• Title/Summary/Keyword: high content screening

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Considering Cell-based Assays and Factors for Genome-wide High-content Functional Screening

  • Chung, Chul-Woong;Kim, In-Ki;Jung, Yong-Keun
    • 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.

Cell-Based Assay Design for High-Content Screening of Drug Candidates

  • Nierode, Gregory;Kwon, Paul S.;Dordick, Jonathan S.;Kwon, Seok-Joon
    • 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.

Screening of High Temperature-Tolerant Oleaginous Diatoms

  • Zhang, Lingxiang;Hu, Fan;Wan, Xiu;Pan, Yufang;Hu, Hanhua
    • Journal of Microbiology and Biotechnology
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    • v.30 no.7
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    • pp.1072-1081
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    • 2020
  • Screening suitable strains with high temperature adaptability is of great importance for reducing the cost of temperature control in microalgae cultivation, especially in summer. To obtain high temperature-tolerant diatoms, water samples were collected in summer from 7 different regions of China across the Northeast, North and East. A total of 731 water samples was collected and from them 131 diatom strains were isolated and identified based on the 18S rRNA sequences. Forty-nine strains out of the 131 diatoms could survive at 30℃, and 6 strains with relatively high biomass and lipid content at high temperature were selected and were found to be able to grow at 35℃. Cyclotella sp. HB162 had the highest dry biomass of 0.46 g/l and relatively high triacylglycerol (TAG) content of 237.4 mg/g dry biomass. The highest TAG content of 246.4 mg/g dry biomass was obtained in Fistulifera sp. HB236, while Nitzschia palea HB170 had high dry biomass (0.33 g/l) but relatively low TAG content (105.9 mg/g dry biomass). N. palea HB170 and Fistulifera sp. HB236 presented relatively stable growth rates and lipid yields under fluctuating temperatures ranging from 28 to 35℃, while Cyclotella HB162 maintained high lipid yield at temperatures below 25℃. The percentage of saturated fatty acids and monounsaturated fatty acids in all the 6 strains was 84-91% in total lipids and 90-94% in TAGs, which makes them the ideal feedstock for biodiesel.

Phytochemical constituent, percentage yield and phenolic content estimation of different solvent system of Carica papaya leaves

  • Sheneni, Victor Duniya;Usman, Oman Salifu;Musa, Quasim
    • The Korean Journal of Food & Health Convergence
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    • v.4 no.2
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    • pp.17-23
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    • 2018
  • Carica papaya is an important medicinal plant used in the management of different disease conditions. Phytochemical screening was carried out using different chemical test, Percentage yield and total phenolic content was evaluated using Folin Ciocalteu method in different solvent system; methanol, ethanol, ethyl acetate, n-butanol and n-hexane respectively. The phytochemical screening of the studies showed the presence of flavonoids, saponins, tannins, terpenoids, glycosides, steroids, carbonhydrate, anthraquinone and alkaloids. The percentage yield of crude extract and total polyphenol content was high in methanol, ethanol and ethyl acetate when compared with n-butanol and n-hexane. The studies show that Carica papaya leave extracts is a potent source of secondary metabolites. The use of the plant in the management of diseases is justified.

Phospholipid polymer can reduce cytotoxicity of poly (lactic acid) nanoparticles in a high-content screening assay

  • Kim, Hyung Il;Ishihara, Kazuhiko
    • 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.

