Some thoughts on systems biology Harvey Rubin, MD, PhD rubinh@maild.upenn

1. Some thoughts on systems biology Harvey Rubin, MD, PhD rubinh@mail.med.upenn.edu

2. The four questions for systems biology • Can biological systems operationalize certain aspects of cyber systems so that we can understand and design advanced biological systems? 2. Can biological systems operationalize certain aspects of cyber systems so that we can understand and design advanced cyber systems? 3. Can cyber systems operationalize certain aspects of biological systems so that we can understand and design advanced biological systems? 4. Can cyber systems operationalize certain aspects of biological systems so that we can understand and design advanced cyber systems?

3. Cyber-Bio comparisons: on the totally arbitrary and arguable scale of 1-5 CyberBio Logic operations 5 1 Programmable 5 2 Parallel processing 3 5 Standardization 5 3 Abstraction 5 2 Modularity 5 5 Predictability of part 5 3 Predictability of part in system 4 2 Stable/durable in the natural environment 4 3 Stable/durable under stress and attack 2 4 Energy efficiency 2 5 Logically reversible 2 4 Thermodynamically reversible 2 4 Scalable 3 3 Evolvable 1 5 Self learning 1 5 Self repair 1 5 Self correcting 1 5 Self assembly 1 5 Self-Replicating (hardware) 0 5 Richness of user interface 2 4 Multi-agent communication 3 4 Aggregate data and predict outcomes 0-1 4 Solve the “inverse problem” 0-1 5 Impact on society 0-4 5

4. 1. Can biological systems operationalize certain aspects of cyber systems so that we can understand and design advanced biological systems? Logic operations 5 1 Programmable 5 2 Parallel processing 3 5 Standardization 5 3 Abstraction 5 2 Modularity 5 5 Predictability of parts 5 3 Predictability of parts in system 4 2 Stable/durable in the natural environment 4 4 Stable/durable under stress and attack 2 4 individuals societies and cultures

5. Answer to Question 1 –YES this is predominantly in the world of molecular and synthetic biology • Cell cycle counter and cell division reporter • Control metabolic pathways and switches • Regulate intracellular communications • Microbial fuel cells • New therapies • Biological sensors Roger Kornberg Arthur Kornberg Andrew Fire Craig Mello

6. Protein synthesis

7. Systems-synthetic biology • synthetic biology seeks both to employ nonnatural molecules to mimic biological behavior and to assemble well-characterized biomolecular components into circuits that perform prescribed functions. • potential to expand our understanding of biomolecular networks and enhance our ability to engineer novel cellular behavior. • most efforts have focused on engineering gene circuits that rely on protein-DNA interactions to control transcription

8. RNA molecules are malleable and attractive molecule that can drive programmable functions • RNA molecules, adopt complex structures • can be generated from potentially all possible sequence combinations • leads to diverse secondary structure and function • causal relationship between sequence, structure and function significantly affects the interaction of RNA molecules with proteins, metabolites and other nucleic acids

9. Programmable functions are the effects of proteins: understand protein synthesis • requires a series of catalytic and regulatory steps involving key cellular machinery. • several factors affect translation initiation, including ribosomal recognition of the mRNA ribosome binding site (RBS) and the start codon • regulate bacterial gene expression by interfering with ribosomal docking at the RBS • create a modular post-transcriptional regulation system that could be integrated into biological networks and implemented with any promoter or gene

10. Example: an engineered Ribo-regulator

11. suppose you can synthesize almost anything…What could/would you do? “The 1918 virus and recombinant H1N1 influenza viruses were generated using the previously described reverse genetics system (8, 14). All viruses containing one or more gene segments from the 1918 influenza virus were generated and handled under high-containment biosafety level 3 enhanced (BSL3) laboratory conditions in accordance with guidelines of the National Institutes of Health and the Centers for Disease Control and Prevention (15).”

12. “1918 Flu and Responsible Science” “I firmly believe that allowing the publication of this information was the correct decision in terms of both national security and public health.” Science Editorial Vol. 310, 7 October 2005 Philip A. Sharp

13. “The 1918 flu genome: Recipe for Destruction” “This is extremely foolish. The genome is essentially the design of a weapon of mass destruction.” New York Times Op-Ed October 17, 2005 Ray Kurzweil and Bill Joy

14. “Mr. President, I feel I have blood on my hands” “I don’t want to see that son of a bitch in this office ever again.”

