Group A Streptococcus: Methods and Protocols (Methods in Molecular Biology, 2136)

✍ Scribed by Thomas Proft (editor), Jacelyn M. S. Loh (editor)

• Publisher: Humana
• Year: 2020
• Tongue: English
• Leaves: 395
• Category: Library

✦ Synopsis

This volume details Group A Streptococcus (GAS) research and provides the reader with an extensive collection of research protocols within this important field. Chapters guide readers through standard genetic protocols such as whole genome sequencing, transcriptome analyses, proteome analysis, application of GAS-derived molecular tools, and methods that are crucial for the evaluation of novel GAS vaccines and GAS virulence factors, including bactericidal assays and animal infection models. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Group A Streptococcus: Methods and Protocols aims to ensure successful results in the further study of this vital field.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Genotyping and Genetic Manipulation of Group A Streptococcus
Chapter 1: Detection of Streptococcus pyogenes Virulence Factors
1 Introduction
1.1 Streptococcus pyogenes Virulence Factors
1.2 Superantigens
1.3 DNAses
1.4 Proteases
1.5 Detection of the Virulence Factors as a Typing Tool
2 Materials
2.1 Reagents and Kits Needed for the DNA Extraction
2.2 Reagents Needed for PCR Reaction (See Note 2)
2.3 Reagents Needed for Product Separation and Visualization
2.4 Equipment
3 Methods
3.1 Template Preparation
3.2 Primer Mix Preparation Reaction Setup (See Note 5)
3.3 Gel Electrophoresis
4 Notes
References
Chapter 2: Closed-Tube Multiplex Real-Time PCR for the Detection of Group A Streptococcal Superantigens
1 Introduction
2 Materials
2.1 Bacterial DNA Isolation
2.2 Multiplex PCR
2.3 SYBR-Green PCR
3 Methods
3.1 Bacterial DNA Isolation
3.2 Multiplex PCR
3.3 Real-Time PCR Using SYBR-Green
4 Notes
References
Chapter 3: The emm-Cluster Typing System
1 Introduction
2 Materials
2.1 DNA Extraction
2.2 PCR
3 Methods
3.1 DNA Template Preparation
3.1.1 Using Heat to Lysate GAS Colonies from a Plate
3.1.2 Using Enzymatic Lysis of GAS Colonies from Plate
3.1.3 Using DNA Extraction of GAS from Liquid Culture
3.2 Emm Amplification by PCR
3.3 Sequencing
3.4 emm-Type and emm-Cluster Assignation
4 Notes
References
Chapter 4: Protocols for Tn-seq Analyses in the Group A Streptococcus
1 Introduction
2 Materials
2.1 Strains, Media, and Solutions
2.2 Reagents, Kits, and Consumables
2.3 Equipment
3 Methods
3.1 Generating Complex Transposon Mutant Pools
3.1.1 Producing the pKRMIT Plasmid
3.1.2 Transformation of pKRMIT into Streptococcus pyogenes
3.1.3 Selection of S. pyogenes Krmit Mutant Libraries for Tn-seq
3.2 Synthesis and Sequencing of Transposon Junctions from Mutant Pools
3.2.1 Preparation of the Adapters
3.2.2 Total Genomic DNA Extraction
3.2.3 Digestion of gDNA by MmeI and Ligation to MmeI Adapter
3.2.4 Production of the Krmit Insertion Tag Library
3.2.5 Illumina Sequencing and Data Analysis
4 Notes
References
Chapter 5: Genetic Manipulation of Group A Streptococcus-Gene Deletion by Allelic Replacement
1 Introduction
2 Materials
2.1 pLZts Vector Preparation
2.2 Insert Preparation and Cloning
2.3 GAS Transformation
2.4 Allelic Exchange
2.5 Mutant Screening
3 Methods
3.1 pLZts Vector Preparation
3.2 Design and Preparation of Mutant Allele Insert DNA
3.3 Gibson Assembly Cloning
3.4 GAS Transformation
3.5 Allelic Exchange
