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Past Issues in 2014

Volume: 4 Issue: 15

Biochemistry

Quantitative Analysis of Cellular Diacylglycerol Content Authors: Fikadu G. Tafesse, Karin Strijbis and Hidde L. Ploegh, date: 08/05/2014, view: 7423, Q&A: 0

Diacylglycerol (DAG) is a bioactive lipid with diverse biological roles. DAG transiently accumulates in a membrane upon receipt of an appropriate stimulus that activates phospholipase C to cleave phospholipids. The resulting hydrolysis product DAG binds to proteins such as protein kinase C to initiate a variety of downstream cellular processes. ...

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Immunology

Generation and Maturation of Human Monocyte-derived DCs Authors: Michela Spadaro, Monica Montone and Federica Cavallo, date: 08/05/2014, view: 15891, Q&A: 0

Dendritic cells (DC) are antigen-presenting cells, which play a critical role in the regulation of the adaptive immune response. They act as a bridge between the innate and the adaptive immune systems. An approach to study their function and potentiality is to generate DC-like cells by culturing CD14⁺ monocyte-enriched peripheral blood ...

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Microbiology

Purification of Herpesvirus Virions and Capsids Authors: Xinghong Dai and Z. Hong Zhou, date: 08/05/2014, view: 9678, Q&A: 4

This protocol was designed for large-scale purification of herpesvirus particles by cell culture. Virions and capsids are isolated from extracellular culture media and cell nuclei, respectively. Purity and concentration of the purified samples are usually sufficient for structural studies with cryo electron microscopy and cryo electron tomography. ...

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Infectious Virus Yield Assay for Hepatitis E Virus Authors: Yannick Debing, Kai Dallmeier and Johan Neyts, date: 08/05/2014, view: 6592, Q&A: 0

Hepatitis E virus (HEV) is one of the main causes of acute hepatitis worldwide. Infections are particularly severe in pregnant women and chronic hepatitis E is known to occur in immunocompromised patients. Current therapy (ribavirin or pegylated alpha interferon) has severe side effects and cannot be employed in all patients. In order to evaluate ...

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Luminescence-based Antiviral Assay for Hepatitis E Virus Authors: Yannick Debing, Kai Dallmeier and Johan Neyts, date: 08/05/2014, view: 6154, Q&A: 0

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Quantification of Flavin Production by Bacteria Authors: Svetlana N. Yurgel, Joseph Lynch, Jennifer Rice, Neil Adhikari and Sanja Roje, date: 08/05/2014, view: 6211, Q&A: 0

This protocol provides a simple and fast method of quantification for intracellular flavin content, and for flavin secretion by bacteria. Intracellular flavins are extracted from bacterial pellets, and secreted flavins are examined in the cell growth medium. Flavins are separated and measured using HPLC with fluorescence detection, and quantified ...

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Assay for GTP Cyclohydrolase II Activity in Bacterial Extracts Authors: Svetlana N. Yurgel, Na Sa, Jennifer Rice and Sanja Roje, date: 08/05/2014, view: 4531, Q&A: 0

Riboflavin is the precursor of flavin nucleotides FMN and FAD, they play significant roles in all organisms. GTP is the initial precursor on riboflavin biosynthesis pathway and GTP cyclohydrolase II catalyzes the first step of this pathway. It converts GTP to 2,5-diamino-6-ribosylamino-4 (3H) -pyrimidinone 5'-phosphate. This protocol ...

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Fluorometric Estimation of Viral Thermal Stability Authors: Vamseedhar Rayaprolu, Shannon Kruse, Ravi Kant, Navid Movahed, Dewey Brooke and Brian Bothner, date: 08/05/2014, view: 6547, Q&A: 1

Differential Scanning Fluorimetry (DSF) is a rapid, economical, and a straightforward technique for estimating the thermal stability of proteins. The principle involves the binding of a fluorescent dye to thermally exposed hydrophobic pockets of a protein. The dyes used in this technique are highly fluorescent in a non-polar environment and are ...

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Plant Science

Isolation of Tomato Fruit Chromoplasts and Determination of ATP Levels Authors: Francesc Hernández-Gras, Rocco Petrizzo, Irini Pateraki, Marta Renato, Maxime Angaman, Joaquín Azcón-Bieto and Albert Boronat, date: 08/05/2014, view: 6254, Q&A: 0

It has recently been reported that tomato fruit chromoplasts can synthesize ATP de novo using an ATP synthase complex harboring an atypical γ-subunit which is also present in a variety of plant species. However many aspects related with the biochemical processes underlying this process remain largely unknown. Here we describe detailed ...

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Quantifying Fruit Dehiscence Using the Random Impact Test (RIT) Authors: Teresa Lenser and Günter Theißen, date: 08/05/2014, view: 5531, Q&A: 0

Fruit dehiscence is an important evolutionary and agronomic trait. For quantifying and comparing the exact fruit dehiscence capability between individual plants, the random impact test has been described (Morgan et al., 1998; Bruce et al., 2002; Arnaud et al., 2010). Here, we describe the random impact test optimized to ...

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Floral Dip Transformation in Lepidium campestre Authors: Teresa Lenser and Günter Theißen, date: 08/05/2014, view: 6715, Q&A: 0

Floral dip is a very common technique to stably transform Arabidopsis thaliana (Clough and Bent, 1998; Martinez-Trujillo et al., 2004; Zhang et al., 2006) and has also been adapted to some other plant species (Curtis and Nam, 2001; Tague, 2001; Bartholmes et al., 2008). Here, we describe this method optimized ...

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DNA Fragmentation Analysis Authors: Kei-ichiro Mishiba, Yukihiro Nagashima, Noriko Hayashi and Nozomu Koizumi, date: 08/05/2014, view: 10711, Q&A: 0

DNA fragmentation with length corresponding to multiple integer of approximately 180 base pairs is a distinct feature of apoptosis in animals and programmed cell death in plants. This feature can simply be detected by DNA gel electrophoresis followed by ethidium bromide staining, although in some cases it is difficult to distinguish the DNA ...

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