• KSBB Journal
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• v.21 no.5
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• pp.360-365
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• 2006

The objectives of this study were to develop nano-pore silica particles and to modify its surface for use as an enzyme immobilization matrix. Sol-gel reaction was used to produce silica particles of various nano pore sizes with hydroxyl groups on their surfaces. The surface was modified with aldehyde that was confirmed by fluorescence imaging. Trypsin was covalently immobilized by reductive amination. Surface density of the immobilized trypsin was ca. $350{\mu}g/m^2$, which was approximately 17- and 35-fold higher than those from the surfaces with hydroxyl and amine group, respectively. About 90% of the initial enzyme activity was maintained after the 12th use of repeated use. When compared with the commercial matrices, the nano-pore silica particle was superior in terms of immobilization yield and specific activity. This study suggests the nano porous silica particles can be used as enzyme immobilization matrix for industrial applications.

• BMB Reports
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• v.51 no.11
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• pp.572-577
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• 2018

Recent studies showed that the PD-1/PD-L1 checkpoint blockade is a dramatic therapy for melanoma by enhancing antitumor immune activity. Currently, major strategies for the PD-1/PD-L1 blockade have mainly focused on the use of antibodies and compounds. Seeking an alternative approach, others employ endogenous proteins as blocking agents. The extracellular domain of PD-1 (ePD1) includes the binding site with PD-L1. Accordingly, we constructed a PD-1-based recombinantly tailored fusion protein (dFv-ePD1) that consists of bivalent variable fragments (dFv) of an MMP-2/9-targeted antibody and ePD1. The melanoma-binding intensity and antitumor activity were also investigated. We found the intense and selective binding capability of the protein dFv-ePD1 to human melanoma specimens was confirmed by a tissue microarray. In addition, dFv-ePD1 significantly suppressed the migration and invasion of mouse melanoma B16-F1 cells, and displayed cytotoxicity to cancer cells in vitro. Notably, dFv-ePD1 significantly inhibited the growth of mouse melanoma B16-F1 tumor cells in mice and in vivo fluorescence imaging showed that dFv-ePD was gradually accumulated into the B16-F1 tumor. Also the B16-F1 tumor fluorescence intensity at the tumor site was stronger than that of dFv. This study indicates that the recombinant protein dFv-ePD1 has an intensive melanoma-binding capability and exerts potent therapeutic efficacy against melanoma. The novel format of the PD-L1-blocked agent may play an active role in antitumor immunotherapy.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.20 no.5
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• pp.1-6
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• 2019

Organic dye-doped silica nanoparticles are used as a promising nanomaterials for bio-labeling, bio-imaging and bio-sensing. Fluorescent silica nanoparticles(NPs) have been synthesized by the modified $St{\ddot{o}}ber$ method. In this study, dye-free fluorescent silica NPs of various sized were synthesized by Sol-Gel process as the modified $St{\ddot{o}}ber$ method. The functional material of APTES((3-aminopropyl)triethoxysilane) was added as an additive during the Sol-Gel process. The as-synthesized silica NPs were calcined at $400^{\circ}C$ for 2 hours. The surface morphology and particle size of the as-synthesized silica NPs were characterized by field-emission scanning electron microscopy. The fluorescent characteristics of the as-synthesized silica NPs was confirmed by UV lamp irradiation of 365 nm wavelength. The photoluminescence (PL) of the as-synthesized silica NPs with different size was analyzed by fluorometry. As the results, the as-synthesized silica NPs exhibits same blue fluorescent characteristics for different NPs size. Especially, as increased of the silica NPs size, the intensity of PL was decreased. The blue fluorescence of dye-free silica NPs was attributed to linkage of $NH_2$ groups of the APTES layer and oxygen-related defects in the silica matrix skeleton.

• Animal Bioscience
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• v.34 no.10
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• pp.1579-1589
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• 2021

