Earticle

Room temperature ionic liquids (ILs) have recently been very popular as green solvents due to their unique physicochemical properties of negligible vapor pressure, non-flammability, excellent thermal stability and a strong ability to dissolve a wide range of organic and inorganic compounds. They also have great potential as reaction media or co-solvent for enzymatic bioconversion including biodiesel production. In this presentation, the application of ILs in biomass pretreatment will be mainly addressed. ILs have been used as alternative solvent for cellulose pretreatment. Pretreatment of biomass is key factors for enhance enzymatic saccharification and fermentation of biomass for biofuel and bio-based chemical industry. The ILs-pretreated celluloses become less crystalline and in somewhat condition have lower degree of polymerization (DP) than that of the nature. Microwave heating could cause a significant decrease in DP of cellulose dissolved in ILs which led to a great improvement on cellulase-catalyzed cellulose hydrolysis. Microwave heating was also effective on the pretreatment of sea algae (Ulva pertusa Kjellman) for enhanced enzymatic hydrolysis.

Various factors affecting yields of Gal, AG, and 5-HMF formationduring saccharification were investigated using agar as a substrate in the presence of several bisulfate-based acidic ionic liquids as catalysts. The result was compared with employing sulfuric acid in saccharification from the viewpoint of sugar yields and 5-HMF formation. [Bmim][HSO4], [Hmim][HSO4], [Morph][HSO4], [Bu4N][HSO4], [Bu4P][HSO4], [Chol][HSO4] showed moderate to high yields of Gal and AG with a remarkable decrease in 5-HMF formation compared with sulfuric acid. Among them, [Chol][HSO4] ionic liquid was found to exhibit the highest yield of sugars with an acceptable concentration of 5-HMF that does not inhibit the fermentation process.

Although polymeric supports are being used for enzyme adsorption and immobilization since more than two decades, there is practically no systemic study on the effects of various functional groups present on the polymers on enzyme structure and function. Present work is primarily focused on the effects of functional groups present on copolymers of allyl glycidyl ether and ethylene glycol dimethacrylate (AGE- EGDM) which are functionalized with different groups such as epoxy, hydroxy, tributyamino etc.) on structural integrity and activity of enzymes. The effects of systematic changes in hydrophobicity, polarity, and ionic groups on activity of different classes of enzymes have been studied to understand relative effects of hydrophobic and hydrophilic interactions involved when a protein comes with contact with a polymer. The enzymes chosen for our studies have been selected from different classes: α-chymotrypsin (an amidase that does not require a co-factor with mol wt of 25 kD), alcohol dehyrogenase (mol wt 144KD) and glucose dehydrogenase (Mol wt 126KD) which need a cofactor NAD(P)H for their reactions, and alkaline phosphatase from shrimp (SAP, mol wt 140KD), a metallo-enzyme in which the metal ion plays a dominant role in reaction. For applications in food and beverage industry, studies have been made with soybean trypsin inhibitor, lipoxidase and pectin methyl esterase. Binding of proteins to the polymers has been studied via adsorption isotherms and activity measurements. Structural changes in the enzymes have been studied by circular dichroism (CD), laser Raman and fluorescence spectroscopy. These studies have shown that hydrophobicity of the polymer plays a dominant role in binding of the protein and polar interactions of the ester groups present in thecopolymer contribute significantly towards protein denaturation. Use of reverse micellar media overcomes the problem of denaturation and it is possible to obtain immobilized enzyme preparations with significantly improved stability.

Despite all the technical progress, our ability to rationally manipulate the structures and functions of proteins is quite limited, because the genetic code specifies the same 20 amino acids building blocks. The development of a method that makes possible the systematic expansion of the genetic code can enable the evolution of proteins with new or enhanced properties. Recent advances in engineering of translation machinery have made it possible to add over 30 novel amino acids to the genetic code, but the repertoire of unnatural functional groups is still limited and their application is quite restricted. There are only a limited number of aminoacyl tRNA synthetases that were successfully engineered to charge tRNA with desired unnatural amino acids, which limits the kind and number of unnatural functional groups. In addition, other factors in translation machinery need to be engineered for efficient incorporation of unnatural amino acids. In an effort to overcome these major obstacles, newly discovered aminoacyl tRNA synthetase and tRNA were characterized in terms of structure and biochemical properties. Based on these studies, the cellular translation system was redesigned and evolved to site-specifically incorporate novel unnatural amino acids. Direct incorporation of unnatural amino acids can make considerable contribution to applied biomedical research areas such as enzyme engineering, click chemistry, and nanobiotechnology, and drug development. Detailed experimental design and results will be presented and application of synthetic biology technology will be discussed.

