• 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.

• The Plant Pathology Journal
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• 제32권4호
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• pp.300-310
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• 2016

Pathogen infection in plants induces complex responses ranging from gene expression to metabolic processes in infected plants. In spite of many studies on biotic stress-related changes in host plants, little is known about the metabolic and phenotypic responses of the host plants to Pseudomonas cichorii infection based on image-based analysis. To investigate alterations in tomato plants according to disease severity, we inoculated plants with different cell densities of P. cichorii using dipping and syringe infiltration methods. High-dose inocula (${\geq}10^6cfu/ml$) induced evident necrotic lesions within one day that corresponded to bacterial growth in the infected tissues. Among the chlorophyll fluorescence parameters analyzed, changes in quantum yield of PSII (${\Phi}PSII$) and non-photochemical quenching (NPQ) preceded the appearance of visible symptoms, but maximum quantum efficiency of PSII ($F_v/F_m$) was altered well after symptom development. Visible/near infrared and chlorophyll fluorescence hyperspectral images detected changes before symptom appearance at low-density inoculation. The results of this study indicate that the P. cichorii infection severity can be detected by chlorophyll fluorescence assay and hyperspectral images prior to the onset of visible symptoms, indicating the feasibility of early detection of diseases. However, to detect disease development by hyperspectral imaging, more detailed protocols and analyses are necessary. Taken together, change in chlorophyll fluorescence is a good parameter for early detection of P. cichorii infection in tomato plants. In addition, image-based visualization of infection severity before visual damage appearance will contribute to effective management of plant diseases.

• 셀메드
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• 제3권1호
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• pp.9.1-9.10
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• 2013

In traditional systems of medicine including homeopathy, the Condurango extract (Con) is often used to cure stomach cancer mainly, without having any scientific validation of its anti-cancer ability. Con has therefore been tested against non-small-cell lung cancer cells (NSCLC) A549 and NCI-H522 (H522) known to contain the KRAS mutation, making them resistant to most chemotherapeutic agents. As cancer cells generally defy cytotoxicity developed by chemopreventive agents and escape cell death, any drug showing the capability of preferentially killing cancer cells through apoptosis is worth consideration for judicious application. A549 and H522 cells were exposed to $0.35{\mu}g/{\mu}l$ and $0.25{\mu}g/{\mu}l$ of Con, respectively, for 48 h and analysed based on various protocols associated with apoptosis and DNA damage, such as MTT assay to determine cell viability, LDH assay, DNA fragmentation assay, comet assay, and microscopical examinations of DNA binding fluorescence stains like DAPI, Hoechst 33258 and acridine orange/ethidium bromide to determine the extent of DNA damage made in drug-treated and untreated cells and the results compared. Changes in mitochondrial membrane potential and the generation of reactive oxygen species were also documented through standard techniques. Con killed almost 50% of the cancer cells but spared normal cells significantly. Fluorescence studies revealed increased DNA nick formation and depolarized membrane potentials after drug treatment in both cell types. Caspase-3 expression levels confirmed the apoptosis-inducing potential of Con in both the NSCLC lines. Thus, overall results suggest considerable anticancer potential of Con against NSCLC in vitro, validating its use against lung cancer by practitioners of traditional medicine including homeopathy.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.1922-1929
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• 2007

A simple whole-cell-based sensing system is proposed for determining the cell mass of H. pluvialis using ultraviolet fluorescence spectroscopy. An emission signal at 368 nm was used to detect the various kinds of green, green-brown, brown-red, and red H. pluvialis cells. The fluorescence emission intensities of the cells were highest at 368 nm with an excitation wavelength of 227 nm. An excitation wavelength of 227 nm was then selected for cell-mass sensing, as the emission fluorescence intensities of the cell suspensions were highest at this wavelength after subtracting the background interference. The emission fluorescence intensities of HPLC-grade water, filtered water, and HPLC-grade water containing a modified Bold's basal medium (MBBM) were measured and the difference was less than 1.6 for the selected wavelengths. Moreover, there was no difference in the emission intensity at 368 nm among suspensions of the various morphological states of the cells. A calibration curve of the fluorescence emission intensities. and cell mass was obtained with a high correlation ($R^2=0.9938$) for the various morphological forms of H. pluvialis. Accordingly, the proposed method showed no significant dependency on the various morphological cell forms, making it applicable for cell-mass measurement. A high correlation was found between the fluorescence emission intensities and the dry cell weight with a mixture of green, green-brown, brown-red, and red cells. In conclusion, the proposed model can be directly used for cell-mass sensing without any pretreatment and has potential use as a noninvasive method for the online determination of algal biomass.

