Annual Report - Iowa State Commerce Commission

Preface
Contents
Contributors
Chapter 1: Using QSAR Models to Predict Mitochondrial Targeting by Small-Molecule Xenobiotics Within Living Cells
	1 Introduction
	2 Materials
		2.1 Information Sources Giving Chemical Structures of Xenobiotics
		2.2 Information Sources Concerning Chemical or Biochemical Modification of Xenobiotics Within, or Adjacent to, Cells
		2.3 Information Sources and Procedures for Specifying Ionized Chemical Species Derived from the Xenobiotic of Interest
		2.4 Procedures for Specifying CBN Values
		2.5 Information Sources and Procedures for Obtaining log P Values
		2.6 Procedure for Obtaining Amphiphilic Index (AI) Values
	3 Methods
		3.1 Establishing Chemical Structures
		3.2 Estimating Structure Parameters for each Compound and each Ionized Species
		3.3 Predicting Mitochondrial Targeting/Nontargeting Using the QSAR Algorithm (See Note 16)
	4 Notes
	References
Chapter 2: DQAsomes as the Prototype of Mitochondria-Targeted Pharmaceutical Nanocarriers: An Update
	1 Introduction
		1.1 DQAsomes-The Early Years
		1.2 DQAsomes: The Recent Years
	2 Materials
	3 Methods
		3.1 Preparation of Empty DQAsomes
		3.2 Quantification of the Dequalinium Chloride Concentration in DQAsomes
		3.3 Transfection of Mammalian Cells with DQAplexes
		3.4 Encapsulation of Paclitaxel into DQAsomes (See Note 8)
		3.5 Quantitative DQA and Paclitaxel Determination in Paclitaxel-Loaded DQAsomes
	4 Notes
	References
Chapter 3: Synthesis and Characterization of Mitochondria-Targeted Triphenylphosphonium Bolaamphiphiles
	1 Introduction
	2 Materials
		2.1 Synthesis of Triphenylphosphonium Bolaamphiphiles TPP1-TPP4
		2.2 Chromatography of Triphenylphosphonium Bolaamphiphiles TPP1-TPP4
		2.3 Conductivity Experiments on Bolaamphiphiles TPP1-TPP4
		2.4 Dialysis Experiments on Aqueous Solutions of TPP1-TPP4 Above the  cac
		2.5 Dynamic and Dielectrophoretic Light Scattering (DLS, DELS) Measurements
		2.6 Transmission Electron Microscopy (TEM)
		2.7 Raman Characterization of Triphenylphosphonium Bolaamphiphiles TPP1-TPP4
	3 Methods
		3.1 Synthesis of Triphenylphosphonium Bolaamphiphiles TPP1-TPP4
			3.1.1 1,16-Dibromohexadecane
			3.1.2 1,20-Dibromoeicosane 5
			3.1.3 1,30-Dibromotricontane 6
			3.1.4 General Procedure for the Quaternization Reaction to Achieve TPP1-TPP4
		3.2 HPLC Analysis for TPP Bolaamphiphiles
			3.2.1 HPLC Analysis for TPP1-TPP3
			3.2.2 HPLC Analysis for TPP4
		3.3 Conductivity Experiments on Bolaamphiphiles TPP1-TPP4
			3.3.1 Krafft Point of TPP1-TPP4
			3.3.2 Determination of the cac of TPP1-TPP4
		3.4 Dialysis Experiments on TPP1-TPP4
		3.5 DLS and DELS Measurements
			3.5.1 Size Measurements
			3.5.2 Zeta Potential Measurements
		3.6 TEM
		3.7 Raman Analysis of Bolaphosphonium TPP1-TPP4 Solutions
	4 Notes
	References
Chapter 4: Synthesis and Evaluation of 18F-Labeled Fluoroalkyl Triphenylphosphonium Salts as Mitochondrial Voltage Sensors in ...
