Hebrew University of Jerusalem 27 articles published in JoVE Developmental Biology Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal Abinaya Sundari Thooyamani*1, Elias Shahin*2, Sanako Takano1, Amnon Sharir2, Jimmy K. Hu1,3 1School of Dentistry, University of California Los Angeles, 2The Institute of Biomedical and Oral Research, Faculty of Dental Medicine, Hebrew University of Jerusalem, 3Molecular Biology Institute, University of California Los Angeles Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors. Biology Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy David Shaul1,2, Gal Sapir1,2, Naama Lev-Cohain1, Jacob Sosna1, J. Moshe Gomori1, Rachel Katz-Brull1,2 1Department of Radiology, Hadassah Medical Organization and Faculty of Medicine, Hebrew University of Jerusalem, 2The Wohl Institute for Translational Medicine We describe an experimental setup for administrating hyperpolarized 13C-labeled metabolites in continuous perfusion mode to an isolated perfused mouse heart. A dedicated 13C-NMR acquisition approach enabled the quantification of metabolic enzyme activity in real-time, and a multiparametric 31P-NMR analysis enabled the determination of the tissue ATP content and pH. Biology Simple Continuous Glucose Monitoring in Freely Moving Mice Doron Kleiman1, Mika Littor1,2, Mahmoud Nawas3, Rachel Ben-Haroush Schyr1, Danny Ben-Zvi1 1Department of Developmental Biology and Cancer Research, Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, 2Department of Military Medicine and “Tzameret”, Faculty of Medicine, Hebrew University of Jerusalem, and Medical Corps, Israel Defense Forces, 3Department of Surgery, Hadassah Medical Center-Ein Kerem Campus Here, we describe a simple method to implant a commercial continuous glucose monitor designed for patients onto mice and provide the scripts to analyze the results. Behavior Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring Liat Morgan1, Tal Raz1 1Koret School of Veterinary Medicine, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress. Biology Inducing Apical Periodontitis in Mice Elisheva Goldman1,2, Eli Reich1, Itzhak Abramovitz*2, Michael Klutstein*1 1Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, 2Department of Endodontics, Hadassah Ein Kerem Medical Center Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse's tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated. Biochemistry Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS) Daniel Some1, Hadar Amartely2, Ayala Tsadok3, Mario Lebendiker2 1Wyatt Technology Corporation, 2Wolfson Centre for Applied Structural Biology, The Alexander Silberman Institute of Life Science, The Hebrew University of Jerusalem, 3Danyel Biotech Ltd. This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins. Chemistry Calcium Carbonate Formation in the Presence of Biopolymeric Additives David N. Azulay1,2, Liraz Chai1,2 1Institute of Chemistry, Edmond J. Safra Campus, The Hebrew University of Jerusalem, 2The Harvey M. Krueger Family Center for Nanoscience and Nanotechnology, Edmond J. Safra Campus, The Hebrew University of Jerusalem We describe a protocol for the precipitation and characterization of calcium carbonate crystals that form in the presence of biopolymers. Behavior A Novel Digital Platform for a Monitored Home-based Cardiac Rehabilitation Program Horesh Dor-Haim1, Sara Katzburg1, David Leibowitz2 1O2 Hadassah Cardiac Rehabilitation Center, Hadassah Medical Center Mount Scopus, Jerusalem, Israel, 2Coronary Care Unit, Hadassah Medical Center Mount Scopus, Jerusalem, Israel The aim is to promote a new approach to cardiac rehabilitation (CR), using a unique remote patient monitoring system that will enable healthcare providers to monitor CR patients at home at low cost, with the intention of making CR services more accessible and improving compliance. The study is currently underway. Biochemistry Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization Hadar Amartely1, Daniel Some2, Ayala Tsadok3, Mario Lebendiker1 1Wolfson Centre for Applied Structural Biology, The Alexander Silberman Institute of Life Science, The Hebrew University of Jerusalem, 2Wyatt Technology Corporation, 3Danyel Biotech Ltd This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples. Biochemistry Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry Rosi Fassler*1, Nufar Edinger*1, Oded Rimon1, Dana Reichmann1 1Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Safra Campus Givat Ram, The Hebrew University of Jerusalem One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS. Chemistry Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy Priyadip Das1, Tal Duanias-Assaf1, Meital Reches1 1Institute of Chemistry and The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase. Genetics Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement Yishai Yehuda1, Britny Blumenfeld1, Dan Lehmann2, Itamar Simon1 1Dept. of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, 2The Core Research Facility, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol. Biology High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications Yulia Fridman1, Neta Holland1, Rivka Elbaum2, Sigal