• Food Science and Biotechnology
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• 제16권3호
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• pp.337-342
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• 2007

Conventional methods for pathogen detection and identification are labor-intensive and take days to complete. Biosensors have shown great potential for the rapid detection of foodborne pathogens. Fiber-optic biosensors have been used to rapidly detect pathogens because they can be very sensitive and are simple to operate. However, many fiber-optic biosensors rely on manual sensor handling and the sandwich assay, which require more effort and are less sensitive. To increase the simplicity of operation and detection sensitivity, a binding inhibition assay method for detecting Listeria monocytogenes in food samples was developed using an automated, fiber-optic-based immunosensor: RAPTOR (Research International, Monroe, WA, USA). For the assay, fiber-optic biosensors were developed by the immobilization of Listeria antibodies on polystyrene fiber waveguides through a biotin-avidin reaction. Developed fiber-optic biosensors were incorporated into the RAPTOR to evaluate the detection of L. monocytogenes in frankfurter samples. The binding inhibition method combined with RAPTOR was sensitive enough to detect L. monocytogenes ($5.4{\times}10^7\;CFU/mL$) in a frankfurter sample.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.2007-2010
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• 2015

A new chemical inhibitor against severe acute respiratory syndrome (SARS) coronavirus helicase, 7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1H-purine-2,6-dione, was identified. We investigated the inhibitory effect of the compound by conducting colorimetry-based ATP hydrolysis assay and fluorescence resonance energy transfer-based double-stranded DNA unwinding assay. The compound suppressed both ATP hydrolysis and double-stranded DNA unwinding activities of helicase with IC₅₀ values of 8.66 ± 0.26 μM and 41.6 ± 2.3 μM, respectively. Moreover, we observed that the compound did not show cytotoxicity up to 80 μM concentration. Our results suggest that the compound might serve as a SARS coronavirus inhibitor.

• 한국가축번식학회지
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• 제16권4호
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• pp.325-333
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• 1993

A competitive type immunoassay method for 17$\beta$-estradiol(E2) based on the idiotypic anti-idiotypic antibody and time-resolved fluorescence is described. The anti-idiotypic antibody(Ab2) produced to E2 binding site of the primary idiotype antibody (Ab1) was labelled with europium and was allowed to compete with E2 standards or serum sample for the binding sites of Ab1 which was bound to 2nd antibody captured ontothe surface of microtitre plates. Fluorescence measured by time-resolved fluorometer was inversely proportional to the concentration of E2 over the range 5~500pg/well. The sensitivity of the assay was 5pg per well which was compatible with that ofradioimmunoassay using the same Ab1 and 3H-E2 as a tracer. One great advantage of this method described here was to enable antibodies to be labelled instead of haptens, and thus makes it easier to develop sensitive and robust immunoassay systems specially for haptens.

• 대한화학회지
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• 제64권3호
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• pp.145-152
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• 2020

Flip-flop rate constants were measured by dithionite assay of NBD-PE fluorescence in DMPC/POPC vesicles made of various DMPC/POPC ratios. The activation energy, enthalpy, entropy, and free energy were determined based on the transition state theory. We found that the activation energy, enthalpy, and entropy increased as the amount of POPC increased, but the activation free energy was almost constant. These experimental results and other similar studies allow us to propose that the POPC molecules included in DMPC vesicles affect the flip-flop motion of NBD-PE in DMPC/POPC vesicles via increasing the packing order of the ground state of the bilayer of the vesicles. The increase in the packing order in the ground state seems to be a result of the effect of the overall molecular shape of POPC with a monounsaturated tail group, rather than the effect of the longer tail group.

• 식물병연구
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• 제29권1호
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• pp.11-22
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• 2023

Resazurin 기반 호흡 측정법으로 strobilurin계 살균제에 대해서 감수성인 고추탄저병균 Colletotrichum acutatum s. lat. 20JDS8에 대한 살균제의 호흡 억제 효과를 조사하였다. Potato dextrose broth에 병원균의 포자를 접종하고 배양하면서 시간별로 상대 형광값(relative fluorescence unit)을 조사한 결과, 12시간 후부터 상승하기 시작하여 24시간 후의 1×10⁴, 1×10⁵, 1×10⁶ spores/ml의 포자 접종구에서 상대 형광값은 1,965.5, 5,412.5, 10,061.0이었다. 병원균을 0, 6, 12, 24시간씩 배양 후에 공시한 살균제를 처리하고, 24시간 후에 상대 형광값을 조사하였다. Dithianon, isopyrazam, pyraclostrobin, fluazinam을 병원균의 포자(0시간), 포자 발아(6시간), 균사 생장(12시간) 단계에 처리할 경우, 각 살균제의 고농도에서 호흡을 90-100% 억제하였다. 하지만 병원균을 24시간 배양한 후에 살균제를 처리할 경우에는 호흡 억제 효과가 크게 감소하였다. Pyraclostrobin 저항성인 C. acutatum s. lat. 20CDJ6에 대해서 pyraclostrobin, azxoystrobin, trifloxystrobin, kresoxim-methyl을 병원균의 모든 생장 단계에 각각 처리하였을 때, 호흡에 대한 억제 효과는 매우 미미하거나, 나타나지 않았다.

• Journal of Veterinary Science
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• 제21권4호
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• pp.68.1-68.8
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• 2020

A fluorescent microsphere-based immunochromatographic strip test (FICT) was developed for the rapid, sensitive, and quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) antibodies at the pen-side. The assay was based on the formation of a sandwich immune-complex (anti-pig IgG-PRRSV antibodies-NSP7/N), which was validated by a comparison with IDEXX-ELISA using 3325 clinical specimens. The diagnostic specificity, sensitivity, and accuracy of FICT were 97.28, 93.41, and 94.95%, respectively. FICT showed a good correlation with the virus neutralization assay. Overall, a promising pen-side diagnostic tool was developed for the rapid and quantitative detection of PRRSV antibodies within 15 min.