• 한국균학회소식:학술대회논문집
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• 2015.11a
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• pp.23-23
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• 2015

Rice blast disease caused by M. oryzae is the most devastating fungal disease in rice. During the infection process, M. oryzae secretes a large number of glycosyl hydrolase (GH) proteins into the apoplast to digest host cell wall and assist fungal ingress into host tissues. In this study, we identified a novel M. oryze arabinofuranosidase B (MoAbfB) which is secreted during fungal infection. Live-cell imaging exhibited that fluorescent labeled MoAbfB was highly accumulated in fungal invasive structures such as appressorium, tips of penetration peg, biotrophic interfacial complex (BIC), as well as invasive hyphal tip. Deletion of MoAbfB mutants extended biotrophic phase followed by enhanced disease severity, whereas, over-expression of OsMoAbfB mutant induced rapid defense responses and enhanced rice resistance to M. oryzae infection. Furthermore, exogenous treatment of MoAbfB protein showed inhibition of fungal infection via priming of defense gene expression. We later found that the extract of MoAbfB degraded rice cell wall fragments could also induce host defense activation, suggesting that not MoAbfB itself but oligosaccharides (OGs) derived from MoAbfB dissolved rice cell wall elicited rice innate immunity.

• Bulletin of the Korean Chemical Society
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• v.31 no.6
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• pp.1474-1478
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• 2010

An efficient method for labeling individual proteins in live cells is required for investigations into biological mechanisms and cellular processes. Here we describe the preparation of small quantum dots (QDs) that target membrane surface proteins bearing a hexahistidine-tag ($His_6$-tag) via specific binding to an nitrilotriacetic acid complex of nickel(II) ($NTA{\cdot}Ni^{2+}$) on the QD surfaces. We showed that the $NTA{\cdot}Ni^{2+}$-QDs bound to His-tag functionalized beads as a cellular mimic with high specificity and that QDs successfully targeted $His_6$-tagged vesicle-associated membrane proteins (VMAP) on cell surfaces. This strategy provides an efficient approach to monitoring synaptic protein dynamics in spatially restricted and confined biological environments.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.526-526
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• 2013

Plasma technology isbeing developed for a range of medical applications including wound healing. However, the effect of plasma on many cells and tissues is unclear. Cell migration and cell proliferation are very important biological processes which are affected by plasma exposure and might be a potential target for plasma therapy during wound healing treatment. In this study, we confirmed the plasma exposure time and incubation time after plasma treatment in skin fibroblast (L-929 cells) to evaluate the optimal conditions forplasma exposure to the cell in-vitro. In addition, we used a scratch method to generate artificial wound for evaluating the cell migration by plasma treatment. Where, the cells were treated with plasma and migration rate was observed by live-cell imaging device. To find the cell proliferation, cell viability assay was executed. The results of this study indicate the increased cell proliferation and migration on mild plasma treatment. The mechanisms for cell migration and cell proliferation after plasma treatment for future studies will be discussed.

• Journal of the Korean Society of Visualization
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• v.5 no.1
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• pp.9-11
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• 2007

We introduce an in vivo imaging flow cytometer, which provides fluorescence images simultaneously with quantitative information on the cell population of interest in a live animal. As fluorescent cells pass through the slit of light focused across a blood vessel, the excited fluorescence is confocally detected. This cell signal triggers a strobe beam and a high sensitivity CCD camera that captures a snap-shot image of the cell as it moves down-stream from the slit. We demonstrate that the majority of signal peaks detected in the in vivo flow cytometer arise from individual cells. The instrument's capability to image circulating T cells and measure their speed in the blood vessel in real time in vivo is demonstrated. The cell signal irradiance variation, clustering percentage, and potential applications in biology and medicine are discussed.

• Molecules and Cells
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• v.44 no.9
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• pp.627-636
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• 2021

The three-dimensional organization of chromatin and its time-dependent changes greatly affect virtually every cellular function, especially DNA replication, genome maintenance, transcription regulation, and cell differentiation. Sequencing-based techniques such as ChIP-seq, ATAC-seq, and Hi-C provide abundant information on how genomic elements are coupled with regulatory proteins and functionally organized into hierarchical domains through their interactions. However, visualizing the time-dependent changes of such organization in individual cells remains challenging. Recent developments of CRISPR systems for site-specific fluorescent labeling of genomic loci have provided promising strategies for visualizing chromatin dynamics in live cells. However, there are several limiting factors, including background signals, off-target binding of CRISPR, and rapid photobleaching of the fluorophores, requiring a large number of target-bound CRISPR complexes to reliably distinguish the target-specific foci from the background. Various modifications have been engineered into the CRISPR system to enhance the signal-to-background ratio and signal longevity to detect target foci more reliably and efficiently, and to reduce the required target size. In this review, we comprehensively compare the performances of recently developed CRISPR designs for improved visualization of genomic loci in terms of the reliability of target detection, the ability to detect small repeat loci, and the allowed time of live tracking. Longer observation of genomic loci allows the detailed identification of the dynamic characteristics of chromatin. The diffusion properties of chromatin found in recent studies are reviewed, which provide suggestions for the underlying biological processes.

