• Molecules and Cells
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• 제23권3호
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• pp.357-362
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• 2007

There is an increasing demand for high throughput (HTP) methods for gene analysis on a genome-wide scale. However, the current repertoire of HTP detection methodologies allows only a limited range of cellular phenotypes to be studied. We have constructed two HTP-optimized expression vectors generated from the red fluorescent reporter protein (RFP) gene. These vectors produce RFP-tagged target proteins in a multiple expression system using gateway cloning technology (GCT). The RFP tag was fused with the cloned genes, thereby allowing us localize the expressed proteins in mammalian cells. The effectiveness of the vectors was evaluated using an HTP-screening system. Sixty representative human C2 domains were tagged with RFP and overexpressed in HiB5 neuronal progenitor cells, and we studied in detail two C2 domains that promoted the neuronal differentiation of HiB5 cells. Our results show that the two vectors developed in this study are useful for functional gene analysis using an HTP-screening system on a genome-wide scale.

• Biomolecules & Therapeutics
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• 제22권4호
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• pp.355-362
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• 2014

We have developed a fully automated high throughput drug screening (HTDS) system based on the microfluidic cell culture array to perform combinational chemotherapy. This system has 64 individually addressable cell culture chambers where the sequential combinatorial concentrations of two different drugs can be generated by two microfluidic diffusive mixers. Each diffusive mixer has two integrated micropumps connected to the media and the drug reservoirs respectively for generating the desired combination without the need for any extra equipment to perfuse the solution such as syringe pumps. The cell array is periodically exposed to the drug combination with the programmed LabVIEW system during a couple of days without extra handling after seeding the cells into the microfluidic device and also, this device does not require the continuous generation of solutions compared to the previous systems. Therefore, the total amount of drug being consumed per experiment is less than a few hundred micro liters in each reservoir. The utility of this system is demonstrated through investigating the viability of the prostate cancer PC3 cell line with the combinational treatments of curcumin and tumor necrosis factor-alpha related apoptosis inducing ligand (TRAIL). Our results suggest that the system can be used for screening and optimizing drug combination with a small amount of reagent for combinatorial chemotherapy against cancer cells.

• The Plant Pathology Journal
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• 제24권1호
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• pp.24-30
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• 2008

Colletotrichum gloeosporioides forms an appressorium, a specialized infection structure, to infect its hosts. Among 400 and 600 culture filtrates from fungi and class Actinomycetes, six methanol extracts (A5005, A5314, A5387, A5560, A5597, and A5598) from the class Actinomycetes significantly inhibited appressorium formation in C. gloeosporioides infecting pepper fruits in a dose-dependent manner, while conidial germination was slightly enhanced. Two (A5005 and A5560) of them also exhibited distinctive inhibitory effect on the disease progress of pepper anthracnose. Water fractions of both culture filtrates also specifically inhibited appressorium formation in C. gloeosporioides and pepper anthracnose disease. Inhibition of appressorium formation by culture filtrate of A5005 was partially restored by the exogenous calcium. This results suggests that chemicals within A5005 extents its biological activity through disturbance of intracellular $Ca^{2+}$ regulation during prepenetration morphogenesis by C. gloeosporioides. Together, cell-based and target-oriented screening system used in this study should be applicable for other plant pathogenic fungi prerequisite appressorium formation to infect their hosts.

• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.329-338
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• 2023

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that produces attaching and effacing lesions on the large intestine and causes hemorrhagic colitis. It is primarily transmitted through the consumption of contaminated meat or fresh produce. Similar to other bacterial pathogens, antibiotic resistance is of concern for EHEC. Furthermore, since the production of Shiga toxin by this pathogen is enhanced after antibiotic treatment, alternative agents that control EHEC are necessary. This study aimed to discover alternative treatments that target virulence factors and reduce EHEC toxicity. The locus of enterocyte effacement (LEE) is essential for EHEC attachment to host cells and virulence, and most of the LEE genes are positively regulated by the transcriptional regulator, Ler. GrlA protein, a transcriptional activator of ler, is thus a potential target for virulence inhibitors of EHEC. To identify the GrlA inhibitors, an in vivo high-throughput screening (HTS) system consisting of a GrlA-expressing plasmid and a reporter plasmid was constructed. Since the reporter luminescence gene was fused to the ler promoter, the bioluminescence would decrease if inhibitors affected the GrlA. By screening 8,201 compounds from the Korea Chemical Bank, we identified a novel GrlA inhibitor named Grlactin [3-[(2,4-dichlorophenoxy)methyl]-4-(3-methylbut-2-en-1-yl)-4,5-dihydro-1,2,4-oxadiazol-5-one], which suppresses the expression of LEE genes. Grlactin significantly diminished the adhesion of EHEC strain EDL933 to human epithelial cells without inhibiting bacterial growth. These findings suggest that the developed screening system was effective at identifying GrlA inhibitors, and Grlactin has potential for use as a novel anti-adhesion agent for EHEC while reducing the incidence of resistance.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2003년도 2003 Annual Meeting, BioExhibition and International Symposium
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• pp.141-142
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• 2003

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.86-87
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• 2002

