Clinical and Preclinical Models for Maximizing Healthspan: Methods and Protocols (Methods in Molecular Biology, 2138)

Preface
Contents
Contributors
Part I: Reviews
	Chapter 1: The Impact of New Biomarkers and Drug Targets on Age-Related Disorders
		1 Introduction
		2 Factors Affecting Aging
			2.1 Gender
			2.2 Diet
			2.3 Physical Exercise
			2.4 Smoking and Alcohol
			2.5 Sociality
			2.6 Telomeres and Telomerase
		3 Dementia and Alzheimer´s Disease
			3.1 Molecular Pathways Affected in Alzheimer´s Disease
				3.1.1 Amyloid Plaques
				3.1.2 Neurofibrillary Tangles
				3.1.3 Oxidative Stress
			3.2 Benefits of Early Diagnosis
			3.3 Interventions
			3.4 New Treatment Approaches
		4 Type 2 Diabetes Mellitus
			4.1 Pathways Affected in Type 2 Diabetes Mellitus
			4.2 Benefits of Early Diagnosis
			4.3 The Need for Biomarkers
				4.3.1 Biomarkers for Disease Risk
				4.3.2 Biomarkers for Cardiovascular Disease Risk
				4.3.3 Biomarkers for Diabetic Nephropathy
		5 Frailty
			5.1 Biological Mechanisms Underlying Frailty
			5.2 Biomarkers of Frailty
			5.3 Management of Frailty
				5.3.1 Exercise
				5.3.2 Nutrition
				5.3.3 Pharmaceuticals
		6 Conclusions
		References
	Chapter 2: Vitamin D and Muscle Sarcopenia in Aging
		1 Introduction
		2 What Is Sarcopenia?
		3 Risk Factors for Sarcopenia
		4 Sarcopenia and Side Effects
		5 Epidemiology of Sarcopenia
		6 Sarcopenia Histopathology
		7 Vitamin D Metabolism and Functions
		8 Vitamin D in Musculoskeletal Tissue
		9 Vitamin D and Muscle Aging
		10 Vitamin D and Muscle Strength
		11 Vitamin D and Muscle Mass
		12 Conclusions
		References
	Chapter 3: Quantitative Assays of Plasma Apolipoproteins
		1 Introduction
		2 Pre-analytical Considerations in the Determination of Apolipoproteins
			2.1 Nature of the Sample
				2.1.1 Example Considerations
				2.1.2 Recommendations
			2.2 Sampling
				2.2.1 Example Considerations
				2.2.2 Recommendations
			2.3 Sample Storage
				2.3.1 Example Considerations
				2.3.2 Recommendations
			2.4 Demographics
				2.4.1 Example Considerations
				2.4.2 Recommendations
			2.5 Lifestyle
				2.5.1 Considerations
				2.5.2 Recommendations
			2.6 Experimental Design
		3 Apolipoprotein Assay Methods
			3.1 Antibody-Based Multiplex Apolipoprotein Assays
				3.1.1 Materials
			3.2 MALDI/ESI-MSIA
				3.2.1 Materials
				3.2.2 Protocol
				3.2.3 Sample Preparation
			3.3 Targeted ESI Mass Spectrometry Assays
				3.3.1 Methods
				3.3.2 Peptide Synthesis
				3.3.3 Calibration, Recovery, and Matrix Effects
				3.3.4 Sample Preparation
			3.4 LC-MSMS Targeted MRM Analysis
		4 Conclusions
		References
	Chapter 4: Protocol for the Use of the Ketogenic Diet in Preclinical and Clinical Practice
		1 Introduction
		2 Best Practices
			2.1 Physiological vs. Pathological Ketosis
			2.2 Prior to Starting the Ketogenic Diet
			2.3 Monitoring the State of Ketosis
		3 Macronutrients
			3.1 Preclinical
			3.2 Clinical
		4 Micronutrients: Is It Just Fat?
		5 Conclusions
		References
Part II: Protocols
	Chapter 5: An In Vivo/Ex Vivo Study Design to Investigate Effects of Chronic Conditions and Therapeutic Compounds on Adipose S...