	1 Introduction
		1.1 The Rationale of [18F]-Labeled Fluoroalkyl Triphenylphosphonium Salts ([18F]FATPs)
		1.2 Limitations
		1.3 Experimental Design
	2 Materials
		2.1 Column Solvents
		2.2 Solutions
		2.3 Other Supplies
	3 Methods
		3.1 Preparation of Fluoroalkyl Triphenylphosphonium Salts
			3.1.1 Synthesis of α, ω-di-Tosyloxyalkane (1a-3a)
			3.1.2 Synthesis of ω-Fluoroalkyl Tosylate (1b-3b)
			3.1.3 Synthesis of Fluoroalkyl Triphenylphosphonium Salts (1c-3c)
		3.2 Radiosynthesis of [18F]Fluoroalkyl Triphenylphosphonium Salts
			3.2.1 Preparation of Activated [18F]Fluoride
			3.2.2 Radiolabeling of Fluoroalkyl Triphenylphosphonium Salts
			3.2.3 HPLC Purification
			3.2.4 Quality Control and Preparation of In Vivo Experiments
		3.3 Preparation of [13N]NH3
		3.4 Perfused Isolated Rat Heart Study
		3.5 Preparation of Animals
			3.5.1 Surgery Preparation and Animal Anesthetization
			3.5.2 Preparation of MI Model
		3.6 Protocol of PET Imaging
			3.6.1 Preparation of Small Animals
			3.6.2 Data Acquisition
		3.7 MI Confirmation by TTC Staining Procedures
	4 Notes
	References
Chapter 5: Novel Mitochondria-Targeted Triphenylphosphonium Conjugates of Linear β-Phosphorylated Nitrones: Preparation, 31P N...
	1 Introduction
	2 Materials
		2.1 Chemical Synthesis and Analysis
		2.2 Cell Culture and Cytotoxicity Studies
		2.3 EPR Spectrometry
		2.4 Mitochondrial Permeation Capacity
		2.5 Apoptosis Determination in Schwann Cells
	3 Methods
		3.1 Synthesis of (4-Iodobutyl)triphenylphosphonium Iodide (IBTP, 3)
		3.2 Synthesis of (6-Iodohexyl)triphenylphosphonium Iodide (IHTP, 4)
		3.3 Synthesis of 8-Bromo-Octanol (5)
		3.4 Synthesis of (8-Hydroxyoctyl)triphenylphosphonium Bromide (6)
		3.5 Synthesis of (8-Iodooctyl)triphenylphosphonium Iodide (IOTP, 7) (Adapted from)
		3.6 General Procedure for the Triphenylphosphonium Nitrone Iodides (8a-d)
		3.7 General Procedure for the Diphenylphosphonyl nitrones (9a-d)
		3.8 Cell Cultures and Cytotoxicity Assays for Nitrones 8(a-d) and 9(a-d)
		3.9 Protocol: EPR Spin Trapping of n-Octyloxyl Radical
		3.10 Protocol: Isolated Liver Experiments and Subcellular Mitochondrial and Cytosolic Fractions Preparation
		3.11 31P NMR on Mitochondrial and Cytosolic Extracts
		3.12 Apoptosis Experiments
	4 Notes
	References
Chapter 6: Insights on Targeting Small Molecules to the Mitochondrial Matrix and the Preparation of MitoB and MitoP as Exomark...