Savaldi-Goldstein1 1Faculty of Biology, Technion-Israel Institute of Technology, 2Smith Institute of Plant Sciences and Genetics in Agriculture, Hebrew University of Jerusalem Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution. Medicine A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies Emily J. Chadwick1, David P. Yang1, Mariella G. Filbin2, Emanuele Mazzola3, Yu Sun1, Oded Behar1,4, Maria F. Pazyra-Murphy1, Liliana Goumnerova5, Keith L. Ligon6, Charles D. Stiles1, Rosalind A. Segal1 1Department of Cancer Biology, Dana-Farber Cancer Institute, 2 Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. Biology Identifying Protein-protein Interaction Sites Using Peptide Arrays Hadar Amartely1, Anat Iosub-Amir1, Assaf Friedler1 1Institute of Chemistry, The Hebrew University of Jerusalem Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays. Biology Reporter-based Growth Assay for Systematic Analysis of Protein Degradation Itamar Cohen1, Yifat Geffen1, Guy Ravid1, Tommer Ravid1 1Department of Biological Chemistry, The Hebrew University of Jerusalem Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker. Neuroscience Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature Lea Ankri1, Yosef Yarom1, Marylka Y. Uusisaari1 1Department of Neurobiology, Hebrew University of Jerusalem In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation. Biology Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example Arava Shatil-Cohen1, Hadas Sibony1, Xavier Draye2, François Chaumont3, Nava Moran1, Menachem Moshelion1 1The RH Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, 2Earth and Life Institute, Université catholique de Louvain, 3Institut des Sciences de la Vie, Université catholique de Louvain Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion. Behavior Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience Hagit Turm1, Diptendu Mukherjee1, Doron Haritan1, Maayan Tahor1, Ami Citri1 1The Alexander Silberman Institute of Life Sciences & Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays. Neuroscience The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye Amy E. Birsner*1, Ofra Benny*2, Robert J. D'Amato1,3 1 The protocol describes the corneal micropocket assay as developed in mice. Immunology and Infection ScanLag: High-throughput Quantification of Colony Growth and Lag Time Irit Levin-Reisman1, Ofer Fridman1, Nathalie Q. Balaban1 1Racah Institute of Physics, The Hebrew University of Jerusalem ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant. Chemistry Exploring the Radical Nature of a Carbon Surface by Electron Paramagnetic Resonance and a Calibrated Gas Flow Uri Green1,2, Yulia Shenberger3, Zeev Aizenshtat1, Haim Cohen2,4, Sharon Ruthstein3 1Chemistry Institute, The Hebrew University of Jerusalem, 2Biological Chemistry Department, Ariel University, 3Chemistry Department, Faculty of Exact Science, Bar Ilan University, 4Chemistry Department, Ben-Gurion University Stable radicals that are present in carbon substrates interact with paramagnetic oxygen through a Heisenberg spin exchange. This interaction can be significantly reduced under STP conditions by flowing a diamagnetic gas over the carbon system. This manuscript describes a simple method to characterize the nature of those radicals. Immunology and Infection Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues Adi Kliot1,2, Svetlana Kontsedalov1, Galina Lebedev1, Marina Brumin1, Pakkianathan Britto Cathrin1, Julio Massaharu Marubayashi1, Marisa Skaljac1,3, Eduard Belausov4, Henryk Czosnek2, Murad Ghanim1 1Department of Entomology, Volcani Center, 2Institute of Plant Sciences and Genetics in Agriculture, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, 3Department of Applied Sciences, Institute for Adriatic Crops and Karst Reclamation, 4The Institute of Plant Sciences, Volcani Center We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections. Chemistry Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity Koli Basu1, Christopher P. Garnham2, Yoshiyuki Nishimiya3, Sakae Tsuda3, Ido Braslavsky4, Peter Davies1 1 Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light. Chemistry Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR Michal S. Shoshan1, Edit Y. Tshuva1, Deborah E. Shalev2 1Department of Chemistry, The Hebrew University of Jerusalem, 2Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described. Engineering Monolayer Contact Doping of Silicon Surfaces and Nanowires Using Organophosphorus Compounds Ori Hazut1,2, Arunava Agarwala1,2, Thangavel Subramani1,2, Sharon Waichman1,2, Roie Yerushalmi1,2 1Institute of Chemistry, The Hebrew University of Jerusalem, 2Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem A detailed procedure for surface doping of Silicon interfaces is provided. The ultra-shallow surface doping is demonstrated by using phosphorus containing monolayers and rapid annealing process. The method can be used for doping of macroscopic area surfaces as well as nanostructures. Chemistry Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides Sivan Yuran1, Meital Reches1 1Institute of Chemistry and The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.