Objective: This study was conducted to generate single stranded DNA oligonucleotides with selective affinity to bovine spermatozoa, assess its binding potential and explore its potential utility in trapping spermatozoa from suspensions. Methods: A combinatorial library of 94 mer long oligonucleotide was used for systematic evolution of ligands by exponential enrichment (SELEX) with bovine spermatozoa. The amplicons from sixth and seventh rounds of SELEX were sequenced, and the reads were clustered employing cluster database at high identity with tolerance (CD-HIT) and FASTAptamer. The enriched nucleotides were predicted for secondary structures by Mfold, motifs by Multiple Em for Motif Elicitation and 5' labelled with biotin/6-FAM to determine the binding potential and binding pattern. Results: We generated 14.1 and 17.7 million reads from sixth and seventh rounds of SELEX respectively to bovine spermatozoa. The CD-HIT clustered 78,098 and 21,196 reads in the top ten clusters and FASTAptamer identified 2,195 and 4,405 unique sequences in the top three clusters from the sixth and seventh rounds, respectively. The identified oligonucleotides formed secondary structures with delta G values between -1.17 to -26.18 kcal/mol indicating varied stability. Confocal imaging with the oligonucleotides from the seventh round revealed different patterns of binding to bovine spermatozoa (fluorescence of the whole head, spot of fluorescence in head and mid- piece and tail). Use of a 5'-biotin tagged oligonucleotide from the sixth round at 100 pmol with 4×10⁶ spermatozoa could trap almost 80% from the suspension. Conclusion: The binding patterns and ability of the identified oligonucleotides confirms successful optimization of the SELEX process and generation of aptamers to bovine spermatozoa. These oligonucleotides provide a quick approach for selective capture of spermatozoa from complex samples. Future SELEX rounds with X- or Y- enriched sperm suspension will be used to generate oligonucleotides that bind to spermatozoa of a specific sex type.

• The Bulletin of The Korean Astronomical Society
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• v.39 no.1
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• pp.68.1-68.1
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• 2014

We present the results of far-ultraviolet (FUV) observations of comet C/2001 Q4 (NEAT) obtained with Far-ultraviolet Imaging Spectrograph (FIMS) on board the Korean microsatellite STSAT-1, which operated at an altitude of 700 km in a sun-synchronous orbit. FIMS is a dual channel imaging spectrograph (S-channel 900-1150 ${\AA}$, L-channel 1350-1710 ${\AA}$, and ${\lambda}/{\Delta}{\lambda}$ ~ 550 for both channels) with large image fields of view (S-channel $4.0^{\circ}{\times}4.6^{\prime}$, L-channel $7.5^{\circ}{\times}4.3^{\prime}$, and angular resolution ~ $5-10^{\prime}$) optimized for the observation of diffuse emission of astrophysical radiation. Comet C/2001 Q4 (NEAT) were made in two campaigns during its perihelion approach between May 8 and 15, 2004. Based on the scanning mode observations in the wavelength band of 1400-1700 ${\AA}$, we have constructed an image of the comet with an angular size of $5^{\circ}{\times}5^{\circ}$, which corresponds to the central coma region. Several important fluorescence emission lines were detected including S I multiplets at 1429 and 1479 ${\AA}$, C I multiplets at 1561 and 1657 ${\AA}$, and the CO $A^1{\Pi}-X^1{\Sigma}^+$ Fourth Positive system; we have estimated the production rates of the corresponding species from the fluxes of these emission lines. The estimated production rate of CO was $Q_{CO}=(2.65{\pm}0.63){\times}10^{28}s^{-1}$, which is 6.2-7.4% of the water production rate and is consistent with earlier predictions. The average carbon production rate was estimated to be $Q_C={\sim}1.59{\times}10^{28}s^{-1}$, which is ~60% of the CO production rate. However, the observed carbon profile was steeper than that predicted using the two-component Haser model in the inner coma region, while it was consistent with the model in the outer region. The average sulfur production rate was $Q_S=(4.03{\pm}1.03){\times}10^{27}s^{-1}$, which corresponds to ~1% of the water production rate.

• Journal of Life Science
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• v.32 no.2
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• pp.148-154
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• 2022

Focal adhesion kinase (FAK) is known to regulate cell adhesion, migration, and mechanotransduction in focal adhesions (FAs). However, studies on how FAK activity is regulated in the plasma membrane microdomains according to the composition of extracellular matrix (ECM) proteins are still lacking. A genetically encoded fluorescence resonance energy transfer (FRET)-based biosensor can provide useful information on the activity of intracellular signals with high spatiotemporal resolution. In this study, we analyzed the FAK activities in lipid raft (detergent-resistant membrane) and non-lipid raft (non-detergent-resistant membrane) microdomains using FRET-based membrane targeting FAK biosensors (FAK-Lyn and FAK-KRas biosensors) under four different ECM protein compositions: glass, type 1 collagen, fibronectin, and laminin. Interestingly, FAK activity in response to laminin in a lipid raft microdomain was lower than that in other ECM conditions. Cells subjected to fibronectin showed higher FAK activity in a lipid raft microdomain than that in a non-lipid raft microdomain. Therefore, this study demonstrates that the FAK activity can be distinctively regulated according to the ECM type and the environment of the plasma membrane microdomains.