	1 Introduction
		1.1 Mitochondria-Targeted Drugs, Prodrugs, and Bioactives
		1.2 Mitochondria-Targeted Sensors
	2 Materials
		2.1 Equipment for Synthesis
		2.2 Synthesis of MitoB
		2.3 Synthesis of d15-MitoB
		2.4 Synthesis of MitoP
		2.5 Synthesis of d15-MitoP
	3 Method
		3.1 Overview of Procedure for the Synthesis of MitoB and d15-MitoB
		3.2 Setup for Preparation of MitoB and d15-MitoB (See Note 1)
		3.3 Purification of MitoB
		3.4 Characterization Data for MitoB
		3.5 Characterization Data for d15-MitoB
		3.6 Overview of Procedure for the Synthesis of MitoP and d15-MitoP (See Note 7)
		3.7 Setting up Reaction A
		3.8 Reaction A
		3.9 Workup of Reaction A (See Note 11)
		3.10 Characterization Data for 3-(Bromomethyl)Phenol
		3.11 Setting up Reaction B
		3.12 Work Up of Reaction B
		3.13 Purification (if Necessary)
		3.14 Characterization Data for MitoP Bromide
		3.15 Characterization Data for d15-MitoP Bromide
	4 Notes
	References
Chapter 7: Synthesis of Triphenylphosphonium Phospholipid Conjugates for the Preparation of Mitochondriotropic Liposomes
	1 Introduction
	2 Materials
		2.1 Synthesis
		2.2 Purification
		2.3 TLC Analysis
		2.4 Miscellaneous Equipment
	3 Methods
		3.1 Dehydration of Solvents
		3.2 Synthesis
		3.3 Purification
	4 Notes
	References
Chapter 8: Imaging Mitochondrial Hydrogen Peroxide in Living Cells
	1 Introduction
	2 Materials
		2.1 Synthetic Chemistry Components
			2.1.1 Step 1: Synthesis of Fmoc-Piperazine Rhodol
			2.1.2 Step 2: Synthesis of Fmoc-Piperazine Rhodol Triflate
			2.1.3 Step 3: Synthesis of Fmoc-Piperazine Rhodol Boronate
			2.1.4 Step 4: Synthesis of MitoPY1
		2.2 Cellular Imaging Components (See Note 3)
	3 Methods
		3.1 Synthetic Chemistry (Fig. 1)
			3.1.1 Synthesis of Fmoc-Piperazine Rhodol
			3.1.2 Synthesis of Fmoc-Piperazine Rhodol Triflate
			3.1.3 Synthesis of Fmoc-Piperazine Rhodol Boronate
			3.1.4 Synthesis of MitoPY1
		3.2 Cellular Imaging with MitoPY1
			3.2.1 Preparation of Aliquots
			3.2.2 Validation of Probe Response to H2O2 In Vitro
			3.2.3 Cell Culture
			3.2.4 Validation of Probe Response to Mitochondrial H2O2 in Cell Culture
			3.2.5 Imaging of Mitochondrial H2O2 in a Cellular Model of Parkinson´s Disease
	4 Notes
	References
Chapter 9: Synthesis and Testing of Novel Isomeric Mitochondriotropic Derivatives of Resveratrol and Quercetin
	1 Introduction
	2 Materials
		2.1 Synthesis of Resveratrol and Quercetin Derivatives
		2.2 Assessment of Accumulation into Mitochondria-TPP+-Selective Electrode with Isolated Rat Liver Mitochondria
		2.3 Assessment of Accumulation into Mitochondria- and Cultured Cells
	3 Methods
		3.1 General Procedure-Alkylation of One Hydroxyl Function to Produce a -(4-O-Chlorobutyl) Derivative
		3.2 General Procedure: -Cl  -I Nucleophilic Substitution to Produce a -(4-O-Iodobutyl) Derivative
		3.3 General Procedure: -I  -P+Ph3I- Nucleophilic Substitution to Produce the -(4-O-Triphenylphosphoniumbutyl) Derivative
		3.4 General Procedure-acetylation of -(4-O-Chlorobutyl) Derivatives
		3.5 General Procedure-Methylation of -(4-O-Chlorobutyl) Derivatives
		3.6 Synthesis of 4′-(4-O-Triphenylphosphoniumbutyl) Resveratrol and 3-(4-O-Triphenylphosphoniumbutyl) Resveratrol
		3.7 Synthesis of 3-(4-O-Triphenylphosphoniumbutyl) Quercetin
		3.8 Synthesis of 5-(4-O-Triphenylphosphoniumbutyl) Quercetin
		3.9 Synthesis of 7-(4-O-Triphenylphosphoniumbutyl) Quercetin
		3.10 Assessment of Accumulation into Mitochondria-TPP+-Selective Electrode with Isolated Rat Liver Mitochondria
