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。     

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.     

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.     

Intracellular Ionic Strength Sensing Using NanoLuc

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.     

ATP生物荧光法在食品微生物检验上的应用

ATP生物荧光法自20世纪50年代开始研究,到了90年代已日趋成熟。由于其快速、灵敏、价廉的特点,现已广泛应用于食品生产清洁度及细菌数量的检测。     

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。     

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。     

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.     

Enhanced Survival of High-Risk Medulloblastoma-Bearing Mice after Multimodal Treatment with Radiotherapy,Decitabine, and Abacavir

Children with high-risk SHH/TP53-mut and Group 3 medulloblastoma (MB) have a 5-year overall survival of only 40%. Innovative approaches to enhance survival while preventing adverse effects are urgently needed. We investigated an innovative therapy approach combining irradiation (RT), decitabine (DEC), and abacavir (ABC) in a patient-derived orthotopic SHH/TP53-mut and Group 3 MB mouse model. MB-bearing mice were treated with DEC, ABC and RT. Mouse survival, tumor growth (BLI, MRT) tumor histology (H/E), proliferation (Ki-67), and endothelial (CD31) staining were analyzed. Gene expression was examined by microarray and RT-PCR (Ki-67, VEGF, CD31, CD15, CD133, nestin, CD68, IBA). The RT/DEC/ABC therapy inhibited tumor growth and enhanced mouse survival. Ki-67 decreased in SHH/TP53-mut MBs after RT, DEC, RT/ABC, and RT/DEC/ABC therapy. CD31 was higher in SHH/TP53-mut compared to Group 3 MBs and decreased after RT/DEC/ABC. Microarray analyses showed a therapy-induced downregulation of cell cycle genes. By RT-PCR, no therapy-induced effect on stem cell fraction or immune cell invasion/activation could be shown. We showed for the first time that RT/DEC/ABC therapy improves survival of orthotopic SHH/TP53-mut and Group 3 MB-bearing mice without inducing adverse effects suggesting the potential for an adjuvant application of this multimodal therapy approach in the human clinic.     

MAEBL Contributes to Plasmodium Sporozoite Adhesiveness

The sole currently approved malaria vaccine targets the circumsporozoite protein—the protein that densely coats the surface of sporozoites, the parasite stage deposited in the skin of the mammalian host by infected mosquitoes. However, this vaccine only confers moderate protection against clinical diseases in children, impelling a continuous search for novel candidates. In this work, we studied the importance of the membrane-associated erythrocyte binding-like protein (MAEBL) for infection by Plasmodium sporozoites. Using transgenic parasites and live imaging in mice, we show that the absence of MAEBL reduces Plasmodium berghei hemolymph sporozoite infectivity to mice. Moreover, we found that maebl knockout (maebl-) sporozoites display reduced adhesion, including to cultured hepatocytes, which could contribute to the defects in multiple biological processes, such as in gliding motility, hepatocyte wounding, and invasion. The maebl- defective phenotypes in mosquito salivary gland and liver infection were reverted by genetic complementation. Using a parasite line expressing a C-terminal myc-tagged MAEBL, we found that MAEBL levels peak in midgut and hemolymph parasites but drop after sporozoite entry into the salivary glands, where the labeling was found to be heterogeneous among sporozoites. MAEBL was found associated, not only with micronemes, but also with the surface of mature sporozoites. Overall, our data provide further insight into the role of MAEBL in sporozoite infectivity and may contribute to the design of future immune interventions.