SUCCESS IN ACADEMIC SURGERY : basic science

This updated volume provides the foundation for starting a basic science research career as an academic surgeon. Taking a practical approach, the book covers the suggested timeline for the initial academic appointment, including how to setup and fund the laboratory and identifying appropriate scientific mentors and lab personnel. It also describes the application of basic and advanced research techniques, including animal models, flow cytometry, gene editing, tissue engineering, and microbiome analysis. __Success in Academic Surgery: Basic Science__ aims to give guidance on the application of basic and advanced techniques in surgical research. This book is relevant to senior residents and fellows approaching their first academic appointment, as well as more senior investigators interested in expanding their research horizons. Preface 6 References 7 Contents 8 1: How to Set Up, Staff, and Fund Your Basic Science or Translational Research Laboratory 10 Introduction 10 Protected Time 11 Schedule and Time Management 12 Research Space 12 Equipment and Supplies: Things You Will Need to Purchase 13 Laboratory Personnel and Technical Assistance 14 Mentorship 15 Collaborators 16 Grant Funding 17 Putting It All Together in “The Package” 19 Responding to Failure 20 Summary 21 Reference 21 2: Choosing a Good Scientific Mentor and Being a Good Mentee 22 Introduction 22 Defining Mentorship 23 Mentorship by Career Stage 23 Choosing a Good Scientific Mentor 24 Defining an Effective Mentor 24 Experience 25 Personal Dedication 25 Generosity 25 Roles of an Effective Mentor 26 Establishing a Team of Mentors 27 Being a Good Mentee 27 Beginning the Mentor-Mentee Relationship 27 Maintenance of the Mentor-Mentee Relationship 28 Summary and Conclusions 29 Suggested Reading 29 3: Effective Time Management Strategies for Conducting Laboratory Research 30 Definition 30 Benefits 31 Failing to Manage Your Time Effectively 31 How to 32 Set Clear Goals 32 Prioritize 33 Spend Time Organizing and Planning 33 Block Time 34 Be Flexible 35 Have a Contingency Plan 35 Review Your Progress 36 Utilize Resources/People 36 Avoid Distractions or Interruptions 37 Conquer Procrastination 37 Say No 38 How Time Management Relates to Laboratory Research 38 References 39 4: Maintaining an Effective Lab Notebook and Data Integrity 40 Introduction 41 The Challenges of Record Keeping 41 Legal Aspects 41 Paper Laboratory Notebooks 42 Electronic Laboratory Notebooks 43 Archiving of Scientific Data 45 Rigor, Reproducibility, and Data Integrity 45 Conclusions 48 Websites for ELN 49 NIH Website for Rigor and Reproducibility 49 References 49 5: Statistics for Bench Research 51 Introduction 51 Types of Data 52 Descriptive Statistics 52 Type 1 and Type 2 Errors 54 Sample Size and Power 54 Data Analysis 54 P Value 54 Confidence Interval 55 Statistical Tests 55 t Test/ANOVA/Mann-Whitney U/Kruskal-Wallis Test 56 Chi-Square/Fishers Exact Test 57 Kaplan-Meier Curve/Log-Rank Test/Cox Proportional Hazard Model 58 Correlation and Regression 58 Sensitivity/Specificity 59 Conclusion 60 Suggested Reading 60 6: Ethics in Laboratory Research 61 Introduction 61 A Personal Introduction to Research 62 Mentoring 62 Case Story: Research Design for Beginners 63 Research Integrity 63 Case Story 64 Honesty 65 Case Story 65 Objectivity 66 Case Story 66 Carefulness and Reproducibility 66 Case Story 66 Openness 67 Case Story 67 Protection of Human Subjects and Animal Research 68 Animal Research 68 Human Subjects Research 69 Responsible Publication and Reporting Corrections 69 Conclusion 70 References 70 7: Modern Techniques for DNA, RNA, and Protein Assessment 72 Introduction 73 Deoxyribonucleic Acid (DNA) Assessment 73 DNA Introduction 73 Measuring and Identifying DNA 74 Southern Blot 74 DNA Sequencing 74 Chemical Degradation Sequencing 74 Chain-Termination Sequencing 74 Massive Parallel Sequencing 75 Whole Exome Sequencing 