3.5.1 Plasmid Integration
3.5.2 Plasmid Excision
3.6 Mutant Screening
4 Notes
References
Chapter 6: Generation of Bioluminescent Group A Streptococcus for Biophotonic Imaging
1 Introduction
2 Materials
2.1 Plasmids
2.2 Electrocompetent GAS
2.3 Electroporation
2.4 Biophotonic Imaging
3 Methods
3.1 Preparation of Electrocompetent GAS
3.2 Electroporation of GAS
3.3 Selection of Bioluminescent Bacteria
3.4 Quantification of Bioluminescence
4 Notes
References
Part II: Application of Group A Streptococcus Omics
Chapter 7: Whole Genome Sequence Analysis and Population Genomics of Group A Streptococci
1 Introduction
1.1 The Streptococcus pyogenes Genome
1.2 Bioinformatic Workflow
1.3 Quality Control (QC)
1.4 Genome Assembly
1.5 Genome Annotation and Curation
1.6 Recombination Detection
1.7 Phylogenetic Analysis
1.8 Population Structure
1.9 Commonly Used Terminology
2 Materials
2.1 Computational Requirements
2.2 File Formats
2.3 Public Genome Sequence Repositories
2.3.1 Reference Sequence (RefSeq) Collection
2.3.2 The Sequence Read Archive (SRA)
2.4 Quality Control
2.4.1 Software Required
2.4.2 Inputs Required
2.5 Genome Assembly
2.5.1 Software Required
2.5.2 Inputs Required
2.6 Genome Annotation and Curation
2.6.1 Software Required
2.6.2 Inputs Required
2.7 Pangenome and Accessory Genome
2.7.1 Software Required
2.7.2 Input Required
2.8 Phylogenetic Analysis
2.8.1 Software Required
2.8.2 Inputs Required
2.9 Population Structure
2.9.1 Software Required
2.9.2 Inputs Required
3 Methods
3.1 Accessing Public Genome Sequence Data
3.2 Quality Control (QC) of Genome Sequence Data
3.2.1 Check Reads
3.2.2 Investigate Read Statistics
3.2.3 Clean Reads
3.2.4 Screen for Contamination
3.2.5 Investigation of Contamination
3.2.6 Other QC Metrics
3.3 Building Draft and Reference Genomes by Genome Assembly
3.3.1 Draft Genome Assembly from Short-Read Sequences
Subsample Reads
Assemble the Genome
Visualize the Assembly Graph (Optional)
Polishing the Genome (Error Correction)
Check Quality
3.3.2 Reference Genome Assembly from Oxford Nanopore Long-Read and Short-Read Genome Assembly Using Unicycler
Convert Nanopore fast5 into fastq Format (See Note 14)
Assemble the Genome
Visualize the Assembly Graph (Optional)
3.4 Annotation and Curation of Genome Assemblies
3.4.1 Gene and Feature Annotation
QC Genome Assembly
Genome Annotation
Check for Errors
Check Annotation Statistics
Relocate GenBank and FASTA for Further Analysis
3.4.2 Assign Multilocus Sequence Type (MLST) Alleles
3.4.3 Assign emm Type
3.4.4 Screen for Bacteriophage
3.4.5 Screen for Integrative Conjugative Elements (ICE)
3.4.6 Screen for Known Virulence Factors
3.4.7 Screen for Known Antimicrobial Resistance Genes
3.4.8 BLAST Screening of Genomes for Genes of Interest
3.5 Defining the Pangenome and Investigating Accessory Genome Variation in a Population
3.5.1 Check Timestamps of Prokka GFF3 Files
3.5.2 Copy GFF3 (.gff) Files to New Directory
3.5.3 Calculate the Pangenome
3.5.4 Pangenome Summary
3.5.5 Investigate the Presence and Absence of Genes
3.5.6 Pangenome-Wide Association
3.5.7 Intergenic Region Investigation (IGR)
3.6 Phylogenetic Analysis
3.6.1 Select a Reference Genome
Haploid Variant Calling
Calculating the Core Genome
Make a Quick Tree
Removal of Recombination
Remove Ambiguous Characters
Calculating the Proportion of SNPs That Are Influenced by Recombination
Examine Recombinogenic Regions
Calculate Constant Sites