		3.11 Assessment of Accumulation into Mitochondria- with Cultured Cells
	4 Notes
	References
Chapter 10: Bridging the Gap Between Nature and Antioxidant Setbacks: Delivering Gallic Acid to Mitochondria
	1 Introduction
	2 Materials
		2.1 Components for Synthesis
		2.2 Rat Mitochondrial Fraction Preparation Components
		2.3 Components to Evaluate the Mitochondriotropic Features of AntiOxBEN3
	3 Methods
		3.1 Development of the Mitochondriotropic Antioxidant AntiOxBEN3 (Scheme 1)
			3.1.1 Synthesis of Tert-Butyl (6-(3,4,5-Trimethoxybenzamido) Hexyl)Carbamate (3)
			3.1.2 Synthesis of N-(6-Aminohexyl)-3,4,5-Trimethoxybenzamide (4)
			3.1.3 Synthesis of [5-(6-(3,4,5-Trimethoxybenzamido)hexylamino) carbonylpentyl]triphenylphosphonium Bromide (6)
			3.1.4 Synthesis of [5-(6-(3,4,5-Trihydroxybenzamido)hexylamino) carbonylpentyl]triphenylphosphonium Bromide (7, AntiOxBEN3)
		3.2 Isolation of Rat Heart Mitochondrial Fractions
		3.3 Uptake of AntiOxBEN3 by Mitochondria
			3.3.1 Preparation of TPP+ Selective Electrode
			3.3.2 Heart Mitochondrial Uptake of AntiOxBEN3
	4 Notes
	References
Chapter 11: Liposomal Delivery of Cyclocreatine Impairs Cancer Cell Bioenergetics Mediating Apoptosis
	1 Introduction
	2 Materials
		2.1 Preparation of CCR-Loaded Liposomes (Plain and PEGylated)
		2.2 Determination of CCR Concentration
		2.3 Physicochemical Characterization of Prepared CCR-Liposomes (Plain and PEGylated)
		2.4 MitoOrange Mitochondrial Polarization Assay
	3 Methods
		3.1 Preparation of Empty and CCR-Loaded Freeze-Thaw Vesicles (FTV)/Liposomes
		3.2 Determination of CCR
		3.3 Physical Characterization of Empty and Drug-Loaded FTV Liposomal Formulations
		3.4 Orange Mitochondrial Polarization Assay in Cultured Human Prostatic Carcinoma Cells
	4 Notes
	References
Chapter 12: Mt-fura-2, a Ratiometric Mitochondria-Targeted Ca2+ Sensor. Determination of Spectroscopic Properties and Ca2+ Ima...
	1 Introduction
	2 Materials
		2.1 Solutions and Reagents
			2.1.1 General Solutions and Reagents
			2.1.2 Solutions and Reagents for Spectroscopic Evaluation
			2.1.3 Solutions and Reagents for Imaging Experiments
		2.2 Equipment
			2.2.1 General Equipment
			2.2.2 Equipment for In Vitro Evaluation
			2.2.3 Equipment for Imaging Experiments
	3 Methods
		3.1 Before Starting: Determination of the Residual [Ca2+] in Solutions
		3.2 Investigation of Absorption Properties of mt-fura-2
		3.3 Investigation of Fluorescence Spectroscopic Properties of mt-fura-2
			3.3.1 Optimization of the Experimental Parameters of the Fluorometer
			3.3.2 Determination of Ca2+ Binding Properties and Dynamic Range (DR) of mt-fura-2
			3.3.3 Determination of Fluorescence Quantum Yield (QY)
		3.4 Determination of the Optimal Excitation Wavelengths at the Microscope
		3.5 Investigation of the Dependence of Fluorescence Properties by Other Interfering Species
			3.5.1 Determination of Dependence of Fluorescence by ROS Species (See Note 35)
			3.5.2 Determination of the Affinity for Other Inorganic Cations
			3.5.3 Determination of pH Dependence of Fluorescence
		3.6 Loading and Imaging Procedures in Cells
			3.6.1 Cell Culturing and Plating: General Considerations
			3.6.2 Loading of mt-fura-2 in Cultured Cells
			3.6.3 Evaluation of Subcellular Localization of mt-fura-2
			3.6.4 Ca2+ Imaging: IP3-Mediated ER Ca2+ Release
			3.6.5 Post Measurement Analysis
	4 Notes
	References
Chapter 13: Sequence-Specific Control of Mitochondrial Gene Transcription Using Programmable Synthetic Gene Switches Called MI...
