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Past Issues in 2017

Volume: 7 Issue: 14

Biochemistry

[2-³H]Mannose-labeling and Analysis of N-linked Oligosaccharides Authors: Marina Shenkman, Navit Ogen-Shtern and Gerardo Z. Lederkremer, date: 07/20/2017, view: 3431, Q&A: 0

Modifications of N-linked oligosaccharides of glycoproteins soon after their biosynthesis correlate to glycoprotein folding status. These alterations can be detected in a sensitive way by pulse-chase analysis of [2-³H]mannose-labeled glycoproteins, with enzymatic removal of labeled N-glycans, separation according to size by HPLC and ...

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Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila Authors: Ines Erdmann, Kathrin Marter, Oliver Kobler, Sven Niehues, Julia Bussmann, Anke Müller, Julia Abele, Erik Storkebaum, Ulrich Thomas and Daniela C. Dieterich, date: 07/20/2017, view: 4158, Q&A: 0

Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent dynamics in subgroups of ...

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In vitro AMPylation Assays Using Purified, Recombinant Proteins Authors: Matthias C. Truttmann and Hidde L. Ploegh, date: 07/20/2017, view: 3398, Q&A: 0

Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly characterized. Research on protein ...

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Mass Spectrometry-based in vitro Assay to Identify Drugs that Influence Cystine Solubility Authors: Neelanjan Bose, Tiffany Zee, Pankaj Kapahi and Marshall L. Stoller, date: 07/20/2017, view: 4103, Q&A: 0

Cystinuria is a rare genetic disorder characterized by recurrent, painful kidney stones, primarily composed of cystine, the dimer of the amino acid cysteine (Sumorok and Goldfarb, 2013). Using a mouse model of cystinuria, we have recently shown that administration of drugs that increase cystine solubility in the urine can be a novel therapeutic ...

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Cancer Biology

Non-radioactive LATS in vitro Kinase Assay Authors: Audrey W. Hong and Kun-Liang Guan, date: 07/20/2017, view: 6182, Q&A: 1

This protocol describes a method to directly measure LATS activity by an in vitro kinase assay using YAP as a substrate.

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Glioma Induction by Intracerebral Retrovirus Injection Authors: Ravinder K Verma , Fanghui Lu and Qing Richard Lu, date: 07/20/2017, view: 4805, Q&A: 0

Glioblastoma (GBM) is the most common primary brain cancer in adults and has a poor prognosis. It is characterized by a high degree of cellular infiltration that leads to tumor recurrence, atypical hyperplasia, necrosis, and angiogenesis. Despite aggressive treatment modalities, current therapies are ineffective for GBM. Mouse GBM models not only ...

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Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader Authors: Magdiel Martínez, Namyr A. Martínez and Walter I. Silva, date: 07/20/2017, view: 15359, Q&A: 0

Intracellular calcium elevation triggers a wide range of cellular responses. Calcium responses can be affected or modulated by membrane receptors mutations, localization, exposure to agonists/antagonists, among others (Burgos et al., 2007; Martínez et al., 2016). Changes in intracellular calcium concentration can be measured ...

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Immunology

Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology Authors: Ran Wang and Sumaira Z. Hasnain, date: 07/20/2017, view: 9772, Q&A: 0

Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 10⁶ Da. These large proteins are densely substituted with short glycan chains, which have many important ...

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Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice Authors: Michael A. Schmid, Elizabeth Kauffman, Anne Payne, Eva Harris and Laura D. Kramer, date: 07/20/2017, view: 8299, Q&A: 2

Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well ...

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Microbiology

Plasmodium Sporozoite Motility on Flat Substrates Authors: Henriette L Prinz, Julia M Sattler and Friedrich Frischknecht, date: 07/20/2017, view: 4584, Q&A: 0

Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro.

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Gene Dosage Experiments in Enterobacteriaceae Using Arabinose-regulated Promoters Authors: Sanchari Bhattacharyya, Shimon Bershtein and Eugene I Shakhnovich, date: 07/20/2017, view: 3764, Q&A: 0

This protocol is used to assay the effect of protein over-expression on fitness of E. coli. It is based on a plasmid expression of a protein of interest from an arabinose-regulated pBAD promoter followed by the measurement of the intracellular protein abundance by Western blot along with the measurement of growth parameters of E. coli ...

