• Animal cells and systems
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• 제13권2호
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• pp.97-103
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• 2009

Recently, great advance is achieved in the field of genome-wide functional screening using cell-based assay. Here, we briefly introduce well-established and typical cell-based assays of GPCR and some parameters which should be considered for genome-wide functional screening. Because of characters and importance of GPCR as drug targets, several ways of assay systems were devised. Among them, high-content screening (HCS) that is based on the analysis of image by confocal microscope is becoming favorite choice. The advances in this technology have been driven exclusively by industry for their convenience. Now, it is turn for academy to define more detail signaling networks via HCS using cDNA or siRNA libraries at genome-wide level. By isolating novel signaling mediators using cDNA or siRNA library, and postulating them as new candidates for therapeutic target, more understanding about life science and more increased chances to develop therapeutics against human disease will be achieved.

• Journal of Microbiology and Biotechnology
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• 제26권2호
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• pp.213-225
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• 2016

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

• Biomaterials and Biomechanics in Bioengineering
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• 제1권2호
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• pp.95-104
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• 2014

The objective of this study was to evaluate the cytotoxicity of poly (lactic acid) (PLA) nanoparticles. We used a water-soluble, amphiphilic phospholipid polymer, poly (2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB30W), as a stabilizer for the PLA nanoparticles. The PLA nanoparticles and PMB30W-modified PLA (PLA/PMB30W) nanoparticles were prepared by evaporating tetrahydrofuran (THF) from its aqueous solution. Precipitation of the polymers from the aqueous solution produced PLA and PLA/PMB30W nanoparticles with a size distribution of $0.4-0.5{\mu}m$. The partial coverage of PMB30W on the surface of the PLA/PMB30W nanoparticles was confirmed by X-ray photoelectron spectroscopy (XPS) and dynamic light-scattering (DLS). A high-content automated screening assay (240 random fields per group) revealed that the PLA nanoparticles induced apoptosis in a mouse macrophage-like cell line (apoptotic population: 73.9% in 0.8 mg PLA/mL), while the PLA/PMB30W nanoparticles remained relatively non-hazardous in vitro (apoptotic population: 13.8% in 0.8 mg PLA/mL). The reduction of the apoptotic population was attributed to the phosphorylcholine groups in the PMB30W bound to the surface of the nanoparticle. In conclusion, precipitation of PLA in THF aqueous solution enabled the preparation of PLA nanoparticles with similar shapes and size distribution but different surface characteristics. PMB30W was an effective stabilizer and surface modifier, which reduced the cytotoxicity of PLA nanoparticles by enabling their avoidance of the mononuclear phagocyte system.

• 대한임상검사과학회지
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• 제52권1호
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• pp.53-61
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• 2020

Cell migration은 embryogenesis 혹은 cancer metastasis 이외에, 물리적 손상에 의한 상처의 수복을 위해서 손상된 부위로 세포가 이동하는 매우 흔하게 관찰되는 현상 중 하나 이다. Wound healing assay는 in vitro의 이차원 평면상에서 세포의 이동을 관찰할 수 있는 기본적인 연구 기법이다. In vitro상에서 물리적 손상을 재현하는 가장 손쉬운 접근법으로서, 세포의 confluent monolayer 표면에 날카로운 도구를 이용하여 기계적인 스크레치를 내는 방법이 사용되고 있다. 완충 스프링이 탑재된 금속 핀을 96-well plate를 기반으로 하는 wound maker에 장착하여 multi-well plate 바닥 표면의 고르지 못한 굴곡과 스크레칭 팁 사이에 직각을 이루는 접촉면에서의 미세한 조절이 가능하도록 하였다. 실험용 팁으로 confluent monolayer위에 스크래치를 내었을 때에는 다양한 지그재그 패턴이 그려진 반면에, 직접 제작한 wound maker에서는 동일한 형태의 선형 wounds가 fibroblast가 seeding된 96-well plate의 각 well의 중심부에 그려짐을 확인하였다. 상용화 되어있는 몇몇 multi-well plate가 본 실험에서 제작된 wound maker와 호환되는 것을 고려하여 보았을 때에, 실시간 wound healing을 관찰하는 high content screening (HCS)실험에 있어서의 활용적인 측면에서 기존의 전형적인 polypropylene 파이펫 팁을 이용한 스크래칭 방법보다 더욱 용이한 방법임을 알 수 있다.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.290-290
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• 2017