	1 Introduction
	2 Materials
		2.1 MITO-PIP-LSP Synthesis
		2.2 Cellular Evaluation of MITO-PIP-LSP
	3 Methods
		3.1 Solid-Phase Synthesis of MITO-PIP-LSP
		3.2 Cleavage and Purification of MITO-PIP
		3.3 Cellular Evaluation of MITO-PIP
	4 Notes
	References
Chapter 14: Targeting the Mitochondrial Genome Via a MITO-Porter: Evaluation of mtDNA and mtRNA Levels and Mitochondrial Funct...
	1 Introduction
	2 Materials
		2.1 Lipids
		2.2 Liposome Preparation
		2.3 Cell Cultures
		2.4 Evaluation of the Levels of mtDNA
		2.5 Evaluation of Mitochondrial Activity
		2.6 Quantification of Target Mitochondrial mRNA Levels
		2.7 Evaluation of Mitochondrial Membrane Potential
	3 Methods
		3.1 Construction of DF-MITO-Porter Encapsulating DNase  I
		3.2 Cell Cultures and the Mitochondrial Delivery of DNase I Using DF-MITO-Porter
		3.3 Evaluation of the Levels of mtDNA After Mitochondrial Delivery of DNase I Using DF-MITO-Porter System
		3.4 Evaluation of Mitochondrial Activity After the Mitochondrial Delivery of DNase  I
		3.5 Construction of a MITO-Porter Encapsulating Nanoparticles of  ASO
		3.6 Cell Culture and the Mitochondrial Transfection of ASO Using MITO-Porter
		3.7 Quantification of Target Mitochondrial mRNA Levels After Mitochondrial Transfection of ASO Using MITO-Porter System
		3.8 Evaluation of Mitochondrial Membrane Potential After Mitochondrial Transfection of  ASO
	4 Notes
	References
Chapter 15: mTRIP, an Imaging Tool to Investigate Mitochondrial DNA Dynamics in Physiology and Disease at the Single-Cell Reso...
	1 Introduction
	2 Materials
		2.1 Probe Preparation and Labeling
		2.2 Cell Treatment
		2.3 mTRIP Hybridization
		2.4 mTRIP Coupled to Immunofluorescence
		2.5 mTRIP Coupled to MitoTracker
		2.6 Imaging Equipment
	3 Methods
		3.1 Extraction of Total Genomic  DNA
		3.2 Preparation and Labeling of the DNA Probe
		3.3 Prehybridization of the DNA Probe
		3.4 Preparation of Cells on Coverslip
		3.5 mTRIP Hybridization
		3.6 mTRIP Washing
		3.7 mTRIP Coupled to Immunofluorescence
		3.8 Imaging Acquisition and Fluorescence Intensity Quantification
	4 Notes
	References
Chapter 16: Development of Mitochondria-Targeted Imaging Nanoplatforms by Incorporation of Fluorescent Carbon Quantum Dots
	1 Introduction
	2 Materials
		2.1 Equipment for Synthesis
		2.2 Synthesis of Fe3O4@mSiO2
		2.3 Synthesis of Fe3O4@mSiO2-TPP
		2.4 Synthesis of Fe3O4@mSiO2-TPP/CQD Nanoplatform
		2.5 Cytotoxicity Assay
		2.6 Cell Imaging
		2.7 Mitochondria Colocalization
	3 Methods
		3.1 Synthesis of Fe3O4@mSiO2
		3.2 Synthesis of Fe3O4@mSiO2-TPP
		3.3 Synthesis of Carbon Quantum Dots
		3.4 Construction of the Fe3O4@mSiO2-TPP/CQD Nanoplatform
		3.5 Physical Characterization the Fe3O4@mSiO2-TPP/CQD Nanoplatform
		3.6 Cytotoxicity Assay
		3.7 Cell Imaging
		3.8 Mitochondria Colocalization
	4 Notes
	References
