Preface
Contents
Contributors
Chapter 1: Sleep Under Preindustrial Conditions: What We Can Learn from It
1 The Conquest of the Night
2 Artificial Light and Its Impact on Sleep
3 Methods to Study the Effects of Electric Light on Human Sleep
4 Lessons from Sleep Under Preindustrial Conditions
5 Limitations of Preindustrial Sleep Studies
6 Conclusions
References
Chapter 2: The Structure-Based Molecular-Docking Screen Against Core Clock Proteins to Identify Small Molecules to Modulate th...
1 Introduction
2 Materials
2.1 Homology Modeling Server
2.2 Programs to Visualize Protein Structures
2.3 Retrieving Protein Structures from Protein Data Bank (PDB)
2.4 NAMD Program and CHARMM Force Field
2.5 Small Molecules
2.6 Autodock Vina and Autodock Tools
3 Methods
3.1 MD Simulation
3.1.1 Homology Modeling of CRY1
3.1.2 Preparing CRY1 for MD Simulation
3.1.3 Running MD Simulation
3.2 Docking
3.2.1 Preparation of CRY1 for Docking Simulations
3.2.2 Preparation of Small Molecules for Docking Simulations
3.2.3 Running and Analyzing AutoDock Vina Simulations
4 Notes
References
Chapter 3: Analysis of Complex Circadian Time Series Data Using Wavelets
1 Introduction
2 Materials
3 Methods
3.1 Graphical User Interface
3.1.1 Example Data
3.1.2 Download Example Data
3.1.3 Launch pyBOAT
3.1.4 Import Data
3.1.5 Detrending
3.1.6 Analysis and Detection of Periodic Signals Using Wavelets
3.1.7 Ridge Analysis Reveals the Main Rhythmic Component
3.1.8 Ensemble Analysis
3.2 Implementing pyBOAT Within a Python Script
3.2.1 Generating Simluated Complex Oscillatory Time Series Data
3.2.2 Import and Initialization of the Wavelet Analyzer
3.2.3 Detrending
3.2.4 Computing the Wavelet Spectrum
3.2.5 Detect and Evaluate the Wavelet Ridge
3.2.6 Import and Analysis of Experimental Datasets
4 Notes
References
Chapter 4: Mathematical Modeling in Circadian Rhythmicity
1 Introduction
2 Clock Modeling Fundamentals: Mathematical Preliminaries, Notations and Basic Concepts
2.1 Ordinary Differential Equations (ODEs)
Box A Formulating a simple ODE
Box B Goodwin model for circadian clocks, part I-Scheme and ODEs
2.2 Limit Cycles
2.2.1 Cooking Recipe for Oscillations
Box C Goodwin model for circadian clocks, part II-Limit cycle oscillations
2.3 Bifurcation Diagrams
Box D Goodwin model for circadian clocks, part III-Bifurcation diagrams
3 Learnings from Modeling Interlocked Feedback Loops
Box E Positive feedback loops promote oscillations in the Goodwin model
4 Interaction of Clocks with the Environment
Box F Amplitude-phase models
4.1 Coupled Oscillators, Synchronization and Entrainment
Box G Coupled circadian oscillators synchronize spontaneously
4.1.1 Entrainment and Arnold Tongues
Box H Entrainment and Arnold tongue of a circadian amplitude-phase model
4.2 Output Regulation
Box I Modeling driven expression of clock-controlled genes
5 Concluding Remarks and Modeling Limitations
References
Chapter 5: Bioinformatics and Systems Biology of Circadian Rhythms: BIO_CYCLE and CircadiOmics
1 Introduction
2 BIO_CYCLE
2.1 The BIO_CYCLE Web Server
2.2 Input Format
