Volume: 10 Issue: 17 —BIO-PROTOCOL

Biophysics

Optogenetic Tuning of Protein-protein Binding in Bilayers Using LOVTRAP Authors: Doug Tischer and Orion D. Weiner, date: 09/05/2020, view: 2040, Q&A: 0

Modern microscopy methods are powerful tools for studying live cell signaling and biochemical reactions, enabling us to observe when and where these reactions take place from the level of a cell down to single molecules. With microscopy, each cell or molecule can be observed both before and after a given perturbation, facilitating better inference ...

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Cell Biology

An in vitro DNA Sensor-based Assay to Measure Receptor-specific Adhesion Forces of Eukaryotic Cells and Pathogens Authors: Maurizio Wack, Tina Wiegand, Friedrich Frischknecht and E. Ada Cavalcanti-Adam, date: 09/05/2020, view: 1311, Q&A: 0

Motility of eukaryotic cells or pathogens within tissues is mediated by the turnover of specific interactions with other cells or with the extracellular matrix. Biophysical characterization of these ligand-receptor adhesions helps to unravel the molecular mechanisms driving migration. Traction force microscopy or optical tweezers are typically ...

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Quantitative Kinetic Analyses of Histone Turnover Using Imaging and Flow Cytometry Authors: Hanae Sato, Robert H. Singer and John M. Greally, date: 09/05/2020, view: 1255, Q&A: 0

Dynamic histone changes occur as a central part of chromatin regulation. Deposition of histone variants and post-translational modifications of histones are strongly associated with properties of chromatin status. Characterizing the kinetics of histone variants allows important insights into transcription regulation, chromatin maintenance and ...

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Developmental Biology

Fluorescent Polysome Profiling in Caenorhabditis elegans Authors: Dan Shaffer and Jarod A Rollins, date: 09/05/2020, view: 1456, Q&A: 0

An important but often overlooked aspect of gene regulation occurs at the level of protein translation. Many genes are regulated not only by transcription but by their propensity to be recruited to actively translating ribosomes (polysomes). Polysome profiling allows for the separation of unbound 40S and 60S subunits, 80S monosomes, and actively ...

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Immunology

Flow-cytometric Detection of Low-level Reactive Oxygen Species in Cell Lines and Primary Immune Cells Authors: Kevin Bode, Corinna Link, Peter H. Krammer and Heiko Weyd, date: 09/05/2020, view: 2259, Q&A: 0

Depending on its concentration and cellular origin the production of reactive oxygen species (ROS) in the organism serves a variety of functions. While high concentrations during an oxidative burst are used to fight pathogens, low to moderate amounts of ROS act as signaling molecules important for several physiological processes such as regulation ...

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Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi Authors: Franziska Mohring, Thomas A. Rawlinson, Simon J. Draper and Robert W. Moon, date: 09/05/2020, view: 1912, Q&A: 0

Malaria remains a major cause of morbidity and mortality globally. Clinical symptoms of the disease arise from the growth and multiplication of Plasmodium parasites within the blood of the host. Thus in vitro assays to determine how drug, antibody and genetic perturbations affect the growth rate of Plasmodium parasites ...

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Microbiology

Live-cell Imaging by Super-resolution Confocal Live Imaging Microscopy (SCLIM): Simultaneous Three-color and Four-dimensional Live Cell Imaging with High Space and Time Resolution Authors: Kazuo Kurokawa and Akihiko Nakano, date: 09/05/2020, view: 2112, Q&A: 0

Many questions in cell biology can be solved by state-of-the-art technology of live cell imaging. One good example is the mechanism of membrane traffic, in which small membrane carriers are rapidly moving around in the cytoplasm to deliver cargo proteins between organelles. For directly visualizing the events in membrane trafficking system, ...

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A High-throughput Interbacterial Competition Platform Authors: Hsiao-Han Lin and Erh-Min Lai, date: 09/05/2020, view: 1334, Q&A: 0

Contact-dependent interbacterial competition is a common strategy used by bacteria to fight for their ecological niches. Interbacterial competition is monitored by a competition assay involving co-culturing the attacker and the recipient bacterial cells on agar, followed by recovery of the surviving recipient cells. Conventional interbacterial ...

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Screening Method for CRISPR/Cas9 Inhibition of a Human DNA Virus: Herpes Simplex Virus Authors: Werner M. Neuhausser, Hyung S. Oh, Pierce Eggan, Magdalena Angelova, Rory Kirchner, Kevin C. Eggan and David M. Knipe, date: 09/05/2020, view: 2160, Q&A: 0

The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and ...

