• Journal of Microbiology and Biotechnology
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• v.30 no.12
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• pp.1919-1926
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• 2020

CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence has been constructed by removing the dependence on the last base G of PAM through protein engineering of Cas9. In this study, a dCas9-NG protein was engineered by introducing two active site mutations in Cas9-NG, and its ability to regulate transcription was evaluated in the gal promoter in E. coli. Analysis of cell growth rate, D-galactose consumption rate, and gal transcripts confirmed that dCas9-NG can completely repress the promoter by recognizing DNA targets with PAM of 5'-NGG, NGA, NGC, NGT, and NAG. Our study showed possible PAM sequences for dCas9-NG and provided information on target-specific sgRNA design for regulation of both gene expression and cellular metabolism.

• BMB Reports
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• v.56 no.11
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• pp.606-611
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• 2023

The main protease (Mpro) of SARS-CoV-2 cleaves 11 sites of viral polypeptide chains and generates essential non-structural proteins for viral replication. Mpro is an important drug target against COVID-19. In this study, we developed a real-time fluorometric turn-on assay system to evaluate Mpro proteolytic activity for a substrate peptide between NSP4 and NSP5. It produced reproducible and reliable results suitable for HTS inhibitor assays. Thus far, most inhibitors against Mpro target the active site for substrate binding. Mpro exists as a dimer, which is essential for its activity. We investigated the potential of the Mpro dimer interface to act as a drug target. The dimer interface is formed of domain II and domain III of each protomer, in which N-terminal ten amino acids of the domain I are bound in the middle as a sandwich. The N-terminal part provides approximately 39% of the dimer interface between two protomers. In the real-time fluorometric turn-on assay system, peptides of the N-terminal ten amino acids, N10, can inhibit the Mpro activity. The dimer interface could be a prospective drug target against Mpro. The N-terminal sequence can help develop a potential inhibitor.

• Journal of the Korean Society of Environmental Restoration Technology
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• v.20 no.3
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• pp.19-31
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• 2017

Plants are the basis for sustainable green growth, and the value of existence and importance of trees including landscape Plants can't be emphasized enough. Therefore, they are precious living things thriving in all sorts of public services, and continuous civil complaints for justifiable compensation of landscape Plants are filed. First, the standard formula of planting intervals according to production target specifications is calculated using root-collar caliper and diameter at breast height, and apply (1) standard medium sized trees which have not yet reached commercialization [deciduous tree production goal (R(B) less than 6cm]= (target standard)= [target standard $R(cm){\times}15{\times}0.7$]. (2) In case of commercialization(R6~R10)= [target standard $R(cm){\times}15{\pm}5%$], (3) In case of more than R12= [target standard $R(cm){\times}15{\times}130%$] shall be applied. In case of using diameter at breast height (4) In case of commercialization(B6~B10)= [target standard $B(cm){\times}20{\times}15{\pm}5%$], (5) In case of more than B12= [target standard $B(cm){\times}20{\times}130%$] shall be applied. Second, appraisal methods based on tree classification of compensation for loss are classified according to planted locations. (1) landscape trees within a house=[price of arrival at the site+planting cost], (2) landscape trees in places such as arboretum=[management technology of tress + relocation expenses considering scarcity of the trees (3) landscape trees in a place of loads= [landscape tree production cost + work out added price. In case of producted landscape threes (4) landscape trees ready to be commercialized as sales loss.

• Journal of the Korean Society of Environmental Restoration Technology
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• v.14 no.5
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• pp.113-125
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• 2011

Since the government of Korea (Ministry of Environment, MOE) introduced the policy applying 'Biotope-Area-Ratio-Indicator (BARI)' to huge residential land developments which Environmental Impact Assessment (EIA) should be performed, MOE has come to have the necessity to apply the indicator concretely at the stage of Prior Environment Review System (PERS) and EIA in various types of large scale land development projects. This study was conducted with the aim of supporting the application of BARI and related decision making in various other types of EIA projects as well as residential development projects through remodeling the system to apply the indicator of the past. Through the analysis of the problems in applying the past BARI and experimental appraisals to 11 types of EIA projects, the results and implications as follows were drawn. First, it's possible to extend the range of applications of BARI, which has been applied to only residential land development project, to all kinds of projects with area-typed land use pattern out of environmental impact assessment target projects. Second, it's also possible to set a target value into which regional characteristics and differences among locational properties are reflected. In addition, it's come to be able to differentially apply the target value of BARI according to the condition of the existing site. Third, it's improved to be able to suggest a macroscopic target value at the stage of PERS and to set detailed target values in each detailed land use at the stage of EIA. The key point underlies inducing methodology to determine target values to secure more permeable land coverage ratio for detailed land use patterns at the stage of EIA by making it possible to calculate BARI of the present land cover condition of the EIA target projects.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10299-10306
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• 2015