2.2.1 Running BIO_CYCLE
2.2.2 Period Selection
2.2.3 Histograms
2.2.4 Trajectory Visualization
3 CircadiOmics
3.1 Dataset Statistics
3.2 Dataset Collection
3.3 CircadiOmics Features
3.3.1 Search
3.3.2 Metabolic Atlas
3.3.3 BIO_CYCLE Web Portal within CircadiOmics
4 Biological Discovery
References
Chapter 6: Cell-Based Phenotypic Screens to Discover Circadian Clock-Modulating Compounds
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Cell Culture Reagents and Supplies
2.3 Compounds
2.4 Equipment
2.5 Software
3 Methods
3.1 Cell Plating (For Sixteen 384-Well Plates)
3.2 Compound Application
3.3 Luminescence Measurement
3.4 Data Analysis
3.5 Hit Compound Validation
4 Notes
References
Chapter 7: Methods for Assessing Circadian Rhythms and Cell Cycle in Intestinal Enteroids
1 Introduction
2 Materials
2.1 Animals
2.2 Growth and Expansion of Enteroids
2.3 Media for Human Intestinal Enteroids (HIEs)
2.4 Lentiviral Transduction of Mouse and Human Enteroids
2.5 RNA Isolation and Processing
2.6 Whole-Mount Staining
3 Methods
3.1 Preparation of R-Spondin and Noggin Conditioned Media [19]
3.2 Isolation, Expansion, and Maintenance of Mouse Enteroids
3.3 Preparation of HIE Culture [22]
3.4 Forty-Eight-Hour Time Course Sample Collection with 2-h Resolution for RNA and/or Protein Isolation
3.5 Bioluminescent Assay
3.6 Time Course Experiments to Measure Cell Proliferation Index Using Immunofluorescence Imaging
3.7 Time Course Live Cell Confocal Microscopy for Cell Cycle Analysis
4 Notes
References
Chapter 8: Using ALLIGATORs to Capture Circadian Bioluminescence
1 Introduction
2 Materials
2.1 Cell Culture and Reagents
2.2 Other Materials
3 Methods
3.1 Seeding and Entrainment
3.2 Recording
3.3 Analysis
4 Notes
References
Chapter 9: Studying Circadian Clock Entrainment by Hormonal Signals
1 Introduction
2 Materials
2.1 Tissue Slice Circadian Reporter Cultures
2.2 Luciferase Clock Gene Promoter Assays
2.3 Circadian Enhancer Motif Chromatin Immunoprecipitation (ChIP)
3 Methods
3.1 Tissue Slice Circadian Reporter Cultures
3.1.1 Tissue Dissection
3.1.2 Assessment of Phase Treatment
3.1.3 Clock Phase Assessment
3.2 Luciferase Clock Gene Promoter Assays
3.2.1 Plasmid Transformation
3.2.2 Plasmid Amplification
3.2.3 Cell Transfection
3.2.4 Luciferase Assay
3.2.5 Gaussia Luciferase Assay
3.2.6 SEAP Assay
3.3 Circadian Enhancer Motif Chromatin Immunoprecipitation (ChIP)
3.3.1 Tissue Collection and Crosslinking
3.3.2 DNA Isolation
3.3.3 qPCR and Analysis
4 Notes
References
Chapter 10: In Vitro Assays for Measuring Intercellular Coupling Among Peripheral Circadian Oscillators
1 Introduction
2 Materials
2.1 Cell Lines (See Note 1)
2.2 Reagents
2.3 Equipment
2.4 Software ChronoStar
3 Methods
3.1 Three-Dimensional Spheroid Cultures: General Considerations
3.1.1 Week 1: Hanging Drop Cultures
3.1.2 Week 2: Prepare Spheroid Cultures
3.1.3 Week 2: Bioluminescence Imaging of Spheroid Cultures
3.2 Time Series Analysis Using ChronoStar
4 Notes
References
Chapter 11: Circadian Control of Transcriptional and Metabolic Rhythms in Primary Hepatocytes