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Molecular Biology

BRIDGE: An Open Platform for Reproducible Protein-Ligand Simulations and Free Energy of Binding Calculations Authors: Tharindu Senapathi, Christopher B. Barnett and Kevin J. Naidoo, date: 09/05/2020, view: 2794, Q&A: 2

Protein-ligand binding prediction is central to the drug-discovery process. This often follows an analysis of genomics data for protein targets and then protein structure discovery. However, the complexity of performing reproducible protein conformational analysis and ligand binding calculations, using vetted methods and protocols can be a ...

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Nonenzymatic RNA-templated Synthesis of N3′→P5′ Phosphoramidate DNA Authors: Derek K. O'Flaherty, Lijun Zhou and Jack W. Szostak, date: 09/05/2020, view: 1601, Q&A: 0

The RNA world hypothesis describes a scenario where early life forms relied on RNA to govern both inheritance and catalyze useful chemical reactions. Prior to the emergence of enzymes capable of replicating the RNA genome, a nonenzymatic replication process would have been necessary to initiate Darwinian Evolution. However, the one-pot ...

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Neuroscience

The Peak Interval Procedure in Rodents: A Tool for Studying the Neurobiological Basis of Interval Timing and Its Alterations in Models of Human Disease Authors: Fuat Balcı and David Freestone, date: 09/05/2020, view: 903, Q&A: 0

Animals keep track of time intervals in the seconds to minutes range with, on average, high accuracy but substantial trial-to-trial variability. The ability to detect the statistical signatures of such timing behavior is an indispensable feature of a good and theoretically-tractable testing procedure. A widely used interval timing procedure is the ...

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Measuring Breathing Patterns in Mice Using Whole-body Plethysmography Authors: Patricia Prada-Dacasa, Andrea Urpi, Laura Sánchez-Benito, Patrizia Bianchi and Albert Quintana, date: 09/05/2020, view: 1846, Q&A: 0

Respiratory dysfunction is among the main cause of severe and fatal pathologies worldwide. The use of effective experimental models and methodologies for the study of the pulmonary pathophysiology is necessary to prevent, control and cure these diseases. Plethysmography, a technique for the assessment of lung function, has been widely applied in ...

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Identification of Socially-activated Neurons Authors: Mary L. Phillips and Lucas Pozzo-Miller, date: 09/05/2020, view: 1947, Q&A: 0

Determining the neuronal circuitry responsible for specific behaviors is a major focus in the field of neurobiology. Activity-dependent immediate early genes (IEGs), transcribed and translated shortly after neurons discharge action potentials, have been used extensively to either identify or gain genetic access to neurons and brain regions ...

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Plant Science

Efficient Agrobacterium-mediated Transformation of the Elite–Indica Rice Variety Komboka Authors: Van T. Luu, Melissa Stiebner, Paula Emmerich Maldonado, Sandra Valdés, Didier Marín, Gerardo Delgado, Virginia Laluz, Lin-Bo Wu, Paul Chavarriaga, Joe Tohme, Inez H. Slamet-Loedin and Wolf B. Frommer, date: 09/05/2020, view: 3511, Q&A: 0

Genetic transformation is crucial for both investigating gene functions and for engineering of crops to introduce new traits. Rice (Oryza sativa L.) is an important model in plant research, since it is the staple food for more than half of the world’s population. As a result, numerous transformation methods have been developed for both ...

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An Efficient Inoculation Technique to Assess the Pathogenicity of Pantoea Species Associated to Bacterial Blight of Rice Authors: Kossi Kini, Raoul Agnimonhan, Issa Wonni and Drissa Silue, date: 09/05/2020, view: 1235, Q&A: 0

Bacteria blight diseases of rice due to several genera of pathogenic bacteria are one of the major constraints worldwide for rice production. The disease can be best managed through host plant resistance sources. For most of these bacteria such as Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, ...

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Ratiometric Measurement of Protein Abundance after Transient Expression of a Transgene in Nicotiana benthamiana Authors: Aashima Khosla and David C. Nelson, date: 09/05/2020, view: 1917, Q&A: 0

Ratiometric reporters are tools to dynamically measure the relative abundance of a protein of interest. In these systems, a target protein fused to a fluorescent or bioluminescent reporter is expressed with fixed stoichiometry to a reference protein fused to a second reporter. Both fusion proteins are encoded on a single transcript but are ...

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Stem Cell

Integration of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neurons into Rat Brain Authors: Xiling Yin, Ted M. Dawson and Valina L. Dawson, date: 09/05/2020, view: 1406, Q&A: 0

Human neuron transplantation offers novel opportunities for modeling human neurologic diseases and potentially replacement therapies. However, the complex structure of the human cerebral cortex, which is organized in six layers with tightly interconnected excitatory and inhibitory neuronal networks, presents significant challenges for in vivo ...

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