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( Yee-seul So ),( Dong-hoon Yang ),( Kwang-woo Jung ),( Won-ki Huh ),( Yong-sun Bahn ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.2
In this study, we aimed to generate a series of versatile tagging plasmids that can be used in diverse molecular biological studies of the fungal pathogen Cryptococcus neoformans. We constructed 12 plasmids that can be used to tag a protein of interest with a GFP, mCherry, 4×FLAG, or 6×HA, along with nourseothricin-, neomycin-, or hygromycin-resistant selection markers. Using this tagging plasmid set, we explored the adenylyl cyclase complex (ACC), consisting of adenylyl cyclase (Cac1) and its associated protein Aca1, in the cAMP-signaling pathway, which is critical for the pathogenicity of C. neoformans. We found that Cac1-mCherry and Aca1-GFP were mainly colocalized as punctate forms in the cell membrane and nonnuclear cellular organelles. We also demonstrated that Cac1 and Aca1 interacted in vivo by coimmunoprecipitation, using Cac1-6×HA and Aca1-4×FLAG tagging strains. Bimolecular fluorescence complementation further confirmed the in vivo interaction of Cac1 and Aca1 in live cells. Finally, protein pull-down experiments using aca1Δ::ACA1-GFP and aca1Δ::ACA1- GFP cac1Δ strains and comparative mass spectrometry analysis identified Cac1 and a number of other novel ACC-interacting proteins. Thus, this versatile tagging plasmid system will facilitate diverse mechanistic studies in C. neoformans and further our understanding of its biology.
Kim, Yee Seul,Ha, Yejin,Sim, Jungeun,Suh, Minah,Lee, Youngmi The Royal Society of Chemistry 2016 The Analyst Vol.141 No.1
<P>In this paper, we report the fabrication of a dual microsensor for sensing nitric oxide (NO) and calcium ions (Ca2+) and its application for simultaneous NO/Ca2+ measurements in living rat kidney tissue. NO and Ca2+ have very important physiological functions and are both intricately involved in many biological processes. The dual NO/Ca2+ sensor is prepared based on a dual recessed electrode possessing Pt (diameter, 25 mu m) and Ag (diameter, 76 mu m) microdisks. The Pt disk surface (WE1) is electrodeposited with porous Pt black and then coated with fluorinated xerogel; and used for amperometric sensing of NO. The Ag disk surface (WE2) is chloridated to AgCl, followed by silanization and then Ca2+ selective membrane loading; and used for potentiometric sensing of Ca2+. The dual sensor exhibits high sensitivity of WE1 to NO (40.8 +/- 6.5 pA mu M-1, n = 10) and reliable Nerntian response of WE2 to Ca2+ changes (25.7 +/- 0.5 mV pCa(-1), n = 10) with excellent selectivity to only NO and Ca2+ over common interferents and reliable stability (up to similar to 4 h tissue experiment). The prepared sensor is employed for real-time monitoring of the dynamic changes of NO and Ca2+ levels of a rat kidney, which is induced by the administration of 10 mM L-NG-nitroarginine methyl ester (L-NAME, a NO synthase inhibitor). Due to the small sensor dimension, location-dependent analyses of NO and Ca2+ are carried out at two different regions of a kidney (renal medulla and cortex). Higher NO and Ca2+ levels are observed at the medulla than at the cortex. This study verifies the feasibility for real-time monitoring of intimately connected Ca2+ and endogenous NO production; and also for localized concentration assessments of both NO and Ca2+.</P>
So, Yee-Seul,Lee, Dong-Gi,Idnurm, Alexander,Ianiri, Giuseppe,Bahn Yong-Sun Genetics Society of America [etc.] 2019 Genetics Vol.212 No.4