1 Introduction
2 Materials
3 Methods
3.1 Isolation and Culture of Mouse Primary Hepatocytes
3.2 De Novo Glucose Production Assay with Synchronized Mouse Primary Hepatocytes
4 Notes
References
Chapter 12: Electrophysiology of the Suprachiasmatic Nucleus: Single-Unit Recording
1 General Introduction
1.1 Electrophysiology of SCN
2 Materials
3 Methods
3.1 Setting Up the Hypothalamic Brain Slice Chamber
3.1.1 Chamber Setup
3.1.2 On the Day of Slicing
3.2 Preparing the Hypothalamic Brain Slice
3.3 Extracellular Recording of Single-Unit Activity
4 Notes
References
Chapter 13: Anatomical Methods to Study the Suprachiasmatic Nucleus
1 Introduction
1.1 Triple Label Immunocytochemistry
1.2 Dual Label In Situ Hybridization
2 Materials
2.1 Triple Label Immunocytochemistry
2.2 Dual Label In Situ Hybridization
3 Methods
3.1 Triple Label Immunocytochemistry
3.1.1 Tissue Preparation
3.1.2 Staining
3.1.3 Mounting
3.2 Dual Label In Situ Hybridization
3.2.1 Tissue Preparation
3.2.2 Probe Preparation by In Vitro Transcription
3.2.3 Hybridization
3.2.4 Post-hybridization
4 Notes
References
Chapter 14: Circadian Analysis of Rodent Locomotor Activity in Home Cages
1 Introduction
2 Materials
3 Methods
3.1 Experiment Setup and Data Collection
3.2 Data Extraction and Analysis
4 Notes
References
Chapter 15: Recording of Diurnal Gene Expression in Peripheral Organs of Mice Using the RT-Biolumicorder
1 Introduction
2 Materials
2.1 Mice and Reporter Gene
2.2 Adenovirus Vector Production and Purification
2.2.1 Adenovirus (Ad) Genome Transfection
2.2.2 Adenovirus Harvest, Rescue, and Amplification
2.2.3 Adenovirus Purification
2.2.4 Adenovirus Titration by Measurement of OD260
2.3 Adenovirus Administration
2.4 Pump Implantation
2.5 Animal Care and Room Disinfection
2.6 Recording
2.6.1 Hardware
2.6.2 Consumables
3 Methods
3.1 Choice of Reporter Gene
3.2 Adenoviral Preparation
3.2.1 Titer quantification
Infectious Titers:
3.2.2 Purity of Vector Preparation
3.3 Tail Vein Injection
3.4 Luciferin Solution Preparation, Pump Filling, and Activation
3.5 Pump Implantation
3.6 Monitoring of Animal Welfare
3.7 Recording
3.8 Recording Parameters: Habituation Period, Feeding Schedules, Skeleton Photoperiods
3.9 Termination of Experiments
3.10 Data Analysis
4 Notes
References
Chapter 16: Isolation and Sorting of Epidermal Interfollicular Stem Cells for the Study of Circadian Rhythms
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagent Preparation
2.3 Other Reagents
3 Methods
3.1 Processing of Mouse Skin
3.2 Isolation of Epidermal Cells from Mouse Skin
3.3 Stem Cell Sorting
3.4 Identification of Rhythmic Genes
4 Notes
References
Chapter 17: Detecting Circadian Rhythms in Human Red Blood Cells by Dielectrophoresis
1 Introduction
2 Materials
2.1 Sample Isolation and Preparation
2.2 Entrainment and Treatment of Cells
2.3 Dielectrophoresis
3 Methods
3.1 Sample Isolation
3.2 Entrainment and Sampling
3.3 DEP Using the 3DEP
4 Notes
References
Chapter 18: Measuring Circadian Neutrophil Infiltration in Tissues by Paired Whole-Mount Tissue Clearing and Flow Cytometry
