往期刊物2015
卷册: 5, 期号: 3
癌症生物学
Hanging Drop Aggregation Assay of Breast Cancer Cells
乳腺癌细胞的悬滴聚集试验
Hanging drop assay can be used to investigate cell-cell cohesion and cell-substratum adhesion through generation of 3D spheroids under physiological conditions. It also can be used to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between two (or more) different cell populations. This simple method requires no specialized equipment and provides a means of generating tissue-like cellular aggregates for measurement of biomechanical properties for molecular and biochemical analysis in a physiologically relevant model.
微生物学
A Filter Binding Assay to Quantify the Association of Cyclic di-GMP to Proteins
膜过滤结合分析法量化环鸟苷二磷酸与蛋白质的关系
Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger that regulates many processes in bacteria including biofilm formation, motility, and virulence (Hengge, 2009). Analysis of c-di-GMP binding properties of bacterial proteins is an important step to characterize c-di-GMP signaling pathways. C-di-GMP binds numerous proteins such as transcription factors, enzymes, and multimeric protein complexes (Hickman and Harwood, 2008, Ryjenkov et al., 2006, Weinhouse et al., 1997). The c-di-GMP binding assay described here is a relatively simple and cost effective method to characterize c-di-GMP binding to a protein using [32P]-labeled c-di-GMP. Radiolabeled c-di-GMP is readily synthesized with a purified GGDEF enzyme [such as WspR from Pseudomonas aeruginosa (P. aeruginosa)] and [32P]-GTP (Srivastava et al., 2013). After incubation of the labeled c-di-GMP with the protein of interest in solution, the resulting mixture is filtered through a nitrocellulose protein binding membrane. The amount of labeled c-di-GMP that is retained on the membrane indicates the interaction between the signal and protein. The specificity of c-di-GMP binding can be tested by competing with unlabeled c-di-GMP or other nucleotides such as GTP in the reaction. By examining binding of a fixed protein concentration to increasing concentrations of c-di-GMP, this method is able to determine the dissociation constant of c-di-GMP-protein interaction.
Infection Experiments (Hepatitis C Virus)
感染实验（丙型肝炎病毒）
The establishment of a cell culture system for hepatitis C virus based on the JFH-1 strain and human hepatoma cell lines has been instrumental for the study of the viral replication cycle. The robustness of the JFH1-based cell culture models enabled many laboratories around the world to perform HCV infections in cell culture, accelerating the identification of cellular and viral targets to develop novel antiviral compounds. Although other robust infection systems based on different molecular clones and different cell lines have been developed since then, here we describe the protocols corresponding to infections with JFH-1 and JFH1-derived viruses carried out in our laboratory to produce virus stocks and persistently infected cell cultures. We also describe the experimental setups used to determine virus spreading capacity (multiple cycle infections) as well as to dissect early and late aspects of HCV infection (single cycle infections).
神经科学
Measuring Anxiety-like Behavior in Crayfish by Using a Sub Aquatic Dark-light Plus Maze
使用水下暗光十字迷宫法测试小龙虾的焦虑样行为
Crayfish are omnivorous freshwater arthropods that naturally explore their environment during day and night, but also frequently hide under a shelter or in a hole in case of danger. They may be submitted to various stressors, including predation, social interactions or changes in environmental parameters (temperature, water quality, oxygen, etc.). It has been recently demonstrated that, as a consequence of stress, crayfish is able to adapt its exploratory behavior by restricting movements to protective areas, a response similar to the anxiety-like behavior (ALB) observed in rodents. To reveal such a behavior in an aquatic species, we designed a plus-shaped sub aquatic maze divided in two protective dark arms and two more aversive illuminated arms. The aim of this paradigm was to place crayfish in a conflicting situation between its innate curiosity for novel environment and its aversion for light (Leo, 2014; Pellow et al., 1985). Unstressed crayfish generally explore the whole maze, including illuminated arms. By contrast stressed crayfish remain preferentially in the dark arms (Fossat et al., 2014). Stressed crayfish injected with anxiolitics (chlordiazepoxide-CDZ), behave as unstressed animals. Several parameters, related to the light arms can be easily measured from video records by commercial software This protocol could be suitable for analyzing the effects of any stressful situation on ALB in crayfish, as well as in many other aquatic species.
植物科学
Application of Silicone Tubing for Robust, Simple, High-throughput, and Time-resolved Analysis of Plant Volatiles in Field Experiments
采用硅胶试管法稳固、简单、高效和快速的分析田间试验植物的挥发性物质
Plant volatiles (PVs) mediate manifold interactions between plants and their biotic and abiotic environments (Dicke and Baldwin, 2010; Holopainen and Gershenzon, 2010). An understanding of the physiological and ecological functions of PVs must therefore be based on measurements of PV emissions under natural conditions. Yet sampling PVs in natural environments is difficult, limited by the need to transport, maintain, and power instruments, or else to employ expensive sorbent devices in replicate. Thus PVs are usually measured in the artificial environments of laboratories or climate chambers. However, polydimethysiloxane (PDMS), a sorbent commonly used for PV sampling (Van Pinxteren et al., 2010; Seethapathy and Górecki, 2012), is available as silicone tubing (ST) for as little as 0.60 €/m (versus 100-550 € apiece for standard PDMS sorbent devices). Small (mm-cm) ST pieces can be placed in any experimental setting and used for headspace sampling with little manipulation of the organism or headspace. ST pieces have absorption kinetics and capacities sufficient to sample plant headspaces on a timescale of minutes to hours, producing biologically meaningful “snapshots” of PV blends. When combined with thermal desorption (TD)-GC-MS analysis - a 40-year-old and widely available technology - ST pieces yield reproducible, sensitive, spatiotemporally resolved, quantitative data from headspace samples taken in natural environments (Kallenbach et al., 2014).