		1 Introduction
		2 Materials
			2.1 Drug-Containing Gelatin Blocks (See Note 1)
			2.2 OGTT
			2.3 Isolation and Culturing of Primary ASCs
		3 Methods
			3.1 Administration of Extracts or Drugs Using Jelly Blocks (See Note 7)
			3.2 Procedure for OGTT
			3.3 Harvesting of Adipose Tissue for Isolation of Primary ASCs (See Note 12)
			3.4 Isolation of ASCs from Excised Fat by Collagenase Digestion
			3.5 Passaging of ASC Cultures (Fig. 2) (See Note 20)
			3.6 Adipogenic Differentiation (See Note 22)
			3.7 ORO Staining (See Note 25)
		4 Notes
		References
	Chapter 6: Model for Studying the Effects of Chronic Metabolic Disease on Endogenous Bone Marrow Stem Cell Populations
		1 Introduction
		2 Materials
			2.1 Solutions
			2.2 Equipment
		3 Methods
			3.1 Harvesting of Femurs
			3.2 Isolation and Culture of Bone Marrow Mesenchymal Stem Cells
			3.3 Harvesting of RNA from Isolated Cells (RNase-Free Laboratory)
			3.4 Harvesting of Protein from Isolated Cells
			3.5 Flow Cytometry
			3.6 Assessing MSC Viability
			3.7 Assessing Proliferation Rate
			3.8 Assessing Differentiation Capacity
		4 Notes
		References
	Chapter 7: Investigating Alcohol Behavior and Physiology Using Drosophila melanogaster
		1 Introduction
		2 Materials
			2.1 Building a FlyBar
			2.2 Fly Husbandry
			2.3 Alcohol Exposure Testing
			2.4 Gut and Brain Permeability Assay
		3 Methods
			3.1 Assembly of the FlyBar (See Note 1)
			3.2 Fly Husbandry and Preparation of Experimental Animals (See Note 7)
			3.3 Loss-of-Righting Reflex and Sedation (See Notes 13 and 14)
			3.4 Recovery from Alcohol-Induced Sedation (See Note 19)
			3.5 Repeat Exposures to Alcohol (Fig. 3b) (See Note 21)
			3.6 Alcohol Tolerance (Fig. 3c, d) (See Note 24)
			3.7 Alcohol Absorbance and Alcohol Clearance (See Note 28)
			3.8 Gut and Brain Permeability Assays (See Note 31)
		4 Notes
		References
	Chapter 8: Using Genome-Editing Tools to Develop a Novel In Situ Coincidence Reporter Assay for Screening ATAD3A Transcription...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Generation of CRISPR-Cas9 Plasmid Targeting ATAD3A
			3.2 Preparation of Coincidence Reporter Gene Construct with Homology Sequences Targeting ATAD3A (Fig. 1)
			3.3 Generate HN12-ATAD3A-FLuc-NLuc Cells (SeeNote 11)
		4 Notes
		References
	Chapter 9: Visualizing and Evaluating Cancer Cell Growth and Invasion by a Novel 3D Culture System
		1 Introduction
		2 Materials
		3 Methods
			3.1 Visualizing and Evaluating Cancer Cell Growth Using a 3D System
			3.2 Visualizing and Evaluating Cancer Cell Invasion Using a 3D System
		4 Notes
		References
	Chapter 10: Exploiting Plug-and-Play Electrochemical Biosensors to Determine the Role of FGF19 in Sorafenib-Mediated Superoxid...
		1 Introduction
		2 Materials (See Note 1)
			2.1 Synthesis of O2- Electrochemical Sensor
			2.2 Synthesis of NO Electrochemical Sensor
			2.3 Polishing of Glassy Carbon Electrode
			2.4 Regents
			2.5 Equipment (See Note 3)
		3 Methods
			3.1 Cell Culture
			3.2 Preparation of O2- Electrochemical Biosensor (Fig. 2a)
			3.3 Preparation of a NO Electrochemical Biosensor (Fig. 3a)
			3.4 Detection of Intracellular O2- Levels in HCC Cells
			3.5 Detection of Intracellular NO Levels in HCC Cells
		4 Notes
		References
	Chapter 11: Proteomic Analysis of the Anoikis-Resistant Human Breast Cancer Cell Lines
		1 Introduction
		2 Materials
			2.1 Anoikis-Resistant Assay
			2.2 Proteomic Analysis
		3 Methods
			3.1 Anoikis Assay
			3.2 Sample Preparation for Proteomics
			3.3 LC-MS/MS Proteomic Analysis
		4 Notes
		References
	Chapter 12: Real-Time PCR Analysis of Metabolism-Related Genes in a Long-Lived Model of C. elegans
		1 Introduction
		2 Materials (See Note 1)
			2.1 Nematode Strains and Synchronized Culture Reagents
			2.2 Reagents and Equipment for Total RNA Preparation
			2.3 Purification of Poly(A)+ RNA
			2.4 Reverse Transcription
			2.5 Real-Time PCR
		3 Methods
			3.1 Synchronized Culture of C. elegans with FUdR
			3.2 Preparation of Total RNA
			3.3 Removal of Genomic DNA by DNase I Treatment
			3.4 Purification of Poly(A)+ RNA
			3.5 cDNA Synthesis
			3.6 Real-Time PCR Using the TaqMan Gene Expression Assay
		4 Notes
		References
	Chapter 13: Clinical Assessment of the NADome as Biomarkers for Healthy Aging
		1 Introduction
		2 Materials
			2.1 Samples
			2.2 Extraction
			2.3 Sample Analysis
		3 Methods
			3.1 Sample Preparation
			3.2 Sample Extraction
			3.3 Standard Preparation
			3.4 Liquid Chromatography
			3.5 Analysis
		4 Notes
		References
	Chapter 14: Two-Dimensional Gel Electrophoresis Combined with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass ...