Rice (Oryza sativa L.) is one of the most consumed staple food crop which is energy source as carbohydrate and also is considered as the important antioxidant sources including various phenolic compounds. According to the increasing demand of healthy life, the concern to antioxidant also is increasing because of its health-promoting effect. Phenolic compounds are one of the plant secondary metabolites class, which shows various benefits to preventing or treating chronic diseases. In this study, we have measured the total phenol content from total 647 rice samples using the Floin-Ciocalteau method, and then were selected 30 rice genetic resources classified with high, middle, and low group on the basis of total phenol content. The average of the total phenol content of each group was high-group ($6892.9{\pm}488.5{\mu}g\;GAE/g$) > middle-group ($1428.1{\pm}76.0{\mu}g\;GAE/g$) > low-group ($97.6{\pm}11.4{\mu}g\;GAE/g$). The selected rice samples were analyzed with LC-MS/MS to find the composition and concentration of individual phenolic in rice grain. High-group and middle-group contained large amounts of protocatechuic acid and (+)-catechin whereas low-group showed limited amount. Among high-group samples, rice samples with black pericarp color (IT 174089, IT 220079, and IT 259958) had high content of peonidin-3-O-glucoside. Further, these black rice samples were special since polydatin, rarely found stilbenoid in rice grain, was detected. Overall, both the sum of phenolic acid and the sum of flavonoid were high-group > middle-group > low-group. Also, each group exhibited different phenolic compositions; high-group consisted of flavonoid more than phenolic acid, middle-group and low-group was comprised of phenolic acid rather than flavonoid, and non-pigmented rice was composed by fully phenolic acid. The total phenol content had positive relationships with the sum of phenolic compound (r = 0.64), the sum of flavonoid (r = 0.74) at the significance level of p < 0.0001. In addition, protocatechuic acid and quercetin showed positive correlation with above phenolic composition parameters; in order, r = 0.98, 0.65 for protocatechuic acid and r = 0.73, 0.78 for quercetin (p < 0.0001). In conclusion, the total phenol content assay showed the possibility of utilization as a phenolic composition indicator in rice grain. Also, this result was suggested study pigment on other material.

• 한국식품영양과학회지
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• 제40권7호
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• pp.1053-1062
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• 2011

건강한 삶에 대한 현대인의 관심이 나날이 고조되고 있으며, 이에 따라 노화와 질병의 예방에 효과가 있는 항산화제의 연구가 활발히 이루어지고 있다. 특히 천연물이나 식품을 소재로 한 식이성 항산화제에 대한 연구는 꾸준히 증가하고 있는 추세이며, 천연물의 소재나 연구 분야의 폭이 매우 넓다. 따라서 다양한 식품 소재의 항산화능을 조사할 수 있는 측정법의 중요성이 크게 부각되고 있다. 현재 약용 식물이나 상용 채소, 과일을 시료로 하여 여러 가지 radical이나 target molecule에 대한 항산화능을 측정할 수 있는 다수의 생체외(in vitro) 측정법이 사용되고 있다. 이에 본 총설에서는 널리 사용되고 있는 항산화능 측정법의 특징을 분석하고 적용 사례를 검토하였다. 식품의 구성 성분은 단일물질이 아닌 복합체로 구성되어 있으므로 하나의 측정법으로 항산화능을 평가할 수가 없다. 그러므로 채소와 과일을 포함한 여러가지 식물성 식품 추출물의 항산화 활성을 정확히 측정하기 위해서는 각 실험에 사용되는 radical과 기전(mechanism), 실험 조건(온도, pH, 실험기기, 시료추출방법, 소요 시간, 비용) 등을 고려하여 적절히 수행되어야 할 것이다. 그러나 이들 측정법의 다양성과 사용 단위의 차이로 인해 각 시료의 항산화능을 객관적으로 비교하는데 어려움이 있는 실정이다. 따라서 향후 항산화능 측정법의 표준화와 표현 단위의 통일이 절실히 요구된다. 또한 in vitro에서 항산화능을 나타내는 채소, 과일 등의 생체 내 활성은 다양한 biomechanism과 polymerphism으로 인해 그 효과를 예측하기가 매우 어렵다. 따라서 in vitro에서의 항산화능 screening 결과를 바탕으로 in vivo에서의 그 효과를 검증하는 연구가 부가적으로 시행되어야 할 것이라 사료된다.

• Nano
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• 제13권10호
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• pp.1850116.1-1850116.14
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• 2018

In this study, we report the mercury ions ($Hg^{2+}$) mediated phosphorus-containing carbon dots (PCDs) as a selective "off-on" fluorescence probe for glutathione (GSH), cysteine (Cys) and homocysteine (Hcys). PCDs obtained by hydrothermal reaction are sensitive to $Hg^{2+}$ ions and its fluorescence can be significantly quenched owing to the electron transfer from the lowest unoccupied molecular orbital (LUMO) of PCDs to $Hg^{2+}$. Interestingly, the weak fluorescence of $Hg^{2+}$-mediated PCDs could be gradually recovered with the addition of GSH, Cys and Hcys. This can be attributed to the formation of $Hg^{2+}-S$ complex due to the super affinity of $Hg^{2+}$-sulfydryl bond. The formation of $Hg^{2+}-S$ complex extremely reduces the oxidation ability of $Hg^{2+}$ that inhibits the electron transfer from LUMO of PCDs to $Hg^{2+}$ and re-opens the native electron transition from LUMO to the highest occupied molecular orbital (HOMO) of PCDs. Thus, the green fluorescence of PCDs is switched on. Furthermore, the present $Hg^{2+}$-mediated PCDs assay exhibits a high selectivity for GSH, Cys and Hcy and has been successfully used to detect the total biothiols content in human urine samples.