76 Third-Generation Sequencing 76 CRISPR/Cas9 Genome Editing 77 Assessment of DNA-Protein Interactions 78 Electrophoretic Mobility Shift Assay (EMSA) 78 Deoxyribonuclease (DNase) I Footprinting 78 Chromatin Immunoprecipitation (ChIP) 79 Systematic Evolution of Ligands by Exponential Enrichment (SELEX) 79 Scanning Probe Microscopy (SPM) 79 Surface Plasmon Resonance (SPR) 80 Laser Flow Cytometry 80 Ribonucleic Acid (RNA) Assessment 81 RNA Introduction 81 Measuring and Identifying RNA 81 Northern Blot 82 Ribonuclease Protection Assay (RPA) 82 In Situ Hybridization (ISH) 82 Measuring Gene Expression 82 cDNA Arrays 83 Oligonucleotide Arrays or Microarrays 83 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 83 RNA Sequencing 84 Single-Cell RNA Sequencing (scRNA-Seq) 84 Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) 85 Analysis and Interpretation of Gene Expression Profiles 85 Normalization 85 Quality Assessment 85 Data Analysis 86 Multiple Comparison Problem 86 Interpretation 86 Clinical Applications of DNA and RNA Analysis 87 Protein Assessment 87 Protein Introduction 87 Protein Sources 88 Protein Extraction 88 Protein Purification 89 Precipitation 89 Centrifugation 90 Chromatography 90 Gel Electrophoresis 90 Two-Dimensional Gel Electrophoresis 93 Protein Identification and Quantification 94 Mass Spectrometry and MALDI-TOF 94 Western Blot 95 Immunoassays 97 Immunohistochemistry 98 Flow Cytometry 99 Immunoassays in Live Animals 99 Establishing Protein Function 100 Yeast Two-Hybrid 100 Protein Microarray 100 Co-immunoprecipitation 102 Immunoaffinity Chromatography 102 Fluorescence Resonance Energy Transfer (FRET) 103 Surface Plasmon Resonance (SPR) 103 Summary 103 References 104 8: Considerations for Immunohistochemistry 112 9: Utilizing Flow Cytometry Effectively 152 Background 153 Fluorochromes 153 Sample Preparation 154 Data Collection 155 Data Analysis 156 Applications 159 Cell Viability, Proliferation, and Death 159 Phenotypic Characterization 159 Intracellular Staining 160 Fluorescence-Activated Cell Sorting 160 Future Directions 160 Summary 161 References 161 10: Effective Cell Culture 163 Introduction: Why Experiment with Cultured Cells? 164 How to: A Guide to Basic Culture Techniques 165 Sterility in the Lab 165 Culture Media 168 Cell Culture: A Word About Adherent and Suspension Cell Culture 168 Primary Culture Versus Subculture Versus Cell Line: What Does It All Mean Anyway? 169 Passaging and Plating Cells 169 Counting Cells for Culture 170 Storage: Freezing and Thawing Cells for Use 172 Cell Types: Choosing the Right Tool for the Right Job 173 Conclusion 174 References 175 11: Gene-Editing Techniques 176 Introduction 176 Applications of CRISPR/Cas Technology 177 Introducing Mutations 177 Insertions/Deletions from Nonhomologous End Joining 178 Knock-Ins Through Homologous Recombination 178 Base Editing 179 Large Deletions and Translocations 179 Controlling for Off-Target Genomic Editing 180 Considerations for Designing the Guide RNA 181 Regulating Gene Transcription 181 High-Throughput Genetic Screens 181 Animal Models 182 References 183 12: Stem Cells and Tissue Engineering 185 Introduction 185 Types of Stem Cells 186 Embryonic Stem Cells 186 Adult Stem Cells 188 Hematopoietic Stem Cells 188 Mesenchymal Stem Cells 188 Neural Stem Cells 189 Intestinal Stem Cells 190 Liver Stem Cells 190 Cardiac Stem Cells 191 Induced Pluripotent Stem Cells 192 Mechanism of Action 193 Cellular Differentiation 193 Heterotopic Cell Fusion 193 Paracrine Mediators 194 Immunomodulation 194 Tissue Restoration 195 Antioxidant Properties 195 Tissue Engineering Principals 195 Scaffolds 196 Guiding Cells to Integrate 196 Bioreactors 197 3D Printers 198 Conclusion 198 References 199 13: Animal Models in Surgical Research 206 Introduction 207 Chicken 207 Zebrafish 208 Rodents 209 Nonhuman Primates 210 Swine 211 Sheep 212 Conclusion 213 References 213 14: Microbiome: Current Status and Future