		1 Introduction
		2 Materials
			2.1 Samples
			2.2 2DGE
			2.3 In-Gel Digestion
			2.4 MALDI-TOF MS
		3 Methods
			3.1 Lens Homogenization
			3.2 2DGE Analysis
			3.3 In-Gel Digestion (See Note 17)
			3.4 MALDI-TOF Mass Fingerprinting Analysis
		4 Notes
		References
	Chapter 15: Testing the Effects of Dietary Seafood Consumption on Depressive Symptoms
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 16: Assays for Monitoring the Effects of Nicotinamide Supplementation on Mitochondrial Activity in Saccharomyces cerev...
		1 Introduction
		2 Materials
			2.1 Yeast Strains and Growth Conditions
			2.2 Reagents
			2.3 Assays
			2.4 Equipment
		3 Methods
			3.1 NAM Supplementation to S. cerevisiae Cells at the Diauxic Shift
			3.2 Determination of Respiratory Parameters of Intact Cells
			3.3 Determination of the Index of Respiratory Competence (IRC)
			3.4 Fluorescence Microscopy to Assess Mitochondrial Membrane Potential and Morphology
			3.5 Fluorescence Microscopy to Analyze O2- Accumulation
		4 Notes
		References
	Chapter 17: Measurement of a Surrogate Biomarker for Arginine Vasopressin Secretion in Association with Physiometric and Molec...
		1 Introduction
		2 Materials
			2.1 Physical and Routine Blood Measurements
			2.2 Copeptin Measurements
			2.3 Multiplex Immunoassay
		3 Methods
			3.1 Physical Measurements
			3.2 Blood Analyses
			3.3 Copeptin Measurement
			3.4 Multiplex Immunoassay (See Note 10)
		4 Notes
		References
	Chapter 18: Liquid Chromatography Tandem Mass Spectrometry Analysis of Proteins Associated with Age-Related Disorders in Human...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Pituitary Protein Extract
			3.2 Analysis of Peptide Digests by LC/MS/MS
		4 Notes
		References
	Chapter 19: Coenzyme Q10 Assessment and the Establishment of a Neuronal Cell Model of CoQ10 Deficiency
		1 Introduction
		2 Materials
			2.1 Preparation of Cell and Tissue Samples (See Note 1)
			2.2 Extraction of Cellular CoQ10
			2.3 HPLC Analysis
			2.4 Total Protein Determination
			2.5 Citrate Synthase (CS) Assay
			2.6 Cell Model of CoQ10 Deficiency
		3 Methods
			3.1 Preparation of Tissue and Cell Samples
			3.2 Extraction of Cellular CoQ10
			3.3 Coenzyme Q10 Quantification Using HPLC-UV Detection (Fig. 2) (See Note 5)
			3.4 Total Protein Determination (See Note 10)
			3.5 CS Assay (See Note 11)
			3.6 Establishment of a Human Neuronal Cell Model of CoQ10 Deficiency (See Note 12)
		4 Notes
		References
	Chapter 20: Analyzing Mitochondrial Function in Brown Adipocytes with a Bioenergetic Analyzer
		1 Introduction
		2 Materials
			2.1 Preadipocyte Isolation and Culture Reagents
			2.2 Mito Stress Test Reagents
			2.3 Equipment
		3 Methods
			3.1 Isolation of Brown Preadipocytes
			3.2 Culture of Brown Preadipocytes and Differentiation into Mature Brown Adipocytes
			3.3 Mito Stress Test
			3.4 Data Analysis and Interpretation
		4 Notes
		References
	Chapter 21: Quantitative Analysis of DNA Methylation by Bisulfite Sequencing
		1 Introduction
		2 Materials
			2.1 DNA Isolation from Solid Tissues and Cells
			2.2 Bisulfite Conversion
			2.3 Amplification of Bisulfite-Converted DNA
			2.4 Sequence Analysis
		3 Methods
			3.1 DNA Purification from Solid Tissues
			3.2 DNA Purification from Cultured Cells
			3.3 Bisulfite Conversion (See Note 12)
			3.4 Amplification of Bisulfite-Converted DNA (See Note 19)
			3.5 PCR Fragments Isolation (See Note 20)
			3.6 DNA Methylation Analysis by Sanger Sequencing of Amplified BS-Converted DNA (See Note 26)
		4 Notes
		References
	Chapter 22: Histomorphometric Analysis of Anti-Aging Properties on Rat Skin
		1 Introduction
		2 Materials
			2.1 Animals, Solutions, and Reagents
			2.2 Equipment and Software
		3 Methods
			3.1 Animals
			3.2 UV Ray Induction
			3.3 Preparation of Curcuma heyneana Rhizome Extract
			3.4 Treatment
			3.5 Biopsy and Histology Analysis
			3.6 Statistical Analysis
		4 Notes
		References
	Chapter 23: Integration of qRT-PCR and Immunohistochemical Techniques for mRNA Expression and Localization of m1AChR in the Br...