Detection and Quantification of Heme and Chlorophyll Precursors Using a High Performance Liquid Chromatography (HPLC) System Equipped with Two Fluorescence Detectors
利用双荧光探测器的高效液相色谱（HPLC）检测和定量血红素和叶绿素前体
Intermediates of tetrapyrrole biosynthetic pathway are low-abundant compounds, and their quantification is usually difficult, time consuming and requires large amounts of input material. Here, we describe a method allowing fast and accurate quantification of almost all intermediates of the heme and chlorophyll biosynthesis, including mono-vinyl and di-vinyl forms of (proto) chlorophyllide, using just a few millilitres of the cyanobacterial culture. Extracted precursors are separated by High Performance Liquid Chromatography system (HPLC) and detected by two ultra-sensitive fluorescence detectors set to different wavelengths.
Phosphatase Protection Assay: 14-3-3 Binding Protects the Phosphate group of RSG from λ Protein Phosphatase
磷酸酶保护试验：14-3-3结合保护λ蛋白磷酸酶中的RSG磷酸根
14-3-3 proteins regulate diverse cellular processes in eukaryotes by binding to phospho-serine or threonine of target proteins. One of the physiological functions of 14-3-3 is to bind and protect phosphate groups of the target proteins against phosphatases. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in the feedback regulation of biosynthetic genes of plant hormone gibberellin. 14-3-3 binds to phospho-Ser-114 in RSG. Ca2+-dependent protein kinase NtCDPK1 was identified as a kinase that phosphorylates Ser-114 of RSG. Our recent study revealed that NtCDPK1 forms a heterotrimer with RSG and 14-3-3 and that 14-3-3 was transferred from NtCDPK1 to phosphorylated RSG (Ito et al., 2014). In the course of the study, we found that 14-3-3 protects the phosphate group of RSG from λ protein phosphatase in vitro. Here, we describe a protocol for in vitro phosphatase protection assay. To detect the phosphorylation state of proteins, we used Phos-tag SDS-PAGE and autoradiography. This protocol can be adapted for the examinations whether the phosphoprotein-binding proteins protect the phosphate group of target proteins from phosphatases although protein kinases may be required for the phosphorylation of target proteins.
Water Deficit Treatment and Measurement in Apple Trees
苹果树的水分亏损处理和测量
Water is considered perhaps the most limiting factor for plant growth and productivity (Boyer, 1982), and climate change predicts more frequent, more severe and longer drought periods for a significant portion of the world in coming years. Unfortunately, drought resistance is particularly difficult to measure due in part to the complexity of the underlying biology that contributes to a plant’s ability to cope with water limitations. For example, water deficit is frequently examined by detaching leaves or withholding water for a set period of time prior to tissue collection. Such approaches may elucidate the early stages of drought response but are generally not physiologically relevant for maintenance of drought resistance over a longer period. A more realistic approach is to impose a gradual water limitation with a sustained soil moisture level, particularly in the case of woody perennials. We describe here a protocol that imposes a long-term water deficit under controlled laboratory conditions that allow a molecular biology approach to understanding how woody plants survive severe water limitations. Representative data can be found in Artlip et al. (1997) and Bassett et al. (2014).
干细胞
In vivo Heterotopic Bone Formation Assay Using Isolated Mouse and Human Mesenchymal Stem Cells
采用分离的小鼠和人间充质干细胞进行体内异位骨形成分析
Exogenous (human or mouse) mesenchymal stem cells (MSCs) seed in HA scaffold and transplant subcutaneously in immunodeficient mice, the cells can finally form bone tissues in the in vivo environment. This protocol describes how to get heterotopic bone formation in mice by mesenchymal stem cells (MSCs) in hydroxyapatite (HA) scaffolds. This is a simple and robust approach to detect the bone formation by tissue engineering approaches in vivo, and it also fits for examining the roles of different factors in bone formation.
系统生物学
A Conceptual Outline for Omics Experiments Using Bioinformatics Analogies
采用生物信息类推法进行组学实验概念概述
Hypothetical proteins (HP) are those that are not characterized in the laboratory and so remain “orphaned” in genomic databases. In recent times there has been a lot of progress in characterizing HPs in the laboratory. Various methods, such as sequence capture and Next Generation Sequencing (NGS), have been used to rapidly identify HP functions and their encoded genes. Applications and methods, such as the isolation of single genes, are greatly facilitated by pull-down assays to characterize proteins. Furthermore, there are methods to extract proteins from either the whole cell or a subcellular fraction. But the weakness is that some assays are fairly expensive and laborious, and characterizing HP function is always imperfect. In the recent past, statistical interpretations of the in silico selection strategies have improved the identification of the most promising candidates, including those from various annotation methods, such as protein interaction networks (PIN). Given the improvements in technology that have permitted a substantial increase in computational annotation, we ask if the prediction of HP function in silico (validation of models through algorithms and data subsets) could likewise be improved. In this work, we apply a bioinformatics analogy to each step of a wet lab experiment performed to predict aspects confirming protein function. Although it may be a less bona fide approach, assigning a putative function from conservation observed in homologous protein sequences might be worthwhile to consider prior to a wet lab experiment.