Some thoughts on systems biology

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• Some thoughts on systems biology
• Harvey Rubin, MD, PhD
• rubinh_at_mail.med.upenn.edu

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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?

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Cyber-Bio comparisons on the totally arbitrary
and arguable scale of 1-5
Cyber Bio
Logic operations 5 1 Programmable 5 2
Parallel processing 3 5
Standardization 5 3 Abstraction 5 2 Modula
rity 5 5 Predictability of part 5 3 Predict
ability 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
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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 Modula
rity 5 5 Predictability of parts 5 3 Predic
tability of parts in system 4 2 Stable/durabl
e in the natural environment 4 4 Stable/durable
under stress and attack 2 4
individuals
societies and cultures
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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
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Protein synthesis
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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

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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

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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

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Example an engineered Ribo-regulator
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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).
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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
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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
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Mr. President, I feel I have blood on my hands
I dont want to see that son of a bitch in
this office ever again.
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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

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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)
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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.

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

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NSABB ex officios

• Exec. Office of the President
• Department of Health and Human Services
• Department of Energy
• Department of Homeland Security
• Department of Veterans 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

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NSABB Charge

• Recommend
• Criteria for identifying dual use research
• National guidelines for oversight of dual use
  research at both local and federal levels,
  including
• Strategies for oversight of new classes of
  experiments and technologies

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NSABB Charge

• Advise on
• Program for biosecurity education and training
  for all scientists and laboratory workers at
  federally funded institutions
• A code of conduct for scientists and laboratory
  workers in life sciences research
• National guidelines on communication and
  dissemination of dual use research methodology
  and research results
• Strategies for fostering international
  collaboration on the oversight of dual use
  research

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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
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2. Can biological systems operationalize certain
aspects of cyber systems so that we can
understand and design advanced cyber systems?
Cyber Bio Logic operations 5 1 Programmabl
e 5 2 Parallel processing 3 5

• Len Adelman DNA computation papershighly
  parallel, solve NP problems

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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-------gt101216 liters!
From Cox, Cohen, Ellington
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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
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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
Cyber Bio 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 a minimum 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 American 253(1)48-56
(July, 1985).
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A Fredkin Gate Logically reversible with no
energy limit on the computation
CAB is a piece of DNA that we can synthesize
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a NAND gate
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Why reversible? Minimal energy
expense Detection and correction of
intrusion Error checking by reversing
computation to recreate inputs Bidirectional
debugging
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In principle it can take minimal energy to go
through a biochemical gate DNAn dNTP
DNAn1 PPi D G kt lndNTP/PPi If dNTPs
are just 1 over the equilibrium value D G kt
ln10.1/10 or about 0.01kT a modification of
an idea in Bennett and Landaurs Sci. Am
papersuggested using RNA
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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.
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The gate works in the lab
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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

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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
• gt 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
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4. Can cyber systems operationalize certain
aspects of biological systems so that we can
understand and design advanced cyber systems?
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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- metabolomemetabolic flux
  models
• supply chain
• Swarm behavior
• Autonomous mobile robots
• Markets
• Data aggregation
• Event prediction

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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.
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Heres one of the big problems for tuberculosis
as well as many other biological
systems PERSISTERS
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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

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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.
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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
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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
  bacteriumkeep 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

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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-p PPi p-p-p-G
pppGpp alters RNAP kinetics and mediates the
transcriptional response to environmental
conditions to which Mtb is exposed
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Systems biology molecular components of
persistence in bacteria
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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, Imielinski,
Marjanovich, Teh, Kumar, Rubin
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(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.
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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-KOKO comp
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The bug must have a mechanism to make energy in
the form of ATPthe role of the respiratory chain
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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
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Research programCan cyber systems
operationalize certain aspects of biological
systems so that we can understand and design
advanced cyber systems?
Cyber Bio Evolvable 1 5Self
learning 1 5Self repair 1 5Self
correcting 1 5Self assembly
1 5Self-Replicating (hardware) 0 5 Ric
hness 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