Septins

 
 
Academic Press
  • 1. Auflage
  • |
  • erschienen am 26. Juli 2016
  • |
  • 382 Seiten
 
E-Book | ePUB mit Adobe DRM | Systemvoraussetzungen
E-Book | PDF mit Adobe DRM | Systemvoraussetzungen
E-Book | ePUB mit Adobe DRM | Systemvoraussetzungen
978-0-12-804029-4 (ISBN)
 

Septins provides established septin and molecular and developmental biologists and researchers new to the field with proven, state-of-art techniques and relevant historical background and theory to aid efficient design and effective implementation of experimental methodologies. Topics include the purification of septin proteins from diverse systems, their visualization in live cells, and their analysis by a variety of cutting-edge microscopy approaches.


  • Provides the latest information on septins
  • Includes both established and new technologies
  • Brings together specialists from the field who contribute their expertise
0091-679X
  • Englisch
  • San Diego
  • |
  • USA
Elsevier Science
  • 10,66 MB
978-0-12-804029-4 (9780128040294)
0128040297 (0128040297)
weitere Ausgaben werden ermittelt
  • Front Cover
  • Methods in Cell Biology: Septins
  • Series Editors
  • Methods in Cell Biology: Septins
  • Copyright
  • Contents
  • Contributors
  • Preface
  • 1 - The nonopisthokont septins: how many there are, how little we know about them, and how we might learn more
  • INTRODUCTION
  • 1. THE WIDE PHYLOGENETIC DISTRIBUTION AND INFERRED ANCIENT ORIGIN OF THE SEPTINS
  • 1.1 Search Methods
  • 1.2 Apparent Absence of Septins From the Amoebozoa
  • 1.3 Presence of Septins in Some, but Not All, Planta Lineages
  • 1.4 Presence of Septins in Cryptophytes and Haptophytes
  • 1.5 Presence of Septins in At Least One Rhizarian Lineage
  • 1.6 Presence of Septins in At Least Some Alveolate Lineages
  • 1.7 Presence of Septins in At Least Some Heterokont Lineages
  • 1.8 Apparent Absence of Septins From the Excavata
  • 1.9 General Conclusions
  • 2. THE NEARLY TOTAL LACK OF FUNCTIONAL INFORMATION ABOUT THE NONOPISTHOKONT SEPTINS
  • 3. THE PROSPECTS FOR INSIGHTS FROM FUNCTIONAL ANALYSES OF NONOPISTHOKONT SEPTINS
  • Acknowledgments
  • REFERENCES
  • 2 - Preparing recombinant yeast septins and their analysis by electron microscopy
  • INTRODUCTION
  • 1. METHODS
  • 1.1 Preparation of Recombinant Budding Yeast Septin Complexes
  • 2. MATERIALS
  • 2.1 Sample Preparation for Electron Microscopy
  • 2.1.1 Sample preparation for negative stain samples
  • 2.1.2 Sample preparation for thin film frozen hydrated samples
  • 3. MATERIALS
  • 3.1 Microscopy and Image Two-Dimensional Analysis
  • 4. MATERIALS
  • 4.1 Tagging Strategies to Label Septin Subunits Within Complexes
  • 4.1.1 Antibody labeling
  • 4.1.2 Covalent bound tags
  • CONCLUSION
  • Acknowledgments
  • REFERENCES
  • 3 - A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins
  • INTRODUCTION
  • 1. PROTEIN PREPARATION AND LABELING
  • 1.1 Expression and Purification of Single-Cys Yeast Septin Heterooctamers
  • 1.2 Single-Cys Septin Subunits and Dye Labeling by Maleimide Chemistry
  • 1.3 Selection of Fluorophores for Förster Resonance Energy Transfer Analysis
  • 2. DATA COLLECTION AND ANALYSIS
  • 2.1 Spectroscopy Data Collection
  • 2.2 Semiquantitative Sensitized Emission
  • 2.3 Principal Component Analysis
  • CONCLUSIONS AND OUTLOOK
  • Acknowledgments
  • REFERENCES
