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Past Issues in 2018

Volume: 8 Issue: 1

Biochemistry

Determining Ribosome Translational Status by Ribo-ELISA Authors: Amandine Bastide, Jonathan W. Yewdell and Alexandre David, date: 01/05/2018, view: 5618, Q&A: 0

The Ribo-ELISA was originally developed to elucidate the basis for the ribopuromycylation method (RPM)-based detection of ribosome bound nascent chains. The Ribo-ELISA enables characterization of the translational status of ribosomes, and can be applied to the discovery of super-ribosomal complexes with novel ribosome associated macromolecules ...

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Cell Biology

High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells Authors: Xingmin Aaron Zhang and Thomas F.J. Martin, date: 01/05/2018, view: 5713, Q&A: 0

Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay using DCV cargo, NPY-Venus, and an orthogonal ³H-serotonin secretion assay. The NPY-Venus ...

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Immunology

Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H₂O₂ Authors: Seeyoung Choi and Paul E. Love, date: 01/05/2018, view: 5508, Q&A: 0

Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a ‘signature’ cysteine residue within the catalytic domain that is maintained in the ...

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Microbiology

Easy and Efficient Permeabilization of Cyanobacteria for in vivo Enzyme Assays Using B-PER Authors: Simon Matthé Erstad and Yumiko Sakuragi, date: 01/05/2018, view: 6098, Q&A: 0

Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO₂ and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. A better understanding ...

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Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9 Authors: Marie-Laurence Lemay, Ariane C. Renaud, Geneviève M. Rousseau and Sylvain Moineau, date: 01/05/2018, view: 14279, Q&A: 1

This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is ...

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Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay Authors: Ana Lechuga, Mónica Berjón-Otero, Margarita Salas and Modesto Redrejo-Rodríguez, date: 01/05/2018, view: 8746, Q&A: 0

This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules ...

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Transplantation of Fecal Microbiota Shaped by Diet Authors: Keiran H. McLeod, Linda Mason and Eliana Mariño, date: 01/05/2018, view: 7878, Q&A: 0

Alterations in diet and gut microbial ecology underlie the pathogenesis of type 1 diabetes (T1D). In the non-obese diabetic (NOD) mouse, we found high concentrations of bacterial metabolites acetate and butyrate in blood and faeces correlated with protection from disease. We reconstituted germ free (GF) NOD mice with fecal bacteria from protected ...

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Bacterial Aggregation Assay in the Presence of Cyclic Lipopeptides Authors: Pengyuan Xiu, Rui Liu, Dechao Zhang and Chaomin Sun, date: 01/05/2018, view: 7401, Q&A: 0

Lipopeptides is an important class of biosurfactants having antimicrobial and anti-adhesive activity against pathogenic bacteria. These include surfactin, fengycin, iturin, bacillomycin, mycosubtilin, lichenysin, and pumilacidin (Arima et al., 1968; Naruse et al., 1990; Yakimov et al., 1995; Steller and Vater, 2000; ...

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Neuroscience

A Rodent Model for Chronic Brain Hypoperfusion Related Diseases: Permanent Bilateral Occlusion of the Common Carotid Arteries (2VO) in Rats Authors: Mei-Ling Yan and Jing Ai, date: 01/05/2018, view: 7632, Q&A: 0

Permanent occlusion of bilateral common carotid arteries (2VO) in rat is considered as a suitable animal model to mimic chronic brain hypoperfusion status, which is proved to be a risk factor to precede the Alzheimer’s disease or/and vascular dementia. In this protocol, we describe how to successfully ligate the bilateral common carotid arteries ...

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The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level Authors: Amandine Bastide, Jonathan W. Yewdell and Alexandre David, date: 01/05/2018, view: 11043, Q&A: 0

While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ribopuromycylation method (RPM) was primarily to map translation sites at the sub-cellular level while avoiding ...

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Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons Authors: Katarina Trajkovic, Hyunkyung Jeong and Dimitri Krainc, date: 01/05/2018, view: 6336, Q&A: 0

Quantitative analysis of proteins secreted from the cells poses a challenge due to their low abundance and the interfering presence of a large amount of bovine serum albumin (BSA) in the cell culture media. We established assays for detection of mutant huntingtin (mHtt) secreted from Neuro2A cell line stably expressing mHtt and rat primary ...