15. Systems biologists- are our hands bloody? Demonstrate how to render human or animal vaccines ineffective Confer resistance to therapeutically useful antibiotics or antiviral agents for humans, animals, or crops Enhance the virulence of human, animal, or plant pathogens, or make nonpathogens virulent Increase the transmissibility of pathogens Alter the host range of pathogens Enable the evasion of diagnostic or detection methods Enable the weaponization of biological agents or toxins Generate novel pathogens or toxins, or reconstitute an eradicated pathogen

16. NRC Report on Dual Use Research Report of the National Research Council of the National Academies: “Biotechnology Research in an Age of Terrorism: Confronting the Dual Use Dilemma” (October 2003)

17. Life Sciences: Need for Biosecurity • “Dual use” potential of certain life sciences research requires consideration of biosecurity measures • Goal (and challenge) is to enhance biosecurity protections for life sciences research while ensuring that any impact to the free flow of scientific inquiry is minimized.

18. National Science Advisory Board on Biosecurity http://www.biosecurityboard.gov

19. National Science Advisory Board for Biosecurity (NSABB) The NSABB is established to provide advice to federal departments and agencies on ways to minimize the possibility that knowledge and technologies emanating from vitally important biological research will be misused to threaten public health or national security. • Advisory to the Secretary of Health and Human Services, Director of National Institutes of Health, and heads of all US federal departments and agencies that conduct or support life science research • Will recommend specific strategies for efficient and effective oversight of US federally conducted or supported dual use biological research

20. Exec. Office of the President Department of Health and Human Services Department of Energy Department of Homeland Security Department of Veteran’s Affairs Department of Defense Environmental Protection Agency United States Department of Agriculture Department of Interior National Sciences Foundation Department of Justice Department of State Department of Commerce National Aeronautics and Space Administration Intelligence community NSABB ex officios

21. Criteria for DUR: Emerging Concepts Research: With agents that possess a high biological threat/risk potential That could increase the potential of an agent to cause harm Apply Criteria Developed from these Categories Dual Use Research That could enhance susceptibility of host populations to harm That yields enabling technologies or facilitating information That may be misused to pose a biologic threat to public health and/or national security

23. 2. Can biological systems operationalize certain aspects of cyber systems so that we can understand and design advanced cyber systems? CyberBio Logic operations 5 1 Programmable 5 2 Parallel processing 3 5 Len Adelman DNA computation papers—highly parallel, solve NP problems

24. Physical Limitations of DNA Computing Directed Hamiltonian path problem 25 nodes….. 1 kilogram of DNA needed 70 nodes….. 1000 kilograms of DNA needed Decryption 101233 strands of DNA at 0.17 uM------->101216 liters! From Cox, Cohen,& Ellington

25. Adleman reported in a meeting that he solved a 20 variable SAT problem using DNA “It is not remarkable that the bear dances well-- It is that the bear dances at all”

26. Not particularly interested in dancing bears, we decided to see if DNA computing had anything to say about some of the fundamental limits of computation CyberBio Energy efficiency 2 5 Logically reversible 2 4 Thermodynamically reversible 2 4 The Fundamental Physical Limits of Computation What constraints govern the physical process of computing? Is aminimum amount of energy required, for example, per logic step?There seems to be no minimum., but some other questions are open by Charles H. Bennett and Rolf Landauer Scientific American253(1):48-56 (July, 1985).

27. A Fredkin Gate: Logically reversible with no energy limit on the computation CAB is a piece of DNA that we can synthesize

28. a NAND gate

29. Why reversible? Minimal energy expense Detection and correction of intrusion Error checking by reversing computation to recreate inputs Bidirectional debugging

30. In principle it can take minimal energy to go through a biochemical gate DNAn + dNTP DNAn+1 + PPi D G = kt ln[dNTP/PPi] If dNTPs are just 1% over the equilibrium value: D G = kt ln[10.1/10] or about 0.01kT a modification of an idea in Bennett and Landaur’s Sci. Am paper—suggested using RNA

31. We synthesized the oligonucleotides and ran the reactions Klein, JP., Leete, TH. & Rubin H. A Biomolecular Implementation of Logically Reversible Computation with Minimal Energy Dissipation. BioSystems 52, 15-23, 1999.