• Asian Pacific Journal of Cancer Prevention
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• 제14권2호
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• pp.1017-1021
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• 2013

The cellular apoptosis susceptibility (CSE1L) gene has been demonstrated to regulate multiple cellular mechanisms including the mitotic spindle check point as well as proliferation and apoptosis. However, the importance of CSE1L in human colon cancer is largely unknown. In the present study, we examined expression levels of CSE1L mRNA by semiquantitative RT-PCR. A lentivirus-mediated small interfering RNA (siRNA) was used to knock down CSE1L expression in the human colon cancer cell line RKO. Changes in CSE1L target gene expression were determined by RT-PCR. Cell proliferation was examined by a high content screening assay. In vitro tumorigenesis was measured by colony-formation assay. Cell cycle distribution and apoptosis were detected by flow cytometric analysis. We found CSE1L mRNA to be expressed in human colon cancer cells. Using a lentivirus based RNAi approach, CSE1L expression was significantly inhibited in RKO cells, causing cell cycle arrest in the G2/M and S phases and a delay in cell proliferation, as well as induction of apoptosis and an inhibition of colony growth capacity. Collectively, the results suggest that silencing of CSE1L may be a potential therapeutic approach for colon cancer.

• 한국식품영양학회지
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• 제25권2호
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• pp.246-252
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• 2012

After a mixed carbohydrate diet, inhibition of ${\alpha}$-amylase and ${\alpha}$-glucosidase involved in the digestion and absorption of carbohydrates can significantly decrease the postprandial increase of blood glucose level. In the course of screening these useful enzyme inhibitors, we selected five kinds of bean, using an in-vitro enzyme inhibition assay method. To evaluate the effect of germination process on the functionality of the bean, we investigated the inhibitory activities of the water extracts of non-germinated bean and germinated bean against ${\alpha}$-amylase and ${\alpha}$-glucosidase, relevant to postprandial hyperglycemia. We also investigated the oxygen radical absorbance capacity(ORAC), total phenolics content, and postprandial blood glucose lowering effect in rats(Sprague-Dawley rat model). Most germinated beans showed significantly higher ${\alpha}$-glucosidase inhibitory activity, compared with non-germinated beans. Among germinated beans, Glycine max had the highest ${\alpha}$-glucosidase inhibitory activity(53.3%). The water extract of germinated Phaseolus vulgaris L. had the highest ${\alpha}$-amylase inhibitory activity(95.1%), followed by Glycine max(58.7%), and Glycine max L. Merr(54.1%). Furthermore, the five germinated beans also showed high antioxidant activities in ORAC assay. Results suggested that the germination process may improve and enhance the anti-hyperglycemia potential and antioxidant activity of the bean.

• Journal of Microbiology and Biotechnology
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• 제26권11호
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• pp.1891-1907
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• 2016

Yeasts that are present in marine environments have evolved to survive hostile environments that are characterized by high exogenous salt content, high concentrations of inhibitory compounds, and low soluble carbon and nitrogen levels. Therefore, yeasts isolated from marine environments could have interesting characteristics for industrial applications. However, the application of marine yeast in research or industry is currently very limited owing to the lack of a suitable isolation method. Current methods for isolation suffer from fungal interference and/or low number of yeast isolates. In this paper, an efficient and non-laborious isolation method has been developed and successfully isolated large numbers of yeasts without bacterial or fungal growth. The new method includes a three-cycle enrichment step followed by an isolation step and a confirmation step. Using this method, 116 marine yeast strains were isolated from 14 marine samples collected in the UK, Egypt, and the USA. These strains were further evaluated for the utilization of fermentable sugars (glucose, xylose, mannitol, and galactose) using a phenotypic microarray assay. Seventeen strains with higher sugar utilization capacity than the reference terrestrial yeast Saccharomyces cerevisiae NCYC 2592 were selected for identification by sequencing of the ITS and D1/D2 domains. These strains belonged to six species: S. cerevisiae, Candida tropicalis, Candida viswanathii, Wickerhamomyces anomalus, Candida glabrata, and Pichia kudriavzevii. The ability of these strains for improved sugar utilization using seawater-based media was confirmed and, therefore, they could potentially be utilized in fermentations using marine biomass in seawater media, particularly for the production of bioethanol and other biochemical products.