• Volume 9, 2019
  • Volume 8, 2018
  • Volume 7, 2017
  • Volume 6, 2016
  • Volume 5, 2015
  • Volume 4, 2014
  • Volume 3, 2013
  • Volume 2, 2012
  • Volume 1, 2011

Biochemistry

Cellular Translational Reporter Assay Authors:  Taishi Kimura and Kiyoshi Takeda, date: 03/20/2014, view: 6972, Q&A: 0
The method described here allows measuring the effect of exogenously introduced modifications to in vitro-transcribed mRNA on the translation in cells. Using cells derived from knockout mice and control littermates, this method enables to compare the results in the presence or absence of specific gene products. In our lab, we used this ...
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Synthesis of the Adenosine A2A Receptor Fluorescent Agonist MRS5424 Authors:  Kenneth A. Jacobson and Francisco Ciruela, date: 03/20/2014, view: 5804, Q&A: 0
MRS5424 is a functional fluorescent agonist for the adenosine A2A receptor (A2AR) in which the fluorescent dye Alexa Fluor 532 is covalently attached to the A2AR agonist 2-[[2-[4-[2-(2-aminoethyl)-aminocarbonyl]ethyl]phenyl]ethylamino]-5'-N-ethyl-carboxamidoadenosine (APEC). This easy-to-synthesize new A2A ...
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Cancer Biology

In vitro Regulatory T cells Differentiation From Naïve T Cells Authors:  Tomás Dalotto-Moreno, Gabriel A. Rabinovich and Marian Salatino, date: 03/20/2014, view: 15855, Q&A: 1
In the past years, a subset of regulatory T cells (Tregs) expressing CD4, CD25 and the transcription factor FoxP3 has gained considerable attention as key regulators of T-cell tolerance and homeostasis (Sakaguchi, 2004). This population of T cells is specifically engaged in the maintenance of immune self-tolerance and the control of aberrant ...
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Immunology

Measurement of CD8 and CD4 T Cell Responses in Mouse Lungs Authors:  Craig Fett, Jincun Zhao and Stanley Perlman, date: 03/20/2014, view: 10022, Q&A: 0
Study of the adaptive immune response to a viral challenge in an animal model often includes analysis of the T cell response. Here we discuss in detail the methods that are used to characterize the CD8 and CD4 T cell response following viral challenge in the lung.
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Microbiology