		1 Introduction
		2 Materials
			2.1 qRT-PCR
			2.2 IHC
		3 Methods
			3.1 Tissue Collection
			3.2 RNA Extraction
			3.3 cDNA Synthesis
			3.4 qRT-PCR
			3.5 Brain Tissue Processing for IHC (See Note 26)
			3.6 IHC Staining
			3.7 IHC Quantification
		4 Notes
		References
	Chapter 24: Randomized Study Design to Test Effects of Vitamin D and Omega-3 Fatty Acid Supplementation as Adjuvant Therapy in...
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 25: Effect of Vitamin D Supplementation on Muscle Strength, Muscle Function, and Body Composition in Vitamin D-Deficie...
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 26: The Association of Food Intake and Physical Activity with Body Composition, Muscle Strength, and Muscle Function i...
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 27: Absolute Quantification of Plasma Apolipoproteins for Cardiovascular Disease Risk Prediction
		1 Introduction
		2 Materials
			2.1 Samples
			2.2 Sample and Standard Preparation
			2.3 Sample Digestion
			2.4 Sample Analysis
		3 Methods
			3.1 Sample Collection
			3.2 Standard Preparation
			3.3 Sample Preparation
			3.4 Online IMER Trypsin Digestion
			3.5 Liquid Chromatography
			3.6 Analysis
		4 Notes
		References
	Chapter 28: Multiplex Analysis of Circulating Hormone Levels in Rat Models of Age-Related Diseases
		1 Introduction
		2 Materials
			2.1 Conjugation
			2.2 Biotinylation
			2.3 Assay
		3 Methods
			3.1 Conjugation
			3.2 Biotinylation
			3.3 Assay
			3.4 Data Analysis
		4 Notes
		References
	Chapter 29: Proteomic Analysis of Brain Tissue from a Chronic Model of Stress Using a Combined 2D Gel Electrophoresis and Mass...
		1 Introduction
		2 Materials
			2.1 Animals, Materials, and Reagents
			2.2 Equipment
		3 Methods
			3.1 Corticosterone Treatment and Tissue Protein Extraction
			3.2 2D-DIGE
			3.3 MALDI-TOF Mass Fingerprinting
		4 Notes
		References
	Chapter 30: Proteomic Analysis of Rat Hippocampus for Studies of Cognition and Memory Loss with Aging
		1 Introduction
		2 Materials
			2.1 Protein Extraction
			2.2 2D LC-MS/MS
		3 Methods
			3.1 Protein Extraction
			3.2 Ion-Exchange Chromatography
			3.3 LC-MS/MS
		4 Notes
		References
	Chapter 31: Brain Proteomic Analysis on the Effects of the Antidepressant Fluoxetine
		1 Introduction
		2 Materials
			2.1 Fluoxetine Treatment (See Note 1)
			2.2 Preparation of Protein Extracts
			2.3 IEF and Second-Dimension Electrophoresis
			2.4 MALDI-TOF Mass Spectrometry
			2.5 Western Blot Validation
		3 Methods
			3.1 Fluoxetine Treatment and Preparation of Cerebral Cortex Protein Extracts (See Note 8)
			3.2 2D-DIGE
			3.3 MALDI-TOF Mass Spectrometry
			3.4 Western Blot Analysis
		4 Notes
		References
	Chapter 32: MK-801 Treatment of Oligodendrocytes as a Cellular Model of Aging
		1 Introduction
		2 Materials
			2.1 Cell Culture and Sample Preparation
			2.2 LC-MS/MS Analysis
		3 Methods
			3.1 Cell Culture and Treatment (See Note 6)
			3.2 Preparation of Cell Proteomes
			3.3 LC-MS/MS Analysis (See Note 15)
		4 Notes
		References
Index