<P> The target of rapamycin (TOR) pathway is an evolutionarily conserved signal transduction system that governs a plethora of eukaryotic biological processes, but its role in <I>Cryptococcus neoformans</I> remains elusive. In this study, we investigated the TOR pathway by functionally characterizing two Tor-like kinases, Tor1 and Tlk1, in <I>C. neoformans.</I> We successfully deleted <I>TLK1</I>, but not <I>TOR1. TLK1</I> deletion did not result in any evident <I>in vitro</I> phenotypes, suggesting that Tlk1 is dispensable for the growth of <I>C. neoformans.</I> We demonstrated that Tor1, but not Tlk1, is essential and the target of rapamycin by constructing and analyzing conditionally regulated strains and sporulation analysis of heterozygous mutants in the diploid strain background. To further analyze the Tor1 function, we constructed constitutive <I>TOR1</I> overexpression strains. Tor1 negatively regulated thermotolerance and the DNA damage response, which are two important virulence factors of <I>C. neoformans. TOR1</I> overexpression reduced Mpk1 phosphorylation, which is required for cell wall integrity and thermoresistance, and Rad53 phosphorylation, which governs the DNA damage response pathway. Tor1 is localized to the cytoplasm, but enriched in the vacuole membrane. Phosphoproteomics and transcriptomics revealed that Tor1 regulates a variety of biological processes, including metabolic processes, cytoskeleton organization, ribosome biogenesis, and stress response. TOR inhibition by rapamycin caused actin depolarization in a Tor1-dependent manner. Finally, screening rapamycin-sensitive and -resistant kinase and transcription factor mutants revealed that the TOR pathway may crosstalk with a number of stress signaling pathways. In conclusion, our study demonstrates that a single Tor1 kinase plays pleiotropic roles in <I>C. neoformans.</I> </P>
만 3세반 교사의 동작교육 교수능력 향상을 위한 협력적 실행연구
김이슬(Kim, Yee-Seul),이혜정(Lee, Hye-Jeong),김은심(Kim, Eun-Shim) 한국영유아교원교육학회 2021 유아교육학논집 Vol.25 No.6
본 연구는 만 3세반 교사의 동작교육 교수능력 향상을 위한 협력적 실행연구 과정에서 나타난 교사와 유아의 변화를 탐색하는 것이다. 이를 위해 만 3세반 담임교사, 연구자, 유아 24명을 연구 참여자로 선정하여 약 12개월간 협력적 실행연구를 실시하였다. 자료수집은 수업관찰 자료, 교사 및 연구자의 저널, 심층면담, 동작수업 관련 자료 등을 기초로 분석 실시하였다. 이에 따른 연구결과를 살펴보면 첫째, 협력적 실행연구에 참여한 교사는 개별 유아에 대해 이해가 높아졌으며, 유아 주도의 동작활동을 지원하고, 다양한 방법과 형태의 동작활동을 지원하는 교사로 변화되었다. 둘째, 협력적 실행연구를 통해 유아는 자신이 주도하는 동작활동에 자유롭게 참여할 수 있었으며, 기본운동능력이 발달하며, 흥미도와 참여도가 높음이 나타났다. 본 연구는 동작교육 교수능력 향상을 위해 효과적이고 실질적 방법으로 교사와 연구자 간의 연구 공동체의 중요성을 제시하였다는 데 의의가 있다. The study explores the changes in teachers and infants in the process of collaborative practical research to improve the teaching ability of motion education of 3-year-old early childhood class teachers. For this purpose, the homeroom teachers, researchers, and 24 infants of 3-year-old were selected as research participants and a collaborative action study was conducted for about 12 months. Data collection was analyzed based on class observation data, journals of teachers and researchers, in-depth interviews, and movement class-related data. First, the teachers who participated in the collaborative practical research improved their understanding of individual infants and evolved into teachers who supported infant-led, and various methods and types of movement activities. Second, through collaborative practical research, infants were able to freely participate in their leading motion activities, develop basic exercise skills, and increase their interest and participation. This study is meaningful in that it suggests the importance of a research community between teachers and researchers in an effective and practically to improve the teaching ability of motion education.