The esophageal squamous cell carcinoma (ESCC) is thought to develop through a multi-stage process. Epigenetic gene silencing constitutes an alternative or complementary mechanism to mutational events in tumorigenesis. Posttranscriptional regulation of human leukocyte antigen class I (HLA-I) and antigen processing machinery (APM) proteins expression may be associated with novel epigenetic modifications in cancer development. In the present study, we determined the expression levels of HLA-I antigen and APM components by immunohistochemistry. Then by a bisulfite-sequencing PCR (BSP) approach, we identified target CpG islands methylated at the gene promoter region of APM family genes in a ESCC cell line (ECa109), and further quantitative analysis of CpG site specific methylation of these genes in cases of Kazakh primary ESCCs with corresponding non-cancerous esophageal tissues using the Sequenom MassARRAY platform. Here we showed that the development of ESCCs was accompanied by partial or total loss of protein expression of HLA-B, TAP2, LMP7, tapasin and ERp57. The results demonstrated that although no statistical significance was found of global target CpG fragment methylation level sof HLA-B, TAP2, tapasin and ERp57 genes between ESCC and corresponding non-cancerous esophageal tissues, there was significant differences in the methylation level of several single sites between the two groups. Of thesse only the global methylation level of LMP7 gene target fragments was statistically higher ($0.0517{\pm}0.0357$) in Kazakh esophageal cancer than in neighboring normal tissues ($0.0380{\pm}0.0214$, p<0.05). Our results suggest that multiple CpG sites, but not methylation of every site leads to down regulation or deletion of gene expression. Only some of them result in genetic transcription, and silencing of HLA-B, ERp57, and LMP7 expression through hypermethylation of the promoters or other mechanisms may contribute to mechanisms of tumor escape from immune surveillance in Kazakh esophageal carcinogenesis.

• Molecules and Cells
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• v.40 no.3
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• pp.211-221
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• 2017

Transforming growth factor ${\beta}1$ $(TGF{\beta}1)/Smad4$ signaling plays a pivotal role in maintenance of the dynamic balance between bone formation and resorption. The microRNA miR-155 has been reported to exert a significant role in the differentiation of macrophage and dendritic cells. The goal of this study was to determine whether miR-155 regulates osteoclast differentiation through $TGF{\beta}1/Smad4$ signaling. Here, we present that $TGF{\beta}1$ elevated miR-155 levels during osteoclast differentiation through the stimulation of M-CSF and RANKL. Additionally, we found that silencing Smad4 attenuated the upregulation of miR-155 induced by $TGF{\beta}1$. The results of luciferase reporter experiments and ChIP assays demonstrated that $TGF{\beta}1$ promoted the binding of Smad4 to the miR-155 promoter at a site located in 454 bp from the transcription start site in vivo, further verifying that miR-155 is a transcriptional target of the $TGF{\beta}1/Smad4$ pathway. Subsequently, TRAP staining and qRT-PCR analysis revealed that silencing Smad4 impaired the $TGF{\beta}1$-mediated inhibition on osteoclast differentiation. Finally, we found that miR-155 may target SOCS1 and MITF to suppress osteoclast differentiation. Taken together, we provide the first evidence that $TGF{\beta}1/Smad4$ signaling affects osteoclast differentiation by regulation of miR-155 expression and the use of miR-155 as a potential therapeutic target for osteoclast-related diseases shows great promise.

