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Past Issues in 2018

Volume: 8 Issue: 2

Biochemistry

Fish Bile Clean-up for Subsequent Zymography and Mass Spectrometry Proteomic Analyses Author: Rachel Ann Hauser-Davis, date: 01/20/2018, view: 2396, Q&A: 0

Biliary excretion offers a way to analyze various contaminants in aquatic organisms, and fish bile has been used as a biomarker for environmental contamination. The use of the fish bile proteome as a tool for monitoring the impact of environmental contaminants has been recently validated. However, scarce studies in this context are available, and ...

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Cell Biology

Generation of Chemically Induced Liver Progenitors (CLiPs) from Rat Adult Hepatocytes Authors: Takeshi Katsuda, Kazunori Hosaka and Takahiro Ochiya, date: 01/20/2018, view: 2939, Q&A: 0

Primary mature hepatocytes (MHs) or their progenitor cells are candidate cell sources for cell transplantation therapy in severe liver diseases. However, stable culture of these cells or generation of equivalent cells from pluripotent stem cells has been limited. Using a cocktail of small molecules that we previously found useful in stable culture ...

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Analysis of Exosome Transfer in Mammalian Cells by Fluorescence Recovery after Photobleaching Authors: Hernán González-King, Nahuel A. García, María Ciria, Sandra T. Gascón, Rafael S. Sánchez, Helena Grueso, Marta Gómez , Rubén M. Cabezuelo, Vanesa L. Cava and Pilar Sepúlveda , date: 01/20/2018, view: 2290, Q&A: 0

During the course of evolution, prokaryote and eukaryote cells have developed elegant and to some extent analogous strategies to communicate with each other and to adapt to their surrounding environment. Eukaryotic cells communicate with each other through direct interaction via juxtracrine signaling and/or by secreting soluble factors. These ...

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Measuring Mitochondrial ROS in Mammalian Cells with a Genetically Encoded Protein Sensor Authors: Xin Zhang, Christine Silvia Gibhardt, Sabrina Cappello, Katharina Maria Zimmermann, Adina Vultur and Ivan Bogeski, date: 01/20/2018, view: 2755, Q&A: 1

Reactive oxygen species (ROS) are not only known for their toxic effects on cells, but they also play an important role as second messengers. As such, they control a variety of cellular functions such as proliferation, metabolism, differentiation and apoptosis. Thus, ROS are involved in the regulation of multiple physiological and ...

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Microbiology

Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli Authors: Enrico König, Francesca Zerbini, Ilaria Zanella, Davide Fraccascia and Guido Grandi, date: 01/20/2018, view: 8406, Q&A: 1

With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the ...

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Isolation and Purification of Viruses Infecting Cyanobacteria Using a Liquid Bioassay Approach Authors: Caroline Chénard and Amy M Chan, date: 01/20/2018, view: 2459, Q&A: 0

The following protocol describes the isolation and purification of viruses infecting cyanobacteria using a liquid bioassay approach. Viruses infecting cyanobacteria are also known as cyanophages. This protocol was written specifically for the isolation of cyanophages infecting freshwater cyanobacteria particularly, cyanobacteria that cannot be ...

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Bacteria-fungal Confrontation and Fungal Growth Prevention Assay Authors: Rahul Kumar, Durga Madhab Swain, Sunil Kumar Yadav, Isha Tyagi, Rajeev Kumar, Joyati Das, Srayan Ghosh and Gopaljee Jha, date: 01/20/2018, view: 2264, Q&A: 0

There are some bacteria which can grow and multiply at the cost of living fungal biomass. They can potentially utilize fungi as a source of nutrients to forage over them. Such phenomenon is known as bacterial mycophagy, however, its mechanistic insights need to be explored to identify the molecules involved in mycophagy for potential utilization ...

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Live-cell Imaging of Neisseria meningitidis Microcolony Dispersal Induced by Lactate or Other Molecules Authors: Sara Sigurlásdóttir, Olaspers Sara Eriksson, Jens Eriksson and Ann-Beth Jonsson, date: 01/20/2018, view: 1743, Q&A: 0

To efficiently colonize the nasopharyngeal epithelium, the human restricted pathogen Neisseria meningitidis follows a multistep adhesion cascade. First, the bacteria adhere to host cells and aggregate into spherical shaped structures called microcolonies. Several hours later, single bacteria start dispersing from the microcolonies and ...

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Detection of Protein Interactions in the Cytoplasm and Periplasm of Escherichia coli by Förster Resonance Energy Transfer Authors: Nils Y. Meiresonne, Svetlana Alexeeva, René van der Ploeg and Tanneke den Blaauwen, date: 01/20/2018, view: 3065, Q&A: 0

This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in Escherichia coli by Förster Resonance Energy Transfer (FRET). The described assay allows for the previously impossible in vivo screening of periplasmic protein-protein interactions. In FRET, excitation of a donor fluorescent ...

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Infectious Subviral Particle to Membrane Penetration Active Particle (ISVP-to-ISVP*) Conversion Assay for Mammalian Orthoreovirus Authors: Anthony J. Snyder and Pranav Danthi, date: 01/20/2018, view: 1598, Q&A: 0

The mammalian orthoreovirus (reovirus) outer capsid undergoes a series of conformational changes prior to or during viral entry. These transitions are necessary for delivering the genome-containing core across host cell membranes. This protocol describes an in vitro assay for monitoring the transition into a membrane penetration-active ...

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Infectious Subviral Particle-induced Hemolysis Assay for Mammalian Orthoreovirus Authors: Anthony J. Snyder and Pranav Danthi, date: 01/20/2018, view: 1552, Q&A: 0

Mammalian orthoreovirus (reovirus) utilizes pore forming peptides to penetrate host cell membranes. This step is essential for delivering its genome containing core particle during viral entry. This protocol describes an in vitro assay for measuring reovirus-induced pore formation.

