1Department of Pediatrics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 2Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa
Disease-causing mutations in actin can alter cytoskeletal function. Cytoskeletal dynamics are quantified through imaging of fluorescently tagged proteins using total internal fluorescence microscopy. As an example, the cytoskeletal protein, Aip1p, has altered localization and movement in cells expressing the mutant actin isoform, R256H.
Published July 30, 2014. Keywords: Developmental Biology, green fluorescent protein, actin, Aip1p, total internal fluorescence microscopy, yeast, cloning
1Architecture et Fonction des Macromolécules Biologiques (AFMB), Aix-Marseille Université, 2iBiTec-S, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France
A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.
Published July 30, 2014. Keywords: Bioengineering, E. coli, expression, recombinant, high throughput (HTP), purification, auto-induction, immobilized metal affinity chromatography (IMAC), tobacco etch virus protease (TEV) cleavage, disulfide bond isomerase C (DsbC) fusion, disulfide bonds, animal venom proteins/peptides
1Department of Biology, KU Leuven
The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).
Published July 28, 2014. Keywords: Cellular Biology, G protein-coupled receptor (GPCR), calcium mobilization assay, reverse pharmacology, deorphanization, cellular expression system, HEK293T, Fluo-4, FlexStation
1Department of Engineering Sciences, Uppsala University, 2Gatan Inc., 3Department of Microbiology, Swedish University of Agricultural Sciences, 4Physics Department, University of Oslo
Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.
Published July 26, 2014. Keywords: Bioengineering, Cryoelectron Microscopy, Life Sciences (General), Cryo-microscopy, Focused ion beam, Sample preparation, TEM, FIB
1Department of Medical Biophysics, University of Toronto, 2Platform Biological Sciences, Sunnybrook Research Institute, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Physical Sciences, Sunnybrook Research Institute, 5Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 6Manitoba Institute of Cell Biology, University of Manitoba
We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.
Published July 26, 2014. Keywords: Bioengineering, Histology Volume Reconstruction, Transgenic Mouse Model, Image Registration, Digital Histology, Image Processing, Mouse Mammary Gland
1Laboratoire Matière et Systèmes Complexes, UFR de Physique, Université Paris Diderot, 2Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, INRA Centre de Versailles-Grignon
We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.
Published July 24, 2014. Keywords: Plant Biology, Tissue growth, Cell wall, Plant mechanics, Elasticity, Young’s modulus, Root, Apical meristem, Hypocotyl, Organ formation, Biomechanics, Morphogenesis
1Max Planck Institute of Molecular Cell Biology and Genetics
Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.
Published July 24, 2014. Keywords: Developmental Biology, Zebrafish, Primary Cell Culture, Biological Clocks, Somitogenesis, Oscillator, In Vitro, Time-lapse Imaging, Primary Culture, Fluorescence
1Laboratory of Pathology, National Cancer Institute, National Institutes of Health, 2Department of Pathology, University of Michigan
SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.
Published July 23, 2014. Keywords: Bioengineering, SIVQ, LCM, personalized medicine, digital pathology, image analysis, ArcturusXT
1Department of Biochemistry, McGill University, 2Institute of Parasitology, McGill University, 3McGill Centre for Bioinformatics, McGill University
A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.
Published July 22, 2014. Keywords: Genetics, RNA editing, Trypanosoma brucei, Editosome, Hammerhead ribozyme (HHR), High-throughput screening, Fluorescence resonance energy transfer (FRET)
1Physical Biosciences Division, Lawrence Berkeley National Laboratory
This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.
Published July 21, 2014. Keywords: Genetics, DNA-Affinity-Purified-chip, response regulator, transcription factor binding site, two component system, signal transduction, Desulfovibrio, lactate utilization regulator, ChIP-chip