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Biochemistry

Measuring Oxidative Stress in Caenorhabditis elegans: Paraquat and Juglone Sensitivity Assays Authors:  Megan M. Senchuk, Dylan J. Dues and Jeremy M. Van Raamsdonk, date: 01/05/2017, view: 3513, Q&A: 0
Oxidative stress has been proposed to be one of the main causes of aging and has been implicated in the pathogenesis of many diseases. Sensitivity to oxidative stress can be measured by quantifying survival following exposure to a reactive oxygen species (ROS)-generating compound such as paraquat or juglone. Sensitivity to oxidative stress is a ...
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Examination of the Interaction between a Membrane Active Peptide and Artificial Bilayers by Dual Polarisation Interferometry Authors:  Jennifer A.E. Payne, Tzong-Hsien Lee, Marilyn A. Anderson and Marie-Isabel Aguilar, date: 01/05/2017, view: 1845, Q&A: 0
Examining the interaction of peptides with lipid bilayers to determine binding kinetics is often performed using surface plasmon resonance (SPR). Here we describe the technique of dual polarisation interferometry (DPI) that provides not only information on the kinetics of the peptide binding to the bilayer, but also how the peptide affects the ...
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Cancer Biology

Measuring Procaspase-8 and -10 Processing upon Apoptosis Induction Authors:  Sabine Pietkiewicz, Clara Wolfe, Jörn H. Buchbinder and Inna N. Lavrik, date: 01/05/2017, view: 2228, Q&A: 0
Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling Complex (DISC), where the initiator caspases-8 ...
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In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease Authors:  Sakshi Chauhan, Gajendra Kumar Azad and Raghuvir Singh Tomar, date: 01/05/2017, view: 2440, Q&A: 0
Histone proteins are subjected to a wide array of reversible and irreversible post-translational modifications (PTMs) (Bannister and Kouzarides, 2011; Azad and Tomar, 2014). The PTMs on histones are known to regulate chromatin structure and function. Histones are irreversibly modified by proteolytic clipping of their tail domains. The proteolytic ...
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Generation of Tumour-stroma Minispheroids for Drug Efficacy Testing Authors:  Mark Watters and Eva Szegezdi, date: 01/05/2017, view: 2115, Q&A: 0
The three-dimensional organisation of cells in a tissue and their interaction with adjacent cells and extracellular matrix is a key determinant of cellular responses, including how tumour cells respond to stress conditions or therapeutic drugs (Elliott and Yuan, 2011). In vivo, tumour cells are embedded in a stroma formed primarily by ...
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In vivo Efficacy Studies in Cell Line and Patient-derived Xenograft Mouse Models Authors:  Elizabeth A. Tovar, Curt J. Essenburg and Carrie Graveel, date: 01/05/2017, view: 2962, Q&A: 0
In vivo xenograft models derived from human cancer cells have been a gold standard for evaluating the genetic drivers of cancer and are valuable preclinical models for evaluating the efficacy of cancer therapeutics. Recently, patient-derived tumorgrafts from multiple tumor types have been developed and shown to more accurately ...
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Relative Stiffness Measurements of Cell-embedded Hydrogels by Shear Rheology in vitro Authors:  Thomas R. Cox and Chris D. Madsen, date: 01/05/2017, view: 2631, Q&A: 0
Hydrogel systems composed of purified extracellular matrix (ECM) components (such as collagen, fibrin, Matrigel, and methylcellulose) are a mainstay of cell and molecular biology research. They are used extensively in many applications including tissue regeneration platforms, studying organ development, and pathological disease models such as ...
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Immunology

FICZ Exposure and Viral Infection in Mice Authors:  Taisho Yamada and Akinori Takaoka, date: 01/05/2017, view: 1938, Q&A: 0
The aryl hydrocarbon receptor (AHR) is known as a sensor for dioxins that mediates their toxicity, and also has important biophysiological roles such as circadian rhythms, cell differentiation and immune responses. 6-formylindolo(3,2-b)carbazole (FICZ), which is derived through the metabolism of L-tryptophan by ultraviolet B irradiation, is one of ...
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In vitro Treatment of Mouse and Human Cells with Endogenous Ligands for Activation of the Aryl Hydrocarbon Receptor Authors:  Taisho Yamada and Akinori Takaoka, date: 01/05/2017, view: 1906, Q&A: 0
Activation of the aryl hydrocarbon receptor (AHR) by endogenous ligands has been implicated in a variety of physiological processes such as cell cycle regulation, cell differentiation and immune responses. It is reported that tryptophan metabolites, such as kynurenine (Kyn) and 6-formylindolo(3,2-b)carbazole (FICZ), are endogenous ligands for AHR ...
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Simultaneous Intranasal/Intravascular Antibody Labeling of CD4+ T Cells in Mouse Lungs Authors:  Yanqun Wang, Jing Sun, Rudragouda Channappanavar, Jingxian Zhao, Stanley Perlman and Jincun Zhao, date: 01/05/2017, view: 2137, Q&A: 0
CD4+ T cell responses have been shown to be protective in many respiratory virus infections. In the respiratory tract, CD4+ T cells include cells in the airway and parenchyma and cells adhering to the pulmonary vasculature. Here we discuss in detail the methods that are useful for characterizing CD4+ T cells in ...
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Microbiology

