Volume: 10 Issue: 9 —BIO-PROTOCOL

Biochemistry

Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer Authors: Regina Wirth, Peng Gao, G. Ulrich Nienhaus, Murat Sunbul and Andres Jäschke, date: 05/05/2020, view: 880, Q&A: 0

Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be ...

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Quantification of Fatty Acids in Mammalian Tissues by Gas Chromatography–Hydrogen Flame Ionization Detection Authors: Fumie Hamano, Suzumi M Tokuoka, Tomomi Hashidate-Yoshida, Hideo Shindou, Takao Shimizu and Yoshihiro Kita, date: 05/05/2020, view: 556, Q&A: 0

In mammalian organisms, fatty acids (FAs) exist mostly in esterified forms, as building blocks of phospholipids, triglycerides, and cholesteryl esters, while some exist as non-esterified free FAs. The absolute quantification of FA species in total lipids or in a specific lipid class is critical in lipid-metabolism studies. To quantify FAs in ...

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Cancer Biology

Colorimetric RhoB GTPase Activity Assay Authors: Kossay Zaoui and Stephanie Duhamel, date: 05/05/2020, view: 763, Q&A: 0

The Ras homologous protein (Rho) GTPase subfamily, including RhoA, RhoB, and RhoC are small molecules (~21 kDa) that act as molecular switches in a wide range of signaling pathways to orchestrate biological processes associated with both physiological and tumorigenic cellular states. The Rho GTPases are crucial regulators of actin cytoskeleton ...

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Developmental Biology

A Modified Semisolid Clonal Culture for Identification of B-1 and B-2 Progenitor Colony Forming Ability of Mouse Embryonic Hemogenic Endothelial Cells Authors: Michihiro Kobayashi and Momoko Yoshimoto, date: 05/05/2020, view: 491, Q&A: 0

The search for the origin of the first hematopoietic stem cells (HSCs) in the mouse embryo has been a hot topic in the field of developmental hematopoiesis. Detecting lymphoid potential is one of the supportive evidence to show the definitive hematopoietic activity of HSCs. However, the first B-lymphoid potential in the mouse embryos are reported ...

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Application of Mechanical Forces on Drosophila Embryos by Manipulation of Microinjected Magnetic Particles Authors: Arturo D’Angelo and Jérôme Solon, date: 05/05/2020, view: 431, Q&A: 0

Cells generate mechanical forces to shape tissues during morphogenesis. These forces can activate several biochemical pathways and trigger diverse cellular responses by mechano-sensation, such as differentiation, division, migration and apoptosis. Assessing the mechano-responses of cells in living organisms requires tools to apply controlled local ...

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Live Cell Imaging of Male Meiosis in Arabidopsis by a Landmark-based System Authors: Maria Ada Prusicki, Emma Mathilde Keizer, Rik Peter van Rosmalen, Christian Fleck and Arp Schnittger, date: 05/05/2020, view: 776, Q&A: 0

Live cell imaging has tremendously promoted our understanding of cellular and subcellular processes such as cell division. Here, we present a step-by-step protocol for a robust and easy-to-use live cell imaging approach to study male meiosis in the plant Arabidopsis thaliana as recently established. Our method relies on the concomitant ...

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Immunology

Evaluation of B Cell Proliferation in vivo by EdU Incorporation Assay Authors: Adi Biram and Ziv Shulman, date: 05/05/2020, view: 549, Q&A: 0

Generation of antibodies is crucial for establishing enduring protection from invading pathogens, as well as for maintaining homeostasis with commensal bacteria at mucosal surfaces. Chronic exposure to microbiota- and dietary- derived antigens results in continuous production of antibody producing cells within the Peyer’s patch germinal center ...

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Assessing in vitro and in vivo Trogocytosis By Murine CD4⁺ T cells Authors: Jim Reed and Scott A. Wetzel, date: 05/05/2020, view: 501, Q&A: 0

Recognition of antigens by lymphocytes (B, T, and NK) on the surface of an antigen-presenting cell (APC) leads to lymphocyte activation and the formation of an immunological synapse between the lymphocyte and the APC. At the immunological synapse APC membrane and associated membrane proteins can be transferred to the lymphocyte in a process called ...

