1	Experimental and Computational Requirements for Post-Genomic Integrative Cellular Physiology John Wikswo
2	Topics The advantages of micro/nanoscale instruments Cellular complexity The need for closed-loop control How to identify early manifestations of disease Modeling Interactive, dynamical analysis Mining dynamics data
3	Topics The advantages of micro/nanoscale instruments Cellular complexity The need for closed-loop control How to identify early manifestations of disease Modeling Interactive, dynamical analysis Mining dynamics data
4	Reductionism in Science
5	Historical Evolution of Spatial Resolution in Biology and Physiology X-Ray / SEM / STM Optical microscope Magnifying glass Unaided eye
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7	Hypotheses I and II I. The explosion in genomic and proteomic knowledge and measurement techniques will revolutionize the early detection of diseases II. Much of the potential lies in the clinical implementation of the instrumentation and techniques that provided the scientific foundation for genomics and proteomics
8	Technologies for Early Disease Detection Analysis of biofluids Molecular profiles that define biological states (Dahl) Disposable plastic lab-on-a-chip devices for point-of-care systems (Luke Lee) Indwelling biosensors and analyzers (Stephen C. Lee) Chemokine and cytokine expression (Barrett Rollins; Philip R. Streeter) Gene expression patterns (Carl W. Cotman; Marti Jett) Detection of mutant alleles (Helmut Zarbl) Protein expression/distribution (Philip R. Streeter; Gordon R. Whiteley) Single-pass analysis of proteins, cells and tissues Detection of small numbers of molecules (Roger Brent) Multispectral cellular imaging (David Basiji) Protein distribution in tissues (Richard Caprioli) Interactive cellular assays for systems biology Disease/pathogen-induced changes in cells (Christopher Chen) Nanoscale sensing of single molecule binding (Michael Roukes) Massively Parallel, Multi-Phasic Cellular Biological activity detectors (John Wikswo)
9	Technologies for Early Disease Detection Analysis of biofluids Molecular profiles that define biological states (Dahl) Disposable plastic lab-on-a-chip devices for point-of-care systems (Luke Lee) Indwelling biosensors and analyzers (Stephen C. Lee) Chemokine and cytokine expression (Barrett Rollins; Philip R. Streeter) Gene expression patterns (Carl W. Cotman; Marti Jett) Detection of mutant alleles (Helmut Zarbl) Protein expression/distribution (Philip R. Streeter; Gordon R. Whiteley) Single-pass analysis of cells and tissues Detection of small numbers of molecules (Roger Brent) Multispectral cellular imaging (David Basiji) Protein distribution in tissues (Richard Caprioli) Key Features: Low temporal bandwidth sensing Slow events, long measurement intervals or single-pass imaging Semi-standard, static biochemical analyses Feature correlation and pattern recognition Will benefit directly from advances in genomics and proteomics May involve significant issues in bioinformatics Will benefit from Micro/Nano
10	Standard Rationale for Micro & Nanoscale Analytical Systems BioMicroElectroMechanical Systems (BioMEMS) Low-cost mass production Automated analysis Reduced instrument footprint Single instruments are very, very small Reduced volumes of analyte and reagents Massively parallel Increased data Lower cost per datum Combinatorics for frontal assault on multivariable systems Enabling new physical/chemical properties Single molecule detection Quantum dots
11	"~2,000 valves to control" ~2,000 valves to control Reagents Samples Wash steps www.fluidigm.com
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15	Genomics Proteomics What is next?