• Journal of Microbiology
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• v.41 no.4
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• pp.340-344
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• 2003

The group I intron from Tetrahymena thermophila has been demonstrated to employ splicing reactions with its substrate RNA in the trans configuration. Moreover, we have recently shown that the transsplicing group I ribozyme can replace HCV-specific transcripts with a new RNA that exerts anti-viral activity. In this study, we explored the potential use of RNA replacement for cancer treatment by developing trans-splicing group I ribozymes, which could replace tumor-associated RNAs with the RNA sequence attached to the 3' end of the ribozymes. Thymidine phosphorylase (TP) RNA was chosen as a target RNA because it is known as a valid cancer prognostic factor. By performing an RNA mapping strategy that is based on a trans-splicing ribozyme library, we first determined which regions of the TP RNA are accessible to ribozymes, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. Next, we assessed the ribozyme activities by comparing trans-splicing activities of several ribozymes that targeted different regions of the TP RNA. This assessment was performed to verify if the target site predicted to be accessible is truly the most accessible. The ribozyme that could target the most accessible site, identified by mapping studies, was the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme reacted with and altered the TP transcripts by transferring an intended 3' exon tag sequence onto the targeted TP RNA in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace TP RNAs in tumors with a new RNA harboring anti-cancer activity, which would revert the malignant phenotype.

• Journal of the Korean earth science society
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• v.38 no.2
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• pp.161-171
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• 2017

Observed ground motions are composed of three factors such as, seismic source, attenuation, and site amplification effect. Among them, the site amplification characteristics should be considered significantly when estimating seismic source and attenuation characteristics with more confidence. The site effect is also necessary when estimating not only seismic hazard in seismic design engineering but also rock mechanical properties. This study uses the method of H/V spectral ratio of observed ground motion between target site and reference site called a reference site method. In addition to using the vertical Fourier spectrum of the reference site, we try out the horizontal Fourier spectrum as a new method in this study. We analyze H/V spectral ratio of six ground motions respectively, observed at four sites close to Yedang Reservoir. We then compare site amplification effects at each site using 3 kinds of seismic energies including S waves, Coda waves energy, and background noise. The results suggest that each site showed similar site amplification patterns in S waves and Coda waves energy. However, the site amplification of background noise shows much different characteristics from those of S waves and Coda wave energy, which suggests that the background noises at each site have their own developing mechanism. Each station shows its own characteristics of specific resonance frequency and site amplification properties in low, high and specific resonance frequency ranges. Comparison of the method used in this study to the others that used different methods can provide us with more information about the dynamic amplification of a site characteristics and site classification.

• Journal of The Korean Digital Architecture Interior Association
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• v.11 no.3
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• pp.5-16
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• 2011

This study aims for improvement and rebuilding of the 'Green Building Certification Criteria Information System;GBCCIS'. To achieve this, improved information system has developed self-assessment program available on the web and to provide data about the target of Green Building Certification based on the 2010 revised criteria. The target building of Green Building Certification is Apartment, Office, Mixed-Use Building, School, property for sale, accommodation, and other facilities. Also, the information system about the Green building related technologies and techniques to take advantage of offer a variety of information was built. Green Building Certification Criteria web-site Established in this study has information and each menu and contents are linked to organic and high-utilization. Each menu is organized according to the used frequency and the group. Also this web-site is composed of information guide field, core service field, and other field. These improvement of Green Building Certification Criteria Information System can be a positive incentive measures for the activation of Green Building Certification Criteria easily by taking advantage of relevant information by users. And these will be possible to planning development of Green building technologies by introducing the latest Green building construction techniques applicable building. Also, to assess in advance by using self-assessment program in the design phase of building will lead to Green building design and provide the convenience of the application process and through database construction and share information of building assessment resources certified Green Building Certification Criteria will lead to the containment and fair assessment between cation authorities.

• Archives of Pharmacal Research
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• v.26 no.6
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• pp.493-498
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• 2003

In this study we have investigated the pharmacokinetics and tissue distribution of GX-12, a multiple plasmid DNA vaccine for the treatment of HIV-1 infection. Plasmid DNA was rapidly degraded in blood with a half-life of 1.34 min and was no longer detectable at 90 min after intravenous injection in mice. After intramuscular injection, plasmid DNA concentration in the injection site rapidly declined to less than 1 ％ of the initial concentration by 90 min post-injection. However, sub-picogram levels (per mg tissue) were occasionally detected for several days after injection. The relative proportions of the individual plasm ids of GX-12 remained relatively constant at the injection site until 90 min post-injection. The concentration of plasmid DNA in tissues other than the injection site peaked at 90 min post-injection and decreased to undetectable levels at 8 h post-injection. The rapid in vivo degradation of GX-12 and absence of persistence in non-target tissues suggest that the risk of potential gene-related toxicities by GX-12 administration, such as expression in non-target tissues, insertional mutagenesis and germline transmission, is minimal.