Park, Jong Woo;Yu, Jeong Hee;Kim, Seong-Wan;Kweon, Hae Yong;Choi, Kwang-Ho;Kim, Seong-Ryul
International Journal of Industrial Entomology and Biomaterials
/
v.37
no.1
/
pp.21-28
/
2018
CRISPR/Cas9 gene editing system is an efficient method to mutation in a sequence specific manner. Here we report the direct transfection of the Cas9 nuclease and gene specific guide RNA can be used in BM-N cell line derived from Bombyx mori ovarian tissue to enfeeble function of endogenous gene in vitro. We have used gene editing system to negative regulation components of major signaling cascade, the Toll pathway, which controls B. mori resistance to microbe infections, such as fungi and gram positive bacteria. We demonstrate that the $I{\kappa}B-like$ protein Cactus may controls the activation of transcription factors such as Rel A and Rel B. The direct transfection of Cas9 nuclease and Cactus-specific guide-RNA complex may be used in BM-N cells to disrupt the function of endogenous genes in vitro. A mutation frequency of 30-40% was observed in the transfected cells, and various mutations caused the target region. Moreover, RT-PCR analysis revealed that Cactus gene was down regulated after these mutations. More importantly, mutation of BmCactus stimulated expression of lysozyme, moricin, and lebocin genes. These results suggest that the CRISPR/Cas9 systems are expected to efficiently induce site-specific mutations and it was possible to produce antimicrobial peptide through the gene editing.
Phospholipase D (PLD) catalyzes the hydrolysis of phospholipid to phosphatidic acid (PA), a lipid secondary messenger. Two forms of PLD isozymes, phosphatidylcholine-specific PLD1 and PLD2, have been identified. PLD has emerged as a critical regulator of cell proliferation and survival signaling, and dysregulation of PLD occurs in a various illnesses, including cancer. PLD activity is essential for cell survival and protection from apoptosis. Overexpression of PLD isozymes or PLD-generated PA attenuates the expression of apoptotic genes and confers resistance to apoptosis. The apoptosis-related molecular mechanisms of PLD remain largely unknown. Recently, the dynamics of PLD turnover during apoptosis have been reported. The cleavage of PLD isozymes as specific substrates of caspase differentially regulates apoptosis. PLD1 is cleaved at one internal site, and PLD2 is cleaved two sites at the front of the N-terminus. The cleavage of PLD1 reduces its enzymatic activity, probably via the dissociation of two catalytic motifs, whereas the cleavage of PLD2 does not affect the catalytic motifs and its activity. Thus, PLD2 maintains antiapoptotic capacity, despite its cleavage. Therefore, the differential cleavage pattern of PLD isozymes by caspase affects its enzymatic activity and antiapoptotic function. Thus, PLD is considered a potential target for cancer therapy. We summarize recent studies regarding the functional role of PLD in apoptosis.
Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant immune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVR-Pii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen.
A suspected biotype of Scirpus planiculmis to be resistant to sulfonylurea(SU) herbicides was identified in Seosan reclaimed paddy fields in Korea, in 2004. The fields have been cultivated for monocultural rice production with wet-direct seeding method and continuously treated with SU-based herbicide mixtures for thirteen years since 1990. In greenhouse studies, 6 different SU herbicides, such as azimsulfuron, bensulfuron-methyl, cinosulfuron, ethoxysulfuron imazosulfuron and pyrazosulfuron-ethyl, completely controlled the Musan assession of Scirpus planiculmis at the recommended dose of each herbicide, however, the Seosan accession of S. planiculmis biotype was survived 20 to 45% even treated with 5 times higher dose of each recommended rate of all herbicides treated. The $GR_{50}$ values of 6 SU herbicides for Seosan accession of S. planiculmis were 47 to 100 times higher than those for Musan accession of S. planiculmis. The $I_{50}$ values pyrazosulfuron-ethyl to acetolactate synthase(ALS) extracted from Sesan and Muan accession of S. planiculmis were 409 nM and 0.8 nM, respectively. The $I_{50}$ value of Seosan was 511 times higher than that of Muan accession. These results suggested that the Seosan accession of S. planiculmis have strong resistant characteristics to 6 SU herbicides, respectively, indicating that resistance might be due to the alteration in the target site of ALS.
