Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC₅₀) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC₅₀ value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC₅₀ values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.     

Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging

New applications for bioluminescence imaging require an expanded set of luciferase enzymes and luciferin substrates. Here, we report two novel luciferins for use in vitro and in cells. These molecules comprise regioisomeric pyridone cores that can be accessed from a common synthetic route. The analogues exhibited unique emission spectra with firefly luciferase, although photon intensities remained weak. Enhanced light outputs were achieved by using mutant luciferase enzymes. One of the luciferin–luciferase pairs produced light on par with native probes in live cells. The pyridone analogues and complementary luciferases add to a growing set of designer probes for bioluminescence imaging.     

虫萤光素酶固定化及其传感器

本文比较了戊二醛法，环氧法、重氮法和ＣＮＢｒ活化等方法固定虫莹光素酶，发现ＣＮＢｒ活化Ｓｅｐｈａｒｏｓｅ４Ｂ后固定该酶的交果最佳．获得较高比活性（７５０ｕ／ｇ）和良好稳定性的固定化虫萤光素酶．我们构建了一套利用光纤传导的虫萤光素酶的测活装置；并以ＣＮＢｒ－Ｓｅｐｈａｒｏｓｅ４Ｂ固定化酶为核心建成光纤生物传感器和柱式流动分析装置．可测定１０－１１～１０－８ｍｏｌ／ｍｌＡＴＰ．     

Near-Infrared Bioluminescence Imaging with a through-Bond Energy Transfer Cassette

A coelenterazine (CTZ) analogue emitting near-infrared (NIR) bioluminescence was synthesized for through-bond energy transfer (TBET)-based imaging modalities. The analogue, named Cy5-CTZ, was prepared by conjugating cyanine-5 (Cy5) dye to CTZ through an acetylene linker. This novel derivative is intrinsically fluorescent and emits NIR-shifted luminescence upon reacting with an appropriate luciferase, the Renilla luciferase. This Cy5-CTZ substrate is optically stable in physiological samples and rapidly permeabilize through the plasma membrane into the cytosolic compartment of live cells.     

Evaluation of cleaning procedures for allergen control in a food industry environment

The hygiene of chicken processing surfaces and retention of the wheat protein gliadin and of protein in general on those surfaces were compared in 15 trials after 3 increasingly rigorous cleaning steps. Eleven different chicken products with wheat derivatives as a batter were prepared on 3 processing lines in 15 production runs selected at random over 6 mo (5 runs were thus replicates). Using surface swabs, surface hygiene was monitored by adenosine triphosphate (ATP) bioluminescence, gliadin by immunoassay, and protein by the Coomassie dye method. Gliadin was monitored in 14 trials, protein in 5, and all trials were monitored by ATP bioluminescence. In a typical trial, gliadin values normalized to uncleaned values fell from 100000 arbitrary units, to 6000 after rinsing, to 30 (foam, rinse), to not detected (sanitize, rinse). Parallel ATP bioluminescence values also decreased, but crucially, the relative gliadin value was less than the relative ATP value after foam and rinse in all 14 trials, a result unchanged after sanitize and rinse. In trials comparing ATP and protein, the relative ATP values exceeded the relative protein values in 4 of 5 trials after foaming and after sanitizing. Thus, for these 11 products, ATP bioluminescence was a surrogate indicator of residual gliadin and probably of residual protein. Absolute gliadin concentration on an uncleaned processing line was also the basis of modeling the risk of cross-contamination of gliadin in follow-up product, where the line was hypothetically left uncleaned between production runs. The results show that all follow-up product could be declared "gluten-free" under proposed legislation, and suggest that some industrial cross-contamination risks are currently overestimated.     

ATP生物发光技术在微生物检测中的应用

ATP(三磷酸腺苷)生物发光法作为一种不断完善成熟的微生物快速检测方法,具有简便、快速、高灵敏度等优点。对该方法的历史发展、检测原理、目标物提取方法、应用领域和最新研究成果等做重点介绍,并对ATP生物发光法的应用现状和发展方向进行总结和展望。     

生物发光快速测定生乳菌落总数的方法

为消除利用ATP生物发光法测定生乳菌落总数时非细菌ATP对测定结果的干扰,建立了一种样品前处理方法。利用ATP生物发光法对经过前处理的生乳样品进行检测,结果表明,生乳菌落总数对数值与生乳细菌ATP发光对数值呈现较好的线性关系(R2=0.982),相关程度为显著相关(P<0.01),说明该前处理方法能够有效排除非细菌ATP的干扰,有利于提高ATP生物发光法定量测定生乳菌落总数准确性。     

Bioluminescence and Nanoluminescence

Electricity's reliance on coal-fired power stations and other limited mineral and petroleum resources makes finding an alternative energy source for lighting buildings' interiors and our external spaces one of the greatest challenges of the 21st century. Ken Yeang finds some clues for its resolution in existing biological and evolving artificial systems. Copyright © 2009 John Wiley & Sons, Ltd.     

基于ATP再生体系快速检测乳品中微生物

基于焦磷酸（pyrophosphoric acid,PPi）再生三磷酸腺苷（adenosine triphosphate,ATP）建立乳品中微生物快速检测方法。通过单因素试验优化PPi再生ATP反应条件,并考察方法的灵敏度、准确度、精密度和稳定性。结果表明,PPi再生ATP最佳反应条件为腺苷酰硫酸（adenosine phosphosulfate,APS）浓度10 μmol/L,ATP硫酸化酶（ATP sulfurylase,ATPS）活力0.15 U/mL、反应pH7.8。在最佳的ATP再生条件下偶联生物发光法,对ATP标准品、大肠杆菌、铜绿假单胞菌的检测限分别为10－17mol/mL、102CFU/mL和102CFU/mL。工作曲线在102～107CFU/mL范围内线性关系良好,对乳品基质的回收率为81.33%～97.78%,变异系数为14.24%～22.17%,与国标平板计数法对比显示两种方法检测结果相关性良好,相关系数为0.96。本方法快速、简单、灵敏、稳定,适用于乳品中微生物快速监测。     

Mesenchymal Stem Cells Delivery in Individuals with Different Pathologies: Multimodal Tracking,Safety and Future Applications

Due to their ease of isolation and their properties, mesenchymal stem cells (MSCs) have been widely investigated. MSCs have been proved capable of migration towards areas of inflammation, including tumors. Therefore, they have been suggested as vectors to carry therapies, specifically to neoplasias. As most of the individuals joining clinical trials that use MSCs for cancer and other pathologies are carefully recruited and do not suffer from other diseases, here we decided to study the safety and application of iv-injected MSCs in animals simultaneously induced with different inflammatory pathologies (diabetes, wound healing and tumors). We studied this by in vitro and in vivo approaches using different gene reporters (GFP, hNIS, and f-Luc) and non-invasive techniques (PET, BLI, or fluorescence). Our results found that MSCs reached different organs depending on the previously induced pathology. Moreover, we evaluated the property of MSCs to target tumors as vectors to deliver adenoviruses, including the interaction between tumor microenvironment and MSCs on their arrival. Mechanisms such as transdifferentiation, MSC fusion with cells, or paracrine processes after MSCs homing were studied, increasing the knowledge and safety of this new therapy for cancer.