  • 4 - In vitro reconstitution of septin assemblies on supported lipid bilayers
  • INTRODUCTION
  • 1. RATIONAL
  • 1.1 TIRF-Based Assays for Visualizing Cytoskeletal Properties
  • 1.2 Supported Lipid Bilayers
  • 2. CLEANING OF GLASS COVERSLIPS
  • 2.1 Rational
  • 2.2 Reagents and Equipment
  • 2.3 Protocol for RCA Cleaning of Glass Coverslips
  • 3. PREPARATION OF SMALL UNILAMELLAR VESICLES
  • 3.1 Rational
  • 3.2 Reagents and Equipment
  • 3.3 Protocol for the Preparation of Small Unilamellar Vesicles
  • 4. FORMATION OF SUPPORTED LIPID BILAYERS
  • 4.1 Reagents and Equipment
  • 4.2 Fusion Protocol
  • 5. VISUALIZING SEPTIN ADSORPTION AND POLYMERIZATION ON SUPPORTED LIPID BILAYERS
  • CONCLUDING REMARKS
  • REFERENCES
  • 5 - Visualization of in vivo septin ultrastructures by platinum replica electron microscopy
  • INTRODUCTION
  • 1. PREPARATION OF CELL CORTICES BY UNROOFING SPHEROPLASTS
  • 1.1 Materials
  • 1.2 Equipment
  • 1.3 Methods
  • 2. PLATINUM REPLICA PRODUCTION
  • 2.1 Materials
  • 2.1.1 Additional chemical fixation
  • 2.1.2 Ethanol dehydration
  • 2.1.3 Critical point drying
  • 2.1.4 Platinum and carbon coating
  • 2.1.5 Replica mounting onto EM grids
  • 2.2 Equipment
  • 2.2.1 Chemical fixation
  • 2.2.2 Ethanol dehydration
  • 2.2.3 Critical point drying
  • 2.2.4 Platinum and carbon coating
  • 2.2.5 Replica mounting onto EM grids
  • 2.3 Methods
  • 2.3.1 Chemical fixation
  • 2.3.2 Ethanol dehydration
  • 2.3.3 Critical point drying
  • 2.3.4 Platinum and carbon coating
  • 2.3.5 Replica mounting onto EM grids
  • 3. IMAGING AND ANALYSIS
  • 4. CORRELATIVE LIGHT AND ELECTRON MICROSCOPY
  • 4.1 Materials
  • 4.2 Equipment
  • 4.3 Methods
  • Acknowledgments
  • REFERENCES
  • 6 - Assays for genetic dissection of septin filament assembly in yeast, from de novo folding through polymerization
  • 1. BACKGROUND
  • 1.1 Septins Identified in Archetypal Genetic Screen
  • 2. THE LOGIC OF OBTAINING AND INTERPRETING SIMPLE PHENOTYPES
  • 2.1 Isolation, Characterization, and Interpretation of TS mutants
  • 2.1.1 Isolation
  • 2.1.2 Characterization
  • 2.1.3 Interpretation
  • 2.2 Extragenic Suppression
  • 2.3 Dosage Suppression
  • 2.4 Synthetic Genetic Interactions
  • 2.5 Septin Overexpression
  • 2.6 Heterologous Septin Expression in Saccharomyces cerevisiae
  • 2.7 Other Important Methodological Considerations
  • 3. SUMMARY AND PERSPECTIVE
  • REFERENCES
  • 7 - Investigation of septins using infection by bacterial pathogens
  • INTRODUCTION
  • 1. METHODS
  • 1.1 Preparation of Bacteria and Tissue Culture Cells
  • 1.1.1 Prepare Shigella flexneri for infection
  • 1.1.2 Prepare HeLa cells for infection
  • 1.1.3 Infection of host cells
  • 1.2 Investigation of Septin-Bacteria Interactions
  • 1.2.1 siRNA depletion
  • 1.2.2 Survival assay
  • 1.3 Microscopy of Infected Cells Using Fixed Samples
  • 1.3.1 Fixing and labeling infected cells for microscopy
  • 1.3.2 Microscopic imaging and analysis of infected cells
  • 1.4 Real-Time Microscopy of Infected Cells
  • 1.4.1 Transient DNA transfection
  • 1.4.2 Stable DNA transfection using recombinant lentivirus
  • 1.4.3 Infect cells with Shigella flexneri for live microscopy
  • 1.4.4 Investigation of actin-based motility
  • 1.4.5 Study of bacteria entrapped in septin cage-like structures
  • CONCLUSION
  • Acknowledgments
  • REFERENCES
  • 8 - In vivo analysis of septin heteropolymer rods and higher-order structures in filamentous fungi