1 Introduction
2 Materials
3 Methods
3.1 Obtaining Tissue Samples for Paired Analyses
3.2 Measuring Neutrophil Infiltration into Tissues by Flow Cytometry
3.2.1 Before You Begin
3.2.2 Processing the Liver
3.2.3 Processing the Lung
3.2.4 Processing the Skin
3.2.5 Processing the Spleen
3.2.6 Processing the Intestine
3.2.7 Processing the Bone Marrow
3.2.8 Processing the Blood
3.2.9 Processing the Heart, Skeletal Muscle and Kidney
3.2.10 Staining
3.2.11 Data Collection and Analysis
3.3 Whole-Mount Tissue Clearing and Immunofluorescence Staining
3.3.1 Tissue Immunofluorescence Staining and Clearing: Day 1-Tissue Permeabilization and Blocking
3.3.2 Tissue Immunofluorescence Staining and Clearing: Day 2 and 3-Staining with Primary Antibodies
3.3.3 Tissue Immunofluorescence Staining and Clearing: Day 4 and 5-Washing and Staining with Secondary Antibodies
3.3.4 Tissue Immunofluorescence Staining and Clearing: Day 6-Washing
3.3.5 Tissue Immunofluorescence Staining and Clearing: Day 7-Tissue Clearing
3.3.6 Image Capture
3.3.7 Quantification Pipeline: Quantification of Total Neutrophils and Infiltrating Neutrophils
3.3.8 Quantification Pipeline: Quantification of NETs
3.3.9 Quantification Pipeline: Measuring Spatial Relationships
3.4 Amplitude vs. Zero Test to Statistically Validate Circadian Behavior
4 Notes
References
Chapter 19: In Vivo Imaging of Circadian NET Formation During Lung Injury by Four-Dimensional Intravital Microscopy
1 Introduction
2 Materials
3 Methods
3.1 TRALI as a Model of Acute Lung Injury Dependent on Neutrophils and NETs
3.2 Method for Intravital Imaging of the Lung
3.2.1 Surgical Procedure for Tracheotomy
3.2.2 Surgical Procedure for Lung Exposure
3.3 In Vivo Staining and Quantification of NET Formation in the Lungs Upon Acute Lung Injury
3.3.1 Capture
3.3.2 Quantification
3.3.3 Analysis
4 Notes
References
Chapter 20: Real-Time Measurement of Energy Metabolism Over Circadian Time Using Indirect Calorimetry-Enabled Metabolic Cages
1 Introduction
2 Materials
2.1 Equipment (Fig. 1)
2.2 Consumables
3 Methods
3.1 Entrainment
3.2 Experimental Setup
3.3 Data Acquisition
3.4 Experiment Shutdown
3.5 Data Analysis
4 Notes
References
Chapter 21: Untargeted and Targeted Circadian Metabolomics Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and...
1 Introduction
2 Materials
2.1 Sample Preparation for Untargeted Metabolomics (See Note 1)
2.2 Hardware for Untargeted LC-MS/MS Analysis
2.3 Targeted Metabolomics
2.4 Hardware for Targeted Metabolomics
2.5 Software for Targeted Metabolomics
3 Methods
3.1 Sample Preparation for LC-MS/MS Based Untargeted Metabolomics
3.2 LC-MS/MS Analysis for Untargeted Metabolomics
3.3 LC-MS/MS and FIA-MS/MS Analysis for Targeted Metabolomics
3.4 Metabolomics Data Analysis
3.4.1 Untargeted Metabolomics Data Pre-processing and Quality Control
3.4.2 Targeted Metabolomics Data Pre-processing and Quality Control
3.5 Data Processing
4 Notes
References
Chapter 22: Time-Restricted Feeding and Caloric Restriction: Two Feeding Regimens at the Crossroad of Metabolic and Circadian ...