Recent technological innovations in the drug discovery process such as combinatorial synthesis and high throughput screening have led to the identification of an increasingly large number of compounds at the hits-to-leads stage. Therefore, rapid and precise pharmacokinetic/metabolic screening is essential to enhance the tractability of selected leads and to minimize the risk of failure in the later stages of drug development. (omitted)

• Journal of Microbiology and Biotechnology
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• 제33권6호
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• pp.831-839
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• 2023

Tylosin is a potent veterinary macrolide antibiotic produced by the fermentation of Streptomyces fradiae; however, it is necessary to modify S. fradiae strains to improve tylosin production. In this study, we established a high-throughput, 24-well plate screening method for identifying S. fradiae strains that produce increased yields of tylosin. Additionally, we constructed mutant libraries of S. fradiae via ultraviolet (UV) irradiation and/or sodium nitrite mutagenesis. A primary screening of the libraries in 24-well plates and UV spectrophotometry identified S. fradiae mutants producing increased yields of tylosin. Mutants with tylosin yield 10% higher than the wild-type strain were inoculated into shake flasks, and the tylosin concentrations produced were determined by high-performance liquid chromatography (HPLC). Joint (UV irradiation and sodium nitrite) mutagenesis resulted in higher yields of mutants with enhanced tylosin production. Finally, 10 mutants showing higher tylosin yield were re-screened in shake flasks. The yield of tylosin A by strains UN-C183 (6767.64 ± 82.43 ㎍/ml) and UN-C137 (6889.72 ± 70.25 ㎍/ml) was significantly higher than that of the wild-type strain (6617.99 ± 22.67 ㎍/ml). These mutant strains will form the basis for further strain breeding in tylosin production.

• International Journal of Oral Biology
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• 제37권2호
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• pp.43-50
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• 2012

The use of high throughput screening (HTS) in drug development is principally for the selection new drug candidates or screening of chemical toxicants. This system minimizes the experimental environment and allows for the screening of candidates at the same time. Umbilical cord-derived stem cells have some of the characteristics of fetal stem cell and have several advantages such as the ease with which they can be obtained and lack of ethical issues. To establish a HTS system, optimized conditions that mimic typical cell culture conditions in a minimal space such as 96 well plates are needed for stem cell growth. We have thus established a novel HTS system using human umbilical cord derived-mesenchymal stem cells (hUC-MSCs). To determine the optimal cell number, hUC-MSCs were serially diluted and seeded at 750, 500, 200 and 100 cells per well on 96 well plates. The maintenance efficiencies of these dilutions were compared for 3, 7, 9, and 14 days. The fetal bovine serum (FBS) concentration (20, 10, 5 and 1%) and the cell numbers (750, 500 and 200 cells/well) were compared for 3, 5 and 7 days. In addition, we evaluated the optimal conditions for cell cycle block. These four independent optimization experiments were conducted using an MTT assay. In the results, the optimal conditions for a HTS system using hUC-MSCs were determined to be 300 cell/well cultured for 8 days with 1 or 5% FBS. In addition, we demonstrated that the optimal conditions for a cell cycle block in this culture system are 48 hours in the absence of FBS. In addition, we selected four types of novel small molecule candidates using our HTS system which demonstrates the feasibility if using hUC-MSCs for this type of screen. Moreover, the four candidate compounds can be tested for stem cell research application.

• Weed & Turfgrass Science
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• 제4권2호
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• pp.118-123
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• 2015

메타게놈(metagenome)은 연구실에서 배양이 불가능한 미생물을 포함한 자연계에 존재하는 미생물의 유전자를 직접 연구하는 학문분야이다. 지구상 거의 모든 자연, 인공 환경에서 살고 있는 미생물 DNA를 분리 정제하는 것이 가능하며, 재조합 DNA 기술 등을 이용하여 배양 가능한 숙주미생물에 메타지놈을 클로닝함으로서 메타지놈 라이브러리를 제작할 수 있다. 최근 메타게노믹스를 통하여 자연계에 존재하고 있는 대다수의 미생물들이 실험실에서 배양되지 않았던 이유를 구명할 수 있게 되었고, 그들이 가지고 있는 생태학적인 의미와 역할에 대한 이해와 더불어 점차 그 응용 분야와 범위도 확대되고 있다. 이와 같은 메타게놈의 응용 분야 확대의 한 방안으로 본 연구에서는 새로운 제초활성 물질 및 제초활성 물질 생산에 필요한 유전자를 확보하기 위해 메타게놈 라이브러리를 대상으로 오이 떡잎절편 검정, 미세조류 생장저해 검정, 종자발아 저해 검정의 HTS (high throughput screening) 시스템을 구축하였고, 구축된 시스템으로부터 선발된 최종 단일 클론인 9-G1과 9-G12의 바랭이에 대한 in vivo assay를 통해 본 연구에서 개발한 HTS 시스템의 유효성을 확인 하였다. 후속연구로서 활성단일클론이 만들어내는 제초활성물질의 동정 및 제초활성물질을 합성하는 유전자군에 대한 연구를 수행 중에 있다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.422-423
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• 2005