Sustainable chemistry, or green chemistry, designs chemicals and reactions that eliminate or reduce hazards like pollutants or dangerous chemicals. It aims to avoid problems before they happen. One of the most promising technologies to create sustainable chemical processes is synthetic biology. Synthetic biology is the redesign of existing, natural biological systems for useful purposes. Enzymes are selective, nontoxic catalysts that eliminate waste and exposure to hazardous chemicals. In the past, chemists have used naturally occurring enzymes, but now chemists are improving enzymes using protein engineering. The goals of this protein engineering may be to increase the stability of an enzyme, to increase its selectivity, and even to change the type of reaction that it catalyzes. One example is the use of enzymes to convert biomass to fuels. A current application is starch hydrolases engineered to remain active at >90°C to convert starch to sugars for fermentations. One current research project is engineering of enzymes to make peracetic acid. Peracetic acid is a reactive oxidant that breaks down lignin in wood. The lignin fragments are potential raw materials for synthesis. The lignin breakdown also exposes the sugar polymers in wood making it easier to convert them to sugars. As protein engineering advances to make the redesign of enzymes easier and more predictable, enzymatic catalysis will be an increasingly important tool for chemical processes. P. Bernhardt, K. Hult, R. J. Kazlauskas (2005) Molecular basis of perhydrolase activity in serine hydrolases. Angew. Chem., Intl. Ed. 44, 2742-6. D. (T.) Yin, P. Bernhardt, K. L. Morley, Y. Jiang, J. D. Cheeseman, J. D. Schrag, R. J. Kazlauskas (2010) Switching catalysis from hydrolysis to perhydrolysis in P. fluorescens esterase. Biochemistry 49, 1931-42.

Self-assembled nanostructured materials have attracted much attention owing to their application to catalyst supports, hosts for biomolcules, and sensors. The first synthesis of ordered mesoporous material stimulated research on the self-assembly of block copolymers with inorganic materials. Herein, we present functional nanostructured materials synthesized by the self-assembly of block copolymers with inorganic materials. Highly crystalline mesoporous transition metal oxides were fabricated through ‘one-pot’ assembly method employing PI-b-PEO block copolymers. We developed intermetallic nanoparticle loaded highly ordered mesoporous carbon and transition metal oxides by the assembly of PI-b-PEO block copolymer. We have developed multifunctional nano-biocatalyst using various designed nanostrucutred materials. Hierarchically ordered mesocellular mesoporous silica materials (HMMS) were synthesized using a single structure-directing agent. To improve the retention of enzymes in HMMS, we adsorbed enzymes, and then employed crosslinking using glutaraldehyde (GA). The resulting crosslinked enzyme aggregates (CLEAs) show an impressive stability with extremely high enzyme loadings. Magnetically separable and highly active enzyme system was developed using nanostrucutred magnetic materials. Magnetically switchable bioelectrocatalytic system was constructed using magnetic mesocellular carbon foam (Mag-MCF-C) with ultra-large mesopores. Large cellular porous silica materials were synthesized by the self-assembly of commercially available block copolymers, P123. One-pot multi-catalyst system, so called "nanofactory", was developed entrapping magnetic nanoparticles and oxidases in large cellular mesoporous silica with high loadings of simultaneously above 40 wt% MNPs and 20 wt% enzymes. Our approach provided highly loaded MNP system and any highly loaded enzymes with superior activity, stability, and reusability, thereby making further applications as versatile sensors for detecting DNA, protein, and cell highly promising.

Nanobiocatalysts, enzymes incorporated with nanostructured materials, have great potential to provide robust biocatalysts with stable and high enzymatic activity for many practical applications. The use of nanostructured materials including nanofibers, carbon nanotubes, and nanoparticles is an intriguing approach for developing nanobiocatalysts since these materials manifest great efficiency in the manipulation of the nanoscale of enzymes and thus provide a large surface area, which can lead to high volumetric enzyme activity and unprecedented stability beyond classical enzyme immobilization methods such as simple adsorption, covalently attachment or entrapment. In order to realize nanobiocatalysts the new immobilization strategy between enzymes and nanostructured materials should be considered. For these concerns, enzyme coating approach was introduced, which is accomplished by cross-linked enzymes via glutaraldehyde mediated coupling with nanostructured materials. Recent results about the improved performance of nanobiocatalysts using various enzymes and nanostructured materials such as nanofibers, nanoporous media, carbon nanotubes, and nanoparticles will be introduced. In addition, the practical applications of nanobiocatalysts in proteomic analysis, biosensors, biofuel cells, and bioconversion will be introduced to show the possible extension of nanobiocatalysts in various practical applications depending on the selection of appropriate enzymes and nanostructured materials. The deep understanding between enzymes and nanostructured materials will help the development of innovative nanobioctalysis approaches, which might guide to numerous practical applications of enzyme technology.