Results of Anxiety Disorder Screening Programat the 2013 Mental Health Exposition in Seoul (2013년 정신건강박람회 불안 장애 선별검사 결과)

  • Choi, Kangrok;Kim, Daeho;Seo, Ho Jun;Huh, Hyu Jung;Lee, Dong-Woo;Chae, Jeong Ho
    • Anxiety and mood
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    • v.9 no.2
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    • pp.147-153
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    • 2013
  • Objective : Despite the chronic nature and high social costs, individuals with anxiety disorders seldom seek treatment. Thus, education for public awareness and screening for the illness is tremendously important for mental health professionals. This study summaries and presents the results from Anxiety Disorder Screening Program during the Mental Health Exposition held in Seoul in April, 2013. Methods : We analyzed the data from 116 participants who agreed and completed the screening questionnaires during their visits to two-day Anxiety Disorder Screening Program prepared by the Korean Academy of Anxiety Disorder. The questionnaire comprised of modified Mobility inventory for agoraphobia, Contents of worries, Penn State Worry Questionnaire, Life Events Checklist, and Abbreviated Posttraumatic Stress Disorder Checklist. Results : Participants demonstrated high rates of anxiety symptoms and possible anxiety disorders. Experience of panic attack was reported by 45%, lifetime and 16% in the past month by respondents. Phobia was reported by 46%. Participants had an average of 3.3 pathological worries and among those, social or interpersonal content was most common (46%). At least one lifetime traumatic event was reported by 64%. By the cut-off scores in the literature, 46% had possible generalized anxiety disorder and 58% possible posttraumatic stress disorder. Conclusion : Our results suggested that many visitors to Anxiety Disorder Screening Program were in fact treatment seeking after experiencing some forms of anxiety symptoms. Further efforts for delivery of medical information and increasing public awareness for anxiety disorders are needed.

Use of DNA-Specific Anthraquinone Dyes to Directly Reveal Cytoplasmic and Nuclear Boundaries in Live and Fixed Cells

  • Edward, Roy
    • Molecules and Cells
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    • v.27 no.4
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    • pp.391-396
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    • 2009
  • Image-based, high-content screening assays demand solutions for image segmentation and cellular compartment encoding to track critical events - for example those reported by GFP fusions within mitosis, signalling pathways and protein translocations. To meet this need, a series of nuclear/cytoplasmic discriminating probes have been developed: DRAQ5$^{TM}$ and CyTRAK Orange$^{TM}$. These are spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. At their most fundamental they provide a convenient fluorescent emission signature which is spectrally separated from the commonly used reporter proteins (e.g. eGFP, YFP, mRFP) and fluorescent tags such as Alexafluor 488, fluorescein and Cy2. Additionally, they do not excite in the UV and thus avoid the complications of compound UV-autofluorescence in drug discovery whilst limiting the impact of background sample autofluorescence. They provide a convenient means of stoichiometrically labelling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Further developments have permitted the simultaneous and differential labelling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions and most recently compound in vitro toxicology testing.

Water Deficit in Salt- and Drought- stressed Rice (Oryza sativa L.) Seedlings (염과 건조처리에 따른 벼 유묘의 수분결핍)

  • Kang, Dong-Jin;Ishii, Ryuichi;Lee, In-Jung
    • Current Research on Agriculture and Life Sciences
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    • v.21
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    • pp.1-9
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    • 2003
  • Plants are often subjected to periods of soil and atmospheric water deficit during their life cycle. To find critical levels for identification of tolerant rice variety to salt- and drought-stresses, we investigated the water deficiency in the leaf of a Dongjinbyeo (DJ) cultivar, identified as intolerant variety, subjected to NaCl- and Polyethylene glycol 6000 (PEG)- treatments. The relative water content and water potential in leaf of DJ plant sharply declined along the high concentration and time after treatment in NaCl- and PEG-treated rice plants. To elucidate the method of simple screening of tolerant variety to salt- and drought-stresses, we examined the relationship between relative water content and water potential of leaves in NaCl- and PEG-treated rice plants. The relationship between relative water content and water potential in leaf of DJ plant showed the highest correlation in 80 mM NaCl-treatment, and showed high correlation only 8% PEG treatment. These results indicate that the critical level of salt stress for screening of tolerant rice was 80 mM NaCl at 48 h after NaCl treatment, and the critical concentration of drought stress for screening of tolerant rice was 8% PEG at 96 h after PEG treatment.

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