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Past Issues in 2017

Volume: 7 Issue: 9

Biochemistry

Biotinylated Micro-RNA Pull Down Assay for Identifying miRNA Targets Authors: Pornima Phatak and James M Donahue, date: 05/05/2017, view: 16919, Q&A: 1

microRNA (miRNA) directly associates with its target transcripts (mRNA). This protocol describes a method for detection of direct interaction between miRNA and mRNA. The result of interaction helps screening the specific target mRNAs for a miRNA.

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Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes Authors: Yoshihiro Adachi, Kie Itoh, Miho Iijima and Hiromi Sesaki, date: 05/05/2017, view: 9186, Q&A: 0

A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial division is mediated by ...

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Metabolic Heavy Isotope Labeling to Study Glycerophospholipid Homeostasis of Cultured Cells Authors: Satu Hänninen, Pentti Somerharju and Martin Hermansson, date: 05/05/2017, view: 6105, Q&A: 0

Glycerophospholipids consist of a glycerophosphate backbone to which are esterified two acyl chains and a polar head group. The head group (e.g., choline, ethanolamine, serine or inositol) defines the glycerophospholipid class, while the acyl chains together with the head group define the glycerophospholipid molecular species. Stable ...

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Cancer Biology

Immunostaining of Formaldehyde-fixed Metaphase Chromosome from Untreated and Aphidicolin-treated DT40 Cells Author: Vibe H. Oestergaard, date: 05/05/2017, view: 9253, Q&A: 0

During mitosis chromosomes are condensed into dense X-shaped structures that allow for microscopic determination of karyotype as well as inspection of chromosome morphology.

This protocol describes a method to perform immunostaining of formaldehyde-fixed metaphase chromosomes from the avian cell line DT40. It was developed to ...

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Relative Stiffness Measurements of Tumour Tissues by Shear Rheology Authors: Chris D. Madsen and Thomas R. Cox, date: 05/05/2017, view: 9259, Q&A: 0

The microenvironment of solid tumours is a critical contributor to the progression of tumours and offers a promising target for therapeutic intervention (Cox and Erler, 2011; Barker et al., 2012; Cox et al., 2016; Cox and Erler, 2016). The properties of the tumour microenvironment vary significantly from that of the original ...

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Cell Biology

Nuclei Isolation from Nematode Ascaris Authors: Yuanyuan Kang, Jianbin Wang and Richard E. Davis, date: 05/05/2017, view: 8258, Q&A: 0

Preparing nuclei is necessary in a variety of experimental paradigms to study nuclear processes. In this protocol, we describe a method for rapid preparation of large number of relatively pure nuclei from Ascaris embryos or tissues that are ready to be used for further experiments such as chromatin isolation and ChIP-seq, nuclear RNA ...

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Immunology

Escherichia coli Infection of Drosophila Authors: Charles Tracy and Helmut Krämer, date: 05/05/2017, view: 7714, Q&A: 0

Following septic insults, healthy insects, just like vertebrates, mount a complex immune response to contain and destroy pathogens. The failure to efficiently clear bacterial infections in immuno-compromised fly mutants leads to higher mortality rates which provide a powerful indicator for genes with important roles in innate immunity. The ...

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Microbiology

Immunoprecipitation of Cell Surface Proteins from Gram-negative Bacteria Authors: Carlos Eduardo Pouey Cunha, Jane Newcombe, Odir Antonio Dellagostin and Johnjoe McFadden, date: 05/05/2017, view: 7897, Q&A: 0

The meningococcus (Neisseria meningitidis) remains an important threat to human health worldwide. This Gram-negative bacterium causes elevated disabilities and mortality in infected individuals. Despite several available vaccines, currently there is no universal vaccine against all circulating meningococcal strains (Vogel et al., ...

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Preparation of Everted Membrane Vesicles from Escherichia coli Cells Author: Marina Verkhovskaya, date: 05/05/2017, view: 7815, Q&A: 0

The protocol for obtaining electrically sealed membrane vesicles from E. coli cells is presented. Proton pumps such as Complex I, quinol oxidase, and ATPase are active in the obtained vesicles. Quality of the preparation was tested by monitoring the electric potential generated by these pumps.

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Incubation of Cyanobacteria under Dark, Anaerobic Conditions and Quantification of the Excreted Organic Acids by HPLC Authors: Chika Yasuda, Hiroko Iijima, Haruna Sukigara and Takashi Osanai, date: 05/05/2017, view: 7651, Q&A: 0

Succinate and lactate are commodity chemicals used for producing bioplastics. Recently, it was found that such organic acids are excreted from cells of the unicellular cyanobacterium Synechocystis sp. PCC 6803 under dark, anaerobic conditions. To conduct the dark, anaerobic incubation, cells were concentrated within a vial that was then ...

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Evaluation of Plasmid Stability by Negative Selection in Gram-negative Bacteria Authors: Damián Lobato Márquez and Laura Molina García, date: 05/05/2017, view: 11016, Q&A: 0

Plasmid stability can be measured using antibiotic-resistance plasmid derivatives by positive selection. However, highly stable plasmids are below the sensitivity range of these assays. To solve this problem we describe a novel, highly sensitive method to measure plasmid stability based on the selection of plasmid-free cells following elimination ...

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Measuring Cyanobacterial Metabolism in Biofilms with NanoSIMS Isotope Imaging and Scanning Electron Microscopy (SEM) Authors: Rhona K. Stuart, Xavier Mayali, Michael P. Thelen, Jennifer Pett-Ridge and Peter K. Weber, date: 05/05/2017, view: 8353, Q&A: 0

To advance the understanding of microbial interactions, it is becoming increasingly important to resolve the individual metabolic contributions of microorganisms in complex communities. Organisms from biofilms can be especially difficult to separate, image and analyze, and methods to address these limitations are needed. High resolution imaging ...