  • 1. PROTOCOL: EXPRESSION, OBSERVATION, AND PURIFICATION OF SEPTIN HOS AND HETEROPOLYMER RODS
  • 1.1 Construct Tagged Strains and Test for Function
  • 1.2 Visualize Fluorescently Tagged Septins (HOS)
  • 1.3 Isolate Crude Protein From Synchronized Cultures
  • 1.4 Affinity-Purify Tagged Septins (Heteropolymer Rods)
  • REFERENCES
  • 9 - Live cell imaging of septin dynamics in Ustilago maydis
  • INTRODUCTION
  • 1. METHODS
  • 1.1 Imaging Subcellular Localization of Septins in Static Structures Such as Septa, Rings, and Filaments
  • 1.2 Imaging of Septins on Dynamically Shuttling Endosomes
  • 1.3 Imaging Septins in Filament Gradients
  • 1.4 RNA Live Imaging of Endosomal Septin mRNA
  • 1.5 FLIM-FRET Analysis of Heteromeric Septin Complexes
  • CONCLUSIONS
  • Acknowledgments
  • SUPPLEMENTARY DATA
  • REFERENCES
  • 10 - Ashbya gossypii as a model system to study septin organization by single-molecule localization microscopy
  • INTRODUCTION
  • 1. CONSIDERATIONS OF ASHBYA GOSSYPII SAMPLE PREPARATION STRATEGY FOR SINGLE-MOLECULE LOCALIZATION MICROSCOPY
  • 2. PREPARATION OF SEPTIN STRUCTURES IN ASHBYA GOSSYPII FOR SINGLE-MOLECULE LOCALIZATION MICROSCOPY
  • 2.1 Material and Reagents
  • 2.2 Choice of Septin Labeling Strategy for Single-Molecule Localization Microscopy
  • 2.3 Ashbya gossypii Spore Isolation and Inoculation
  • 2.4 Ashbya gossypii Mycelia Fixation
  • 2.5 Preparation of poly-l-lysine-Coated Cover Glass for Ashbya ggossypii Immobilization
  • 2.6 Ashbya gossypii Cell Wall Digestion and Immobilization
  • 2.7 Cell Membrane Permeabilization and Reduction of Unspecific Staining
  • 2.8 Labeling of Ashbya gossypii Septin Structure
  • 2.9 Sample Mounting
  • 3. SINGLE-MOLECULE LOCALIZATION IMAGE ACQUISITIONS
  • 3.1 Interpreting Septin Structures Obtained by Single-Molecule Localization Microscopy
  • 4. EXAMPLE IMAGES
  • SUMMARY
  • Acknowledgments
  • REFERENCES
  • 11 - Visualizing septins in early Drosophila embryos
  • INTRODUCTION
  • 1. PREPARATION OF EARLY DROSOPHILA EMBRYOS FOR IMMUNOFLUORESCENCE DETECTION OF SEPTINS
  • 1.1 Embryo Collection and Dechorionation
  • 1.2 Embryo Fixation
  • 1.2.1 Slow formaldehyde fixation and methanol devitellinization
  • 1.2.2 Slow formaldehyde fixation and hand devitellinization
  • 1.2.3 Methanol fixation
  • 1.2.4 Heat-methanol fixation
  • 1.3 Embryo Staining With Antibodies
  • 2. PREPARATION OF EARLY DROSOPHILA EMBRYOS FOR LIVE FLUORESCENCE IMAGING OF FLUORESCENT PROTEIN FUSIONS OF SEPTINS
  • 2.1 Embryo Preparation and Mounting
  • 2.2 Embryo Imaging
  • REFERENCES
  • 12 - Purification of recombinant human and Drosophila septin hexamers for TIRF assays of actin-septin filament assembly
  • INTRODUCTION
  • 1. CLONING STRATEGY FOR RECOMBINANT SEPTIN COMPLEX PRODUCTION IN BACTERIA
  • 2. EXPRESSION OF RECOMBINANT SEPTIN COMPLEXES IN BACTERIA
  • 2.1 Materials and Reagents
  • 2.2 Day 0. Heat-Shock Transformation of Bacteria
  • 2.3 Day 1. Bacterial Preculture
  • 2.4 Day 2. Bacterial Culture for Producing Dark (Unlabeled) Septin Complexes
  • 2.5 Days 2-3. Bacterial Culture for Producing GFP-Labeled Septin Complexes
  • 3. PURIFICATION AND CHARACTERIZATION OF RECOMBINANT SEPTIN COMPLEXES FROM BACTERIA
  • 3.1 Materials and Reagents
  • 3.2 Day 1. Septin Complex Purification
  • 3.3 Day 2a. Concentration of Purified Septin Complexes