Stem cells have been widely studied as a powerful source for cell therapy because of their high self-renewal activity and high telomerase activity that may provide an unlimited supply of target cells. Despite the clinical prospect, it is necessary that several problems be resolved prior to clinical application. For example, establishment of clinical grade stem cells, efficient differentiation toward specific cell lines, functional recovery, and regulation of immune rejection are all pressing issues in the field. Recently we have not only developed a novel culture technique using porous membranes for human embryonic stem cell (hESC) expansion[1], but also shown the neovascularization[2] and bone formation[3] potential of hESCs in vivo using a mouse model. Herein, I will present my recent studies about more improved culture technique as well as therapeutic effect of stem cell using scaffold[4] and gene delivery system[5] for clinical applications. These studies would provide critical platform to address the clinical application of stem cells in the future of regenerative medicine.

Significant improvements have been reported in development of biomaterials to engineer stem cell therapeutics. Despite these advances in biomaterial development for stem cell engineering, the diversity of functional materials still remains limited, in part due to their slow, multistep syntheses, and complicated modifications. The customization of each synthetic reaction and multiple steps limits the ability to generate a significant library size with diversity. To overcome these limitations, we have established combinatorial biomaterial libraries with chemical approaches which allow the simple, rapid, and parallel generation of materials and screened biomaterials optimal for stem cell engineering. Compared with commercially available reagents, some of identified materials have shown higher efficiency of delivering DNA, mRNA or siRNA for genetic modification of stem cells. These genetically engineered stem cells have exhibited enhanced therapeutic efficacy of treating cardiovascular or bone diseases. Interestingly, some of screened biomaterials could support clonal growth or specific differentiation of pluripotent stem cells. The high-throughput screening technology reported here could provide efficient tools for developing stem cell therapeutics for various types of destructive diseases.

Insulin-secreting pancreatic islet transplantation is one of the most promising strategies for patients suffering from diabetes mellitus, but they must be eliminated by host’s immune graft rejection. To protect transplanted islets in host, bioengineered pancreatic islets are being developed. To this end, surface modification with biocompatible polymer and cellular reconstruction technology are introduced. Here PEG-based chemical immunoprotection can provide an effective therapy that protects transplanted islets at least for one year when CsA was administered. Also, when the structure of pancreatic islets was reconstructed with bio-MEMS technology, the immunity of them was attenuated without any modification or treatment of any agent. Finally, these bioengineered pancreatic islets could cure the hyperglycemia in diabetic animals. Therefore, these technologies could strongly support the successful pancreatic islet transplantation for hunting diabetes mellitus.

Tissue engineering is a medical technology intended to replace or regenerate the damaged or lost tissue or organs by utilizing three elements of cells, biological/physical signals and a scaffold, Regenerations of bones, vessels, cartilages, bladders and skins have been reported to be successful in clinical by employing tissue engineering technology, We developed a new scaffold for its application in tissue engineering by employing biocompatible polymers such as hyaluronic acid, chondroitin sulfate and poly(ethylene oxide). Bone tissue engineering has been tried by employing glycosaminoglycan based hydrogel as a scaffold. The hydrogel has been fabricated through in situ Michael type addition reaction between acrylate of hyaluronic acid and thiols of poly (ethylene oxide). After characterization of derivatives of hyaluronic acid and poly(ethylene oxide) with various analytical tools such as FTIR, NMR, XPS, the synthesized hydrogel was evaluated for its application in tissue engineering. Swelling behaviors, gel formation, compression test were performed for its physical evaluations. In vitro tests such as cell viability, cell toxicity have shown its excellent biocompatibilty. In vivo in rat calvarial defect model demonstrated induction of bone tissue formation over several weeks. Analysis of the regenerated bone was performed with histological methods.

Stimulating angiogenesis could improve perfusion and function in ischemic tissues and could be utilized to treat myocardial infarction and peripheral arterial diseases. Therapeutic angiogenesis uses strategies of stem cell implantation or angiogenic gene or protein delivery. The major contribution of stem cell implantation for therapeutic angiogenesis is secretion of paracrine angiogenic growth factors from implanted stem cells rather than differentiation of implanted stem cells into vascular cells and subsequent incorporation into vasculature. Poor survival of stem cells implanted into ischemic tissues limits the angiogenic efficacy of the stem cell therapy. We have demonstrated that PLGA nanospheremediated growth factor delivery to cell transplantation site can enhance the survival of transplanted human adipose-derived stromal cells (hADSCs) and secretion of human angiogenic growth factors, and substantially improve angiogenic efficacy of hADSCs (1). Meanwhile, gene delivery systems that can sustain expression of angiogenic gene may enhance therapeutic angiogenesis. We have also demonstrated that PLGA nanospheres sustained release of pDNA encoding VEGF gene for a long term and that gene therapy using PLGA nanospheres resulted in more extensive neovascularization at ischemic sites than both naked pDNA and PEI/pDNA (2).

