A genetically encoded metallothionein tag enabling efficient protein detection by electron microscopy

The observation of biological materials by transmission electron microscopy (TEM) frequently requires the use of negative staining and/or immuno-gold labeling to visualize or identify the specimen, but these techniques are limited. In contrast, genetic labeling with green fluorescent protein (GFP) and its homologs have led to rapid advances in the observation of proteins of interest in cells by light microscopy (LM). These fluorescent tags allow for the visualization of dynamic processes in live cells without the use of secondary reagents. Here, we report the development of an artificial metalloprotein fusion protein expressed in Escherichia coli grown in Cd(2+)-containing medium that allows for efficient protein detection by TEM without additional staining steps. We linked the subunits of the bacterial 14-mer protein GroEL with three repeats of metallothionein (3MT). The 3MT-fused GroEL (GroEL-14(3MT)) was successfully expressed in E. coli, and the purified protein included 250 cadmium atoms per molecule on average. Cd(2+)-bound GroEL-14(3MT) was detected by TEM in the absence of negative staining on a carbon grid, and the particle densities of GroEL-14(3MT) were much greater than those of untagged GroEL in vitreous ice. Taken together, our data indicate that the 3MT tag provides a promising means of allowing the identification of oligomeric proteins isolated from cells in the absence of other detection techniques.     

Enhanced detection efficiency of genetically encoded tag allows the visualization of monomeric proteins by electron microscopy

A cadmium-binding, genetically encoded protein tag, consisting of three repeats of metallothionein (3MT), can be used in electron microscopy for the visualization of multimeric- but not monomeric-tagged proteins due to insufficient electron density in monomeric proteins. Here, we present a technique for detecting monomeric 3MT-tagged green fluorescent protein (GFP-3MT) using a platinum compound to intensify the electron density. Substitution of cadmium by platinum as a result of incubating purified cadmium-binding 3MT-tagged GFP (GFP-Cd-3MT) with cis-diamminedichloroplatinum(II) (cisDDP) was assessed by a UV absorption band centered at 284?nm thereby indicating platinum-sulfhydryl bonds. The incubation time and the concentration of cisDDP to reach maximal absorption were 2?h and 36-fold molar equivalent of cisDDP, respectively. GFP-Pt-3MT isolated by gel filtration chromatography contained 29 platinum atoms per single GFP-3MT molecule. Electron-dense particles were observed in a GFP-Pt-3MT sample by electron microscopy without negative staining. Further image processing and image analysis demonstrated that particles with higher density relative to their surroundings were detectable in both GFP-Cd-3MT and GFP-Pt-3MT samples. These results demonstrate that replacement of cadmium with platinum, together with proper image analyses, improve detection efficiency and enable the visualization of 3MT-tagged monomeric protein by electron microscopy.     

胶体Cu2CdSnS4纳米晶的合成与其性质研究

用简易的溶液化学方法合成了胶体Cu2CdSnS4纳米晶.透射电子显微镜(TEM),扫描电子显微镜(SEM),能谱(EDS),X射线衍射(XRD),X射线光电子谱(XPS)和UV-vis-NIR吸收光谱测试表明Cu2CdSnS4胶体纳米晶具有均一的尺寸分布和良好的结晶性,并具有四面体结构.纳米晶中Cu/Cd/Sn/S的化学计量比约为2.07:0.75:1.26:3.92,Cu、Cd、Sn和S四种元素的化学态分别为+1、+2、+4和-2价,与Cu2CdSnS4分子式中的化学态一致.通过外推法估算Cu2CdSnS4纳米晶的禁带宽度为~1.3 eV.     

硫化镉纳米微晶掺杂Na2O—B2O3—SiO2系统玻璃的制备及光学性质 …

本文采用Ｓｏｌ－ｇｅｌ方法制备了掺杂ＣｄＳ纳米微晶的Ｎａ２Ｏ－Ｂ２Ｏ３－ＳｉＯ２系统玻璃,研究了这种玻璃材料的合成反应机理和结构特性。利用各种温度下玻璃中掺杂纳米尺寸ＣｄＳ半导体微晶的光致发光光谱,研究了其光致发光带的峰值能量和线宽对温度的信赖关系,拟合了其带隙发光带的线宽随温度的变化曲线,获得样品的非均匀线宽。ＣｄＳ微晶的非均匀线宽主要是由于微晶的尺寸分布引起的,可以利用ＣｄＳ微晶的非均匀宽化系     

Bi3+共掺和能量传递对BaY2Si3O10: Eu3+发光的调制和影响

荧光粉BaY2Si3O10: Bi3+, Eu3+经高温固相法制备并由X-ray衍射谱仪分析其物相结构。实验结果显示Bi3+共掺下BaY2Si3O10: Eu3+的激发光谱呈现一个有明显增强的宽电荷转移带和系列Eu3+的 f – f 窄吸收峰,发射谱为Eu3+的5D0-7FJ橙-红光发射。当用285 nm 紫外光激发时,Bi3+到Eu3+间存在有效的能量传递,导致Bi3+的宽带紫外发射(中心345 nm)强度减弱,而Eu3+的橙-红光发射显著增强;随着Eu3+浓度的增加,能量传递效率也随之提高。最佳Eu3+浓度为0.4摩尔百分比,此后荧光粉发射强度发生浓度猝灭。结果表明Bi3+共掺时明显改善和提升荧光粉在电荷转移带(200 – 350 nm)的激发效率。Bi3+到Eu3+间主要的能量传递机制是通过四极–四极相互作用实现,并且能量传递的临界作用距离是1.604 nm     

