Gene, Drug, and Tissue Engineering

Dedication
Preface
Contents
Contributors
Part I: Molecular Genetics and Genomics
Chapter 1: Gene Alterations Induced by Glutamine (Q) Encoding CAG Repeats Associated with Neurodegeneration
1 Introduction
2 Molecular Cascading Behind PolyQ Pathologies
2.1 Proteolytic Processing
2.2 Aggregation and Nuclear Transport
2.3 Clearance
2.4 Mitochondrial Dysfunction
3 Altered Interaction of the PolyQ-Extended Protein with Chromatin-Related Factors
4 Role of Epigenetic Modifications in the Etiology of PolyQ Tract Disorders
5 Outlook and Discussion
6 Conclusion and Future Perspectives
References
Chapter 2: Latest Trends in Nucleic Acids´ Engineering Techniques Applied to Precision Medicine
1 Introduction
2 Isolation, Replication, Transcription, and Translation
3 Latest Trends in Genetic Engineering
4 Current Advances in Nucleic Acids´ Engineering Applied to Precision Medicine
5 Conclusion and Future Perspectives
References
Chapter 3: Nanotechnology-Driven Delivery Systems in Inoculation Therapies
1 Introduction
2 Insights Into Nanotechnological Design
3 Innovation in Nanodelivery Platforms
4 Nanodelivery Systems in Nucleic Acids-Based Vaccine Technology
5 Conclusion and Future Perspectives
References
Part II: Molecular Biology in Drug Development and Therapeutics
Chapter 4: Chronic Kidney Disease: Challenges in Translational Medicine
1 Chronic Kidney Disease
1.1 Definition and Classification of CKD
1.2 Causes of CKD
1.3 Mechanisms of CKD Progression
1.4 Clinical Features of CKD
1.5 Remarks About CKD
2 Cell-Based Therapy for Kidney Diseases
2.1 Cell Source
2.2 Route of Administration
2.3 Therapeutic Doses
2.4 Cell-Based Therapy Clinical Trials in CKD
2.4.1 Lupus Nephritis
2.4.2 Diabetic Kidney Disease
2.4.3 Renovascular Disease
2.4.4 Autosomal-Dominant Polycystic Kidney Disease
3 Challenges and Future Perspectives
References
Chapter 5: Mass Spectroscopy as an Analytical Tool to Harness the Production of Secondary Plant Metabolites: The Way Forward f...
1 Introduction
2 Role of Plants in Drug Discovery
2.1 Historically Important Plant-Based Natural Drugs
2.2 Plant Cell Cultures: An Alternate Method for Secondary Metabolite Production
2.3 Challenges in Plant-Based Drug Discovery
3 Metabolomics in Plant-Based Secondary Metabolites Identifications
3.1 Methodologies
3.2 Challenges
3.3 Prospects
4 Analytical Techniques Employed in Metabolomics
4.1 Gas Chromatography-Mass Spectrometry (GC-MS)
4.2 Liquid Chromatography-Mass Spectrometry (LC-MS)
4.3 Capillary Electrophoresis Mass Spectrometry (CE-MS)
4.4 Nuclear Magnetic Resonance (NMR) Spectroscopy
5 Metabolomic Data Processing and Interpretation for Drug Discovery
6 Conclusion and Future Perspectives
References
Chapter 6: Assessing the Challenges of Nanotechnology-Driven Targeted Therapies: Development of Magnetically Directed Vectors ...
1 Introduction
2 Targeting Using Magnetic Attraction
2.1 Superparamagnetic Beads
2.2 Spatial Dependence of Magnetic Attraction
2.3 Development of Magnetic Attraction Therapies
3 Swimming Magnetic Robots
3.1 Microscale Fluidics
3.2 Swarming Ensembles
3.3 Shape-Changing Robots
3.4 Flagellum-Beating Microswimmers
3.5 Corkscrew Microswimmers
4 Conclusion and Future Perspectives
References
Part III: Molecular, Cellular, Tissue Engineering, and AI-Based Computational Design
Chapter 7: Advances in Regenerative Medicine and Biomaterials
1 Introduction
2 Current Strategies for Tissue Engineering and Regenerative Medicine
2.1 Cell Therapies
2.2 Scaffolds
2.3 Growth Factors
2.4 Platelet-Rich Plasma
3 Biomaterials in Regenerative Medicine
3.1 Naturally Derived Biomaterials
3.1.1 Extracellular Matrix-Derived Biomaterials
3.1.2 Protein-Based Biomaterials
3.1.3 Polysaccharide-Based Biomaterials
3.1.4 Blood-Derived Biomaterials
3.2 Synthetic Biomaterials
3.2.1 Polymeric Biomaterials
3.2.2 Ceramic Biomaterials
3.2.3 Metallic Biomaterials
4 Toward Bioartificial Tissue and Organ Models
4.1 Microfluidic Systems
4.2 Three-Dimensional Bioprinting
5 Outlook and Discussion
5.1 Biomaterials
5.2 Stem Cell Therapies
5.2.1 Multipotent and Pluripotent Stem Cell Therapies
5.2.2 Genetically Modified and/or Xenogeneic Cell Therapies
5.3 Encapsulation and Scaffold Strategies
6 Conclusion and Future Perspectives
References
Chapter 8: A Brief Review of Plant Cell Transfection, Gene Transcript Expression, and Genotypic Integration for Enhancing Comp...