16	Topics The advantages of micro/nanoscale instruments Cellular complexity The need for closed-loop control How to identify early manifestations of disease Modeling Interactive, dynamical analysis Mining dynamics data
17	Hypotheses III and IV III. Historically, dynamical studies of cellular metabolism and signaling pathways have been limited by the bandwidth of laboratory biochemistry IV. BioMicroElectroMechanical Systems (BioMEMS) offer promise to extend the measurement bandwidth for both research and clinical diagnosis
18	Technologies for Early Disease Detection Interactive cellular assays for systems biology Disease/pathogen-induced changes in cells (Christopher Chen) Nanoscale sensing of single molecule binding (Michael Roukes) Massively Parallel, Multi-Phasic Cellular Biological Activity Detectors (John Wikswo) Key Features: More closely related to experimental physiology than classical clinical biochemistry Can involve rapid sensing of physiological dynamics Measurement bandwidth << physiological bandwidth Real-time intervention is REQUIRED to probe the dynamics Internal vs. external feedback “Bandwidth is everything” May require models for interpretation of complex interactions There may be significant computational contraints to multiscale dynamical models
19	Post-Reductionism
20	What about dynamic processes? Physiology is dynamic Cell cycle Developmental differentiation Growth Voltage- and ligand-gates ion channels Propagating waves Signaling cascades Closed-loop feedback and control
21	Cytion Planar Patch Clamp Courtesy of Christian Schmidt and Will Lachnit
22	Array of Ion Channels Courtesy of Ananta Krishnan, DARPA
23	Physical and Biological Time Constants, Seconds Mixing time to homogenize liquid in a large-scale bioreactor (10-100 m³) 10⁴-10⁸ 90% liquid volume exchange in in a continuous reactor 10⁵-10⁶ Oxygen transfer (forced not free diffusion) 10²-10³ Heat transfer (forced convection) 10³- 10⁴ Cell proliferation, DNA replication 10²-10⁴ Response to environmental changes (temperature, oxygen) 10³-10⁴ Messenger RNA synthesis 10³-10⁴ Translocation of substances into cells (active transport) 10¹-10³ Protein synthesis 10¹-10² Allosteric control of enzyme action 1 Glycolysis 10^-1-10^-2 Oxidative phosphorylation in mitochondria 10^-2 Intracellular quiescent mass & heat transfer (dimension 10^-5 m) 10^-5-10^-3 Enzymatic reaction and turnover 10^-6-10^-3 Bonding between enzyme & substrate, inhibitor 10^-6 Receptor-ligand interaction 10^-6
24	The Systems Physiology Challenge Use experimental measurements, numerical simulations, and knowledge of the genome and proteome to unravel the complex, multiscale interactions and dynamics in normal physiology, toxic exposures, and disease Metabolic networks Intracellular and extracellular signaling Gene expression Protein interactions Cell-cell interactions Active transport Development, growth, aging, death
25	Biological Modeling and Analysis
26	Postgenomic Integrative/Systems Physiology/Biology Specify concentrations and Rate constants Add gene expression, Protein interactions, and Signaling pathways; Include intracellular spatial distributions, diffusion, and transport, … and calculate how the target cell behaves in response to a toxin or pathogen
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28	Molecular Interaction Map: DNA Repair
29	Molecular Interaction Map: Cell Cycle
30	The Catch Modeling of a single mammalian cell may require 100,000 variables and equations Cell-cell interactions are critical to system function 10⁹ interacting cells in some organs Models may be leibnitz-class The data don’t yet exist to drive the models! Hence we need to experiment… *1 leibnitz = 1 mole of PDEs ~ 1 etaFLOPS-year
31	Topics The advantages of micro/nanoscale instruments Cellular complexity The need for closed-loop control How to identify early manifestations of disease Modeling Interactive, dynamical analysis Mining dynamics data
32	The Experimental Problem Most chemical and metabolic sensors and actuators Are too slow to track biochemical events at the cellular level Are made one at a time Biological systems contain extensive closed-loop, multilevel, feedback and control Simple, single-step observations cannot discern how control is distributed through the system. Closed-loop metabolic control is today possible only at the animal and organ level, e.g., glucose clamp Chemical control is limited by diffusion, stirring, uncaging rates, or the time required to move a cell from one medium to another Post-genomic physiology needs multiparameter, wide-bandwidth cellular metabolic and signaling sensing and control
33	What do we need? Simultaneous, fast sensors (transducers) that detect a variety of changes within and outside the cell Actuators that control the microenvironment within and outside the cell Openers for the internal feedback loops System algorithms and models that allow you to close and stabilize the external feedback loop …
34	The Challenge: Instrument and Control the Cell Develop the tools and techniques for integrative, post-genomic cellular biology Genes Proteins Metabolic and signaling pathways Instruments Models Wide-bandwidth dynamic control theory for cellular systems How do normal and diseased cells function?