Western Blotting for Staphylococcus aureus AgrA Authors:  Chikara Kaito and Kazuhisa Sekimizu, date: 03/20/2014, view: 8877, Q&A: 0
Staphylococcus aureus has a quorum sensing system to regulate the expression of various virulence factors, which is exerted by the agr locus that encodes agrBDCA and a regulatory RNA called RNAIII. AgrB, AgrD, and AgrC proteins are involved in producing and recognizing extracellular quorum sensing molecules and transduce ...
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Catalase Activity Assay in Candida glabrata Authors:  Emmanuel Orta-Zavalza, Marcela Briones-Martin-del-Campo, Irene Castano and Alejandro De Las Penas, date: 03/20/2014, view: 13727, Q&A: 0
Commensal and pathogenic fungi are exposed to hydrogen peroxide (H2O2) produced by macrophages of the host. Pathogenic fungi counteract the harmful effects of H2O2 with the enzyme catalase (EC 1.11.1.6), which decomposes two molecules of H2O2 to two molecules of H2O and O ...
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Measuring Genetic Robustness in Vesicular Stomatitis Virus Author:  Isabel S. Novella, date: 03/20/2014, view: 6212, Q&A: 0
Genetic robustness is the ability of a genome to incorporate mutations with the result of no fitness changes. Thus, more robust viruses have an increased neutral mutation rate. This property is particularly important in RNA viruses due to their high mutation rates. The most direct way of measuring robustness in vesicular stomatitis virus (VSV) is ...
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Fitness Determinations in Vesicular Stomatitis Virus Author:  Isabel S. Novella, date: 03/20/2014, view: 5343, Q&A: 0
Fitness is defined as the overall replicative ability. Testing whether a mutation (or combination of mutations) has an effect on fitness often relays on determining virus production as a surrogate measurement. However, viruses do not usually replicate in a void, and evolutionary speaking, it is key to determine replicative ability compared to ...
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Determination of Mutation Frequency During Viral DNA Replication Authors:  Modesto Redrejo-Rodríguez, Javier M. Rodríguez, Cristina Suárez and María L. Salas, date: 03/20/2014, view: 6332, Q&A: 0
This protocol is a simple method for evaluating mutation frequency during African swine fever virus (ASFV) replication, although it could be used also for other DNA viruses (poxvirus, herpesvirus, mimivirus, etc) with minor modifications. In the original Carrascosa et al. (1982), the protocol was carried out with two cloned ...
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RNA Isolation and Northern Blot Analysis Authors:  Ying Liao, To Sing Fung, Mei Huang, Shouguo Fang, Yanxin Zhong and Dingxiang Liu, date: 03/20/2014, view: 20156, Q&A: 0
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Here, we examine ATF3, ATF4, and GADD153 gene expression ...
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Human Astrovirus Propagation, Purification and Quantification Authors:  Shauna Marvin, Victoria Meliopoulos and Stacey Schultz-Cherry, date: 03/20/2014, view: 8157, Q&A: 0
Astrovirus are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. Here, we describe astrovirus propagation, purification and titration. The Caco-2 human intestinal adenocarcinoma cell line is most widely used for studying astrovirus, although other cell lines, such as 293, T84 and LLC-MK2 ...
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Helicase Assays Authors:  Xin Wang, Jing Li, Jason Diaz and Jianxin You, date: 03/20/2014, view: 9977, Q&A: 0
Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair ...
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Virus Infection and Titration of SARS-CoV in Mouse Lung Authors:  Craig Fett, Jincun Zhao and Stanley Perlman, date: 03/20/2014, view: 7624, Q&A: 0
Two critical steps when investigating an animal model of a virus infection are consistently successfully infecting animals and accurately determining viral titers in tissue throughout the course of infection. Here we discuss in detail how to infect mice with SARS-CoV and then quantify the titer of virus in the lung.
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Neuroscience

Adenosine A2A Receptor Ligand Binding Experiments by Using Real-time Single-cell FRET Authors:  Víctor Fernández-Dueñas, Kenneth A. Jacobson and Francisco Ciruela, date: 03/20/2014, view: 7417, Q&A: 0
We designed a fluorescence resonance energy transfer (FRET)-based approach to study the ligand binding constants of the adenosine A2A receptor (A2AR). Our assay is based in the interaction of a fluorescent A2AR agonist ligand (MRS5424) with an A2AR tagged with the cyan fluorescent protein (CFP) at the ...
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Transpharyngeal Exposure of GnRH Neurons Authors:  Stephanie Constantin and Allan E. Herbison, date: 03/20/2014, view: 6716, Q&A: 0
The neurons secreting gonadotropin-releasing hormone (GnRH) control fertility in all mammalian species. However, investigations of this neuronal population are difficult as the cell bodies of the approximately 800 GnRH neurons are scattered through the basal forebrain ranging from the olfactory bulbs through to the base of the hypothalamus. While ...
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Isolation and Culture of Neurospheres for the Study of Pathogenesis of Prion Disease Authors:  Yoshifumi Iwamaru, Takato Takenouchi and Hiroshi Kitani, date: 03/20/2014, view: 16219, Q&A: 0
Neurosphere contains neural stem cells that are capable of self-renewal and multilineage differentiation including neurons, astrocytes, and oligodendrocytes (Gage, 2000). Cell culture model using differentiated neurosphere cultures are suggested to be a valuable tool for studying the pathogenesis of prion disease at the cellular level (Iwamaru et ...
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Neural Stem Cell Differentiation and Prion Infection Authors:  Yoshifumi Iwamaru, Takato Takenouchi and Hiroshi Kitani, date: 03/20/2014, view: 8002, Q&A: 0
Prion diseases are transmissible, fatal, neurodegenerative diseases in human and animals. The molecular basis of neurodegeneration in prion diseases is largely unclear. Developing a cellular model capable of monitoring prion-induced cytotoxicity would be a promising approach for better understanding the prion pathogenesis. One candidate cellular ...
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