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Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions Authors: C. Cheng Kao, Ella Chuang, James Ford, Jie Huang, Ram Podicheti and Doug B. Rusch, date: 07/20/2017, view: 4351, Q&A: 0

We have adapted the methodology of CLIP-seq (Crosslinking-Immunoprecipitation and DNA Sequencing) to map the segments of encapsidated RNAs that contact the protein shells of virions. Results from the protocol report on the RNA sequences that contact the viral capsid.

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Quantitative Determination of Poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803 Authors: Yvonne Zilliges and Ramon Damrow, date: 07/20/2017, view: 4226, Q&A: 0

Cyanobacteria synthesize a variety of chemically-different, high-value biopolymers such as glycogen (polyglucose), poly-β-hydroxybutyrate (PHB), cyanophycin (polyamide of arginine and aspartic acid) and volutin (polyphosphate) under excess conditions. Especially under unbalanced C to N ratios, glycogen and in some cyanobacterial genera ...

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The Sulfur Oxygenase Reductase Activity Assay: Catalyzing a Reaction with Elemental Sulfur as Substrate at High Temperatures Authors: Patrick Rühl and Arnulf Kletzin, date: 07/20/2017, view: 3549, Q&A: 0

The sulfur oxygenase reductase (SOR) reaction is a dioxygen-dependent disproportionation of elemental sulfur (S⁰), catalyzed at optimal temperatures between 65 °C and 85 °C. Thiosulfate and sulfite are formed as oxidized products as well hydrogen sulfide as reduced product. External co-factors are not required. Usually, the SOR assay is ...

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Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus Authors: Eleonora Sendersky, Ryan Simkovsky, Susan S. Golden and Rakefet Schwarz, date: 07/20/2017, view: 5123, Q&A: 0

A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements as a proxy for biomass accumulation ...

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Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC Authors: Sheo Shankar Pandey, Prashantee Singh, Biswajit Samal, Raj Kumar Verma and Subhadeep Chatterjee, date: 07/20/2017, view: 4359, Q&A: 0

Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas ...

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Determination of Survival of Wildtype and Mutant Escherichia coli in Soil Authors: Yinka Somorin and Conor O'Byrne, date: 07/20/2017, view: 3876, Q&A: 0

E. coli resides in the gastrointestinal tract of humans and other warm-blooded animals but recent studies have shown that E. coli can persist and grow in various external environments including soil. The general stress response regulator, RpoS, helps E. coli overcome various stresses, however its role in soil survival ...

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Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae Authors: Huanhuan Hao, Jing Huang, Tongtong Liu, Hui Tang and Liping Zhang, date: 07/20/2017, view: 6453, Q&A: 0

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very ...

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Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis Authors: Liku B Tezera, Magdalena K Bielecka and Paul T Elkington, date: 07/20/2017, view: 4688, Q&A: 0

Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the ...

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Neuroscience

Aldicarb-induced Paralysis Assay to Determine Defects in Synaptic Transmission in Caenorhabditis elegans Authors: Kelly H. Oh and Hongkyun Kim, date: 07/20/2017, view: 3982, Q&A: 0

Aldicarb treatment causes an accumulation of acetylcholine in the synaptic cleft of the neuromuscular junction, resulting in sustained muscle activation and eventually paralysis. Aldicarb-induced paralysis assay is an easy and fast method to determine whether synaptic transmission of a C. elegans mutant of interest is altered. This assay ...

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Ciberial Muscle 9 (CM9) Electrophysiological Recordings in Adult Drosophila melanogaster Authors: Benjamin A. Eaton and Rebekah E. Mahoney, date: 07/20/2017, view: 3401, Q&A: 0

The complexity surrounding presynaptic recordings in mammals is a significant barrier to the study of presynaptic mechanisms during neurotransmission in the mammalian central nervous system (CNS). Here we describe an adult fly neuromuscular junction (NMJ), the ciberial muscle 9 (CM9) NMJ, which allows for the recording of both evoked (EPSPs) and ...

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Isolation and Culturing of Rat Primary Embryonic Basal Forebrain Cholinergic Neurons (BFCNs) Authors: Wei Xu and Chengbiao Wu, date: 07/20/2017, view: 3328, Q&A: 0

The basal forebrain is located close to the medial and ventral surfaces of the cerebral hemispheres that develop from the sub-pallium. It regulates multiple processes including attention, learning, memory and sleep. Dysfunction and degeneration of basal forebrain cholinergic neurons (BFCNs) are believed to be involved in many disorders of the ...

