1. Molecular Biology C SSheng Zhao ( 赵晟 ), Biochemistry and Molecular Department of Medical school in Southeast University CCouse QQ Club: 112342994 ( 分子生物学 C ) WWeb: http://teaching.ewindup.info/ EEmail: shengzhao@seu.edu.cn or windupzs@gmail.com QQQ /MSN/Skype/gChat: windupzs@gmail.com MMobile:18551669724 or 13675130010 Conception, theory, research, and application ——Logic and LIY (Learn It Yourself)

2. Section 1: From breaking up the whole into parts to assembling the parts into a whole. —— Reductionism vs. Holism (the hierarchy of neuroendocrine system axis) Section 2: From inside to outside and then from in vitro to in vivo ——Internal vs. External causes (Construction of the in vitro and in vivo models) Case 1 : Sugar-coated bullets and the freshness date of brain —— Insulin outside Islet (the intrinsic relationship of Diabetes and Alzheimer disease) Chapter 1: Integrative biology 2 2

3. In vitro and in vivo models In vivo In vitro

4. Assay Guidance Manual NIH bookshelf: http://www.ncbi.nlm.nih.gov/books/NBK5 3196/ Structure Activity Relationship (SAR) measurements High Throughput Screening (HTS) 1.Optimal assay reagents. 2.Optimization of assay protocols Sensitivity Dynamic range Signal intensity Stability. 3.Statistical concepts and tools

5. BioAssay Ontology Ontology: theory about the nature of existence In a subject domain. Organize information Formalize knowledge Bioassay Ontology (BAO) Chemical biology and drug screening assays Standardize, organize and annotate assays and results Across multiple and diverse data sets Goal: infer new knowledge about the molecular mechanism in the assay model systems.

6. BioAssay Ontology

7. BioAssay Ontology: Organization Organizing bioassays into interpretable categories to identify the related assays Hierarchies: assay target, format, design, detection technology Questions need to be answer: 1.What is known about the target of assay? 2.What is the assay format, in vitro or in vivo? 3.What are the characteristics of the perturbing agent? 4.How was the perturbation converted into a detectable signal? 5.What was the principle for detection?

8. BioAssay Hierarchy Assay Format BioAssay component EndpointAssay designMeta targetDetection

9. Assay Guidance: What, Where, When, How Question based: 1.What (Target) molecules, pathways, diseases, behaviors, etc. 2.Where (Format) Cytoplasmid vs. neclear Cellular vs. extracellular Tissue and organ Organisms Geography specific 3.When (Format) Embryo, infant, puberty, or adult Circadian 4.How (Design and detection)

10. BioAssay Hierarchy Assay Format Binding BioAssay component EndpointAssay designMeta targetDetection MorphologyMembrane potential Inducible Enzyme Viability Redistribution Conformation Cell-based Biochemical Cell-free Tissue-based Organism-based Physicochemical Molecules Pathways Diseases Behaviors

11. In vitro vs. in vivo assay In vitro In vivo In vitro Cell-free vs. cell-based Cell-based vs. tissue/organ-based Tissue/organ-based vs. organism-based Question overlap Logic overlap Technique overlap In vitro In vivo In vitro

12. In vitro assay (Reductionism) For molecules, pathways at cellular level 1.Cell-Based Assays: Cell Viability Assays Morphology analysis Nucleic acid Analysis Protein analysis Other molecules analysis 2.Biochemical/Molecular biology Assays Enzymatic Assays Immunoassay Methods Fluorescent based assay Electrophysiological assay Gene manipulation

13. Cell-Based Assays Cell Viability Assays 1.cell proliferation 2.cytotoxic effects of compounds 3.Multiplexing as an internal control to determine viable cell number during other cell-based assays 4.multi-well formats using a plate reader for screening 5.measure some aspect of general metabolism or an enzymatic activity as a marker of viable cells

14. Cell Viability Assays 1.Tetrazolium reduction MTT: positively charged and readily penetrates viable eukaryotic cells MTS, XTT, and WST-1: negatively charged and do not readily penetrate cells 2.Resazurin reduction: cell permeable redox indicator 3.Protease markers: constitutive protease activity within live cells 4.ATP detection: Firefly luciferase

15. In vitro assay (Reductionism) For molecules, pathways at cellular level 1.Cell-Based Assays: Cell Viability Assays Morphology analysis Nucleic acid Analysis Protein analysis Other molecules analysis 2.Biochemical/Molecular biology Assays Enzymatic Assays Immunoassay Methods Fluorescent based assay Electrophysiological assay Gene manipulation

16. Morphology analysis 1.Morphology 2.Migration 3.Cell cycle 4.Cell death 5.Live imaging 6.High Content Analysis

17. In vitro assay (Reductionism) For molecules, pathways at cellular level 1.Cell-Based Assays: Cell Viability Assays Morphology analysis Nucleic acid Analysis Protein analysis Other molecules analysis 2.Biochemical/Molecular biology Assays Enzymatic Assays Immunoassay Methods Fluorescent based assay Electrophysiological assay Gene manipulation

18. DNA analysis 1.Genome analysis 2.Methylation 3.Promoter activity 4.Mutagenesis 5.Note: some cell do not have mRNA (e.g. red blood cells)

