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High-Frequency Targeted Mutagenesis in Pseudomonas stutzeri Using a Vector-Free Allele-Exchange Protocol

  • Gomaa, Ahmed E.;Deng, Zhiping;Yang, Zhimin;Shang, Liguo;Zhan, Yuhua;Lu, Wei;Lin, Min;Yan, Yongliang
    • Journal of Microbiology and Biotechnology
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    • 제27권2호
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    • pp.335-341
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    • 2017
  • The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus Pseudomonas. Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes (ncRNA31, rpoN, rpoS) were knocked-out from the root-associative bacterium Pseudomonas stutzeri A1501. The results showed that the P. stutzeri A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in P. stutzeri, which may also be applicable for other gram-negative bacteria.

Development of a Fluoride-Selective Electrode based on Scandium(III) Octaethylporphyrin in a Plasticized Polymeric Membrane

  • Kang, Young-Jea;Lutz, Christopher;Hong, Sung-A;Sung, Da-Yeon;Lee, Jae-Seon;Shin, Jae-Ho;Nam, Hak-Hyun;Cha, Geun-Sig;Meyerhoff, Mark E.
    • Bulletin of the Korean Chemical Society
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    • 제31권6호
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    • pp.1601-1608
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    • 2010
  • A scandium(III) porphyrin-based fluoride-selective potentiometric sensor and its application in the analysis of hydrofluoric acid is described. Scandium(III) octaethylporphyrin, an ionophore recently developed for the optical fluoride sensor, was employed as a host molecule for the selective binding with fluoride in the plasticized PVC membrane. Nernstian response for $F^-$ between $10^{-4.6}$ to $10^{-1}$ M was observed at a glycine-phosphate buffer (pH 3.0). The selectivity pattern was observed as $F^-$, salicylate $\gg$ $SCN^-$ > $Cl^-$, $Br^-$, $NO_3{^-}$, $ClO_4{^-}$, which is consistent with the binding constant data measured in the plasticized PVC membrane based on a sandwich membrane method. This highly selective and reversible fluoride-sensitive electrode was employed for the analysis of hydrofluoric acid (HF). A disposable differential-type HF sensor was fabricated on the screen-printed electrode and demonstrated its ability to detect the neutral HF in the acidic solution.

Hydrogen Passivation for the Enhancement of Poly-Si Performance Crystallized By Double-Frequency YAG Laser

  • Li, Juan;Chong, Luo;Ying, Yao;He, Li;Meng, Zhiguo;Chunya, Wu;Xiong, Shaozhen;Kwok, Hoi-Sing
    • 한국정보디스플레이학회:학술대회논문집
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    • 한국정보디스플레이학회 2009년도 9th International Meeting on Information Display
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    • pp.1608-1611
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    • 2009
  • Here the hydrogen passivation treatment has been adopted to enhance the performance of poly-Si crystallized by YAG laser annealing (LA poly-Si). We have investigated the effects of passivation time, passivation power and passivation temperature on the hall mobility of the LA poly-Si and analyzed the mechanism of the hydrogen passivation preliminary. It has been found that the quality of the poly-Si annealed by YAG laser could be improved after proper hydrogen plasma treatment.

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Production and Characterization of DDT Antibodies and Its Application to Enzyme Immunoassay: Relation of Response and Affinity to Coating Ligand

  • Hong, Ji-Youn;Kim, Jong-Hyun;Park, Song-Ja;Lho, Dong-Seok;Choi, Myung-Ja
    • Bulletin of the Korean Chemical Society
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    • 제24권11호
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    • pp.1605-1608
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    • 2003
  • To development an immunodetection method for DDT, 1,1,1-trichloro-2,2-bis(4-chorophenyl)ethane (p,p'-DDT) and its metabolites (p,p'-DDA, p,p'-DDE, p,p'-DDD), five derivatives of DDT haptens have been synthesised and characterized as coating ligands for antibody evaluation. The appropriate lengths of linkers were introduced to investigate a matching pair of coating ligand and antibody. Among these hapten derivatives, 2,2-bis(4-chlorophenyl)acetic acid (DDA), 5,5-bis(4-chlorophenyl)-5-hydroxypentanoic acid (DDHP) and 5,5-bis(4-chlorophenyl)-5-chloropentanoic acid (DDCP) were conjugated with keyhole limpet hemocyanin (KLH) for its use as an immunogen. The bovine serum albumin (BSA) conjugates of these derivatives were prepared as a coating ligand for monoclonal antibody screening. Fifteen monoclonal antibody clones were screened using these probes. 6,6-Bis(4-chlorophenyl)-6-hydroxyhexanoic acid (DDHH) and 3-[6,6-Bis(4-chlorophenyl)-6-hydroxyhexanoylamino]propanoic acid (DDHHAP), in addition to the above hapten derivatives, were conjugated to ovualbumin (OVA) and bovine serum albumin (BSA) for their use as coating ligands to measure the titration level of the antibody and the displacement of free analytes. The indirect competitive ELISA results indicate that the titration level and free analyte displacement were greatly influenced by the DDT derivatives and carrier proteins used. Three matching pairs of monoclonal antibodies and coating ligands were selected for the DDT immunoassay: antibody clone 1A3 and coating ligand DDA-OVA, 1A1 and DDHHAP-BSA, and 1A4 and DDHP-OVA.

