• Animal cells and systems
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• v.13 no.2
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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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• v.26 no.2
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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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• v.1 no.2
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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.

• Korean Journal of Clinical Laboratory Science
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• v.52 no.1
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• pp.53-61
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• 2020

Cell migration is a central process for recovering from wounds triggered by physical distress besides embryogenesis and cancer metastasis. Wound healing assay is widely used as a fundamental research technique for investigation of two-dimensional cell migration in vitro. The most common approach for imitating physical wound in vitro is mechanical scratching on the surface of the confluent monolayer by using sharp materials. The iron metal pin with a suspension spring for fine adjustment of the orthogonal contact surface between the scratching point and the individual bottom of multi-well plate with planar curvatures were adopted for the creative invention of a 96-well plate wound maker. While classic tips drew diverse and zigzag scratching patterns on the confluent monolayer, our wound maker displayed synchronized linear wounds in the middle of each well of a 96-well plate that was seeded with several cell lines. Given that several types of multi-well plates commercially available are compatible with our lab-made wound maker for creating uniform scratches on the confluent monolayer for the collective cell migration in wound healing assay, it is certain that the application of this wound maker to the real-time wound healing assay in high content screening (HCS) is superior than utilization of typical polypropylene pipette tips.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.40 no.7
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• pp.1053-1062
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• 2011

Reactive oxygen species (ROS), including singlet oxygen (${O_2}^1$), superoxide anion radical ($O_2{\cdot}^-$), hydroxyl radical ($HO{\cdot}$), peroxyl radical ($ROO{\cdot}$), hydrogen peroxide ($H_2O_2$), and hypochlorous (HOCl), are generated as byproducts of normal cellular metabolism. ROS induce damage to many biological molecules, such as lipids, proteins, carbohydrates, and DNA. It is widely believed that some degenerative diseases caused by ROS can be prevented by the high intake of fruits and vegetables due to their antioxidant activities. Recently, research on natural antioxidants has become increasingly active in various fields. Several assays have been developed to measure the total antioxidant capacity of antioxidants in fruits and vegetables in vitro. These assays include those for DPPH radical scavenging activity, SOD-like activity, total polyphenol content, oxygen radical absorbance capacity, reducing power, trolox equivalent antioxidant capacity (ABTS assay), single-cell gel electrophoresis (comet assay), and a cellular antioxidant activity assay. Because different antioxidant compounds may act through different mechanisms in vitro, no single assay can fully evaluate the total antioxidant capacity of foods. Due to the complexity of the composition of foods, it is important to be able to measure antioxidant activity using biologically relevant assays. In this review, recently used assays were selected for extended discussion, including a comparison of the advantages and disadvantages of each assay and their application to fruits and vegetables.

• Nano
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• v.13 no.10
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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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• v.14 no.2
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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.

• The Korean Journal of Food And Nutrition
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• v.25 no.2
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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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• v.26 no.11
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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.