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Bo Xian Jie Shen Weiyang Chen Na Sun Nan Qiao Dongqing Jiang Tao Yu Yongfan Men Zhijun Han Yuhong Pang Matt Kaeberlein Yanyi Huang Jing‐Dong J. Han 《Aging cell》2013,12(3):398-409
Caenorhabditis elegans is a leading model organism for studying the basic mechanisms of aging. Progress has been limited, however, by the lack of an automated system for quantitative analysis of longevity and mean lifespan. To address this barrier, we developed ‘WormFarm’, an integrated microfluidic device for culturing nematodes. Cohorts of 30–50 animals are maintained throughout their lifespan in each of eight separate chambers on a single WormFarm polydimethylsiloxane chip. Design features allow for automated removal of progeny and efficient control of environmental conditions. In addition, we have developed computational algorithms for automated analysis of video footage to quantitate survival and other phenotypes, such as body size and motility. As proof‐of‐principle, we show here that WormFarm successfully recapitulates survival data obtained from a standard plate‐based assay for both RNAi‐mediated and dietary‐induced changes in lifespan. Further, using a fluorescent reporter in conjunction with WormFarm, we report an age‐associated decrease in fluorescent intensity of GFP in transgenic worms expressing GFP tagged with a mitochondrial import signal under the control of the myo‐3 promoter. This marker may therefore serve as a useful biomarker of biological age and aging rate. 相似文献
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Yubin Zhou Wen-Pin Tzeng Hing-Cheung Wong Yiming Ye Jie Jiang Yanyi Chen Yun Huang Suganthi Suppiah Teryl K. Frey Jenny J. Yang 《The Journal of biological chemistry》2010,285(12):8855-8868
The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca2+- and Zn2+-binding papain-like cysteine protease domain within the nonstructural replicase polyprotein precursor, is responsible for the self-cleavage of the precursor into two mature products, P150 and P90, that compose the replication complex that mediates viral RNA replication; the NS protease resides at the C terminus of P150. Here we report the Ca2+-dependent, stoichiometric association of calmodulin (CaM) with the RUBV NS protease. Co-immunoprecipitation and pulldown assays coupled with site-directed mutagenesis demonstrated that both the P150 protein and a 110-residue minidomain within NS protease interacted directly with Ca2+/CaM. The specific interaction was mapped to a putative CaM-binding domain. A 32-mer peptide (residues 1152–1183, denoted as RUBpep) containing the putative CaM-binding domain was used to investigate the association of RUBV NS protease with CaM or its N- and C-terminal subdomains. We found that RUBpep bound to Ca2+/CaM with a dissociation constant of 100–300 nm. The C-terminal subdomain of CaM preferentially bound to RUBpep with an affinity 12.5-fold stronger than the N-terminal subdomain. Fluorescence, circular dichroism and NMR spectroscopic studies revealed a “wrapping around” mode of interaction between RUBpep and Ca2+/CaM with substantially more helical structure in RUBpep and a global structural change in CaM upon complex formation. Using a site-directed mutagenesis approach, we further demonstrated that association of CaM with the CaM-binding domain in the RUBV NS protease was necessary for NS protease activity and infectivity. 相似文献
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Dongqing Jiang Xiannian Zhang Yuhong Pang Jianyun Zhang Jianbin Wang Yanyi Huang 《蛋白质与细胞》2019,10(3):229-233
Dear Editor,Since its invention,next generation sequencing(NGS)has greatly facilitated biomedical research and clinical diagnosis(Sikkema-Raddatz et al.,2013).Continuous dropping of the cost further accelerated the adaptation of sequencing as a standard analytical tool,from identification of drug candidates(Walker et al.,2015)to deciphering the complex biological systems(McConnell et al.,2013). 相似文献
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Yanyi Liu Michael Clark Qifeng Zhang Danmei Yu Dawei Liu Jun Liu Guozhong Cao 《Liver Transplantation》2011,1(2):194-202
Nanostructured V2O5 thin films have been prepared by means of cathodic deposition from an aqueous solution made from V2O5 and H2O2 directly on fluorine‐doped tin oxide coated (FTO) glasses followed by annealing at 500°C in air, and studied as film electrodes for lithium ion batteries. XPS results show that the as‐deposited films contained 15% V4+, however after annealing all the vanadium is oxidized to V5+. The crystallinity, surface morphology, and microstructures of the films have been investigated by means of XRD, SEM, and AFM. The V2O5 thin film electrodes show excellent electrochemical properties as cathodes for lithium ion intercalation: a high initial discharge capacity of 402 mA h g?1 and 240 mA h g?1 is retained after over 200 cycles with a discharging rate of 200 mA g?1 (1.3 C). The specific energy density is calculated as 900 W h kg?1 for the 1st cycle and 723 W h kg?1 for the 180th cycle when the films are tested at 200 mA g?1 (1.3 C). When discharge/charge is carried out at a high current density of 10.5 A g?1 (70 C), the thin film electrodes retain a good discharge capacity of 120 mA h g?1, and the specific power density is over 28 kW kg?1. 相似文献
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Zhou Y Tzeng WP Ye Y Huang Y Li S Chen Y Frey TK Yang JJ 《The Biochemical journal》2009,417(2):477-483
The protease domain within the RUBV (rubella virus) NS (non-structural) replicase proteins functions in the self-cleavage of the polyprotein precursor into the two mature proteins which form the replication complex. This domain has previously been shown to require both zinc and calcium ions for optimal activity. In the present study we carried out metal-binding and conformational experiments on a purified cysteine-rich minidomain of the RUBV NS protease containing the putative Zn(2+)-binding ligands. This minidomain bound to Zn(2+) with a stoichiometry of approximately 0.7 and an apparent dissociation constant of <500 nM. Fluorescence quenching and 8-anilinonaphthalene-1-sulfonic acid fluorescence methods revealed that Zn(2+) binding resulted in conformational changes characterized by shielding of hydrophobic regions from the solvent. Mutational analyses using the minidomain identified residues Cys(1175), Cys(1178), Cys(1225) and Cys(1227) were required for the binding of Zn(2+). Corresponding mutational analyses using a RUBV replicon confirmed that these residues were necessary for both proteolytic activity of the NS protease and viability. The present study demonstrates that the CXXC(X)(48)CXC Zn(2+)-binding motif in the RUBV NS protease is critical for maintaining the structural integrity of the protease domain and essential for proteolysis and virus replication. 相似文献
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Mahmood Khan Yanyi Xu Serena Hua Jed Johnson Andriy Belevych Paul M. L. Janssen Sandor Gyorke Jianjun Guan Mark G. Angelos 《PloS one》2015,10(5)
IntroductionDilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates.MethodshiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes.ResultsSEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro.ConclusionsOverall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch. 相似文献