1 Introduction
2 General Considerations
2.1 Agrobacterium-Mediated Gene Transfer
2.2 Integration of Transgenes
2.3 Integration of Transgene: Analysis and Confirmation
2.4 Summary
3 Enhancement of Bioactive Compound Based Upon Metabolic Engineering
3.1 Production of Natural Bioactive Compounds
3.2 Production of Natural Compounds in Targeted Plant Cell Compartments
3.3 Upregulation/Downregulation of Biosynthetic Pathway Genes
4 Current Trends in Metabolic Engineering of Bioactive Compounds
4.1 miRNAs in Biosynthesis of Bioactive Compounds
4.2 CRISPR/Cas9-Mediated Gene Editing
5 Outlook and Discussion
6 Conclusion and Future Perspectives
References
Chapter 9: A Review of Stem Cell Technology Targeting Hepatocyte Growth as an Alternative to Organ Transplantation
1 Introduction
2 Liver Tissue Engineering
3 Stem Cells
4 Bioprinting
5 Organoid
6 Decellularization
7 Conclusion and Future Perspectives
References
Chapter 10: Novel Nanotechnology-Driven Prototypes for AI-Enriched Implanted Prosthetics Following Organ Failure
1 Introduction
2 Sensors and Material Science
3 Nanotechnology Empowering Implanted Prosthetics
4 Artificial Intelligence Orchestrating Signal-Response-Driven Bioactivity
5 Current Challenges in Organ Donation and Transplantation
6 Reasons for Proposing Nanotechnology-Driven Prototypes for AI-Enriched Implanted Prosthetics Following Organ Failure
7 The Prototypes
7.1 Cardiovascular
7.2 Renal
7.3 Neuronal
8 Comparison with Existing Technology and Results
8.1 Cardiovascular Prosthetics
8.2 Renal Prosthetics
8.3 Neuronal Implants
9 Conclusion and Future Perspectives
References
Part IV: Methods and Protocols
Chapter 11: Simplified CRISPR-Mediated DNA Editing in Multicellular Eukaryotes
1 Introduction
2 Materials
2.1 Plasmids and gRNA Preparation
2.2 Cloning of gRNA
2.3 Lentivirus Production and Mammalian Cell Culture
2.4 Equipment
3 Methods
3.1 Preparation of CRISPR-Cas9 Plasmids
3.2 Identify gRNA Targeting Sequence
3.3 Target Sequence Cloning Methods
3.4 Transfection and Lentiviral Productions in 293FT Cells
3.5 Transduction of Lentiviral Particles into Jurkat Cells
3.6 Assessment of Genome Editing
4 Notes
References
Chapter 12: Design of SaCas9-HF for In Vivo Gene Therapy
1 Overview of CRISPR Cas9 System
1.1 Introduction
1.2 General Guidelines for Guide RNA Design and Delivery
1.3 SaCas9 for In Vivo Gene Editing
1.4 SaCas9-HF for In Vivo Genome Editing
1.5 efSaCas9 for Genome Editing
2 SaCas9 for In Vivo Genome Editing
2.1 Materials
2.2 Method
3 SaCas9-HF for In Vivo Genome Editing
3.1 Materials
3.2 Method
4 Notes
References
Chapter 13: Stem Cell Technology in Organ Transplantation: A Novel Method for 3D Bioprinting Functional and Stable Liver Graft...
1 Introduction
2 Materials
2.1 Human iPS Culture Media
2.2 Endodermal Differentiation Media
2.3 Hepatoblast Induction Media
2.4 Hepatocyte Induction Media
2.5 Alginate Hydrogel
2.6 Pluronic F-127 Hydrogel
2.7 Ionic Polymerization Solution
2.8 Bioprinted Construct Media
3 Methods
3.1 iPS Culture and Quality Control
3.2 iPS-Derived Hepatocyte Spheroid Differentiation
3.3 Bioink Formulation
3.4 3D-Bioprinting
3.5 Bioprinted Constructs Culture
4 Notes
References
Chapter 14: In Vitro Assay for the Assessment of Oxygen Depletion Triggers in Human Cell Lines, Associated with Improving Resp...