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HIV-CRISPR: A CRISPR/Cas9 Screening Method to Identify Genes Affecting HIV Replication Authors: Ferdinand Roesch and Molly OhAinle, date: 05/05/2020, view: 488, Q&A: 0

Screening with CRISPR/Cas9 technology has already led to significant discoveries in the fields of cancer biology, cell biology and virology. Because of the relatively low false discovery rates and the ability to perform high-throughput, pooled approaches, it has rapidly become the assay of choice for screening studies, including whole-genome ...

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Microbiology

Viral Double-Stranded RNA Detection by DNase I and Nuclease S1 digestions in Leishmania parasites Authors: Nathalie Isorce and Nicolas Fasel, date: 05/05/2020, view: 299, Q&A: 0

Many RNA viruses are found in protozoan parasites. They can be responsible for more serious pathology or treatment failure. For the detection of viral double-stranded RNA (dsRNA), sequence-dependent and -independent methods are available, such as quantitative real-time PCR and immunofluorescence, dot blot, ELISA or sequencing. The technique ...

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A Sensitive Coupled Enzyme Assay for Measuring Kinase and ATPase Kinetics Using ADP-Specific Hexokinase Authors: Ciaran R. McFarlane and James W. Murray, date: 05/05/2020, view: 396, Q&A: 0

Kinases and ATPases perform essential biological functions in metabolism and regulation. Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay.

Using the ...

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Quantification of Bacteria Residing in Caenorhabditis elegans Intestine Authors: M. Fernanda Palominos and Andrea Calixto, date: 05/05/2020, view: 631, Q&A: 0

Quantification of intestinal colonization by pathogenic or commensal bacteria constitute a critical part of the analysis to understand host-microbe interactions during different time points of their interplay. Here we detail a method to isolate non-pathogenic and pathogenic bacteria from C. elegans intestines, and classify gut phenotypes ...

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Molecular Biology

Split Nano Luciferase-based Assay to Measure Assembly of Japanese Encephalitis Virus Authors: Simon Goto, Kotaro Ishida, Ryosuke Suzuki and Eiji Morita, date: 05/05/2020, view: 279, Q&A: 0

Cells infected with flavivirus release various forms of infectious and non-infectious particles as products and by-products. Comprehensive profiling of the released particles by density gradient centrifugation is informative for understanding viral particle assembly. However, it is difficult to detect low-abundance minor particles in such ...

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Neuroscience

Rapid Generation of Human Neuronal Cell Models Enabling Inducible Expression of Proteins-of-interest for Functional Studies Authors: Xinzhu Wang, Erik Friesen, Iris Müller, Mackenzie Lemieux, Ramona Dukart, Isabella BL Maia, Suneil Kalia and Gerold Schmitt-Ulms, date: 05/05/2020, view: 506, Q&A: 0

CRISPR-Cas9 technology has transformed the ability to edit genomic sequences and control gene expression with unprecedented ease and scale. However, precise genomic insertions of coding sequences using this technology remain time-consuming and inefficient because they require introducing adjacent single-strand cuts through Cas9 nickase action and ...

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Systems Biology

Sequence Alignment Using Machine Learning for Accurate Template-based Protein Structure Prediction Authors: Shuichiro Makigaki and Takashi Ishida, date: 05/05/2020, view: 593, Q&A: 0

Template-based modeling, the process of predicting the tertiary structure of a protein by using homologous protein structures, is useful when good templates can be available. Indeed, modern homology detection methods can find remote homologs with high sensitivity. However, the accuracy of template-based models generated from the ...

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Past Issues in 2020

Volume: 10 Issue: 9

Biochemistry

Cancer Biology

Developmental Biology

Immunology

Microbiology

Molecular Biology

Neuroscience

Systems Biology