A severely injured anterior cruciate ligament (ACL) does not heal well, and can cause knee instability, meniscal damage, and osteoarthritis. Patellar tendon autografts and allografts are widely used but are not suited for an ACL reconstruction. The problems associated with an autograft include lengthy rehabilitation as well as persistent patellar pain. Allografts carry the risk of disease transmission, and their procurement is difficult and costly. Permanent synthetic ACL prostheses, including the Gore Tex prosthesis, Stryker-Dacoron ligament, and Kennedy ligament augmentation device, may show satisfactory performance in the short term but they tend to break down and fail in the long term. Recently, a tissue engineering strategy has been attempted for an ACL reconstruction. The ideal ACL replacement scaffold should be biodegradable, porous, biocompatible, show adequate mechanical strength, and be able to promote the formation of ligament tissue. Several groups have reported the potential ACL scaffold using collagen, silk, and biodegradable polymers such as polyglycolic acid (PGA) and polylactic acid (PLA). Over the past 20 years, silk and a variety of degradable synthetic materials including PGA have been used as biomedical suture materials. In addition, silk and PGA fibers were studied to regenerate tendon and ligament defects. The biocompatibility of implant materials is of major concern in orthopedic surgery. After implantation, host-material interactions occurring at the implant site can be cause adverse inflammatory reactions.

Non-invasive detection of lymph node metastasis is an extremely important medical procedure mandatory for the determination of the most relevant clinical intervention for individual cancer patients. Nonetheless, lymph node biopsy remains the current gold-standard for this procedure, which is highly invasive. To address this urgent clinical issue, this study has explored the potential utility of Fe-phytate complex as a novel magnetic resonance imaging (MRI) contrast agent for non-invasive metastatic lymph node imaging. The proposed parent compound, phytate, has already proven to be clinical applications in the form of 99Tc-phytate for the mapping of various types of cancer patients, and MRI is a well-known, safe imaging modality free of ionizing radiation. The efficacy of the Fe-phytate complex in non-invasive metastatic lymph node imaging was tested in a mouse model of melanoma for lymph node metastases. The isothermal titration calorimeter (ITC) analyses for the thermodynamic property of Fe-phytate clearly suggested that the proposed contrast agent is biologically safe and clinically applicable for the detection of metastatic lymph node in cancer patients. All MRI experiments were performed on a 9.4 T Bruker animal MR scanner. For histopathological analyses, tissue sections were examined with hematoxylin and eosin (H&E) staining, analyzed for tumor metastases of lymph nodes with fluorescence microscopy, and stained with Prussian blue to examine the biodistribution of Fe-phytate particles. The results strongly suggested that Fe-phytate complex can detect metastatic lymph node by MRI in the mice model of melanoma, that the potential applicability of the proposed contrast agent to different cancer models.

AD is the most common neurodegenerative disease. The pathological hallmark of extra-cellular β-amyloid (Aβ)deposit is considered as one of the primary factors in inducing AD. However, the mechanism of Aβ deposition on the cell membrane are the induced cytotoxicity is still unclear. In our studies, we proposed a “recruiting hypothesis” to describe the binding mechanism of Aβ or Aβ aggregates with cell membrane and the induced cell toxicity from molecular level. This presentation presents basically the isothermal titration calorimetry (ITC) studies on the Aβ aggregations, Aβ binding with liposomes with various compositions to mimic the binding with cell, and Aβ binding with PC12 cell. All these thermodynamics information obtained were discussed with the measurements of circular dichroism, surface plasmon resonance, monolayer (trough), flowcytometry, and cell viability measurements. In summary, the recruiting hypothesis is verified by this investigation and can be adapted as a molecular level understanding of the mechanism of AD and the basis of treatment developments of AD, such as drug design.