Chapter 17: Norbormide-Based Probes and Their Application for Mitochondrial Imaging in Drosophila Melanogaster
	1 Introduction
	2 Materials
		2.1 Norbormide-Based Probes Synthesis
		2.2 Drosophila Larva Dissection
		2.3 Image Acquisition
	3 Methods
		3.1 Norbormide Probe Synthesis
		3.2 Drosophila Larva Dissection
		3.3 Image Acquisition
	4 Notes
	References
Chapter 18: Live-Cell Assessment of Reactive Oxygen Species Levels Using Dihydroethidine
	Abbreviations
	1 Introduction
	2 Materials
		2.1 General
		2.2 Dihydroethidium (HEt)
		2.3 Mito-Dihydroethidium (Mito-HEt)
	3 Methods
		3.1 Microscopy Imaging of Dihydroethidium (HEt) Oxidation
		3.2 Microscopy Imaging of Mito-Dihydroethidium (Mito-HEt) Oxidation
		3.3 Image Analysis
	4 Notes
	References
Chapter 19: Protein Supercomplex Recording in Living Cells Via Position-Specific Fluorescence Lifetime Sensors
	1 Introduction
	2 Materials
		2.1 Confocal Laser Scanning Microscopes
		2.2 Chemicals
		2.3 Plasmids
		2.4 Cells
		2.5 Media and Buffers
		2.6 Software
		2.7 Specific Equipment
	3 Methods
		3.1 Cell Culture
			3.1.1 Cultivation
			3.1.2 Passaging
			3.1.3 Cell Transfection
			3.1.4 Generation of Stable Transfected Cell Lines
		3.2 Preparation of Stable Transfected Cells for Imaging
		3.3 Fluorescence Lifetime Imaging
		3.4 FLIM Evaluation
	4 Notes
	References
Chapter 20: Identification of Peroxynitrite by Profiling Oxidation and Nitration Products from Mitochondria-Targeted Arylboron...
	1 Introduction
	2 Materials
		2.1 Components for the Synthesis of o-MitoPhB(OH)2
		2.2 Cell Incubation Components
		2.3 Cell Extraction Components
		2.4 HPLC Analysis Components
	3 Methods
		3.1 Preparation of o-MitoPhB(OH)2
		3.2 Cell Incubation with the Probe
		3.3 Extraction of the Products
			3.3.1 Cell Pellets
			3.3.2 Media
		3.4 HPLC-MS/MS Analysis of the Extracts
	4 Notes
	References
Chapter 21: Mitochondrial Coenzyme Q10 Determination Via Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry
	1 Introduction
	2 Materials
		2.1 Mitochondrial Isolation
		2.2 Assay of CoQ10
		2.3 Assay of Citrate Synthase Activity
	3 Methods
		3.1 Isolation of Mitochondria
		3.2 Sample and Calibrator Preparation
		3.3 LC-MS/MS Analysis
		3.4 Determination of Citrate Synthase Activity
			3.4.1 Procedure
		3.5 Calculation of the Results
			3.5.1 CoQ10
			3.5.2 Citrate Synthase
	4 Notes
	References
Chapter 22: Janus-Type Mesoporous Silica Nanoparticles for Sequential Tumoral Cell and Mitochondria Targeting
	1 Introduction
	2 Materials
		2.1 Solutions and Buffers
		2.2 Cell Lines and Cell Culture Reagents
		2.3 Cell Culture Equipment
	3 Methods
		3.1 MSN Synthesis
			3.1.1 Synthesis of APTES-FITC
			3.1.2 Synthesis of MSN Labeled with FITC
			3.1.3 Removal of Surfactant in MSN
		3.2 Asymmetric Functionalization of MSN (Fig. 1)
			3.2.1 Step 1: Attachment of Amino Groups Employing a Pickering Emulsion Method (Fig. 2)
			3.2.2 Paraffin Removal