硫化镉纳米微晶掺杂Na2O-B2O3-SiO2系统玻璃的制备及光学性质的研究

本文采用Sol－gel方法制备了掺杂CdS纳米徽晶的Na2O－B2O3－SiO2系统玻璃,研究了这种玻璃材料的合成反应机理和结构特性．利用各种温度下玻璃中掺杂纳米尺寸CdS半导体微晶的光致发光光谱,研究了其光致发光带的峰值能量和线宽对温度的依赖关系,拟合了其带隙发光带的线宽随温度的变化曲线,获得样品的非均匀线宽．CdS微晶的非均匀线宽主要是由于微晶的尺寸分布引起的,可以利用CdS微晶的非均匀宽化系数来评价微晶的尺寸分布．报道了此种玻璃的三阶非线性极化率X（3）＝6.5×10－12esu．简要分析了影响这种材料三阶非线性极化率的因素．     

Essential Protein Prediction Based on Shuffled Frog-Leaping Algorithm

Essential proteins are integral parts of living organisms.The prediction of essential proteins facilitates to discover disease genes and drug targets.The prediction precision and robustness of most of existing identification methods are not satisfactory.In this paper,we propose a novel essential proteins prediction method(EPSFLA),which applies Shuffled frog-leaping algorithm(SFLA),and integrates several biological information with network topological structure to identify essential proteins.Specifically,the topological property and several biological properties (function annotation,subcellular localization,protein complex,and orthology) are integrated and utilized to weight protein-protein interaction networks.Then the position of a frog is encoded and denotes a candidate essential protein set.The frog population continuously evolve by means of local exploration and global exploration until termination criteria for algorithm are satisfied.Finally,those proteins contained in the best frog are regarded as predicted essential proteins.The experimental results show that EPSFLA outperforms some well-known prediction methods in terms of various criteria.The proposed method aims to provide a new perspective for essential protein prediction.     

Si~(4+)掺杂对BaZr(BO_3)_2:Eu荧光粉能量传递的影响

采用高温固相法合成了Si4+掺杂的BaZr(BO3)2:Eu红色发光荧光粉。激发光谱表明,不同Si4+掺杂浓度明显使电荷迁移态(CTS)向高能量的位置移动,且改善了样品的发光强度。分析认为,这是由于Si4+的电负性大于所取代的Zr4+,且Si4+的进入影响了Eu3+的配位数,提高了CTS向发光中心的能量传递几率。依据Judd-Ofelt理论计算的强度参数表明,随着Si4+掺杂浓度的增加,Eu3+所处格位的对称性明显降低,增大了Eu3+的跃迁几率,从而改善了发光强度。计算Eu3+间的能量传递几率发现,在掺杂浓度为5%时,Eu3+间的能量传递几率很小,其对荧光粉的发光影响不大。     

Effects of Pb-Doping on Phase Structural and Optical Properties of CdZnS Nano-Powders

Cadmium zinc lead sulfide[Cd0.8(Zn1-x,Pbx)0.2S] nano-powders were prepared by an improved coprecipitation method. The effect of Pb2+ concentration at 500℃ on the phase and crystalline structure of the Cd0.8(Zn1-x,Pbx)0.2S powders were investigated by X-ray diffraction (XRD). According to the transmission electron microscopy (TEM) images, the particles size are in the range of 58 nm to 72 nm. In addition, optical band gap energy and optical constants of nano-powders were determined using the ultraviolet (UV) spectrum, Fournier transform infrared spectroscopy (FTIR), and Kramers-Kronig analysis, respectively. We calculate the refractive index n, extinction coefficient k, and dielectric function as a function of the wavenumber. The experiment results demonstrate that the amount of Pb+2 has been playing an increasingly important role on optical properties of CZPS nanocrystals.     

Li+增强Ho3+/Yb3+:Y2O3纳米晶的上转化发光研究

采用凝胶溶胶法制备了不同浓度的Y2O3:Ho3+/Yb3+/Li+纳米晶,并且系统研究了不同掺杂浓度对上转化荧光现象的影响。首先,通过XRD图形判断了晶体结构的生成,再通过980 nm的激光器和荧光光谱仪测得的光谱图,发现了位于520~579 nm的绿光、635~674 nm的红光等两条很强的可见光,还有一条较弱的位于743~775 nm的近红外发光。最后,通过与能级图比较和分析可以得出:它们分别是5F4/5S2→5I8,5F5→5I8和5F4/5S2→5I7荧光跃迁。可以看出:在掺杂入Li+之后,上转化荧光得到了极大增强。