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Identification and Quantification of Secondary Metabolites by LC-MS from Plant-associated Pseudomonas aurantiaca and Pseudomonas chlororaphis Authors: Izzah Shahid, Muhammad Rizwan and Samina Mehnaz, date: 01/20/2018, view: 2788, Q&A: 0

Increased antibiotic resistance of plants and human pathogens and continuous use of chemical fertilizers has pushed microbiologists to explore new microbial sources as potential antagonists. In this study, eight strains of Pseudomonas aurantiaca and Pseudomonas chlororaphis, have been isolated from different plant sources and ...

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Molecular Biology

Characterising Maturation of GFP and mCherry of Genomically Integrated Fusions in Saccharomyces cerevisiae Authors: Sviatlana Shashkova, Adam JM Wollman, Stefan Hohmann and Mark C Leake, date: 01/20/2018, view: 2461, Q&A: 0

Single-molecule fluorescence microscopy enables unrivaled sub-cellular quantitation of genomically encoded fusions of native proteins with fluorescent protein reporters. Fluorescent proteins must undergo in vivo maturation after expression before they become photoactive. Maturation effects must be quantified during single-molecule ...

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Neuroscience

Mechanical Allodynia Assessment in a Murine Neuropathic Pain Model Authors: Serena Notartomaso, Pamela Scarselli, Paola Di Pietro, Giuseppe Battaglia, Amadeu Llebaria, Francisco Ciruela and Ferdinando Nicoletti, date: 01/20/2018, view: 2784, Q&A: 0

Experimental animal models are unique tools (i) to study pain transmission and pathophysiology of neuropathic pain, (ii) to identify novel molecular targets and (iii) to test the potential analgesic effect of specific molecules. The chronic constriction injury (CCI) model of neuropathic pain is the first model of post-traumatic painful peripheral ...

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Fluorescent Measurement of Synaptic Activity Using FM Dyes in Dissociated Hippocampal Cultured Neurons Authors: Roman M. Lazarenko, Claire E. DelBove and Qi Zhang, date: 01/20/2018, view: 2697, Q&A: 0

Release and recycling of synaptic vesicles are essential for neurotransmission and synaptic plasticity. To gain mechanistic understanding of these processes, direct measurements of vesicle release and retrieval is indispensable. Styryl dyes like FM1-43 and FM4-64 have been widely used for this purpose and their loading and unloading are reliable ...

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D-serine Measurements in Brain Slices or Other Tissue Explants Authors: Thomas Papouin and Philip G. Haydon, date: 01/20/2018, view: 2109, Q&A: 0

D-serine is an atypical amino acid present in the mammalian body (most amino acids in the mammalian body are L-isomers) that is mostly known in neuroscience for its role as a co-agonist controlling the N-methyl D-aspartate receptor (NMDAR). D-serine levels are decreased in patients with schizophrenia and this is thought to mediate, at least in ...

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Obtaining Acute Brain Slices Authors: Thomas Papouin and Philip G. Haydon, date: 01/20/2018, view: 4292, Q&A: 0

Obtaining acute brain slices for electrophysiology or amperometric recordings has become a routine procedure in most labs in the field of neuroscience. Yet, protocols describing the step by step process are scarce, in particular for routine acute preparations such as from the mouse hippocampus. Here we provide a detailed protocol for the ...

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Common Carotid Arteries Occlusion Surgery in Adult Rats as a Model of Chronic Cerebral Hypoperfusion Authors: Dandan Cao, Yunfei Bai and Liang Li, date: 01/20/2018, view: 3062, Q&A: 0

Chronic cerebral hypoperfusion (CCH) is an important risk factor of vascular dementia (VaD) and Alzheimer’s disease (AD). Hypoxia/ischemia in the whole brain induced by CCH causes serious damage to brain structure and function, which can lead to cognitive impairment. Two-vessel occlusion (2-VO), also known as permanent, bilateral common carotid ...

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Plant Science

Determination of Boron Content Using a Simple and Rapid Miniaturized Curcumin Assay Authors: Thotegowdanapalya C. Mohan and Alexandra M. E. Jones, date: 01/20/2018, view: 2465, Q&A: 0

To determine boron quantity in soil, water and biological samples, several protocols are available. Colorimetric assays are the simplest and cheapest methods which can be used to determine boron concentration. However, published protocols do not give straightforward guidance for beginners to adopt these protocols for routine use in the laboratory. ...

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Stem Cell

Human Endometrial Stem Cell Isolation from Endometrium and Menstrual Blood Authors: Shima Tavakol, Freshteh Azedi, Elham Hoveizi, Jafar Ai and Mohammad Taghi Joghataei, date: 01/20/2018, view: 2725, Q&A: 0

Human endometrial stem cell/stromal cells (hEnSCs) are isolated from endometrium or menstrual blood and are recognized as a valuable cell type in tissue engineering and cell therapy. Furthermore, hEnSCs, which have CD90 (a mesenchymal marker), CD105 (endoglin), CD44, CD146 (endometrial stem cell markers) and lack CD31 (Endothelial marker), CD34 ...

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Mouse Satellite Cell Isolation and Transplantation Authors: Elia Angelino, Simone Reano, Michele Ferrara, Emanuela Agosti, Hana Sustova, Valeria Malacarne, Sara Clerici, Andrea Graziani and Nicoletta Filigheddu, date: 01/20/2018, view: 2740, Q&A: 0

Satellite cell (SC) transplantation represents a powerful strategy to investigate SC biology during muscle regeneration. We described here a protocol for SC isolation from green fluorescent protein (GFP)-expressing mice and their transplantation into murine muscles. This procedure was originally used to assess the effects of the hormone unacylated ...

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