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Behavioral Assays to Study Oxygen and Carbon Dioxide Sensing in Caenorhabditis elegans Authors: Teresa Rojo Romanos, Leelee Ng, Manuel Zimmer and Roger Pocock, date: 01/05/2018, view: 5826, Q&A: 0

Animals use behavioral strategies to seek optimal environments. Population behavioral assays provide a robust means to determine the effect of genetic perturbations on the ability of animals to sense and respond to changes in the environment. Here, we describe a C. elegans population behavioral assay used to measure locomotory responses ...

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Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method for Visual Detection of Protein-protein Interactions Authors: Yohei Katoh, Kentaro Nakamura and Kazuhisa Nakayama, date: 01/05/2018, view: 11078, Q&A: 0

The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation (Katoh et al., 2015). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation with GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads, protein-protein interactions can ...

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Plant Science

Rolling Circle Amplification to Screen Yam Germplasm for Badnavirus Infections and to Amplify and Characterise Novel Badnavirus Genomes Authors: Moritz Bömer, Aliyu A. Turaki, Ajith I. Rathnayake, Gonçalo Silva, P. Lava Kumar and Susan E. Seal, date: 01/05/2018, view: 8706, Q&A: 0

Since the first discovery of badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 2008; Bousalem ...

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In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6 Authors: Kyungmin Baeg, Yukihide Tomari and Hiro-oki Iwakawa, date: 01/05/2018, view: 8884, Q&A: 0

RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant ...

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Fatty Acid Content and Composition of Triacylglycerols of Chlorella kessleri Authors: Motohide Aoki and Norihiro Sato, date: 01/05/2018, view: 7743, Q&A: 0

Triacylglycerols (TAGs) are esters formed from one glycerol and three fatty acids. TAGs are induced to accumulate in algal cells under environmental stress conditions including nutrient-limitation, hyperosmosis, and low temperature, for the storage of metabolic energy and carbon, and also for the consumption of excess energy (e.g., Hirai ...

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Chromatin Affinity Purification (ChAP) from Arabidopsis thaliana Rosette Leaves Using in vivo Biotinylation System Authors: Weronika Sura and Piotr A. Ziolkowski, date: 01/05/2018, view: 7459, Q&A: 0

Chromatin Affinity Purification (ChAP) is widely used to study chromatin architecture and protein complexes interacting with DNA. Here we present an efficient method for ChAP from Arabidopsis thaliana rosette leaves, in which in vivo biotinylation system is used. The chromatin is digested by Micrococcal Nuclease (MNase), hence ...

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Electron Tomography to Study the Three-dimensional Structure of Plasmodesmata in Plant Tissues–from High Pressure Freezing Preparation to Ultrathin Section Collection Authors: William J. Nicolas, Emmanuelle Bayer and Lysiane Brocard, date: 01/05/2018, view: 8184, Q&A: 0

Plasmodesmata (PD) are nanometric (~20 nm wide) membrane lined pores encased in the cell walls of the adjacent plant cells. They allow the cells to exchange all types of molecules ranging from nutrients like sugar, hormones, to RNAs and various proteins. Unfortunately, they are also hijacked by phyto-viruses, enabling them to spread from ...

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Micro-computed Tomography to Visualize Vascular Networks in Maize Stems Authors: Akiteru Maeno and Katsutoshi Tsuda, date: 01/05/2018, view: 8740, Q&A: 0

Plant vascular systems in the stem connect roots with aerial organs to move solutes containing minerals, nutrients as well as signaling molecules, and therefore, they play pivotal roles in plant growth and development. However, stem vascular systems, especially in crop species, have been poorly described since they are deeply embedded in the ...

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Real-time Analysis of Auxin Response, Cell Wall pH and Elongation in Arabidopsis thaliana Hypocotyls Authors: Lanxin Li, S. F. Gabriel Krens, Matyáš Fendrych and Jiří Friml, date: 01/05/2018, view: 8083, Q&A: 0

The rapid auxin-triggered growth of the Arabidopsis hypocotyls involves the nuclear TIR1/AFB-Aux/IAA signaling and is accompanied by acidification of the apoplast and cell walls (Fendrych et al., 2016). Here, we describe in detail the method for analysis of the elongation and the TIR1/AFB-Aux/IAA-dependent auxin response in ...

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