The bare carbon nanotube (CNT) was applied for the channel to detect the large size antigen of IgE as shown in Fig. 1. The aptamer, which is a single strand DNA and selectively reacts with IgE, was immobilized on CNT using linker. As the size of aptamer is as small as 1~2 nm, the reaction of aptamer with IgE occurred within the Debye length. Therefore, the electrical charge of IgE could be detected as the change of the CNT channel conductance. Using this method, IgE of as small as 250 pM could be detected as shown in Fig. 2. Another approach of the biosensor is to use CNT as a working electrode for the electrochemical reaction as shown in Fig. 3. As CNT electrode has a far larger surface area compared to the conventional planar type electrode such as a Pt electrode, the oxidized current of the biomolecule at the working electrode increased in proportional to the surface area size, which means the increase of the sensitivity. For the detection of the PSA (Prostate Specific Antigen), the surface of the CNT is modified by the anti-PSA using the linker. When the PSA/a-PSA reaction occurs, the oxidized current of the PSA/a-PSA molecule is few times larger than that of a-PSA only as shown in Fig. 3. The lowest PSA concentration of 0.5 ng/mL could be detected. 　When the input signal is too small to detect, noise power is added to the input signal to CNT FET which is under the threshold condition. Then, the input signal could reach the threshold of CNT FET and it becomes on state and detects the input signal. This is referred to as stochastic resonance phenomena, which we have succeeded in observing using the CNT FET as shown in Fig. 4. By increasing the number of the channel, the correlation factor between the output/input becomes increased. For this purpose, CNT multi channel FET was realized using the direction controlled growth on the patterned SiO2/Si substrate. The stochastic resonance phenomena could be applicable for the biosensing in the large noise circumstances. Graphene is surface sensitive material and can be applicable to the biosensor. We have first succeeded in detecting the different concentrations of BSA as the shifts of the Dirac point of the graphene FET as shown in Fig. 5, which are linearly proportional to the BSA concentration. As small as 300 pM BSA could be detected. Carbon based biosensors showed the superior characteristics compared to the conventional biosensors because of the high FET characteristics and large surface area and will be applicable in the real world in near future.

Cerebral cortex is a few mm thick and it has both layered and column structures whose size is submillimeter scale. When the neural activity in the small region of cerebral cortex is augmented, blood flow to the region increases to supply sufficient oxygen, which is called neuro-vascular coupling. To elucidate such coupling it is necessary to know microvascular structure and function 3-dimensionally with sufficient spatial resolution. However, microvessels deeper than about 100 μm such as the penetrating arterioles are difficult to observe by light microscope. Although MRI can observe living structures throughout the whole body, its spatial resolution is in the order of mm. Optical coherence tomography (OCT) has a capability of imaging living tissues up to a few mm depth with around 10 μm spatial resolution. It is still difficult to discriminate deeply embedded microvessels from the surrounding tissue by OCT structural image alone. The Doppler effects due to the blood flow can be used as a marker to find out blood vessels embedded within the tissue. In this study, the Doppler-OCT technique was applied to detect and visualize microvessels deeply embedded in the cerebral cortex of the rat. The OCT system used in our study incorporates a broad band near infrared light source with central wavelength of 1310 nm. Its axial and lateral resolutions are about 10 μm and 14 μm, respectively. The male Wistar rats of 150-200 g were used for cerebral preparation. After they were anesthetized, the cranial window about 3x5 mm2 was created in the parietal region while the dura mater was kept intact. The OCT signal obtained from within the cortical tissue was analyzed using short time Fourier transform (STFT) to get spectrogram, which displays the change in power spectrum of the signal with time. The OCT signal originating from the flowing blood suffers from the Doppler frequency shift and is clearly distinguished from the signal of surrounding tissue at rest in the spectrogram. Furthermore the blood flow velocity can be measured with a time resolution of a few msec, which is sufficient for analysis of pulsatile blood flow. As a result, microvessels penetrating into the cerebral cortex from the surface as well as microvessels running deep in the cortex were unveiled up to over 1 mm depth. The blood velocity distribution was also obtained inside the microvessel and was superimposed on the OCT structural image, where the position and passage of microvessels were visualized on the tomographic image of OCT. The information of structure and blood flow of microvessels in the direction of depth can help us to understand blood flow regulation among the layers of cerebral cortex.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Yet, elucidation of molecular pathogenesis of HCC development is still limited, and therefore, new developments to identify important prognostic factors and novel molecular targets of HCC are greatly needed. Our study was conducted to characterize gene expression profile of prognosis in patients with liver cancer by real-time reverse-transcription PCR (RT-PCR). The gene expression levels were obtained from of 128 primary HCCs and 40 non-cancerous surrounding livers. Several risk genes and survival genes were identified which showed significant deregulation in HCC tissues compared to noncancerous surrounding tissues. The expression levels of these genes were independent prognostic factors for overall survival (OS) time in HCC patients. Subsequently, the significant gene signature was validated in an independent cohort of 231 patients from three institutions, in which high risk score was significantly correlated with shorter overall survival (P = 0.00011). In conclusion, we suggest that the proposed gene signature may improve the prediction accuracy for survival of hepatocellular carcinoma patients, and complement prognostic essment based on important clinicopathologic parameters such as tumor stage.