			3.2.3 Step 2: Attachment of Carboxylic Acid Groups
			3.2.4 Step 3: Attachment of Amino Groups
		3.3 Attachment of Targeting moieties on NH2-MSN-CO2H
			3.3.1 Attachment of Folic Acid to Prepare Folic-MSN-CO2H
			3.3.2 Attachment of Triphenylphosphine to Prepare H2N-MSN-TPP
			3.3.3 Synthesis of Dual-Targeted Nanoparticles Folic-MSN-TPP
		3.4 Loading Nanoparticles with Topotecan
		3.5 In Vitro Nanoparticle Uptake and cytotoxicity Evaluation
			3.5.1 Laminar Flow Cabinets Use
			3.5.2 Cell Line Culture
			3.5.3 Nanoparticle Uptake Studies by Flow Cytometry
			3.5.4 Nanoparticle Uptake Studies by Fluorescent Microscopy
			3.5.5 Cell Viability Evaluation After Incubation with Topotecan-Loaded Mesoporous Silica Nanoparticles
		3.6 Characterization Techniques
			3.6.1 Zeta Potential and DLS Measurements
			3.6.2 Thermogravimetric Analysis (TGA)
			3.6.3 Fourier Transform Infrared Spectroscopy
			3.6.4 Analysis of Textural Properties of MSN by N2 Adsorption
			3.6.5 Scanning Electron Microscopy (SEM)
			3.6.6 Asymmetrization Characterization by Transmission Electronic Microscopy (TEM)
	4 Notes
	References
Chapter 23: Split Green Fluorescent Protein-Based Contact Site Sensor (SPLICS) for Heterotypic Organelle Juxtaposition as Appl...
	1 Introduction
		1.1 Old and New Approaches to Detect Organelle Contact Sites
		1.2 The Split GFP Approach: Fiat  Lux
	2 Materials
		2.1 Cell Culture and Transfection
		2.2 DNA Purification
	3 Methods
		3.1 Protocol for Monitoring Basal ER-Mitochondria Contact Sites with SPLICSS and SPLICSL
		3.2 Protocol for Quantifying Basal ER-Mitochondria Contact Sites with SPLICSS and SPLICSL
	4 Notes
	References
Chapter 24: Qualitative Characterization of the Rat Liver Mitochondrial Lipidome Using All Ion Fragmentation on an Exactive Be...
	1 Introduction
	2 Materials
		2.1 Liver Mitochondria Isolation [23]
		2.2 Mitochondrial Lipid Extraction
		2.3 LC-MS Analysis
	3 Methods
		3.1 Liver Mitochondria Isolation
		3.2 Mitochondrial Lipid Extraction
		3.3 LC-MS Conditions
		3.4 Full Scan Profiling Experiments
		3.5 Lipid Identification Studies
		3.6 Data Analysis
	4 Notes
	References
Chapter 25: Live Imaging of Mitochondria in Kidney Tissue
	1 Introduction
	2 Materials
		2.1 Kidney Slices
			2.1.1 Kidney Slice Buffer
			2.1.2 Anesthesia
			2.1.3 Setup for Slicing and Imaging
			2.1.4 Mitochondrial Toxins
			2.1.5 Mitochondrial Dyes
		2.2 Intravital Imaging of the Kidney
			2.2.1 Agarose
			2.2.2 Surgery
			2.2.3 Anesthesia
			2.2.4 Dyes
	3 Methods
		3.1 Kidney Slices
			3.1.1 Slicing
			3.1.2 Setup at the Microscope
			3.1.3 Imaging
		3.2 Intravital Imaging of the Kidney
			3.2.1 Surgery
			3.2.2 Externalization of the Kidney
			3.2.3 Imaging
	4 Notes
	References
Chapter 26: Time-Resolved Imaging of Mitochondrial Flavin Fluorescence and Its Applications for Evaluating the Oxidative State...