Journal of the Korean Society of Environmental Restoration Technology
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v.25
no.1
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pp.25-38
/
2022
Urban problems are constantly occurring around the world due to rapid industrialization and population decline. In particular, as the number of vacant houses is gradually increasing as the population decreases, it is necessary to prepare countermeasures. A plan to utilize vacant houses has emerged to restore the natural environment of the urban ecosystem where forest destruction, damage to habitats of wild animals and plants, and disconnection have occurred due to large-scale development. Through connectivity analysis, it is possible to understand the overall ecosystem flow based on the movement of species and predict the effect when vacant houses are converted into green spaces. Therefore, this study analyzed the green area network to confirm the possibility of greening of vacant houses neglected in Jeonju based on circuit theory. Using Circuitscape and Least-cost path, we tried to identify the connectivity of green areas and propose an ecological axis based on the analysis. In order to apply the resistance values required for analysis based on previous studies, the 2020 subdivision land cover data were integrated into the major classification evaluation items. When the eight forests in the target site were analyzed as the standard, the overall connectivity and connectivity between forests in the area were high, so it is judged that the existing green areas can perform various functions, such as species movement and provision of habitats. Based on the results of the connectivity analysis, the importance of vacant houses was calculated and the top 20 vacant houses were identified, and it was confirmed that the higher the ranking, the more positive the degree of landscape connectivity was when converted to green areas. In addition, it was confirmed that the results of analyzing the least-cost path based on the resistance values such as connectivity analysis and the existing conceptual map showed some differences when comparing the ecological axes in the form. As a result of checking the vacant houses corresponding to the relevant axis based on the width standards of the main and sub-green areas, a total of 30 vacant houses were included in the 200m width and 6 vacant houses in the 80m width. It is judged that the conversion of vacant houses to green space can contribute to biodiversity conservation as well as connectivity between habitats of species as it is coupled with improved green space connectivity. In addition, it is expected to help solve the problem of vacant houses in the future by showing the possibility of using vacant houses.
Kim, Jin-Seog;Lee, Byung-Hoi;Kim, So-Hee;Min, Suk-Ki;Choi, Jung-Sup
Journal of Plant Biotechnology
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v.33
no.1
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pp.57-62
/
2006
Several methods for determining the response of corn to glyphosate were investigated to provide a fast and reliable method for identifying glyphosate-resistant corn in vivo. Two bioassays were developed. One assay is named 'whole plant / leaf growth assay', in which the herbicide glyphosate is applied on the upper part of 3rd leaf and the growth of herbicide-untreated 4th leaf is measured at 3 day after treatment. in this assay, the leaf growth of conventional corn was inhibited in a dose dependent from 50 to $1600{\mu}g/mL$ of glyphosate and growth inhibition at $1600{\mu}g/mL$ was 55% of untreated control. The assay has the potential to be used especially in the case that the primary cause of glyphosate resistance is related with a reduction of the herbicide translocation. Another assay is named 'leaf segment / shikimate accumulation assay', in which the four excised leaf segments ($4{\times}4mm$) are placed in each well of a 48-well microtiter plate containing $200{\mu}L$ test solution and the amount of shikimate is determined after incubation for 24 h in continuous light at $25^{\circ}C$. In this assay, 0.33% sucrose added to basic test solution enhanced a shikimate accumulation by 3 to 4 times and the shikimate accumulation was linearly occurred from 2 to $8{\mu}g/mL$ of glyphosate, showing an improved response to the method described by Shaner et al. (2005). The leaf segment / shikimate accumulation assay is simple and robust and has the potential to be used as a high throughput assay in the case that the primary cause of glyphosate resistance is related with EPSPS, target site of the herbicide. Taken together, these two assays would be highly useful to initially select the lines obtained after transformation, to investigate the migration of glyphosate-resistant gene into other weeds and to detect a weedy glyphosate-resistant corn in field.