Produce Maximum-Likelihood (ML) Phylogeny
Visualize the Tree
3.7 Analysis of Population Structure
3.7.1 Mash and Mashtree
3.7.2 PopPUNK
4 Notes
References
Chapter 8: Group A Streptococcus Transcriptome Analysis
1 Introduction
2 Materials
2.1 Cell Growth and Harvest
2.2 RNA Extraction and Processing (Quantification and Evaluation of Integrity)
2.3 rRNA Removal and Sequencing Library Preparation
3 Methods
3.1 Cell Growth and Harvest
3.2 RNA Extraction and Processing (Quantification and Evaluation of RNA Integrity)
3.2.1 RNA Extraction
3.2.2 RNA Quantification and Quality Control
3.3 Ribosomal RNA Depletion
3.3.1 Bead Washing Procedure
3.3.2 Treatment of Total RNA Samples with Ribo-Zero rRNA Removal Solution
3.3.3 Purification of the rRNA-Depleted Sample
3.4 Sequencing Library Preparation (See Note 11)
3.4.1 Fragmentation of RNA and cDNA Synthesis Primer Anneal
3.4.2 Purification of di-Tagged cDNA
3.4.3 Amplification of the RNA-seq Library
3.4.4 Purification of the RNA-Seq Library
3.4.5 RNA-Seq Library Quantification and Quality Assessment
3.4.6 RNA-seq Library Quality and Size Distribution Assessment
3.5 Library Sequencing
3.5.1 Single-Versus Paired-End
3.5.2 Higher Coverage (Reads) Versus More Replicates
3.6 RNA-seq Data Analysis
4 Notes
References
Chapter 9: Protocol for Proteome Analysis of Group A Streptococcus
1 Introduction
2 Materials
2.1 Bacterial Culture
2.2 Preparation of Secreted Extracellular Proteins
2.3 Subcellular Fractionation: Preparation of Cell Wall Proteins, Cytoplasmic Proteins, and Crude Membranes
2.4 Isolation of Membrane-Associated Peripheral Proteins
2.5 Sample Preparation for Mass Spectrometry
3 Methods
3.1 Bacterial Culture
3.2 Preparation of Secreted Extracellular Proteins
3.3 Subcellular Fractionation: Preparation of Cell Wall Proteins, Cytoplasmic Proteins, and Crude Membranes
3.4 Isolation of Membrane-Associated Peripheral Proteins
3.5 Sample Preparation for Mass Spectrometry
4 Notes
References
Chapter 10: Investigation of Group A Streptococcal Interactions with Host Glycan Structures Using High-Throughput Techniques: ...
1 Introduction
2 Materials
2.1 Preparation of GAS for Whole-Cell Analysis
2.2 Preparation of Purified Protein
2.3 Preparation of Glycan Microarray Slides
3 Methods
3.1 Preparing GAS for Whole-Cell Glycan Array Analysis
3.2 Preparing Purified Protein for Glycan Array Analysis
3.3 Preparation of Glycan Slides
3.4 Affixing Gene Frames to Slide
3.5 Incubating Sample with Slide
3.6 Analysis
4 Notes
References
Part III: Assays to Investigate Group A Streptococcus Virulence Factors and Virulence Mechanisms
Chapter 11: Using Lactococcus lactis as Surrogate Organism to Study Group A Streptococcus Surface Proteins
1 Introduction
2 Materials
2.1 Electrocompetent L. lactis
2.2 Electroporation
2.3 Cell Wall Protein Extraction
2.4 Flow Cytometry Analysis of Cell Surface Protein Expression
3 Methods
3.1 Production of Electrocompetent L. lactis
3.2 Electroporation of L. lactis
3.3 L. Lactis Cell Wall Protein Extraction
3.4 Quantitation of Cell Surface Protein Expression by Flow Cytometry
4 Notes
References
Chapter 12: Expression and Purification of Collagen-Like Proteins of Group A Streptococcus
1 Introduction
2 Materials
2.1 Protein Expression
2.1.1 General Equipment
2.1.2 Bacterial Growth Reagents
2.2 Protein Purification
2.2.1 General Equipment
2.2.2 Affinity Chromatography
2.3 Protein Characterization
3 Methods