• Molecules and Cells
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• v.42 no.4
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• pp.356-362
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• 2019

The binding of MS2 bacteriophage coat protein (MCP) to MS2 binding site (MBS) RNA stem-loop sequences has been widely used to label mRNA for live-cell imaging at single-molecule resolution. However, concerns have been raised recently from studies with budding yeast showing aberrant mRNA metabolism following the MS2-GFP labeling. To investigate the degradation pattern of MS2-GFP-labeled mRNA in mammalian cells and tissues, we used Northern blot analysis of ${\beta}$-actin mRNA extracted from the Actb-MBS knock-in and $MBS{\times}MCP$ hybrid mouse models. In the immortalized mouse embryonic cell lines and various organ tissues derived from the mouse models, we found no noticeable accumulation of decay products of ${\beta}$-actin mRNA compared with the wild-type mice. Our results suggest that accumulation of MBS RNA decay fragments does not always happen depending on the mRNA species and the model organisms used.

• Journal of Microbiology and Biotechnology
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• v.26 no.4
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• pp.790-798
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• 2016

Chlamydiae, obligate intracellular bacteria, are associated with a variety of human diseases. The chlamydial life cycle undergoes a biphasic development: replicative reticulate bodies (RBs) phase and infectious elementary bodies (EBs) phase. At the end of the chlamydial intracellular life cycle, EBs have to be released to the surrounded cells. Therefore, the interactions between Chlamydiae and cell death pathways could greatly influence the outcomes of Chlamydia infection. However, the underlying molecular mechanisms remain elusive. Here, we investigated host cell death after Chlamydia infection in vitro, in L929 cells, and showed that Chlamydia infection induces cell necrosis, as detected by the propidium iodide (PI)-Annexin V double-staining flow-cytometric assay and Lactate dehydrogenase (LDH) release assay. The production of reactive oxygen species (ROS), an important factor in induction of necrosis, was increased after Chlamydia infection, and inhibition of ROS with specific pharmacological inhibitors, diphenylene iodonium (DPI) or butylated hydroxyanisole (BHA), led to significant suppression of necrosis. Interestingly, live-cell imaging revealed that Chlamydia infection induced lysosome membrane permeabilization (LMP). When an inhibitor upstream of LMP, CA-074-Me, was added to cells, the production of ROS was reduced with concomitant inhibition of necrosis. Taken together, our results indicate that Chlamydia infection elicits the production of ROS, which is dependent on LMP at least partially, followed by induction of host-cell necrosis. To our best knowledge, this is the first live-cell-imaging observation of LMP post Chlamydia infection and report on the link of LMP to ROS to necrosis during Chlamydia infection.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.277.2-277.2
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• 2013

Petri dishes and glass slides have been widely used as general substrates for in vitro mammalian cell cultures due to their culture viability, optical transparency, experimental convenience, and relatively low cost. Despite the aforementioned benefit, however, the flat two-dimensional substrates exhibit limited capability in terms of realistically mimicking cellular polarization, intercellular interaction, and differentiation in the non-physiological culture environment. Here, we report a protocol of culturing embryonic rat hippocampal neurons on the electro-spun polymeric network and the results from examination of neuronal cell behavior and network formation on this culture platform. A combinatorial method of laser-scanning confocal fluorescence microscopy and live-cell imaging technique was employed to track axonal outgrowth and synaptic connectivity of the neuronal cells deposited on this model culture environment. The present microfiber-based scaffold supports the prolonged viability of three-dimensionally-formed neuronal networks and their microscopic geometric parameters (i.e., microfiber diameter) strongly influence the axonal outgrowth and synaptic connection pattern. These results implies that electro-spun fiber scaffolds with fine control over surface chemistry and nano/microscopic geometry may be used as an economic and general platform for three-dimensional mammalian culture systems, particularly, neuronal lineage and other network forming cell lines.

• Biomedical Science Letters
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• v.18 no.3
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• pp.218-226
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• 2012

Dopamine is an enteric neurotransmitter that regulates gastrointestinal motility. This study was done to investigate whether dopamine modulates spontaneous pacemaker activity in cultured interstitial cells of Cajal (ICCs) from mouse using whole cell patch clamp technique, RT-PCR and live $Ca^{2+}$ imaging analysis. ICCs generate pacemaker inward currents at a holding potential of -70 mV and generate pacemaker potentials in current-clamp mode. Dopamine did not change the frequency and amplitude of pacemaker activity in small intestinal ICCs. On the contrary dopamine reduced the frequency and amplitude of pacemaker activity in large intestinal ICCs. RT-PCR analysis revealed that Dopamine2 and 4-receptors are expressed in c-Kit positive ICCs. Dopamine2 and 4 receptor agonists inhibited pacemaker activity in large intestinal ICCs mimicked those of dopamine. Domperidone, dopamine2 receptor antagonist, increased the frequency of pacemaker activity of large intestinal ICCs. In $Ca^{2+}$-imaging, dopamine inhibited spontaneous intracellular $Ca^{2+}$ oscillations of ICCs. These results suggest that dopamine can regulate gastrointestinal motility through modulating pacemaker activity of large intestinal ICCs and dopamine effects on ICCs are mediated by dopamine2 receptor and intracellular $Ca^{2+}$ modulation.

• Journal of the Korea Institute of Information and Communication Engineering
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• v.18 no.2
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• pp.431-436
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• 2014

Cell segmentation which extracts cell objects from background is one of basic works in bio-imaging which analyze cell images acquired from live cells in cell culture. In the case of clear images, they have a bi-modal histogram distribution and segmentation of them can easily be performed by global threshold algorithm such as Otsu algorithm. But In the case of degraded images, it is difficult to get exact segmentation results. In this paper, we developed a cell segmentation system that it classify input images by the type of their histogram distribution and then apply a proper segmentation algorithm. If it has a bi-modal distribution, a global threshold algorithm is applied for segmentation. Otherwise it has a uni-modal distribution, our algorithm is performed. By experimentation, our system gave exact segmentation results for uni-modal cell images as well as bi-modal cell images.