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Plant Science

Non-invasive Protocol for Kinematic Monitoring of Root Growth under Infrared Light Authors: François Bizet, Lionel X. Dupuy, Anthony Glyn Bengough, Alexis Peaucelle, Irène Hummel and Marie-Béatrice Bogeat-Triboulot, date: 07/20/2017, view: 4054, Q&A: 0

Phenotyping the dynamics of root responses to environmental cues is necessary to understand plant acclimation to their environment. Continuous monitoring of root growth is challenging because roots normally grow belowground and are very sensitive to their growth environment. This protocol combines infrared imaging with hydroponic cultivation for ...

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Isolation of Ustilago bromivora Strains from Infected Spikelets through Spore Recovery and Germination Authors: Jason Bosch and Armin Djamei, date: 07/20/2017, view: 3595, Q&A: 0

Ustilago bromivora is a biotrophic smut fungus infecting Brachypodium sp. It is closely related to the barley-infecting smut Ustilago hordei, and related to the well-studied, gall-inducing model pathogen Ustilago maydis. Upon flowering, the spikelets of U. bromivora-infected plants are filled with black ...

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Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis Authors: Francisco J Corpas, Larisse de Freitas-Silva, Nuria García-Carbonero, Alba Contreras, Fátima Terán, Carmelo Ruíz-Torres and José M. Palma, date: 07/20/2017, view: 4191, Q&A: 1

6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyzes the third and irreversible reaction of the pentose phosphate pathway (PPP). It carries out the oxidative decarboxylation of the 6-phosphogluconate to yield ribulose-5-phosphate, carbon dioxide and NADPH. In higher plants, 6PGDH has several subcellular localizations including cytosol, ...

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Assessing Plant Tolerance to Acute Heat Stress Authors: Minsoo Kim, Fionn McLoughlin, Eman Basha and Elizabeth Vierling, date: 07/20/2017, view: 5542, Q&A: 1

It is well-established that plants are able to acclimate to temperatures above or below the optimal temperature for their growth. Here, we provide protocols for assays that can be used quantitatively or qualitatively to assess the relative ability of plants to acquire tolerance to high temperature stress. The hypocotyl elongation assay described ...

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Isolation of Fucus serratus Gametes and Cultivation of the Zygotes Authors: Amandine Siméon and Cécile Hervé, date: 07/20/2017, view: 3973, Q&A: 0

Zygotes of the Fucale species are a powerful model system to study cell polarization and asymmetrical cell division (Bisgrove and Kropf, 2008). The Fucale species of brown algae grow in the intertidal zone where they reproduce by releasing large female eggs and mobile sperm in the surrounding seawater. The gamete release can be induced from ...

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Rice Lamina Joint Inclination Assay Authors: Hsing-Yi Li, Hsin-Mei Wang and Seonghoe Jang, date: 07/20/2017, view: 5991, Q&A: 0

Brassinosteroids (BRs) promote rice lamina inclination. Recently, we showed that OsBUL1 knockout mutant rice (osbul1) is defective in brassinosteroid signaling (Jang et al., 2017). To show that lamina joint inclination of osbul1 is less-sensitive than WT to exogenous brassinolide (BL) treatment in the lamina ...

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GUS Staining of Guard Cells to Identify Localised Guard Cell Gene Expression Authors: Zhao Liu, Wei Wang, Chun-Guang Zhang, Jun-Feng Zhao and Yu-Ling Chen , date: 07/20/2017, view: 10739, Q&A: 0

Determination of a gene expression in guard cells is essential for studying stomatal movements. GUS staining is one means of detecting the localization of a gene expression in guard cells. If a gene is specially expressed in guard cells, the whole cotyledons or rosette leaf can be used for GUS staining. However, if a gene is expressed in both ...

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Stem Cell

Implantation of Human Peripheral Corneal Spheres into Cadaveric Human Corneal Tissue Authors: Jeremy John Mathan, Salim Ismail, Jennifer Jane McGhee and Trevor Sherwin, date: 07/20/2017, view: 3519, Q&A: 0

Stem and progenitor cells isolated from human limbal tissue can be cultured in vitro as spheres. These spheres have potential for use as transplantable elements for the repopulation of corneal tissue (Mathan et al., 2016). Herein we describe the detailed protocol for the implantation of human corneal spheres into cadaveric human ...

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