There was a great variation in insecticide susceptibilities among field and laboratory populations of the beet armyworm, Spodoptera exigua (Hiibner). Unselected laboratory population, which had been reared for 6-7 generations in our laboratory without exposure to insecticides, was more susceptible than its parental field population in all tested insecticides. Two selected laboratory populations with parathion or deltamethrin showed much higher insecticide tolerance than did the unselected laboratory population in their own selection insecticide. The variation of the insecticide susceptibilities was highly correlated with esterase and acetylcholinesterase activities. Field and the selected laboratory populations had lower acetylcholinesterase activities and higher esterase activities than did the unselected laboratory population. Acetylcholinesterase of the field and the selected laboratory populations had higher Km values than did that of the unselected. In a population, Km values were varied among different developmental stages; acetylcholinesterase of the fifth instar larvae had the highest Km value among those of the other larval stages. Twenty one esterase bands were separated on 6.5% nondenaturing polyacrylamide gel from the whole body extracts of the fifth instar larvae. E2, E7, E8, Ell, El6, and El7 esterase bands were developed more frequently in the insecticides-selected populations than in the unselected population. These results suggest that the variation of insecticide susceptibilities of the beet armyworm includes both biochemical mechanisms: target site insensitivity and enhanced activity of detoxification enzyme.
Ahn, Myeonghui;Jang, Eun-kyung;Bae, Inhyeok;Ji, Un
KSCE Journal of Civil and Environmental Engineering Research
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v.40
no.6
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pp.571-581
/
2020
Vegetation affects water level change and flow resistance in rivers and impacts waterway ecosystems as a whole. Therefore, it is important to have accurate information about the species, shape, and size of any river vegetation. However, it is not easy to collect full vegetation data on-site, so recent studies have attempted to obtain large amounts of vegetation data using terrestrial laser scanning (TLS). Also, due to the complex shape of vegetation, it is not easy to obtain accurate information about the canopy area, and there are limitations due to a complex range of variables. Therefore, the physical structure of vegetation was analyzed in this study by reconfiguring high-resolution point cloud data collected through 3-dimensional terrestrial laser scanning (3D TLS) in a voxel. Each physical structure was analyzed under three different conditions: a simple vegetation formation without leaves, a complete formation with leaves, and a patch-scale vegetation formation. In the raw data, the outlier and unnecessary data were filtered and removed by Statistical Outlier Removal (SOR), resulting in 17%, 26%, and 25% of data being removed, respectively. Also, vegetation volume by voxel size was reconfigured from post-processed point clouds and compared with vegetation volume; the analysis showed that the margin of error was 8%, 25%, and 63% for each condition, respectively. The larger the size of the target sample, the larger the error. The vegetation surface looked visually similar when resizing the voxel; however, the volume of the entire vegetation was susceptible to error.
Park, Chul-Soo;Mok, Young-Jin;Choi, Chan-Yong;Lee, Tai-Hee
Journal of the Korean Geotechnical Society
/
v.25
no.9
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pp.45-55
/
2009
The quality of railroad trackbed fills has been controlled by field measurements of density and bearing resistance of plate-load tests. The control measures are compatible with the design procedures whose design parameter is $k_{30}$ for both ordinary-speed railways and high-speed railways. However, one of fatal flaws of the design procedures is that there are no simple laboratory measurement procedures for the design parameters ($k_{30}$ or, $E_{v2}$ and $E_{v2}/E_{v1}$) in design stage. To overcome the defect, the compressional wave velocity was adopted as a control measure, in parallel with the advent of the new design procedure, and its measurement technique was proposed in the preliminary investigation. The key concept of the quality control procedure is that the target value for field compaction control is the compressional wave velocity determined at optimum moisture content using modified compaction test, and direct-arrival method is used for the field measurements during construction, which is simple and reliable enough for practice engineers to access. This direct-arrival method is well-suited for such a shallow and homogeneous fill lift in terms of applicability and cost effectiveness. The sensitivity of direct-arrival test results according to the compaction quality was demonstrated at a test site, and it was concluded that compressional wave velocity can be effectively used as quality control measure. The experimental background far the companion study (Park et al., 2009) was established through field and laboratory measurements of the compressional wave velocity.
