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Cancer Biology

Induction of Colitis and Colitis-associated Colorectal Cancer (CAC) Authors:  Jalaj Gupta and Angel R. Nebreda, date: 11/20/2014, view: 7755, Q&A: 0
Inflammatory bowel disease (IBD) including Crohn’s disease and ulcerative colitis are characterized by chronic, progressive and relapsing inflammatory disorders. Existing evidence indicate that IBD is associated with a higher risk of developing CAC, which is directly related to the duration and extent of colitis. Thus, animal models have been ...
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Purification of Tumor-Associated Macrophages (TAM) and Tumor-Associated Dendritic Cells (TADC) Authors:  Damya Laoui, Eva Van Overmeire, Jiri Keirsse, Kiavash Movahedi and Jo A Van Ginderachter, date: 11/20/2014, view: 12838, Q&A: 1
Tumors are heterogeneous microenvironments where complex interactions take place between neoplastic cells and infiltrating inflammatory cells, such as tumor-associated macrophages (TAM) and tumor-associated dendritic cells (TADC). The relevance of tumor-infiltrating mononuclear myeloid cells is underscored by clinical studies showing a correlation ...
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Cell Biology

Analysis of Intestinal Permeability in Mice Authors:  Jalaj Gupta and Angel R. Nebreda, date: 11/20/2014, view: 21065, Q&A: 5
The intestinal epithelial layer serves as a barrier against pathogens and ingested toxins, which are present in the lumen of the intestine. The importance of the intestinal epithelial barrier is emphasized by the alterations in paracellular permeability and tight junction functions observed in inflammatory bowel disease (IBD) and colon cancer.
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Preparation of Golgi Membranes from Rat Liver Authors:  Carmen Valente, Gabriele Turacchio, Stefania Spanò, Alberto Luini and Daniela Corda, date: 11/20/2014, view: 3719, Q&A: 0
This protocol details the isolation of enriched Golgi membranes from rat liver, using discontinuous density gradient centrifugation. This high-yield extraction method is useful for several applications, including immunoprecipitation of solubilised Golgi membrane proteins (preparation included) and electron microscopy. Protocol adapted from ...
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Separation of the Inner and Outer Mitochondrial Membrane in HeLa Cells Authors:  Naotaka Nishimura and Masato Yano, date: 11/20/2014, view: 11737, Q&A: 0
Mitochondria are organelles that have important functions in oxidative phosphorylation, fatty acid oxidation and apoptosis signaling. They have two distinct membranes, outer membrane (OM) and inner membrane (IM). IM contains respiratory chain complexes that produce ATP. IM is rich in cardiolipin, a specific phospholipid reportedly having a ...
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Assay of Ornithine Decarboxylase and Spermidine/Spermine N1-acetyltransferase Activities Authors:  Mervi T. Hyvönen, Tuomo Keinänen and Leena Alhonen, date: 11/20/2014, view: 4730, Q&A: 0
The polyamines, spermidine (Spd) and spermine, and their diamine precursor putrescine, are important regulators of various cellular functions, such as proliferation and differentiation. Polyamine homeostasis is tightly regulated on the level of uptake, excretion, biosynthesis, interconversion and terminal catabolism. The rate-controlling enzymes ...
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Microbiology

Purification and Sequencing of DNA Guides from Prokaryotic Argonaute Authors:  Daan C. Swarts, Edze R. Westra, Stan J. J. Brouns and John van der Oost, date: 11/20/2014, view: 5324, Q&A: 0
Some proteins utilize nucleic acids to guide them to complementary nucleic acid targets. One example is prokaryotic Argonaute protein, which, binds small single stranded DNA molecules as guides (Swarts et al., 2014). This protocol describes a method to purify DNA guides from these proteins. It also describes a PCR-based method to enrich ...
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Infectious Focus Assays and Multiplicity of Infection (MOI) Calculations for Alpha-herpesviruses Authors:  Anna Sloutskin and Ronald S. Goldstein, date: 11/20/2014, view: 18547, Q&A: 0
Titration of viral stocks is a critical process before any experimental use of the virus. Here we describe an infectious focus assay for several alphaherpesviruses, a titration method for fluorescently labeled viruses, based on the original plaque assay. In addition, the calculation of multiplicity of infection (MOI) is presented.
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Semi-denaturing Detergent Agarose Gel Electrophoresis (SDD-AGE) Authors:  Laura Molina-García and Fátima Gasset-Rosa, date: 11/20/2014, view: 7637, Q&A: 0
Pathological proteins in neurodegenerative diseases suffer a conformational change to a misfolded amyloid state. Such pathological event leads to the aggregation of these proteins that indefinitely propagates as an altered form of itself, and harbor prion-like properties (Wickner, 1994; Prusiner, 2012). In addition to diseases, prions can also ...
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Competitive ELISA for Protein-lipopolysaccharide (LPS) Binding Authors:  Victoria Martínez-Sernández and Florencio M. Ubeira, date: 11/20/2014, view: 5968, Q&A: 1
Lipopolysaccharide is the major constituent of the outer membrane of gram-negative bacteria and, once released from the bacterial surface into the bloodstream, is a potent activator of the host immune system, which can lead to septic shock. LPS has a hydrophilic region consisting of a repeating oligosaccharide that is strain-specific (O-antigen) ...
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Isolation and Characterisation of Dendritic Cells from Peripheral Blood Authors:  Matthew Reeves and John Sinclair, date: 11/20/2014, view: 8122, Q&A: 0
Latency and reactivation of human cytomegalovirus (HCMV) is intimately associated with the myeloid lineage. Multiple studies have used in vitro protocols to generate dendritic cells (DCs) from myeloid precursors. Here we describe the direct isolation of DCs from peripheral blood to study HCMV latency directly in this cell type.
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Secretion Assay in Shigella flexneri Authors:  Jonathan Reinhardt and Michael Kolbe, date: 11/20/2014, view: 5008, Q&A: 0
Shigella flexneri (S. flexneri) is a Gram-negative bacterium that causes gastroenteritis and shigellosis in humans. In order to establish and maintain an infection, S. flexneri utilises a type three secretion system (T3SS) to deliver virulence factors called effector proteins into the cytoplasm of host cells, ...
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Plant Science

In vitro Detection of S-acylation on Recombinant Proteins via the Biotin-Switch Technique Authors:  Dong Qi and Roger W. Innes, date: 11/20/2014, view: 4310, Q&A: 0
Protein palmitoylation is the post-translational modification of proteins via the attachment of palmitate through acyl linkages. The nucleophile sulfhydryl group of cysteines is the common palmitoylation site. Covalent attachment of palmitate occurs on numerous proteins and is usually associated with directing protein localization to the ...
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Stem Cell

Isolation of FAP Cells from Mouse Dystrophic Skeletal Muscle Using Fluorescence Activated Cell Sorting Authors:  Nicoletta Cordani, Viviana Pisa, Laura Pozzi and Clara Sciorati, date: 11/20/2014, view: 5480, Q&A: 1
A population of muscle resident CD45-, CD31- cells expressing the mesenchymal PDGF receptor alpha (PDGFRα) as well as Sca-1 was first isolated in healthy mouse muscles in Uezumi et al. (2010). In the same year, Joe et al. (2010) identified and purified fibro-adipogenic precursors (FAPs), cells located into ...
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