Liposome is one of popular materials in biotechnology due to the special characteristics: hydrophilic inner space inside liposome and hydrophobic space between liposome bilayers. In this study, liposome was used for two applications: liposome immunoassay and active cell targeting. For both applications, the surface of liposome was biofunctionalized with antibody or recombinant human epidermal growth factor (EGF). Liposome was prepared based on DPPC, DCP, and cholesterol by film rehydration method. Liposome immunoassay (LIA) is based on enzyme immunoassay (EIA) but the detection sensitivity could be significantly enhanced by biofunctionalized immunoliposomes encapsulating horse radish peroxidase (HRP). Therefore, enzyme encapsulated liposomes are very useful to detect a small amount of analyte with enhanced signal. As a model antibody, anti-rabbit IgG antibody was immobilized on the liposome surface by iminothiolane reaction. Unbound anti-rabbit IgG antibody was removed by gel permeation chromatography. 4-chloro-naphtol was used as a HRP substrate. This substrate can be penetrated into liposome inner space and be accumulated inside of liposome after nzyme reaction. This causes denser signal than conventional EIA (6 times higher signal density at 1 μg/mL rabbit IgG concentration and 16 times higher LOD). By decreasing the spot volume, signal density from LIA did not changed. This shows that LIA can be applied to small amount analyte detection in a small volume. Another application of liposome in this study was a carrier for active cell targeting. Liposome was biofunctionalized with EGF by BAM (biological anchor for cell membrane) molecule. BAM is consisted of oleyl group conjugated to NHS (N-hydroxysuccinimide) and PEG2000 (polyethylene glycol). EGF was reacted with NHS moiety on BAM and unreacted EGF was removed by gel permeation chromatography. Then EGF-BAM complex was incubated with liposome particle; this complex could be effectively inserted to the bilayers of a liposomal vesicle through lipophilc interaction. By this post-insertion method, EGF is only immobilized on the outside of liposome particle. Uptake of BAM inserted liposome to macrophage cell (RAW 264.7) was investigated by confocal microsopy. Compared with unmodified liposome, BAM inserted liposome has 2 times lower uptaken level; this is resulted from PEG moiety of BAM. Also, EGF-BAM inserted liposome was incubated with breast cancer cell line (MCF-7 and MDA-MB-231 cell). EGF-BAM inserted liposome has specificity only to EGF receptor overexpressed cell (MDA-MB-231 cell). In addition, biofunctionalized liposome encapsulating an anti-cancer drug (doxorubicin) selectively killed only MDA-MB-231 cell.

In this presentation, we report a quick and reproducible surface-enhanced Raman scattering (SERS)-based immunoassay technique, using hollow gold nanospheres (HGNs) and magnetic beads. HGNs show strong SERS enhancement effects from individual particles because hot spots can be localized within pinholes in the hollow particle structure. Accordingly, HGNs can be used for highly reproducible immunoanalysis of cancer markers. Magnetic beads were used as supporting substrates for the formation of the immunocomplex. In addition, we have implemented this SERS-based immunoassay, performed in a microtube, into a microfluidic platform. The SERSbased optofluidic sensor is composed of three compartments consisting of the gradient channel that serially dilutes the target marker, the injection and mixing area of antibody-conjugated hollow gold nanospheres and magnetic beads and the trapping area of sandwich immunocomplexes using multiple solenoids. Quantitative analysis of a specific target marker is performed by analyzing its characteristic SERS signals. For validation, a well-known lung cancer marker, carcinoembryonic antigen (CEA), was used as a target. Based on experimental results, the limit of detection (LOD) was determined to be 1–10 pg/mL, this value being about 100–1000 times more sensitive than the LOD of conventional enzyme-linked immunosorbent assay (ELISA). Our proposed SERS-based optofluidic sensing technology has many advantages over previously reported SERS detection methods, such as good reproducibility, low limits of detection and fast assay times. Accordingly, this technique is expected to be a powerful clinical tool for fast and reliable cancer diagnosis.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Yet, elucidation of molecular pathogenesis of HCC development is still limited, and therefore, new developments to identify important prognostic factors and novel molecular targets of HCC are greatly needed. Our study was conducted to characterize gene expression profile of prognosis in patients with liver cancer by real-time reverse-transcription PCR (RT-PCR). The gene expression levels were obtained from of 128 primary HCCs and 40 non-cancerous surrounding livers. Several risk genes and survival genes were identified which showed significant deregulation in HCC tissues compared to noncancerous surrounding tissues. The expression levels of these genes were independent prognostic factors for overall survival (OS) time in HCC patients. Subsequently, the significant gene signature was validated in an independent cohort of 231 patients from three institutions, in which high risk score was significantly correlated with shorter overall survival (P = 0.00011). In conclusion, we suggest that the proposed gene signature may improve the prediction accuracy for survival of hepatocellular carcinoma patients, and complement prognostic assessment based on important clinicopathologic parameters such as tumor stage.