  • 3.4 Day 2b. Characterization of Purified Septin Complexes
  • 4. LABELING SEPTINS FOR TOTAL INTERNAL REFLECTION FLUORESCENCE IMAGING
  • 4.1 Generating Septin-GFP Fusions
  • 4.2 Chemical Labeling of Purified Septin Complexes With Alexa Fluor Dyes
  • 4.2.1 Materials and reagents
  • 4.2.2 Day 1. Reaction with NHS ester and pelleting of polymerization-competent septins
  • 4.2.3 Day 2. Separation of AF488-septins from unreacted AF488 with a PD-10 column
  • 5. BUILDING FLOW CELLS FOR TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY
  • 5.1 Materials and Reagents
  • 5.2 Cleaning and Silanizing Microscope Glass Slides/Coverslips
  • 5.3 Constructing Flow Cells
  • 6. IN VITRO RECONSTITUTION OF ACTIN-SEPTIN FILAMENT ASSEMBLY
  • 6.1 Materials and Reagents
  • 6.2 Preparing Protein Solutions
  • 6.3 Buffers and Components
  • 6.4 Reconstituting Actin-Septin Filament Assembly
  • 7. TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY
  • Acknowledgments
  • REFERENCES
  • 13 - Investigation of septin biology in vivo using zebrafish
  • INTRODUCTION
  • 1. METHODS
  • 1.1 Zebrafish Husbandry
  • 1.1.1 Preparation of zebrafish larvae
  • 1.2 Detection of Septin Expression In Vivo
  • 1.2.1 Whole-mount in situ hybridization
  • 1.2.2 Western blot
  • 1.2.3 Reverse-transcription quantitative polymerase chain reaction
  • 1.2.4 Immunostaining of zebrafish larvae
  • 1.3 Manipulation of Septins In Vivo
  • 1.3.1 Pharmacological manipulation of septins
  • 1.3.2 Transient septin depletion using morpholino oligonucleotide
  • 1.3.3 Inducing mRNA-mediated expression
  • 1.3.4 Targeted gene editing of septins using CRISPR-Cas9
  • 1.4 Septin Rearrangements During Bacterial Infection
  • 1.4.1 Prepare Shigella flexneri
  • 1.4.2 Intravenous and local infection of zebrafish
  • 1.4.3 Quantification of inoculum and bacterial load
  • 1.5 Visualization of Septins In Vivo Using Microscopy
  • 1.5.1 Microscopy of fixed zebrafish larvae
  • 1.5.2 Live microscopy of infected zebrafish larvae
  • CONCLUSION
  • Acknowledgments
  • REFERENCES
  • 14 - Fluorescence microscopy of actin- and microtubule-associated septins in mammalian cells
  • INTRODUCTION
  • 1. METHODS
  • 1.1 Cell Culture and Plating
  • 1.2 Fixation and Permeabilization
  • 1.3 Staining With Antibodies
  • 1.4 Mounting and Imaging of Fixed Cells
  • 1.5 Fluorescent Probes and Stable Cell Lines for Time-Lapse Imaging
  • 1.6 Time-Lapse Imaging of Actin- and Microtubule-Associated Septins
  • 2. MATERIALS
  • 2.1 Tissue Culture Reagents, Materials, and Protocols
  • 2.2 Reagents and Buffers for Cell Fixation and Permeabilization
  • 2.3 Antibodies, Reagents, and Procedures for Immunostaining
  • 2.4 Reagents for Mounting and Imaging Fixed Cells
  • 2.5 Expression of Fluorescent Proteins for Live Cell Imaging
  • 2.6 Reagents for Live Cell Imaging
  • 2.7 Microscopes and Imaging Software
  • Acknowledgments
  • REFERENCES
  • 15 - Immunofluorescent staining of septins in primary cilia
  • INTRODUCTION
  • 1. MATERIALS
  • 2. METHODS AND RESULTS
  • 2.1 Primary Cilia Induction in hTert-RPE1
  • 2.2 Fixation Conditions for Cilia Antigen Preservation
  • 2.2.1 Methanol fixation
  • 2.2.2 Paraformaldehyde fixation
  • 2.2.3 "Transition zone" fixation
  • 2.3 Immunostaining and Microscopy
  • 2.4 Immunofluorescent Staining of Ciliary Proteins Under Different Fixation Conditions