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Adhesion and Invasion Assay Procedure Using Caco-2 Cells for Listeria monocytogenes Authors: Swetha Reddy and Frank Austin, date: 05/05/2017, view: 13786, Q&A: 0

Listeria monocytogenes is an important Gram-positive foodborne pathogen that is a particular problem in ready-to-eat food. It has an ability to survive in harsh conditions like refrigeration temperatures and high salt concentrations and is known to cross intestinal, placental and blood-brain barriers. Several cancerous cell lines like ...

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Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting Authors: Martino Bardelli, Francisco Zarate-Perez, Leticia Agundez, Nelly Jolinon, R. Michael Linden, Carlos R. Escalante and Els Henckaerts, date: 05/05/2017, view: 8612, Q&A: 0

Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol ...

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In vitro Assays for Measuring Endothelial Permeability by Transwells and Electrical Impedance Systems Authors: Hong-Ru Chen and Trai-Ming Yeh, date: 05/05/2017, view: 23715, Q&A: 2

Vascular leakage is an important feature in several diseases, such as septic shock, viral hemorrhagic fever, cancer metastasis and ischemia-reperfusion injuries. Thus establishing assays for measuring endothelial permeability will provide insight into the establishment or progression of such diseases. Here, we provide transwell permeability assay ...

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Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci Authors: Forrest C. Walker and Asma Hatoum-Aslan, date: 05/05/2017, view: 11091, Q&A: 0

CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between ...

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Neuroscience

Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice Authors: Lubna Hindi, Joseph D. McMillan, Dil Afroze, Sajedah M. Hindi and Ashok Kumar, date: 05/05/2017, view: 22067, Q&A: 4

Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many ...

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Olfactory Cued Learning Paradigm Authors: Gary Liu, Cynthia K. McClard, Burak Tepe, Jessica Swanson, Brandon Pekarek, Sugi Panneerselvam and Benjamin R. Arenkiel, date: 05/05/2017, view: 7330, Q&A: 0

Sensory stimulation leads to structural changes within the CNS (Central Nervous System), thus providing the fundamental mechanism for learning and memory. The olfactory circuit offers a unique model for studying experience-dependent plasticity, partly due to a continuous supply of integrating adult born neurons. Our lab has recently implemented an ...

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Peripheral Nerve Injury: a Mouse Model of Neuropathic Pain Authors: Takahiro Masuda, Yuta Kohro, Kazuhide Inoue and Makoto Tsuda, date: 05/05/2017, view: 10700, Q&A: 0

Neuropathic pain is one of the highly debilitating chronic pain conditions, for which, currently, there is no therapeutic treatment. In order to reveal the underlying mechanism for neuropathic pain, various animal models have been established (Burma et al., 2016). This protocol describes how to prepare spinal nerve injury model (Kim and ...

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Electroshock Induced Seizures in Adult C. elegans Authors: Monica G Risley, Stephanie P Kelly and Ken Dawson-Scully, date: 05/05/2017, view: 7633, Q&A: 0

The nematode Caenorhabditis elegans is a useful model organism for dissecting molecular mechanisms of neurological diseases. While hermaphrodite C. elegans contains only 302 neurons, the conserved homologous neurotransmitters, simpler neuronal circuitry, and fully mapped connectome make it an appealing model system for ...

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Plant Science

Mating Based Split-ubiquitin Assay for Detection of Protein Interactions Authors: Wijitra Horaruang and Ben Zhang, date: 05/05/2017, view: 13833, Q&A: 1

The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are ...

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Haustorium Induction Assay of the Parasitic Plant Phtheirospermum japonicum Authors: Juliane K. Ishida, Satoko Yoshida and Ken Shirasu, date: 05/05/2017, view: 8372, Q&A: 0

Phtheirospermum japonicum is a facultative root parasitic plant in the Orobanchaceae family used as a model parasitic plant. Facultative root parasites form an invasive organ called haustorium on the lateral parts of their roots. To functionally characterize parasitic abilities, quantification of haustorium numbers is required. However, ...

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Pathogenicity Assay of Penicillium expansum on Apple Fruits Authors: Yong Chen, Boqiang Li, Zhanquan Zhang and Shiping Tian, date: 05/05/2017, view: 8951, Q&A: 0

Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and leads to huge economic losses during postharvest storage and shipping. Furthermore, it produces mycotoxin on the infected fruits that may cause harmful effects to human health. This pathogenicity assay involves a stab inoculation procedure ...

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Comprehensive Methods for Leaf Geometric Morphometric Analyses Authors: Laura L. Klein and Harlan T. Svoboda, date: 05/05/2017, view: 13563, Q&A: 0

Leaf morphometrics are used frequently by several disciplines, including taxonomists, systematists, developmental biologists, morphologists, agronomists, and plant breeders to name just a few. Leaf shape is highly variable and can be used for identifying species or genotypes, developmental patterning within and among individuals, assessing plant ...

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Stem Cell

Explant Methodology for Analyzing Neuroblast Migration Authors: Kirsty J. Dixon, Alisa Turbic, Ann M. Turnley and Daniel J. Liebl, date: 05/05/2017, view: 8450, Q&A: 0

The subventricular zone (SVZ) in the mammalian forebrain contains stem/progenitor cells that migrate through the rostral migratory stream (RMS) to the olfactory bulb throughout adulthood. SVZ-derived explant cultures provide a convenient method to assess factors regulating the intermediary stage of neural stem/progenitor cell migration. Here, we ...

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Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC) Authors: Megan V. Jackson and Anna D. Krasnodembskaya, date: 05/05/2017, view: 11542, Q&A: 0

Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The ...

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