Pilot-scale Columns Equipped with Aqueous and Solid-phase Sampling Ports Enable Geochemical and Molecular Microbial Investigations of Anoxic Biological Processes Authors:  Dina M. Drennan, Robert Almstrand, Ilsu Lee, Lee Landkamer, Linda Figueroa and Jonathan O. Sharp, date: 01/05/2017, view: 1729, Q&A: 0
Column studies can be employed to query systems that mimic environmentally relevant flow-through processes in natural and built environments. Sampling these systems spatially throughout operation, while maintaining the integrity of aqueous and solid-phase samples for geochemical and microbial analyses, can be challenging particularly when redox ...
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Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis Authors:  Brenda S. Pratte and Teresa Thiel, date: 01/05/2017, view: 1896, Q&A: 0
One of the most successful fluorescent proteins, used as a reporter of gene expression in many bacterial, plant and animals, is green fluorescent protein and its modified forms, which also function well in cyanobacteria. However, these fluorescent proteins do not allow rapid and economical quantitation of the reporter gene product, as does the ...
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Bacterial Intracellular Sodium Ion Measurement using CoroNa Green Authors:  Yusuke V. Morimoto, Keiichi Namba and Tohru Minamino, date: 01/05/2017, view: 2320, Q&A: 0
The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure ...
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Measurements of Free-swimming Speed of Motile Salmonella Cells in Liquid Media Authors:  Yusuke V. Morimoto, Keiichi Namba and Tohru Minamino, date: 01/05/2017, view: 1733, Q&A: 0
Bacteria such as Escherichia coli and Salmonella enterica swim in liquid media using the bacterial flagella. The flagellum consists of the basal body (rotary motor), the hook (universal joint) and the filament (helical screw). Since mutants with a defect in flagellar assembly and function cannot swim smoothly, motility assay is ...
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Neuroscience

Optogenetic Mapping of Synaptic Connections in Mouse Brain Slices to Define the Functional Connectome of Identified Neuronal Populations Authors:  Susana Mingote, Nao Chuhma and Stephen Rayport, date: 01/05/2017, view: 2579, Q&A: 0
Functional connectivity in a neural circuit is determined by the strength, incidence, and neurotransmitter nature of its connections (Chuhma, 2015). Using optogenetics the functional synaptic connections between an identified population of neurons and defined postsynaptic target neurons may be measured systematically in order to determine the ...
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Primary Culture of Mouse Neurons from the Spinal Cord Dorsal Horn Authors:  De-Li Cao, Peng-Bo Jing, Bao-Chun Jiang and Yong-Jing Gao, date: 01/05/2017, view: 3004, Q&A: 0
Primary afferents of sensory neurons mainly terminate in the spinal cord dorsal horn, which has an important role in the integration and modulation of sensory-related signals. Primary culture of mouse spinal dorsal horn neuron (SDHN) is useful for studying signal transmission from peripheral nervous system to the brain, as well as for developing ...
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Plant Science

In vitro Ubiquitin Dimer Formation Assay Authors:  Sheng Wang, Ling Cao and Hong Wang, date: 01/05/2017, view: 2001, Q&A: 0
The process of protein ubiquitination typically consists of three sequential steps to add an ubiquitin (Ub) or Ub chain to a substrate protein, requiring three different enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Most E2s possess the classical E2 activity in forming E2-Ub ...
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Cation (Ca2+ and Mn2+) Partitioning Assays with Intact Arabidopsis Chloroplasts Authors:  Anna Harms, Iris Steinberger and Anja Schneider, date: 01/05/2017, view: 2220, Q&A: 0
Determination of the relative distribution of Ca2+ and Mn2+ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative distribution of calcium ...
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Dot Blot Analysis of N6-methyladenosine RNA Modification Levels Authors:  Lisha Shen, Zhe Liang and Hao Yu, date: 01/05/2017, view: 2941, Q&A: 0
N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). The total amount of m6A can be detected by several methods, such as dot blot analysis using specific m6A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fu et al ...
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Stem Cell

Ex vivo Culture of Adult Mouse Antral Glands Authors:  Valeria Fernandez Vallone, Morgane Leprovots, Gilbert Vassart and Marie-Isabelle Garcia, date: 01/05/2017, view: 1933, Q&A: 0
The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker et al. (2010), who ...
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Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors Authors:  Valeria Fernandez Vallone, Morgane Leprovots, Gilbert Vassart and Marie-Isabelle Garcia, date: 01/05/2017, view: 2413, Q&A: 0
Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata et al. described the isolation of ...
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