Proceedings of the Korean Vacuum Society Conference
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2013.08a
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pp.88-89
/
2013
A variety of influenza A viruses from animal hosts are continuously prevalent throughout the world which cause human epidemics resulting millions of human infections and enormous industrial and economic damages. Thus, early diagnosis of such pathogen is of paramount importance for biomedical examination and public healthcare screening. To approach this issue, here we propose a fully integrated Rotary genetic analysis system, called Rotary Genetic Analyzer, for on-site detection of influenza A viruses with high speed. The Rotary Genetic Analyzer is made up of four parts including a disposable microchip, a servo motor for precise and high rate spinning of the chip, thermal blocks for temperature control, and a miniaturized optical fluorescence detector as shown Fig. 1. A thermal block made from duralumin is integrated with a film heater at the bottom and a resistance temperature detector (RTD) in the middle. For the efficient performance of RT-PCR, three thermal blocks are placed on the Rotary stage and the temperature of each block is corresponded to the thermal cycling, namely $95^{\circ}C$ (denature), $58^{\circ}C$ (annealing), and $72^{\circ}C$ (extension). Rotary RT-PCR was performed to amplify the target gene which was monitored by an optical fluorescent detector above the extension block. A disposable microdevice (10 cm diameter) consists of a solid-phase extraction based sample pretreatment unit, bead chamber, and 4 ${\mu}L$ of the PCR chamber as shown Fig. 2. The microchip is fabricated using a patterned polycarbonate (PC) sheet with 1 mm thickness and a PC film with 130 ${\mu}m$ thickness, which layers are thermally bonded at $138^{\circ}C$ using acetone vapour. Silicatreated microglass beads with 150~212 ${\mu}L$ diameter are introduced into the sample pretreatment chambers and held in place by weir structure for construction of solid-phase extraction system. Fig. 3 shows strobed images of sequential loading of three samples. Three samples were loaded into the reservoir simultaneously (Fig. 3A), then the influenza A H3N2 viral RNA sample was loaded at 5000 RPM for 10 sec (Fig. 3B). Washing buffer was followed at 5000 RPM for 5 min (Fig. 3C), and angular frequency was decreased to 100 RPM for siphon priming of PCR cocktail to the channel as shown in Figure 3D. Finally the PCR cocktail was loaded to the bead chamber at 2000 RPM for 10 sec, and then RPM was increased up to 5000 RPM for 1 min to obtain the as much as PCR cocktail containing the RNA template (Fig. 3E). In this system, the wastes from RNA samples and washing buffer were transported to the waste chamber, which is fully filled to the chamber with precise optimization. Then, the PCR cocktail was able to transport to the PCR chamber. Fig. 3F shows the final image of the sample pretreatment. PCR cocktail containing RNA template is successfully isolated from waste. To detect the influenza A H3N2 virus, the purified RNA with PCR cocktail in the PCR chamber was amplified by using performed the RNA capture on the proposed microdevice. The fluorescence images were described in Figure 4A at the 0, 40 cycles. The fluorescence signal (40 cycle) was drastically increased confirming the influenza A H3N2 virus. The real-time profiles were successfully obtained using the optical fluorescence detector as shown in Figure 4B. The Rotary PCR and off-chip PCR were compared with same amount of influenza A H3N2 virus. The Ct value of Rotary PCR was smaller than the off-chip PCR without contamination. The whole process of the sample pretreatment and RT-PCR could be accomplished in 30 min on the fully integrated Rotary Genetic Analyzer system. We have demonstrated a fully integrated and portable Rotary Genetic Analyzer for detection of the gene expression of influenza A virus, which has 'Sample-in-answer-out' capability including sample pretreatment, rotary amplification, and optical detection. Target gene amplification was real-time monitored using the integrated Rotary Genetic Analyzer system.
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