One of the main goals of research activies in Korean Universities is to commercialize the reseach results through licensing to suitale partners in an industrial field. Actually, licensing activies in Korea are not yet active due to some reasons or hurdles. Today, I will present preferable exmaples for licensing, helping researchers elevate understanding in and disires for licensing of their own research results.

This presentation will provide the history of the licensing and commercialization of the Cohen-Boyer patents. This license is considered among the most successful examples of university technology licensing. The presentation begins by providing the context for university's approach to licensing and then examines the implementation of the licensing practices and procedures. The final section examines the business model of the company "Barr Laboratories". We also demonstrate the importance of the "Quality" of patents and explain the concept of "I-pyramid".

The global pharma market size is estimated to be upwards of US$ 770 Billion as of 2008 and new pharmaceuticals makes a great influence on a pharmaceutical company itself and the whole country. However, considering the R&D cost and time for a new drug into the market, developing a new drug is not an easy decision even to a big pharmaceutical company. In addition, despite all the R&D cost and time, a new drug has a strong possibility of failing in the market and approval process. Therefore, pharmaceutical companies have tried a technology licensing, which would be one of major sources to find a lead compound for a new drug. Since the technology transfer act was enacted in 2000 in Korea, there have been so many cases of licensing between a company and public R&D organization including a University. Nevertheless the R&D investment of government and the amount of patent is increasing, total amount of licensing cases and fees never match up to our expectation. There could be several reasons, however the difference in understanding of drug development process between company and R&D organization would be a major reason. A successful licensing in a bio and pharmaceutical field depends on how well a company expresses its unmet needs and people in R&D understand the company's drug development process and prepare to meet that needs.

Biotechnology in Korea is growing fast as a core industrial technology which is able to create the national resources in the 21st century, and the government has recently selected biotechnology as a growth power technology and concentrated the effort to secure international competitiveness in biotechnology area through nation-wide investment and support. In Korea Research Institute of Bioscience & Biotechnology (KRIBB), which is the only government-funded life science research institute, has been established in 1985, and successfully operating in both technology transfer and spin-off by accomplishing the annual out-licensing contract fee of USD 35 million in 2009, and 6 companies out of total 23 spin-offs were listed in stock market. Once the IP or technology is ready for market, KRIBB TLO(Technology Licensing Office) determines whether to create out-licensing, joint-venture, and spin-off by the consideration of scientific value, market situation and business opportunity for the IP or technology. This presentation introduces the detailed examples of a KRIBB’s experience and strategy for successful technology commercialization including out-licensing, joint-venture and spin-off.

Surface plasmon resonance (SPR) is one of the most versatile techniques for the observation of biochemical interactions such as antigen-antibody bindings. In principle, SPR is highly sensitive to changes in the refractive index close to metal surfaces. Therefore, one can sensitively detect the binding of biochemical molecules to the surface without any labels in real time. Gold surface-based SPR has been used to detect diverse biological substances (e.g., proteins, DNA, small organic molecules, toxins and viruses) for a wide range of applications (e.g., drug screening, disease immunoassays, process control, and environmental monitoring). So, we had a good technology transfer from KRIBB to commercialize about a palm-sized SPR sensor using rotating mirror system. In this system, the beam from a diode laser (as the incident light source) is modulated using a rotating mirror, and a part of the reflected light that diverges from the centre of the rotating mirror is focused on the gold surface. The reflected light from the gold chip is then captured using a CMOS image sensor controlled by a notebook PC via a USB interface. This provides a portable SPR sensor device for POCT via label-free and real-time analyses that it can be put to practical use in the on-site field detection of a variety of substances that are of interest to biology, diagnosis, the environment, and defense.