  • 2.4.1 Different tubulin antibodies mark distinct ciliary substructures
  • 2.4.2 Axonemal, basal body, and transition zone immunostaining in primary cilia
  • 2.4.3 Intraflagellar transport immunostaining in primary cilia
  • 2.4.4 Immunofluorescent staining of mammalian septins under different fixation conditions
  • 2.5 Live Imaging of GFP-Septin in Primary Cilia
  • 3. SUMMARY
  • Acknowledgments
  • REFERENCES
  • 16 - Methods for immunoblot detection and electron microscopic localization of septin subunits in mammalian nervous .
  • INTRODUCTION
  • 1. METHODS
  • 1.1 Discontinuous Semidry Transfer for Immunoblot
  • 1.2 Preembedding Immunogold Labeling and 3D Reconstruction of Serial Section Immunoelectron Microscopic Images
  • 1.2.1 Transcardial perfusion fixation, dissection, and postfixation of the brain
  • 1.2.2 Vibratome sectioning of the fixed brain
  • 1.2.3 Membrane permeabilization by repeated freeze and thaw process
  • 1.2.4 Immunogold labeling
  • 1.2.5 Postfixation, silver enhancement, and negative staining
  • 1.2.6 Resin embedding
  • 1.2.7 Ultrathin serial sectioning and TEM imaging
  • 1.2.8 3D reconstruction of immunoelectron microscopic images
  • 1.2.9 Materials
  • CONCLUSION
  • REFERENCES
  • 17 - Visualizing septin and cell dynamics in mammalian brain slices
  • INTRODUCTION
  • 1. METHODS
  • 1.1 Immunohistochemistry
  • 1.1.1 Preparation and immunostaining of vibratome sections from embryonic mouse brain
  • 1.1.1.1 Preparation of sections
  • 1.1.1.1.1 Materials
  • 1.1.1.2 Immunostaining procedure
  • 1.1.1.2.1 Materials
  • 1.1.2 Preparation and immunostaining of frozen sections of embryonic mouse brains
  • 1.1.2.1 Preparation of sections
  • 1.1.2.1.1 Materials
  • 1.1.2.2 Immunostaining procedure
  • 1.1.2.2.2 Materials
  • 1.2 In Utero Electroporation Into the Cerebral Cortex of Embryonic Mouse Brain
  • 1.2.1 Notes
  • 1.2.2 Materials
  • 1.3 Time-Lapse Imaging of Migrating Neurons During Corticogenesis
  • 1.3.1 Notes
  • 1.3.2 Materials
  • 1.4 In Vivo Electroporation Into the Granule Cells of Dentate Gyrus of Neonatal Mouse Hippocampus
  • 1.4.1 Note
  • 1.4.2 Materials
  • CONCLUSION
  • Acknowledgments
  • REFERENCES
  • 18 - Small molecule perturbations of septins
  • 1. FORCHLORFENURON
  • 1.1 Background and Summary of Cellular Effects
  • 1.2 Properties of Forchlorfenuron
  • 1.3 Genetic Manipulation of Septin Dosage in Budding Yeast Alters Forchlorfenuron Sensitivity
  • 2. 1-ETHYL-3-(4-METHOXYPHENYL)-6-METHYLPYRIMIDO[5,4-E][1,2,4]TRIAZINE-5,7-DIONE
  • 3. SUMMARY AND PERSPECTIVE
  • REFERENCES
  • 19 - Septin crystallization for structural analysis
  • INTRODUCTION
  • 1. SEPTINS AND THEIR STRUCTURES
  • 2. METHODS
  • 2.1 Septin Construct
  • 2.2 Expression Vector and Cell Strain
  • 2.3 Protein Expression
  • 2.4 Cell Lysis and Lysis Buffer
  • 2.5 Affinity Chromatography
  • 2.6 Affinity Tag Cleavage
  • 2.7 Size Exclusion Chromatography
  • 2.8 Septin Concentration
  • 2.9 Crystallization Assay
  • 2.10 Crystal Handling, Data Collection, Processing, Structure Solution, and Refinement
  • 3. GENERAL SEPTIN EXPRESSION, PURIFICATION, AND CRYSTALLIZATION PROTOCOL
  • 4. FINAL COMMENTS
  • REFERENCES
  • Index
  • A
  • B
  • C
  • D
  • E
  • F
  • G
  • H
  • I
  • K
  • L
  • M
  • N
  • O
  • P
  • R
  • S
  • T
  • U
  • V
  • W
  • Y
  • Z
  • Volumes in Series
  • Color Plate
  • Back Cover