1 Introduction
2 A Hypoxia Sensor Based on HIF-1α
2.1 Materials
2.2 Methods
2.2.1 Synthesis of the Peptide/Probe
2.2.2 Cell Culture and Treatments
3 A Dual Tracer Fusion Protein Sensor for Hypoxia Imaging
3.1 Materials
3.2 Methods
3.2.1 Plasmid Construction
3.2.2 Cell Culture Treatments
4 A Nitroreductase-Based Fluorescent Probe, 7[(5-Nitrofuran-2-yl)methoxy]3Hphenoxazin-3-One for Hypoxia Imaging
4.1 Materials
4.2 Methods
4.2.1 Synthesis of Probe 1
4.2.2 Cell Culture and Treatments
5 A Nitroreductase (NTR) Probe, FBN-1 for Monitoring Hypoxic Conditions in Cancer Cells
5.1 Materials
5.2 Methods
5.2.1 Synthesis of Probe
5.2.2 Cell Culture and Treatments
6 A Nitroreductase-Based Fluorescence Probe (NBP) for Hypoxia Detection
6.1 Materials
6.2 Methods
6.2.1 Synthesis of Probe
6.2.2 Cell Culture and Treatments
7 A Nitroreductase-Based Fluorescent Probe (Semi-CyHP) for the Detection of Hypoxia
7.1 Materials
7.2 Methods
7.2.1 Synthesis of Probe
7.2.2 Cell Culture and Treatments
8 A Nitroreductase-Based Ratiometric Fluorescent Sensor for the Detection of Hypoxia Microenvironment
8.1 Materials
8.2 Methods
8.2.1 Synthesis of Probe
8.2.2 Cell Culture and Treatments
9 A Nitroreductase-Based Two-Photon Fluorescent Probe for Hypoxia Imaging
9.1 Materials
9.2 Methods
9.2.1 Synthesis of Probe
9.2.2 Cell Culture and Treatments
10 Hypoxia Determination by a pH-Based Fluorescent Probe UTX-12
10.1 Materials
10.2 Methods
10.2.1 Synthesis of Probe
10.2.2 Cell Culture and Treatments
11 Hypoxia Detection by Azo-Based Fluorescent Probes
11.1 Materials
11.2 Method
11.2.1 Synthesis of Probe
11.2.2 Cell Culture and Treatments
12 A Fluorescent Probe: Indolequinone-Rhodol Conjugate for the Determination of Cellular Hypoxia
12.1 Materials
12.2 Methods
12.2.1 Synthesis of Probe
12.2.2 Cellular Imaging
12.2.3 IQ-R Activation by A549 Cell Lysate
13 A Chemiluminescent Probe: Acetoxymethyl Ester for Hypoxia Sensing
13.1 Materials
13.2 Methods
13.2.1 Synthesis of Probe
13.2.2 Cell Culture and Treatments
14 Biocompatible Fiber Sensors for Hypoxia Detection
14.1 Materials
14.2 Methods
14.2.1 Design of Core-Shell Fibers
14.2.2 Cell Culture and Treatments
15 Notes
References
Chapter 15: The Use of 99mTc-Labeled Stem Cells for Evaluation of Chronic Kidney Disease
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 16: An Automated Strategy to Handle Antigenic Variability in Immunisation Protocols, Part I: Nanopore Sequencing of In...
1 Introduction
2 Materials
3 Methods
3.1 RT Sample Library Preparation Reverse Transcription (70 min)
3.2 Multiplex PCR (10 min Prep per RT Sample + 7 min in Thermocycler)
3.3 PCR Amplicons Dilution (1 min per Sample)
3.4 End Prep and Native Barcoding (1.5 h)
3.5 Adapter Ligation and Sequencing
4 Notes
References
Chapter 17: An Automated Strategy to Handle Antigenic Variability in Immunisation Protocols, Part II: In Vitro Transcribed mRN...
1 Introduction
2 Materials
3 Methods
3.1 In-Vitro mRNA Co-transcriptional 5′ Capping (3-4 h)
3.2 3′ Poly(A) Tailing (1 h)
3.3 Purification of IVT Final Products Via High-Performance Liquid Chromatography HPLC (2 h)
4 Notes
References
Index