32. The gate works in the lab

33. How fast is the gate? t1/2 annealing: 3 sec. DNA polymerization rate: 15 bases/sec For 60 bases pair input: 10 sec • Can biological systems operationalize certain aspects of cyber systems so that we can understand and design advanced cyber systems? • ---NO

34. 3. Can cyber systems operationalize certain aspects of biological systems so that we can understand and design advanced biological systems? • Nano-bio • Medical devices • Lab on a chip • NSF workshop on high confidence medical devices and software systems last year • Subject of Tele-Physical services and applications working group at this meeting • > $3 billion invested already 2007 NSTI Nanotechnology Conference and Trade Show – May 2007 - Santa Clara Life Sciences & Medicine Bio-nano Materials & Tissues Bio Sensors & Diagnostics Biomarkers & Nanoparticles Cancer Nanotechnology Cellular & Molecular Dynamics Drug Delivery & Therapeutics Imaging Nano Medicine Nanotech to Neurology Answer to Question 3--YES

35. CyberBio Evolvable 1 5Self learning 1 5Self repair 1 5Self correcting 1 5Self assembly 1 5Self-Replicating (hardware) 0 5 Richness of user interface 2 4 Multi-agent communication 3 4 Aggregate data and predict outcomes 0-1 4 Solve the “inverse problem” 0-1 5 Impact on society 0-4 5 4. Can cyber systems operationalize certain aspects of biological systems so that we can understand and design advanced cyber systems?

36. Can cyber systems operationalize certain aspects of biological systems so that we can understand and design advanced cyber systems? examples abound from molecular level to societal level • Persistence in bacteria as hedge strategy against attack • Quorum sensing • Cellular metabolism- metabolome:metabolic flux models • supply chain • Swarm behavior • Autonomous mobile robots • Markets • Data aggregation • Event prediction

37. Problem to solve: tuberculosis TB infection is spreading at the rate of one person per second. The disease kills more young people and adults than any other infectious disease and is the world's biggest killer of women. Each year, an estimated eight million to 10 million people contract the disease and about two million people die from it. An estimated 33% of the 40 million people living with HIV/AIDS worldwide are co-infected with TB. Without treatment, 90% of people living with HIV/AIDS die within months of contracting TB. The majority of people who are co-infected with both diseases live in sub-Saharan Africa.

38. Here’s one of the big problems for tuberculosis as well as many other biological systems…… PERSISTERS

39. Bio-systems under potential attackPersistence in bacteria • microorganisms often encounter an environment with limited nutrients or certain other stress related stimuli • they enter a dramatically slowed growth state until a new equilibrium is established

40. “However, no further additional bactericidal activity was found during further incubation with isoniazid alone or when gatifloxacin was added to either isoniazid or rifampin. This suggested that the stationary- phase culture contained a mixture of occasionally dividing bacilli that were killed during the first 2 days and true static persisters in the residual population that mimicked those in human lesions.”

41. The bug must have a mechanism(s) to sense and respond to hard times…...sense the environment and couple inhibition of transcription to inhibition of translation

42. Stringent Response System in Mtb • Regulation of a key component of the pathogenesis of Mycobacterium tuberculosis is determined by the ability of the bacterium to sense and respond to adverse growth conditions …...couple inhibition of transcription to inhibition of translation • Deletion of this response will disable the bacterium—keep metabolism going to kill the cells in the presence of antibiotics! • Insertion of this response will enable an organism to maintain viability under stress conditions • The survival response comprises the interplay of a complex network of genes and proteins

43. Two opposing RelMtb activities 1) pppGpp synthesis: p-p-p-G + p-p-p-A p-p-p-G-p-p + p-A GTP + ATP G5 + AMP 2) pppGpp hydrolysis: p-p-p-G-p-pPPi + p-p-p-G pppGpp alters RNAP kinetics and mediates the transcriptional response to environmental conditions to which Mtb is exposed

44. Systems biology: molecular components of persistence in bacteria

46. Persistence in bacteria Kill curves in the presence of ampicillin E. COLI PERSISTENCE LINKED TO (p)ppGpp BY A MIXED STOCHASTIC AND DETERMINISTIC MECHANISM Halász, Buckstein, Imieliński, Marjanovich, Teh, Kumar, Rubin

47. (B) Histograms obtained by sampling the growth rates of one single-cell simulation over approximately 1000 hours. The thin line marked "(p)ppGpp knockout 2" corresponds to a shorter sampling period which does not include a large shutdown event. (C) Kill curves derived from the growth rate histograms. Both versions of the knockout exhibit fewer persisters.

48. Lungs from mice infected with wild type MTb Lungs from mice infected with Rel KO strain Lungs from mice infected with Rel KO strain complemented with plasmind containing rel Spleens from mice infected with WT-KO—KO + comp

49. The bug must have a mechanism to make energy in the form of ATP—the role of the respiratory chain

50. TUBERCULOSIS — METABOLISM AND RESPIRATION IN THE ABSENCE OF GROWTH Helena I. M. Boshoff and Clifton E. Barry, 3rd NATURE REVIEWS | MICROBIOLOGY VOLUME 3 | JANUARY 2005 |