19. RNA analysis 1.Transcriptomics 2.Rnomics: e.g. microRNomics

20. In vitro assay (Reductionism) For molecules, pathways at cellular level 1.Cell-Based Assays: Cell Viability Assays Morphology analysis Nucleic acid Analysis Protein analysis Other molecules analysis 2.Biochemical/Molecular biology Assays Enzymatic Assays Immunoassay Methods Fluorescent based assay Electrophysiological assay Gene manipulation

21. Protein analysis 1.Proteomics 2.Metabolomics 3.Signaling

22. In vitro assay (Reductionism) For molecules, pathways at cellular level 1.Cell-Based Assays: Cell Viability Assays Morphology analysis Nucleic acid Analysis Protein analysis Other molecules analysis 2.Biochemical/Molecular biology Assays Enzymatic Assays Immunoassay Methods Fluorescent based assay Electrophysiological assay Gene manipulation

23. Other Molecule analysis 1.Glycomics 2.Lipidomics 3.Small active molecules

24. In vitro assay For molecules, pathways at cellular level 1.Cell-Based Assays: Cell Viability Assays Morphology analysis Nucleic acid Analysis Protein analysis Other molecules analysis 2.Biochemical/Molecular biology Assays Enzymatic Assays Immunoassay Methods Fluorescent based assay Electrophysiological assay Gene manipulation

25. Enzymatic Assays

26. In vitro assay For molecules, pathways at cellular level 1.Cell-Based Assays: Cell Viability Assays Morphology analysis Nucleic acid Analysis Protein analysis Other molecules analysis 2.Biochemical/Molecular biology Assays Enzymatic Assays Immunoassay Methods Fluorescent based assay Electrophysiological assay Gene manipulation

27. Immunoassay Methods

28. In vitro assay For molecules, pathways at cellular level 1.Cell-Based Assays: Cell Viability Assays Morphology analysis Nucleic acid Analysis Protein analysis Other molecules analysis 2.Biochemical/Molecular biology Assays Enzymatic Assays Immunoassay Methods Fluorescent based assay Electrophysiological assay Gene manipulation

29. Fluorescent based assay XFPs Labeled Endoplasmatic Reticulum

30. In vitro assay For molecules, pathways at cellular level 1.Cell-Based Assays: Cell Viability Assays Morphology analysis Nucleic acid Analysis Protein analysis Other molecules analysis 2.Biochemical/Molecular biology Assays Enzymatic Assays Immunoassay Methods Fluorescent based assay Electrophysiological assay Gene manipulation

31. Electrophysiological assay

32. In vitro assay For molecules, pathways at cellular level 1.Cell-Based Assays: Cell Viability Assays Morphology analysis Nucleic acid Analysis Protein analysis Other molecules analysis 2.Biochemical/Molecular biology Assays Enzymatic Assays Immunoassay Methods Fluorescent based assay Electrophysiological assay Gene manipulation

33. Optogenetics

34. In vitro vs. in vivo assay In vitro In vivo In vitro Cell-free vs. cell-based Cell-based vs. tissue/organ-based Tissue/organ-based vs. organism-based Question overlap Logic overlap Technique overlap In vitro

35. In vivo assay

36. In vivo assay (Holism)

37. Project Design 1.Objectives and/or Hypotheses 2.Strategy: all meaningful biological effects should be statistically significant Manipulated variable (independent) Response variable (dependent) Extraneous variables (uncontrolled/random, Outlier) 3.Endpoints: where to stop? 4.Control Groups: positive vs. negative 5.Statistical Analysis Plan and Implementation Parallel group Randomized block Repeated measures Cross-over design

38. Data and Compound Management Result Levels 1.Raw data: Individual measurements 2.Normalized data: raw data that have been transformed to provide a consistent, biologically relevant context. 3.Aggregated data: median (preferred) or mean normalized data. 4.Derived data: Results calculated from groups normalized or aggregate data based upon mathematical model fitting. 5.Summarized data: Statistical summarization

39. Statistical Analysis Considerations 1.Parallel groups 1.One factor with only two levels: t-test or ANCOVA 2.One factor with more than two levels: one-way ANOVA or ANCOVA 3.Two factors: two-way ANOVA or ANCOVA 2.Randomized blocks 1.One factor: two-way ANOVA, Block as the second factor 2.Two factors: three-way ANOVA, Block as the third factor 3.Crossover & repeated measures: two-way and repeated- measures ANOVA 4.Stratified designs: two-way ANOVA

40. How to Deal with High Assay Variability 1.The between- and within-run sources of variability. 2.The animal-to-animal variability 3.The variability in measuring the response of the subject 4.Use variance to find the sources of variability. 5.Simply increasing the sample may not necessarily reduce between-run variability.

41. Example: Analyzing Variability 1.5 animals (multiplue test tubes for each animal) in each of three studies 2.study-to-study variability 3.animal-to-animal variability 4.tube-to-tube variability Source of Variation Estimated Variance Estimated Std Dev Pct of Total (%) Study373088610.868.3 Animal72819269.913.3 Tube100033316.318.3 Total545941100