Applications 216 Introduction 216 The Human Microbiome 217 Studying the Human Microbiome 220 High-Level Community Profiling 221 Functional Profiling 224 Metatranscriptomics 227 Metaproteomics 227 Rodent Models 229 Clinical Implications of Microbiome Science for Surgeons 230 Future Applications 231 Summary 231 References 232 15: Systems Biology: Generating and Understanding Big Data 236 Introduction 237 Systems Biology Resources 237 Generating Big Data 240 Genomic and Transcriptomic Experiments 240 Proteomic and Metabolomic Experiments 241 Data Analysis 241 Data Integration 243 High-Throughput Drug Screening 244 Conclusion 245 References 245 Index 247 Front Matter ....Pages i-viii How to Set Up, Staff, and Fund Your Basic Science or Translational Research Laboratory (Jashodeep Datta, J. Joshua Smith)....Pages 1-12 Choosing a Good Scientific Mentor and Being a Good Mentee (Mark L. Kovler, David J. Hackam)....Pages 13-20 Effective Time Management Strategies for Conducting Laboratory Research (Evie Carchman)....Pages 21-30 Maintaining an Effective Lab Notebook and Data Integrity (Andrew J. Murphy)....Pages 31-41 Statistics for Bench Research (Timothy W. King)....Pages 43-52 Ethics in Laboratory Research (Sidd Dalal, Luke Brewster)....Pages 53-63 Modern Techniques for DNA, RNA, and Protein Assessment (Jurgis Alvikas, Matthew D. Neal)....Pages 65-104 Considerations for Immunohistochemistry (Swathi Balaji, Hui Li, Emily Steen, Sundeep G. Keswani)....Pages 105-144 Utilizing Flow Cytometry Effectively (Yue Guan, Jonathan B. Mitchem)....Pages 145-155 Effective Cell Culture (Patrick B. Schwartz, Sean M. Ronnekleiv-Kelly)....Pages 157-169 Gene-Editing Techniques (Kevin W. Freeman)....Pages 171-179 Stem Cells and Tissue Engineering (Troy A. Markel)....Pages 181-201 Animal Models in Surgical Research (Morgan L. Hennessy, Allan M. Goldstein)....Pages 203-212 Microbiome: Current Status and Future Applications (Rafael G. Ramos-Jimenez, Michael J. Morowitz)....Pages 213-232 Systems Biology: Generating and Understanding Big Data (Stephanie S. Kim, Timothy R. Donahue)....Pages 233-243 Back Matter ....Pages 245-252 Academic surgeons play an essential role in advancing the field and improving the care of patients with surgical disease. As the Association for Academic Surgery (AAS) Fall Courses (www.aasurg.org) and international courses continue to evolve to address the rapidly expanding scope and complexity of academic surgery, there is a greater need for an accompanying textbook to supplement the material presented in the courses. Success in Academic Surgery: Basic Science is a unique and portable handbook that focuses on the basic and translational research. It includes new educational materials that are necessary to address not only the rapid evolution and rise of novel research methodologies in basic science and translational research, but also the changing environment for academic surgeons. Success in Academic Surgery: Basic Science is a valuable text for medical students, surgical residents, junior faculty and others considering a career in surgical research. "This updated volume provides the foundation for starting a basic science research career as an academic surgeon. Taking a practical approach, the book covers the suggested timeline for the initial academic appointment, including how to setup and fund the laboratory and identifying appropriate scientific mentors and lab personnel. It also describes the application of basic and advanced research techniques, including animal models, flow cytometry, gene editing, tissue engineering, and microbiome analysis. Success in Academic Surgery: Basic Science aims to give guidance on the application of basic and advanced techniques in surgical research. This book is relevant to senior residents and fellows approaching their first academic appointment, as well as more senior investigators interested in expanding their research horizons"--Publisher's description