35	Instrumenting the Single Cell Arrays of instrumented single or multiple cells: Rapid, sensitive, and accurate differential diagnosis of cellular pathophysiology Cellular dynamics: Discrimination between causal and secondary events Functional biopsy: Determine the state of specific physiological pathways and mechanisms affected by an as-yet undetected disease, and thus define a prophylaxis or therapy. Artificial, minimal cells: engineered to serve as dedicated, configurable, robust on-chip biosensors. “Quantitative physiology at the speed of life,” C. Kovac
36	Possible Approaches A biological cell or molecule inserted into a microinstrument, e.g., a single-cell spectrophotometer or a whole-cell patch clamp A nanoinstrument inserted into the cell/molecule, e.g., caged ATP Combine the two approaches to form an FAST integrated, closed-loop bio/nano/micro system
37	MicroBottle Goal – utilize proven gigaOhm seals to biological membranes Result – high-speed microfluidics and silicon microelectronics can be placed “inside” a living cell
38	Hypotheses V and VI V. Great advances in physiology have been made through opening physiological feedback loops and applying external control Frank-Starling cardiovascular regulation Glucose/insulin regulation Hodgkin-Huxley model of the nerve action potential VI. There will exist a class of diseases or susceptibilities to drugs or toxins that can be diagnosed through altered cellular dynamics
39	Physical and Biological Time Constants, Seconds Mixing time to homogenize liquid in a large-scale bioreactor (10-100 m³) 10⁴-10⁸ 90% liquid volume exchange in in a continuous reactor 10⁵-10⁶ Oxygen transfer (forced not free diffusion) 10²-10³ Heat transfer (forced convection) 10³- 10⁴ Cell proliferation, DNA replication 10²-10⁴ Response to environmental changes (temperature, oxygen) 10³-10⁴ Messenger RNA synthesis 10³-10⁴ Translocation of substances into cells (active transport) 10¹-10³ Protein synthesis 10¹-10² Allosteric control of enzyme action 1 Glycolysis 10^-1-10^-2 Oxidative phosphorylation in mitochondria 10^-2 Intracellular quiescent mass & heat transfer (dimension 10^-5 m) 10^-5-10^-3 Enzymatic reaction and turnover 10^-6-10^-3 Bonding between enzyme & substrate, inhibitor 10^-6 Receptor-ligand interaction 10^-6
40	A Key Rationale for Micro & Nanoscale Analytical Systems Wide measurement bandwidth, i.e., good response to high frequencies, is required to track fast cellular events Stable control of fast systems requires high bandwidth Small is the best way to beat the time for diffusional mixing in large-scale assays Small lets one look at individual cellular events rather than ensemble averages
41	Lactate Diffusion Times
42	What do we gain by small and fast? Electrochemical sensitivity scale-invarient; frequency response improves as size is decreased Decreased mixing times for mass and heat transfer Reduced reagent volumes for rapid injection Many nanocultures within a single device Monitor known, small (N=1?) number of cells in each nanoculture Array of NanoBioReactors, in parallel, in series, and with redundancy for high-content screening
43	Chemical Clamp Sensors: Advanced micro and nanosensors can quantify the extra- cellular and intra-cellular environments with unprecedented temporal resolution Actuators: Microfluidics can control extracellular and intracellular concentrations of key chemicals with millisecond-response picoliter pumps Openers: RNAi, genetic knockouts, and blockers will allow opening of the internal feedback loop Controllers: It will be possible to create high-speed extracellular and intracellular chemical clamps functionally equivalent to voltage clamp for V_m
44	High-Content Toxicology Screening Using Massively Parallel, Multi-Phasic Cellular Biological Activity Detector (MP²-CBAD) Vanderbilt University Departments of Biomedical Engineering, Chemical Engineering, Chemistry, Mechanical Engineering, Molecular Physiology & Biophysics, Physics & Astronomy
45	Cell Metabolism
46	Discrimination
47	Vanderbilt Instrumenting the Single Cell
48	Coupled Modeling of Cell and Environment
49	The Challenge: Convert Steady-State Metabolic Flux Balances to Dynamic Metabolic Network Responses
50	Bio-Molecular Devices/Systems Courtesy of Ananta Krishnan, DARPA
51	Bio-Molecular Devices/Systems
52	Topics The advantages of micro/nanoscale instruments Cellular complexity The need for closed-loop control How to identify early manifestations of disease Modeling Interactive, dynamical analysis Mining dynamics data
53	Biological Modeling and Analysis
54	Signal Classification: Feature Extraction
55	Agent Discrimination Algorithms
56	Data Mining/Exploration
57	Conclusions Micro/Nano will “increase throughput and automation, reducing cost per analysis, and enabling entirely new applications.” C. Dahl Understanding cellular dynamics is key to understanding cellular physiology Micro/Nano will enable closed-loop control of certain cellular functions Biology and biochemistry can serve as preamplifiers for biological, biochemical, and biophysical detectors PCR of course, but what else? Cell harvesting may be a problem for many tissues Physiological biopsy Pretransplant certification (pancreatic beta cells in islet transplants) Well suited for probing drug interactions for particular phenotypes Dynamic model complexity is a major challenge Specification Verification