3.1 Recombinant Protein Expression and Purification
3.1.1 Protein Expression-Small-Scale Induction
3.1.2 Protein Expression-10-mL Medium-Scale Induction
3.1.3 Protein Expression-Large-Scale Induction
3.2 Protein Purification-Affinity Purification
3.3 Protein Characterization (Quality Control)
3.3.1 Circular Dichroism Spectroscopy
4 Notes
References
Chapter 13: Detection of Fibronectin-Binding Proteins of Streptococcus pyogenes Using Ligand Blot Analysis
1 Introduction
2 Materials
2.1 Enzymatic Cell Wall Extraction
2.2 Extraction of Surface Proteins Under Denaturing Condition
2.3 Fn Biotinylation
2.4 Immunoblotting (Ligand Blot Assay)
3 Methods
3.1 Preparation of Cell-Surface Protein Fraction
3.1.1 Preparation of Cell Wall Extracts with Mutanolysin
3.1.2 Preparation of 8 M Urea Extracts
3.2 Biotinylation of Fn
3.3 Fbp Detection with Biotinylated Fn (Ligand Blot Assay)
4 Notes
References
Chapter 14: Morphology and Ultrastructure of Group A Streptococcus Biofilms
1 Introduction
2 Materials
2.1 Formation of Biofilms
2.2 Light Microscopy
2.3 Transmission Electron Microscopy
2.4 Scanning Electron Microscopy
3 Methods
3.1 Formation of Biofilms on Coverslips (See Note 3)
3.2 Formation of Biofilms in Petri Dishes
3.3 Transmission Electron Microscopy
3.4 Detection of Exopolysaccharides on Ultrathin Sections by Alcian Blue Staining
3.5 Preparation of Ultrathin Sections
3.6 Scanning Electron Microscopy
4 Notes
References
Chapter 15: Dynamic Interactions of Group A Streptococcus with Host Macrophages
1 Introduction
2 Materials
2.1 GAS Medium Preparation and Bacteria Culture
2.2 Growth and Differentiation of THP-1 Cells
2.3 Isolation of Bone Marrow-Derived Macrophages (BMDMs)
2.4 Isolation and Differentiation of Human Monocyte-Derived Macrophages (HMDMs)
2.5 Infection of Macrophages with GAS and Assessing GAS Survival
2.6 Assessing GAS Differential Protein Expression
2.7 Assessing Cytokine Release
2.8 Assessing Macrophage Host Cell Death
2.9 Assessing Differential Macrophage Protein Expression
2.10 Fluorescence Microscopy
3 Methods
3.1 GAS Culture
3.2 GAS Inoculum Preparation for Macrophage Infection
3.3 Growth and Differentiation of THP-1 Cells
3.4 Isolation and Differentiation of Bone Marrow-Derived Macrophages (BMDMs)
3.5 Isolation and Differentiation of Human Monocyte-Derived Macrophages (HMDMs)
3.6 Infection of Macrophages with GAS
3.7 Assessing GAS Outputs
3.7.1 GAS Total Bacteria Survival
3.7.2 GAS Intracellular Bacteria Survival
3.7.3 GAS Differential Protein Expression
3.8 Assessing Macrophage Outputs
3.8.1 Cytokine Release
3.8.2 Assessing Macrophage Host Cell Death Using the LDH Release Assay
3.8.3 Assessing Macrophage Host Cell Death Using Propidium Iodide Uptake Assay
3.8.4 Macrophage Differential Protein Expression
3.8.5 Fluorescence Microscopy to Observe GAS-Macrophage Encounters and to Perform Colocalization Analysis
4 Notes
References
Chapter 16: Identification of Group A Streptococcus-Containing Autophagosome-Like Vacuoles
1 Introduction
1.1 Group A Streptococcus Infection of Epithelial Cells
1.2 Autophagy
1.3 Autophagy During GAS Infection
2 Materials
3 Methods
3.1 Immunostaining for LC3
3.2 Fluorescent Protein-Tagged LC3
4 Notes
References
Chapter 17: Flow Cytometry-Based Assays to Quantify Complement Deposition and Neutrophil Uptake of Group A Streptococcus
1 Introduction
2 Materials
2.1 Phlebotomy
2.2 C3 Deposition Assay
2.3 Neutrophil Purification