산업화 시대 퇴직 노인을 위한 치매예방프로그램의 효과에 관한 연구 (Research on the Effects of the Dementia Prevention Program on the Retired Seniors in the Industrial Age)

  • 손연희;박지온;황현숙
    • 한국전자통신학회논문지
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    • 제8권10호
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    • pp.1601-1608
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    • 2013
  • 의학계는 2020년 국내노인성 치매환자가 60만명을 넘을 것으로 예상하고 있다. 현재 연령별 치매 환자 비율은 70대 3%, 85~89세 23%, 95세 이상이 58%로 나이에 따라 급격히 증가한다는 것도 주목해야 할 일이다. "21세기의 병"이라 불리우고 있는 치매는 환자 자신뿐 아니라 가족들의 삶까지도 심각한 영향을 미치고 있으며 삶의 질을 떨어뜨려 결국은 가족을 황폐화시킬 정도로 많은 부담과 고통을 동반하고 사회적으로도 심각한 문제가 되고 있다는 지적도 있다. 본 논문은 치매예방센터를 운영하는 대학의 치매예방센터의 프로그램에 참여하여 일정기간 노인들과 함께 프로그램에 참여하며 치매예방 프로그램에 대한 적극적인 개발과 치매예방 인력의 전문화를 위해 노인들과 함께 했다.

Enhancing the Antioxidant Activities of Wines by Addition of White Rose Extract

  • Seong, Hyunbin;Heo, Jieun;Lee, Kyun Hee;Lee, Yoon Bok;Kim, Yun Bae;Han, Nam Soo
    • Journal of Microbiology and Biotechnology
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    • 제27권9호
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    • pp.1602-1608
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    • 2017
  • White rose petal extract (WRE) contains large amounts of phenolic compounds and is considered edible. In this study, red and white wines were prepared by the addition of WRE (0.10% or 0.25% (w/v)), followed by fermentation at $25^{\circ}C$ for 15 days. The fermentation profiles, colors, sensory test results, and antioxidant activities of the wines were compared. As reported herein, the fermentation profiles of the pH, $CO_2$ production rate, and final ethanol concentration were not affected by the addition of WRE, but a slow consumption rate of sugar was observed in 0.25% WRE-added wine. In contrast, the total polyphenol concentrations in WRE-added wines increased significantly (p < 0.05) in a dose-dependent manner, resulting in appreciable enhancement of the antioxidant activities of the wines. Chromaticity tests showed slight changes in the redness and yellowness, but sensory tests showed that the overall flavor qualities of the WRE-added wines were acceptable to the panels. This study demonstrates that addition of WRE to wine confers beneficial health effects and this treatment results in better outcome in white wine.

The role of calmodulin in regulating calcium-permeable PKD2L1 channel activity

  • Park, Eunice Yon June;Baik, Julia Young;Kwak, Misun;So, Insuk
    • The Korean Journal of Physiology and Pharmacology
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    • 제23권3호
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    • pp.219-227
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    • 2019
  • Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when co-expressed with CaM and $CaM{\triangle}N$. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ${\triangle}EF$-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.

혼합현실을 활용한 교통 안전교육 애플리케이션 개발 (A Development of Traffic Safety Education Application Using Mixed Reality)

  • 김강호;이대웅
    • 한국정보통신학회논문지
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    • 제23권12호
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    • pp.1602-1608
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    • 2019
  • 본 연구에서 우리는 혼합현실 애플리케이션을 활용해 어린이들에게 다양한 교통상황을 간접적으로 체험하게 하고, 사고의 위험에서 스스로 방어 행동을 할 수 있도록 도움을 주는 "Zetton 어린이 교통안전교육" 애플리케이션을 개발했다. 개발된 애플리케이션은 어린이 교통사고 중 사망률이 높은 사고 유형을 분석해 학습 목표를 정하였고, 목표 달성을 재미있게 체험을 할 수 있도록 개발되었다. 플레이어는 여러 교통상황을 정해진 시나리오를 플레이하는 과정에서 신호체계와 교통정보를 자연스럽게 습득할 수 있도록 체험 중심 콘텐츠가 되도록 개발하였다. 개발된 애플리케이션의 교육 효과성 검증을 위해 방과 후 교육 활동으로 어린이들에게 교통안전교육을 했다. 그 결과 교육 전보다 교육 후에 교통안전에 대한 인식과 학습 목표와 관련된 질문에 올바른 대답 빈도가 높아졌다.