• Journal of Biomedical Engineering Research
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• v.36 no.1
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• pp.16-21
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• 2015

Indocyanine green(ICG) and 5-aminolevulinic acid(5-ALA) have been widely used to mark blood vessels or tumors. However, fluorescent dye detection systems were designed to use one type of dyes only. In this study, we proposed a detection system capable of detecting Indocyanine green and 5-aminolevulinic acid. Multiple filters and light sources are integrated into a single system. In this study, we performed analysis of fluorescent dyes and configured a detection system. During the analysis, it was found that Indocyanine green and 5-aminolevulinic acid have the maximum intensity at $40{\mu}M$. We designed light source for fluorescent dyes and conducted compatibility test using a commercial surgical microscope. The fluorescent dye detection system was configured based on the experimental results. The developed system successfully detects Indocyanine green and 5-aminolevulinic acid. Therefore, more efficient surgical operations can be achieved using both fluorescent dyes at the same time. We expect that the developed system can increase the survival rate of patients.

• JSTS:Journal of Semiconductor Technology and Science
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• v.1 no.4
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• pp.239-247
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• 2001

The Fluorescence-Activated Cell Sorter (FACS) is a well-established instrument used for identifying, enumerating, classifying and sorting cells by their physical and optical characteristics. For a miniaturized FACS device, a disposable plastic microchip has been developed which has a hydrodynamic focusing chamber using soft lithography. As the characteristics of the spatially confined sample stream have an effect on sample throughput, detection efficiency, and the accuracy of cell sorting, systematic fluid dynamic studies are required. Flow visualization is conducted with a laser scanning confocal microscopy (LSCM), and three-dimensional flow structure of the focused sample stream is reconstructed from 2D slices acquired at $1\mutextrm{m}$ intervals in depth. It was observed that the flow structure in the focusing chamber is skewed by unsymmetrical velocity profile arising from trapezoidal cross section of the microchannel. For a quantitative analysis of a microscopic flow structure, Confocal Micro-PIV system has been developed to evaluate the accelerated flow field in the focusing chamber. This study proposes a method which defines the depth of the measurement volume using a detection pinhole. The trajectories of red blood cells (RBCs) and their interactions with surrounding flow field in the squeezed sample stream are evaluated to find optimal shape of the focusing chamber and fluid manipulation scheme for stable cell transporting, efficient detection, and sorting

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4263-4266
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• 2012

Colorectal cancer has become a major disease threatening human health. To establish animal models that exhibit the characteristics of human colorectal cancer will not only help to study the mechanisms underlying the genesis and development effectively, but also provide ideal carriers for the screening of medicines and examining their therapeutic effects. In this study, we established a stable, colon cancer nude mouse model highly expressing green fluorescent protein (GFP) for spontaneous metastasis after surgical orthotopic implantation (SOI). GFP-labeled colon cancer models for metastasis after SOI were successfully established in all of 15 nude mice and there were no surgery-related complications or deaths. In week 3, primary tumors expressing GFP were observed in all model animals under fluoroscopy and two metastatic tumors were monitored by fluorescent imaging at the same time. The tumor volumes progressively increased with time. Seven out of 15 tumor transplanted mice died and the major causes of death were intestinal obstruction and cachexia resulting from malignant tumor growth. Eight model animals survived at the end of the experiment, 6 of which had metastases (6 cases to mesenteric lymph nodes, 4 hepatic, 2 pancreatic and 1 mediastinal lymph node). Our results indicate that our GFP-labeled colon cancer orthotopic transplantation model is useful with a high success rate; the transplanted tumors exhibit similar biological properties to human colorectal cancer, and can be used for real-time, in vivo, non-invasive and dynamic observation and analysis of the growth and metastasis of tumor cells.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4751-4756
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• 2012

Colon cancer continues to be one of the most common cancers, and the importance and necessity of new therapies needs to be stressed. The most important proto-oncogen factors for colon cancer appear to be epidermal growth factor receptor, EGFR, and c-Src with high expression and activity leading to tumor growth and ultimately to colon cancer progression. Application of c-Src and EGFR antisense agents simultaneously should theoretically therefore have major benefit. In the present study, anti-EGFR and c-Src specific antisense oligodeoxynucleotides were combined in a formulation using PAMAM dendrimers as a carrier. Nano drug entry into cells was confirmed by flow cytometry and fluorescence microscopy imaging and real time PCR showed gene expression of c-Src and EGFR, as well as downstream STAT5 and MAPK-1 with the tumor suppressor gene P53 to all be downregulated. EGFR and c-Src protein expression was also reduced when assessed by western blotting techniques. The effect of the antisense oligonucleotide on HT29 cell proliferation was determined by MTT assay, reduction beijng observed after 48 hours. In summary, nano-drug, anti-EGFR and c-Src specific antisense oligodeoxynucleotides were effectively transferred into HT-29 cells and inhibited gene expression in target cells. Based on the results of this study it appears that the use of antisense EGFR and c-Src simultaneously might have a significant effect on colon cancer growth by down regulation of EGFR and its downstream genes.