기업의 특허기술 역량이 글로벌 경쟁력의 원천으로 등장하면서 특허기술사업화 등이 고려되지 않은 기업 연구 및 경영 활동은 그 경쟁력이 점차 약화되고 있는 것이 현실인데 비해 아직도 우리나라의 중소 기업은(상당수 중견기업 포함)은 해외의 선진기업들에 비해 특허기술사업화에 대한 인식이 저조한 실정이다. 따라서 우리나라 기업들의 특허기술사업화 촉진 유도 및 중장기적으로 세계 최고 수준의 “지식재산 강국 실현”을 위한 방안의 일환으로 정부의 특허기술사업화 정책자금 (융자․출연․보조금 등)지원 정책과 특허기술사업화 과정(창업전․창업 및 도약기․성장기 등) 등을 살펴보고 이에 대한활용전략 방안 제시를 통해 기업들의 특허기술 사업화 성공률을 제고하고자 한다.

Quorum sensing (QS) is a cell density-dependent signaling system used by bacteria to coordinate gene expression within their population. In the QS mechanism of many gram-negative bacteria, acyl homoserine lactones (AHLs) are known to be the triggering molecules which form a complex with a transcriptional activator protein and promote the binding of the complex to DNA regulatory site thus activates the transcription of many virulence genes. In this study, we describe the development and characterization of in vivo (cell-based) and in vitro (protein-based) bioassay systems for detecting QS inhibitors for the three members of the LuxR family proteins, TraR, LasR and the recently identified QscR. For TraR and LasR, soluble proteins could be obtained from the cells which were grown in AHL-deficient medium but they did not show binding affinity to the corresponding promoter sequences. Furthermore, the active LasR, presumably produced as LasR-AHL complex, was not dissociated into its components (LasR and AHLs) in vitro. These findings indicate that the development of biochemical in vitro assay which requires pure and active TraR/LasR proteins might not be possible unless the structure of proteins and/or theirs folding processes are modified. Unlike the LasR and TraR, QscR could be obtained as an apo-protein that reversibly forms an active complex in vitro with its cognate signal molecule and, subsequently, binds to the target promoter DNA sequences. Both the in vitro assay and the in vivo assay using QscR-overproducing recombinant strains were applied for the screening process. Among furanones tested, several compounds showed strong and dose-dependent inhibitory effects against QS activity. These results suggest that (i) the in vivo and in vitro assay are equally sensitive and reliable tools for screening QS inhibitors, and (ii) furanones are potentially important QS inhibitors for many LuxR-type receptor proteins.

The GA733-2 antigen is a 40 kDa transmembrane glycoprotein that functions as a homotypic cell-cell adhesion molecule. The GA733-2 gene encodes a 314-amino acid polypeptide that includes a 23-amino acid signal sequence, a 242-amino acid extracellular domain, a 23-amino acid transmembrane domain, and a 26-amino acid cytoplasmic domain. The extracellular domain of GA733-2 antigen has three potential N-linked glycosylation sites and an N-terminal cystein-rich region containing 12 cysteines [1]. The extracellular domain of antigen GA733-2 has been used as a target for cancer vaccination in several clinical trials. When used as a recombinant protein in cancer patients, tumor cell-specific humoral and cellular immune responses were observed [2]. Currently, the vaccinia virus is used to deliver GA733-2 to cancer patients in ongoing clinical trials [3]. In this study, we examined the expression of recombinant GA733-2 using a BCTV (Beet curly top virus) vector in plants. The formation of replicating molecules was confirmed in plants by Southern hybridization analysis. Expression of recombinant GA733-2 was analyzed by RT-PCR and Western blot analysis. Plant-derived recombinant GA733-2 elicited the production of specific IgG in serum by intraperitoneal immunization. The sera derived from mice immunized with recombinant GA733-2 have a specific binding activity on colorectal cancer cells. Our findings show that plant-derived recombinant GA733-2 can be used as an effective experimental immunogen for research in vaccine development. This work was supported by a grant from the Biogreen 21 project (20070401034026).

Eutrophication of a closed water system, usually associated with an excess influx of nitrogen and phosphate from domestic and industrial discharge, results in severe adverse effects on the environment of the system. The demand for compliance with discharge regulations has yielded many wastewater treatment plants that remove nitrogen compounds from wastewater using biological processes. Over the past several decades, biological nutrient removal processes have become a more widely-applied method to treat wastewater containing nitrogen and phosphorus, because these processes have economic advantages over chemical processes. In this study, we performed pilot-scale verification experiments to evaluate the performance of A2O4 and YPNR process concerning the removal of total nitrogen components in wastewater. These processes harness several mechanistic elements of the nitrogen cycle: In the aerobic stages, nitrifiers oxidize ammonia and organic nitrogen (include amines) to nitrite and nitrate; in the subsequent anaerobic stages, denitrifiers convert the nitrate to N2gas.