Probing phospholipase a(2) with fluorescent phospholipid substrates

The Foerster resonance energy transfer-based sensor, PENN, measures intracellular phospholipase A(2) (PLA(2)) activity in living cells and small organisms. In an attempt to modify the probe for the detection of particular isoforms, we altered the sn-2 fatty acid in such a way that either one or three of the Z double bonds in arachidonic acid were present in the sensor molecule. Arachidonic-acid-mimicking fatty acids were prepared by copper-mediated coupling reactions. Probes with a single double bond in the 5-position exhibited favorable substrate properties for secretory PLA(2)s. In vitro experiments with the novel unsaturated doubly labeled phosphatidylethanolamine derivatives showed preferred cleavage of the sensor PENN2 (one double bond) by the physiologically important group V sPLA(2), while the O-methyl-derivative PMNN2 was accepted best by the isoform from hog pancreas. For experiments in living cells, we demonstrated that bioactivation via S-acetylthioethyl (SATE) groups is essential for probe performance. Surprisingly, membrane-permeant versions of the new sensors that contained double bonds, PENN2 and PENN3, were only cleaved to a minor extent in HeLa cells while the saturated form, PENN, was well accepted.     

生物免疫传感器检测迟缓爱德华氏菌研究

迟缓爱德华氏菌(E.tarda)是最为严重的水产动物致病菌之一,准确、即时的检测手段是预防控制该菌传播的关键所在.通过量子点(QDs)标记E.tarda单克隆抗体(Ab),利用生物免疫传感器技术实现E.tarda的快速、特异性检测.结果显示,QDs-Ab的荧光通过加入氧化石墨烯(GO)产生淬灭,构建了捕获目标细菌的探针,GO最适淬灭浓度为60μg/L.细菌捕获探针中加入E.tarda后,能够检测到重新恢复强度的橙色荧光.针对E.tarda设计的生物免疫传感器的特异性,选取灿烂弧菌、溶藻胶弧菌、副溶血弧菌和嗜水气单胞菌作为对照,结果显示,对照组不能明显引起荧光强度的改变,而实验组却能显著提高荧光强度.本研究建立的基于荧光能量共振转移(FRET)的具有高灵敏度和特异性的生物传感器检测方法在细菌的早期诊断中有良好的应用潜质.     

Intravital FRET: Probing Cellular and Tissue Function in Vivo

The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice.     

Isolation of the effect of the hairy layer length on the mechanical properties of waterborne coatings

The effect of the acidic hairy layer length on the interdiffusion of polymer between particles and as a consequence on the mechanical properties of the films produced from waterborne coatings has been studied. In order to isolate this effect, latexes with the same particle diameter and molecular weight but stabilized with poly(acrylic acid)-block-poly(butyl acrylate) (PAA-b-PBA) block copolymers of controlled and different lengths were prepared. Tensile strength measurements showed at the macroscopic level that the presence of AA chains in the particle surface reduced the mechanical properties of the films dried at room temperature, being its effect worse the longer the AA chain length. Higher annealing temperatures erased the negative effect of the acidic hairy layer on mechanical properties. The neutralization with NaOH instead of with NH₄OH also led to worse mechanical properties. These macroscopic results were supported by Fluorescence Resonance Energy Transfer (FRET) experiments that showed that at the microscopic level, the extent of interdiffusion occurred slower when the AA chains in the particles surface increased, the annealing temperature was lower and when NaOH was used as neutralizing agent instead of NH₄OH.     

Proposal of a New Method for Measuring F?rster Resonance Energy Transfer (FRET) Rapidly,Quantitatively and Non-Destructively

The process of radiationless energy transfer from a chromophore in an excited electronic state (the “donor”) to another chromophore (an “acceptor”), in which the energy released by the donor effects an electronic transition, is known as “Förster Resonance Energy Transfer” (FRET). The rate of energy transfer is dependent on the sixth power of the distance between donor and acceptor. Determining FRET efficiencies is tantamount to measuring distances between molecules. A new method is proposed for determining FRET efficiencies rapidly, quantitatively, and non-destructively on ensembles containing donor acceptor pairs: at wavelengths suitable for mutually exclusive excitations of donors and acceptors, two laser beams are intensity-modulated in rectangular patterns at duty cycle ½ and frequencies f₁ and f₂ by electro-optic modulators. In an ensemble exposed to these laser beams, the donor excitation is modulated at f₁, and the acceptor excitation, and therefore the degree of saturation of the excited electronic state of the acceptors, is modulated at f₂. Since the ensemble contains donor acceptor pairs engaged in FRET, the released donor fluorescence is modulated not only at f₁ but also at the beat frequency Δf: = |f₁ − f₂|. The depth of the latter modulation, detectable via a lock-in amplifier, quantitatively indicates the FRET efficiency.     

New inhibitors of the Tat-TAR RNA interaction found with a "fuzzy" pharmacophore model

TAR RNA is a potential target for AIDS therapy. Ligand-based virtual screening was performed to retrieve novel scaffolds for RNA-binding molecules capable of inhibiting the Tat-TAR interaction, which is essential for HIV replication. We used a "fuzzy" pharmacophore approach (SQUID) and an alignment-free pharmacophore method (CATS3D) to carry out virtual screening of a vendor database of small molecules and to perform "scaffold-hopping". A small subset of 19 candidate molecules were experimentally tested for TAR RNA binding in a fluorescence resonance energy transfer (FRET) assay. Both methods retrieved molecules that exhibited activities comparable to those of the reference molecules acetylpromazine and chlorpromazine, with the best molecule showing ten times better binding behavior (IC50 = 46 microM). The hits had molecular scaffolds different from those of the reference molecules.     

人脑中脱碘酶相互作用蛋白的筛选与验证

阐述人脑内硒蛋白在维护细胞正常功能、抵御氧化损伤和预防脑疾病方面的重要作用,指出脱碘酶3是人脑中一种重要的硒蛋白.克隆了人脱碘酶3的开放读码框,将其编码区中编码硒代半胱氨酸的TGA码突变为编码半胱氨酸的密码,以脱碘酶3突变体为诱饵,利用酵母双杂交系统从人胎脑cD-NA文库中筛选一个能与脱碘酶3相互作用的蛋白,即人丝氨酸蛋白酶抑制剂A族蛋白3.采用荧光共振能量转移技术中的敏化发射法和荧光寿命法,验证了人脑中脱碘酶的相互作用.     

Effect of Bilayer Phospholipid Composition and Curvature on Ligand Transfer by the α-Tocopherol Transfer Protein

We report here our preliminary investigations on the mechanism of α-TTP-mediated ligand transfer as assessed using fluorescence resonance energy transfer (FRET) assays. These assays monitor the movement of the model α-tocopherol fluorescent derivative ((R)-2,5,7,8-tetramethyl-chroman-2-[9-(7-nitro-benzo[1,2,5]oxadiazol-4-yl amino)-nonyl]-chroman-6-ol; NBD-Toc) from protein to acceptor vesicles containing the fluorescence quencher TRITC-PE. We have found that α-TTP utilizes a collisional mechanism of ligand transfer requiring direct protein–membrane contact, that rates of ligand transfer are greater to more highly curved lipid vesicles, and that such rates are insensitive to the presence of anionic phospholipids in the acceptor membrane. These results point to hydrophobic features of α-TTP dominating the binding energy between protein and membrane. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at