2.4 Neutrophil Uptake Assays
3 Methods
3.1 GAS Culture
3.2 Preparation of Fresh Human Serum
3.3 Complement Deposition Assay
3.4 Neutrophil Purification
3.5 Neutrophil Uptake Assays
3.5.1 Preparation of GAS
3.5.2 Neutrophil Uptake
4 Notes
References
Chapter 18: Live Cell Microscopy and Flow Cytometry to Study Streptolysin S-Mediated Erythrocyte Hemolysis
1 Introduction
2 Materials
2.1 Bacteria Culture
2.2 SLS Preparations
2.3 Blood Preparation
2.4 Chloride Indicator Loading of Red Blood Cells
2.5 Live Imaging
2.6 Flow Cytometry
3 Methods
3.1 Bacteria Culture
3.2 SLS Preparation
3.3 Blood Preparation
3.4 Chloride Indicator Loading of Red Blood Cells
3.5 Live Imaging
3.6 Quantitating Water Influx by Flow Cytometry
4 Notes
References
Chapter 19: Isolation of Monoclonal Antibodies to Group A Streptococcus Antigens Using Phage Display
1 Introduction
2 Materials
2.1 Biotinylation of GAS Antigens
2.2 Biopanning
2.3 Screening Clones Using Monoclonal Phage ELISA
2.4 Fingerprinting to Identify Unique Clones
3 Methods
3.1 Biotinylation of GAS Antigens and Immobilization on Streptavidin Resin
3.2 Preparation of Phage Library
3.3 First Round of Biopanning
3.3.1 Phage Binding and Elution
3.3.2 Infection of X-L1 Blue E. coli and Amplification of the Eluted Phage
3.3.3 Preparation of Phage for Subsequent Panning Rounds
3.4 Subsequent Rounds of Panning to Enrich for Antibodies That Bind to GAS Antigens
3.5 Screening Individual Clones
3.5.1 Small-Scale Phage Rescue
3.5.2 Monoclonal Phage ELISA
3.5.3 Restriction Digestion Fingerprinting to Identify Unique Clones
4 Notes
References
Part IV: Group A Streptococcus Killing Assays and Infection Models
Chapter 20: Assays to Analyze Adhesion of Group A Streptococcus to Host Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Bacteria
2.3 Adhesion Assay
3 Methods
3.1 Preparation of Cell Monolayer
3.2 Determine the Number of GAS Colony-Forming Units with Respect to OD600
3.3 Host-Cell Adhesion Assay
3.3.1 Adhesion Assay with Bioluminescent GAS Expressing Luciferase
3.3.2 Adhesion Assay with Nonbioluminescent GAS
4 Notes
References
Chapter 21: The Use of Galleria mellonella (Wax Moth) as an Infection Model for Group A Streptococcus
1 Introduction
2 Materials
2.1 Bacterial Culture
2.2 G. mellonella Larvae
2.3 Materials for Injection and Monitoring
2.4 Materials for Assessing Bacteria Burden by Homogenization
3 Methods
3.1 Preparing Bacterial Suspension for Injection
3.2 Preparation and Infection of G. mellonella Larvae
3.3 Monitoring of G. mellonella Larvae
3.4 Assessment of Post-infection Bacterial Burden
4 Notes
References
Chapter 22: A Superficial Skin Scarification Method in Mice to Mimic Streptococcus pyogenes Skin Infection in Humans
1 Introduction
2 Materials
2.1 Bacterial Preparation and Mice Procedures
2.2 Media and Solutions
3 Methods
3.1 Bacterial Inoculum
3.2 Animal Adaptation of S. pyogenes Isolates
3.3 Superficial Skin Infection
3.3.1 Anesthetization of Mice
3.3.2 Skin Site Scarification and Infection
3.4 Sample Collection and Preparation
3.5 Sample Processing and Bacterial Burden Determination
3.6 Quantification of Bacterial Burden
4 Notes
References
Chapter 23: A Mouse Nasopharyngeal Colonization Model for Group A Streptococcus
1 Introduction
2 Materials
2.1 Bacteria Preparation
2.2 Mouse Challenge and Monitoring
3 Methods
3.1 GAS Preparation
3.2 Nasopharyngeal Challenge
3.3 Monitoring of Bacterial Shedding
