indicated that the relative quantity of α‐Gal epitope was in the region of 50% of the complex glycans. High‐mannose type glycans were also abundant (35–43%) but appeared to be ionized more efficiently than the complex glycans by ESI than by MALDI.