Dateiformat: EPUB
Kopierschutz: Adobe-DRM (Digital Rights Management)

Systemvoraussetzungen:

Computer (Windows; MacOS X; Linux): Installieren Sie bereits vor dem Download die kostenlose Software Adobe Digital Editions (siehe E-Book Hilfe).

Tablet/Smartphone (Android; iOS): Installieren Sie bereits vor dem Download die kostenlose App Adobe Digital Editions (siehe E-Book Hilfe).

E-Book-Reader: Bookeen, Kobo, Pocketbook, Sony, Tolino u.v.a.m. (nicht Kindle)

Das Dateiformat EPUB ist sehr gut für Romane und Sachbücher geeignet - also für "fließenden" Text ohne komplexes Layout. Bei E-Readern oder Smartphones passt sich der Zeilen- und Seitenumbruch automatisch den kleinen Displays an. Mit Adobe-DRM wird hier ein "harter" Kopierschutz verwendet. Wenn die notwendigen Voraussetzungen nicht vorliegen, können Sie das E-Book leider nicht öffnen. Daher müssen Sie bereits vor dem Download Ihre Lese-Hardware vorbereiten.

Weitere Informationen finden Sie in unserer E-Book Hilfe.


Dateiformat: PDF
Kopierschutz: Adobe-DRM (Digital Rights Management)

Systemvoraussetzungen:

Computer (Windows; MacOS X; Linux): Installieren Sie bereits vor dem Download die kostenlose Software Adobe Digital Editions (siehe E-Book Hilfe).

Tablet/Smartphone (Android; iOS): Installieren Sie bereits vor dem Download die kostenlose App Adobe Digital Editions (siehe E-Book Hilfe).

E-Book-Reader: Bookeen, Kobo, Pocketbook, Sony, Tolino u.v.a.m. (nicht Kindle)

Das Dateiformat PDF zeigt auf jeder Hardware eine Buchseite stets identisch an. Daher ist eine PDF auch für ein komplexes Layout geeignet, wie es bei Lehr- und Fachbüchern verwendet wird (Bilder, Tabellen, Spalten, Fußnoten). Bei kleinen Displays von E-Readern oder Smartphones sind PDF leider eher nervig, weil zu viel Scrollen notwendig ist. Mit Adobe-DRM wird hier ein "harter" Kopierschutz verwendet. Wenn die notwendigen Voraussetzungen nicht vorliegen, können Sie das E-Book leider nicht öffnen. Daher müssen Sie bereits vor dem Download Ihre Lese-Hardware vorbereiten.

Weitere Informationen finden Sie in unserer E-Book Hilfe.


Download (sofort verfügbar)

145,18 €
inkl. 19% MwSt.
Download / Einzel-Lizenz
ePUB mit Adobe DRM
siehe Systemvoraussetzungen
PDF mit Adobe DRM
siehe Systemvoraussetzungen
Hinweis: Die Auswahl des von Ihnen gewünschten Dateiformats und des Kopierschutzes erfolgt erst im System des E-Book Anbieters
E-Book bestellen

Unsere Web-Seiten verwenden Cookies. Mit der Nutzung dieser Web-Seiten erklären Sie sich damit einverstanden. Mehr Informationen finden Sie in unserem Datenschutzhinweis. Ok