3.4 Monitoring by Biophotonic Imaging
3.5 Enumeration of Bacterial Load
4 Notes
References
Chapter 24: Humanized Plasminogen Mouse Model to Study Group A Streptococcus Invasive Disease
1 Introduction
2 Materials
2.1 Preparation of Frozen Stock GAS Inoculum
2.2 Preparation of Fresh GAS Inoculum
2.3 Mouse Infection
2.4 Determination of Bacterial Burden
3 Methods
3.1 Preparation of Frozen Stock GAS Inoculum
3.2 Preparation of Fresh GAS Inoculum
3.3 Mouse Infection
3.4 Determination of Bacterial Burden
4 Notes
References
Chapter 25: Lancefield Whole Blood Killing Assay to Evaluate Vaccine Efficacy
1 Introduction
2 Materials
3 Method
4 Notes
References
Chapter 26: An Opsonophagocytic Killing Assay for the Evaluation of Group A Streptococcus Vaccine Antisera
1 Introduction
2 Materials
2.1 HL-60 Cell Culture
2.2 Materials for Bacterial Growth, Optimum Dilution, and OPK Assay
2.3 Plates and Medium Preparation
3 Methods
3.1 Preparation of GAS TGG Working Stocks
3.2 Preparation of Bacteria for Use in Optimum Dilution and OPK Assays
3.3 HL-60 Cell Expansion from ATCC Master Stock
3.4 HL-60 Working Stock Preparation
3.5 HL-60 Cell Differentiation and Preparation for Use in Assays
3.6 Optimum Dilution Assay
3.7 Opsonophagocytic Killing Assay (OPK Assay)
4 Notes
References
Part V: Group A Streptococcus Therapies
Chapter 27: Validation of Suitable Carrier Molecules and Target Genes for Antisense Therapy Using Peptide-Coupled Peptide Nucl...
1 Introduction
2 Materials
2.1 PNA Stock Solution
2.2 PNA Killing Assay
2.3 PNA Minimal Inhibitory Concentration (MIC) Assay
3 Methods
3.1 PNA Killing Assay
3.2 PNA MIC Assay
4 Notes
References
Chapter 28: Interfering with the Folding of Group A Streptococcal pili Proteins
1 Introduction
2 Materials
2.1 Protein Engineering and Isopeptide-Blocker Design
2.2 Protein Expression
2.3 Protein Purification
2.4 Protein Labeling and SDS-PAGE
2.5 AFM-Based Force Spectroscopy
3 Methods
3.1 Preparation of the Pili Protein and Isopeptide Blocker
3.1.1 Pilus Polyprotein Design
3.1.2 Isopeptide-Blocker Design
3.1.3 Isopeptide-Blocker + Pili-Protein System
3.2 Culturing and Protein Purification
3.2.1 Expression of the Isopeptide-Blocker-Pili System
3.2.2 Purification of the Pilus Protein
3.2.3 SDS-Polyacrylamide Gel Electrophoresis
3.3 Single-Molecule Force Spectroscopy (smFS)
3.3.1 AFM Measurements
3.3.2 Data Analysis
4 Notes
References
Part VI: Application of Group A Streptococcus-Derived Methods
Chapter 29: Use of Streptolysin O (SLO) to Study the Function of Lipid Rafts
1 Introduction
2 Materials
2.1 BCR Stimulation and SLO Treatment
2.2 Cell Fixation and Fluorescent Staining
3 Methods
3.1 SLO Treatment and Labeling BCRs and Lipid Rafts for Microscopy
3.2 Tyrosine Phosphorylation Staining for Microscopy
3.3 Tyrosine Phosphorylation Staining for Flow Cytometry
3.4 BCR Endocytosis Assay Using Flow Cytometry
4 Notes
References
Chapter 30: Engineering of Group A Streptococcus Isopeptide Bonds into Immunoglobulin-Like Protein Domains
1 Introduction
2 Materials
2.1 Modeling Software and Web Tools
2.2 Cloning
2.3 Expression and Purification
2.4 SDS-Page
2.5 Mass Spectrometry
2.6 DSF
3 Methods
3.1 Domain Searching
3.2 Isopeptide Bond Modeling
3.3 Cloning
3.4 Expression
3.5 Characterization of Isopeptide Bond Formation by Mass Spectrometry
3.6 Thermal Stability by Differential Scanning Fluorimetry (DSF)
3.7 X-Ray Crystallography
4 Notes
References
Index