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Drug Preparation
	3 Methods
		3.1 Microscopy
		3.2 Cell Preparation for Microscopy
		3.3 Fluorescence Lifetime Imaging of Flavin Fluorescence in Living Cells
		3.4 Testing Mitochondrial Presence
		3.5 Reducing Mitochondrial Oxidation by OXPHOS Inhibition
		3.6 Increasing Mitochondrial Oxidation by Uncoupling of Respiratory Chain
		3.7 Analysis
	4 Notes
	References
Chapter 27: Mito-SinCe2 Approach to Analyze Mitochondrial Structure-Function Relationship in Single Cells
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Confocal Microscope (Parameters Described Herein Were Optimized on a Zeiss LSM700) Equipped  with
		2.3 Software
		2.4 Plasmids
		2.5 Plasmid Expression System
		2.6 Drugs or Metabolites for Validation
	3 Methods
		3.1 Expressing Mito-SinCe2 Probes
		3.2 Confocal Microscopy
			3.2.1 Turn on Microscope
			3.2.2 Perform 3D Confocal Microscopy to Obtain and Validate Mito-SinCe2 Energetics and Dynamics Metrics
			3.2.3 Validate Probes for New Cell Lines
			3.2.4 Perform Pulse-Chase Microscopy Experiment to Measure Mitochondrial Matrix Continuity (Fig. 2) in Conjunction with Redox ...
			3.2.5 Validate Matrix-Continuity Metric for New Cell Lines
		3.3 Image Processing and Analysis
			3.3.1 Obtain Energetics Metrics, [ATP] and [Oxidation], from 3D Images of Ratiometric Probes as Follows
			3.3.2 Obtain Value of Dynamics Metrics of Steady-State [Fission], [Fusion(1-10)], and [Diameter] (Fig. 1b) from 3D Images Usin...
			3.3.3 Obtain [Matrix-Continuity] from Pulse-Chase Time-Lapse Images as Follows
			3.3.4 To Measure the Impact of Exogenous Agents (EA) on Mitochondrial Energetics and Dynamics Use Similar Steps to Validation ...
			3.3.5 For Mito-SinCe2 Analyses, Plot [Dynamics] Against [Energetics] Metrics for Single Cells in Control and Experimental Grou...
		3.4 Limitations of Mito-Since2
	4 Notes
	References
Chapter 28: Computer-Aided Prediction of Protein Mitochondrial Localization
	1 Introduction
		1.1 Targeting Peptides and Mitochondrial Localization
		1.2 Available Computational Methods for Targeting peptide Detection
		1.3 Available Computational Methods for Discrimination of Submitochondrial Localization
	2 Materials
		2.1 Benchmark Dataset
		2.2 Length and Residue Composition of Targeting Peptides
		2.3 Features of the Cleavage  Site
		2.4 Compositions of Proteins Residing in Different Mitochondrial Compartments
	3 Methods
		3.1 TPpred3
		3.2 Benchmarking TPpred3 on the Human Protein Datasets
		3.3 DeepMito
		3.4 Benchmarking DeepMito on the Human Protein Datasets
		3.5 TPpred3 and DeepMito at Work on the Human Genome
		3.6 How to Predict Mitochondrial Localization with TPpred3
		3.7 How to Read the Results of TPpred3
		3.8 How to Predict Submitochondrial Localization with DeepMito
		3.9 How to Read DeepMito Results
	References
Index