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1
High-Frequency Targeted Mutagenesis in Pseudomonas stutzeri Using a Vector-Free Allele-Exchange Protocol

( Ahmed E. Gomaa ),( Zhiping Deng ),( Zhimin Yang ),( Liguo Shang ),( Yuhua Zhan ),( Wei Lu ),( Min Lin ),( Yongliang Yan ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.2
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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 selfligation (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.
2
Culture-Positive Spontaneous Ascitic Infection in Patients with Acute Decompensated Cirrhosis: Multidrug-Resistant Pathogens and Antibiotic Strategies

Jing Liu,Yanhang Gao,Xianbo Wang,Zhiping Qian,Jinjun Chen,Yan Huang,Zhongji Meng,Xiaobo Lu,Guohong Deng,Feng Liu,Zhiguo Zhang,Hai Li,Xin Zheng 연세대학교의과대학 2020 Yonsei medical journal Vol.61 No.2
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Purpose: This study investigated multidrug-resistant (MDR) pathogens and antibiotic strategies of culture-positive spontaneousascitic infection (SAI) in patients with acute decompensated cirrhosis. Materials and Methods: We retrospectively analyzed 432 acute decompensated cirrhotic patients with culture-positive SAI from11 teaching hospitals in China (January 2012 to May 2018). A Cox proportional hazards model analysis was conducted to identifyindependent predictors of 28-day mortality. Results: A total of 455 strains were isolated from 432 ascitic culture samples. Gram-negative bacteria (GNB), gram-positive bacteria(GPB), and fungi caused 52.3, 45.5, and 2.2% of all SAI episodes, respectively. Episodes were classified as nosocomial (41.2%), healthcare-related (34.7%), and community-acquired (24.1%). Escherichia coli (13.4%) and Klebsiella pneumoniae (2.4%) were extendedspectrumβ-lactamase producing isolates. The prevalence of methicillin-resistant Staphylococcus aureus was 1.1%. Ceftazidime,cefepime, aztreonam, and amikacin were recommended as first-line antibiotics agents for non-MDR GNB infections; piperacillin/tazobactam and carbapenems for MDR GNB in community-acquired and healthcare-related or nosocomial infections, respectively;and vancomycin or linezolid for GPB infections, regardless of drug-resistance status. Multivariate analysis revealed days ofhospital stay before SAI, upper gastrointestinal bleeding, white blood cell count, alanine aminotransferase, serum creatinine concentration,total bilirubin, and international normalized ratio as key independent predictors of 28-day mortality. Conclusion: MDR pathogens and antibiotic strategies were identified in patients with acute decompensated cirrhosis with culture-positive SAI, which may help optimize therapy and improve clinical outcomes.
3
An Essential Role for 14-3-3 Proteins in Brassinosteroid Signal Transduction in Arabidopsis

Gampala, Srinivas S.,Kim, Tae-Wuk,He, Jun-Xian,Tang, Wenqiang,Deng, Zhiping,Bai, Mingyi-Yi,Guan, Shenheng,Lalonde, Sylvie,Sun, Ying,Gendron, Joshua M.,Chen, Huanjing,Shibagaki, Nakako,Ferl, Robert J. Elsevier 2007 DEVELOPMENTAL CELL Vol.13 No.2
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SummaryBrassinosteroids (BRs) are essential hormones for plant growth and development. BRs regulate gene expression by inducing dephosphorylation of two key transcription factors, BZR1 and BZR2/BES1, through a signal transduction pathway that involves cell-surface receptors (BRI1 and BAK1) and a GSK3 kinase (BIN2). How BR-regulated phosphorylation controls the activities of BZR1/BZR2 is not fully understood. Here, we show that BIN2-catalyzed phosphorylation of BZR1/BZR2 not only inhibits DNA binding, but also promotes binding to the 14-3-3 proteins. Mutations of a BIN2-phosphorylation site in BZR1 abolish 14-3-3 binding and lead to increased nuclear localization of BZR1 protein and enhanced BR responses in transgenic plants. Further, BR deficiency increases cytoplasmic localization, and BR treatment induces rapid nuclear localization of BZR1/BZR2. Thus, 14-3-3 binding is required for efficient inhibition of phosphorylated BR transcription factors, largely through cytoplasmic retention. This study demonstrates that multiple mechanisms are required for BR regulation of gene expression and plant growth.