Structure of a Copper(Ⅱ) Hexaazamacrotricyclic Complex : (1,3,6,9,11,14-Hexaazatricyclo[12.2.1.16,9]octadecane)-copper(Ⅱ) Perchlorate

  • Cheon Manseog;Suh Paik Myunghyun;Shin Whanchul
    • Bulletin of the Korean Chemical Society
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    • 제13권4호
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    • pp.363-367
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    • 1992
  • The crystal structure of (1,3,6,9,11,14-hexaazatricycol[12.2.1.$1^{6,9}$]octadecane)copper(Ⅱ) perchlorate, Cu($C_{12}H_{26}N_6$)$(ClO_4)_2$, has been determined by the X-ray diffraction methods. The crystal data are as follows: Mr=516.9, triclinic, ${\alpha}=8.572\;(2)$, b=8.499 (3), c=15.204 (3) ${\AA}$, ${\alpha}=80.42\;(5),\;{\beta}=73.57\;(3),\;{\gamma}=69.82\;(4)^{\circ},\;V=994.2\;{\AA}^3,\;D_C=1.726\;gcm^{-3}$, space group $P{\tilde{1}},\;Z=2,\;{\mu}=21.27\;cm^{-1}&, F(000)=534 and T=297 K. The structure was solved by direct methods and refined by full-matrix least-squares methods to and R value of 0.081 for 1608 observed reflections measured with graphite-mono-chromated Mo Ka radiation on a diffractometer. There are two independent complexes in the unit cell. The two copper ions lie at the special positions (1/2, 0, 0) and (0, 1/2, 1/2)and each complex possesses crystallographic center of symmetry. Each Cu ion is coordinated to four nitrogen donors if the hexaazamacrotricyclic ligand and weakly interacts with two oxygen atoms of the perchlorate ions to form a tetragonally distorted octahedral coordination geometry. The Cu_N (sec), Cu_N(tert) and Cu_O coordination distances are 1.985 (14), 2.055 (14) and 2.757 (13) ${\AA}$ for the complex A and 1.996 (10), 2.040 (11) and 2.660 (13) ${\AA}$ for the complex B, respectively. The macrocycles in the two independent cations assume a similar conformation with the average r.m.s. deviation of 0.073 ${\AA}$. Two 1,3-diazacyclopentane ring moieties of the hexaazamacrotricyclic ligand are placed oppositely and almost perpendicularly to the square coordination plane of the ruffled 14-membered macrocycle. The secondary N atoms are hydrogen-bonded to the perchlorate O atoms with distances of 3.017 (23) and 3.025 (19) ${\AA}$ for the complexes A and B, respectively.

Double magnetic entropy change peaks and high refrigerant capacity in Gd1-xHoxNi compounds in the melt-spun form

  • Jiang, Jun-fan;Ying, Hao;Feng, Tang-fu;Sun, Ren-bing;Li, Xie;Wang, Fang
    • Current Applied Physics
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    • 제18권12호
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    • pp.1605-1608
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    • 2018
  • $Gd_{1-x}Ho_xNi$ melt-spun ribbons were fabricated by a single-roller melt spinning method. All the compounds crystallize in an orthorhombic CrB-type structure. The Curie temperature ($T_C$) was tuned between 46 and 99 K by varying the concentration of Gd and Ho. A spin reorientation (SRO) transition is observed around 13 K. Different from $T_C$, the SRO transition temperature is almost invariable for all compounds. Two peaks of magnetic entropy change (${\Delta}S_M$) were found. One at the higher temperature range was originated from the paramagnet-ferromagnet phase transition and the other at the lower temperature range was caused by the SRO transition. The maximum of ${\Delta}S_M$ around $T_C$ is almost same. The other maximum of ${\Delta}S_M$ around SRO transition, however, had significantly positive relationship with x. It reached a maximum about $8.2J\;kg^{-1}\;K^{-1}$ for x = 0.8. Thus double large ${\Delta}S_M$ peaks were obtained in $Gd_{1-x}Ho_xNi$ melt-spun ribbons with the high Ho concentration. And the refrigerant capacity power reached a maximum of $622J\;kg^{-1}$ for x = 0.6. $Gd_{1-x}Ho_xNi$ ribbons could be good candidate for magnetic refrigerant working in the low temperature especially near the liquid nitrogen temperature range.