1 Introduction
1.1 Nutrition, Metabolic Health, and the Circadian Clock
1.2 Feeding Regimen Studies
2 Materials
2.1 Manual Implementation of TRF and CR
2.2 Twenty-four Hour Time-Series Collection (See Note 1)
2.3 ipGTT
3 Methods
3.1 Manual Implementation of TRF Studies in Rodents (See Note 2)
3.2 Manual Implementation of Caloric Restriction (CR) in Rodents (See Note 2)
3.3 Evaluation of the Activity of the Molecular Circadian Clock in Rodents from 24 h Time-Series Collection (See Note 8)
3.4 Performing an Intra-peritoneal Glucose Tolerance Test (ipGTT) in Mice Under Different Feeding Regimen
4 Notes
References
Chapter 23: Chromatin Immunoprecipitation and Circadian Rhythms
1 Introduction
2 Materials
2.1 Cross-linking
2.2 DNA Shearing
2.3 Immunoprecipitation
2.4 Reverse Cross-link
2.5 DNA Fragment Check
3 Methods
3.1 Cross-linking for the Liver Tissue
3.2 Cross-linking for Cells
3.2.1 Sonication for the Liver Tissues
3.2.2 Sonication for Cells
3.3 Immunoprecipitation
3.4 Reverse-Cross-linking
3.5 DNA Fragment Check
4 Notes
References
Chapter 24: Fluorescent Reporters for Studying Circadian Rhythms in Drosophila melanogaster
1 Introduction
1.1 The Circadian Clock
1.2 Drosophila as a Model to Understand Intestinal Stem Cell Function
1.3 Reporters of Circadian Rhythms
2 Materials
2.1 Fly Strains and Food
2.2 Solutions
2.3 Dissection Tools
2.4 Immunostaining Reagents
3 Methods
3.1 Clock Reporter Design
3.2 Fly Strain Preparation
3.3 Sample Collection
3.4 Midgut Dissection
3.5 Fixation and Staining
3.5.1 Fixation and DAPI Counterstaining
3.5.2 Immunostaining
3.6 Microscopy
3.7 Quantification
4 Notes
References
Chapter 25: Visualization of Mutant Aggregates from Clock Neurons by Agarose Gel Electrophoresis (AGERA) in Drosophila melanog...
1 Introduction
2 Materials
2.1 Protein Extraction
2.2 Agarose Gel for Protein Separation
2.3 Immunoblotting
2.4 Locomotor Activity Analysis
3 Methods
3.1 Flies: Experimental Set Up
3.2 Flies: Locomotor Activity Analysis
3.3 Gel Preparation
3.4 Protein Extraction and Gel Electrophoresis
3.5 Immunoblotting and Visualization
4 Notes
References
Chapter 26: Methods for Delivery of dsRNAi Against Canonical Clock Genes and Immunocytodetection of Clock Proteins in Crustacea
1 Introduction
2 Materials
2.1 Design, Synthesis, Storage, and Delivery of dsRNA
2.2 Immunocytochemistry
3 Methods
3.1 Design, Synthesis, and Storage of dsRNA
3.2 Preparation and Loading of Capillaries
3.3 Injection Delivery of dsRNA
3.4 Immunohistochemistry
3.4.1 Tissue Preparation and Sectioning
3.4.2 Immunodetection
4 Notes
References
Chapter 27: In Vivo Bioluminescence Analyses of Circadian Rhythms in Arabidopsis thaliana Using a Microplate Luminometer
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
2.3 Bacteria
2.4 Plants
3 Methods
3.1 Luciferase Vectors and Bioluminescent Reporter Constructs
3.2 Transformation of Arabidopsis thaliana
3.2.1 Transformation of Agrobacterium
3.2.2 Agrobacterium-Mediated Transformation of Arabidopsis thaliana by the Floral Dip Method
3.3 Seed Sterilization, Seedling Growth, and Entrainment
3.4 Preparation of 96-Well Plates
3.5 Setting the Luminometer and Chamber Conditions
3.6 Setting Measurement Conditions
3.7 Data Analyses
3.7.1 Upload Data Sets
3.7.2 Analysis of Data
3.7.3 Further Information About Using BioDare2
4 Notes
References
Index