The document describes a rule-based modeling approach to simulate the signaling of interferon-gamma (IFN-γ) mediated by the JAK-STAT pathway. The model accounts for combinatorial complexity by allowing all possible protein-protein interactions and reactions according to defined rules. Simulation of the full model generated over 50,000 species and 500,000 reactions before running out of memory. Splitting the model into receptor binding and signal transduction subnetworks produced time courses that better matched experimental data compared to previous models. Further optimization is needed to fully simulate the model.
Take your RNA research to the next level with QIAGEN LNA tools!QIAGEN
Download the flyer!
Experience truly exceptional RNA research with QIAGEN's next-generation, LNA®-enhanced tools. LNA (Locked Nucleic Acid) oligos bind with much higher affinity and specificity to RNA targets than standard DNA and RNA oligos – This enables specific and sensitive detection of small RNAs and discrimination between highly similar
sequences.
FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network - Mol. Biol. Cell. 2010 21(17): 3041 – 3053
This document describes a study that aimed to quantify sodium toxin gene activity in the venom gland of the striped bark scorpion (Centruroides vittatus). Total RNA was extracted from venom gland and body tissue samples of female scorpions. Real-time PCR was performed to compare levels of sodium toxin mRNA between the tissues and determine which sodium toxin genes are most highly expressed. The results will help characterize venom composition and toxicity variations across the geographic range of C. vittatus.
The document describes the steps of Illumina sequencing. Genomic DNA is first fragmented and adapters are ligated to create single-stranded DNA fragments. These fragments are attached to a flow cell and undergo bridge amplification to create clusters of identical DNA fragments. Sequencing occurs through cycles of reversible terminator-based sequencing using fluorescently labeled dNTPs, imaging of the fluorescence, and cleavage of the label and terminator to allow the next cycle. After multiple cycles, the sequenced reads are aligned to the reference genome to determine the original sequence.
Fred Hoyle: Life and Science A biography of the British cosmologist who coine...Simon Mitton
Fred Hoyle (1915-2001) was a pioneering British astrophysicist known for his contributions to stellar and cosmological theory. Some of his key accomplishments included developing theories of stellar nucleosynthesis and the origin of chemical elements within stars. He also proposed the steady state theory of the universe and opposed the Big Bang model, though the Big Bang is now widely accepted. Throughout his career, Hoyle engaged in significant scientific debates and controversies while also establishing the UK's Institute of Theoretical Astronomy.
Take your RNA research to the next level with QIAGEN LNA tools!QIAGEN
Download the flyer!
Experience truly exceptional RNA research with QIAGEN's next-generation, LNA®-enhanced tools. LNA (Locked Nucleic Acid) oligos bind with much higher affinity and specificity to RNA targets than standard DNA and RNA oligos – This enables specific and sensitive detection of small RNAs and discrimination between highly similar
sequences.
FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network - Mol. Biol. Cell. 2010 21(17): 3041 – 3053
This document describes a study that aimed to quantify sodium toxin gene activity in the venom gland of the striped bark scorpion (Centruroides vittatus). Total RNA was extracted from venom gland and body tissue samples of female scorpions. Real-time PCR was performed to compare levels of sodium toxin mRNA between the tissues and determine which sodium toxin genes are most highly expressed. The results will help characterize venom composition and toxicity variations across the geographic range of C. vittatus.
The document describes the steps of Illumina sequencing. Genomic DNA is first fragmented and adapters are ligated to create single-stranded DNA fragments. These fragments are attached to a flow cell and undergo bridge amplification to create clusters of identical DNA fragments. Sequencing occurs through cycles of reversible terminator-based sequencing using fluorescently labeled dNTPs, imaging of the fluorescence, and cleavage of the label and terminator to allow the next cycle. After multiple cycles, the sequenced reads are aligned to the reference genome to determine the original sequence.
Fred Hoyle: Life and Science A biography of the British cosmologist who coine...Simon Mitton
Fred Hoyle (1915-2001) was a pioneering British astrophysicist known for his contributions to stellar and cosmological theory. Some of his key accomplishments included developing theories of stellar nucleosynthesis and the origin of chemical elements within stars. He also proposed the steady state theory of the universe and opposed the Big Bang model, though the Big Bang is now widely accepted. Throughout his career, Hoyle engaged in significant scientific debates and controversies while also establishing the UK's Institute of Theoretical Astronomy.
Review of Cell Press Article titled:
"Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module."
Jared Toettcher, Orion Weiner, Wendell Lim
Cell. 2013 Dec 5;155(6):1422-34. doi: 10.1016/j.cell.2013.11.004.
ESS-Bilbao Initiative Workshop. RF structure comparison for low energy accele...ESS BILBAO
This document compares different radio frequency (RF) structures that can be used for particle acceleration in the low energy range of 3 MeV to 200 MeV. It discusses figures of merit like shunt impedance and beam dynamics constraints for different structure types, including drift tube linacs (DTLs), coupled cavity drift tube linacs (CCDTLs), separated drift tube linacs (SDTLs), spoke cavities, and traveling wave structures. A comparison performed by the HIPPI collaboration assessed the performance of these structures based on factors like shunt impedance, beam dynamics acceptance, and mechanical complexity. Overall, no consensus structure exists for the low energy range, and the optimal design depends on project-specific parameters and future technology developments.
Randomizing genome-scale metabolic networksAreejit Samal
The document proposes a new Markov Chain Monte Carlo (MCMC) based method to generate randomized metabolic networks that impose biochemical and functional constraints. The method successively constrains the networks by (1) fixing the number of reactions, (2) fixing the number of metabolites, (3) excluding blocked reactions, and (4) requiring growth on a specified environment. Imposing these constraints causes the randomized networks to more closely match properties of real metabolic networks like E. coli. The approach generates an ensemble of diverse yet meaningful randomized networks to help identify design principles in metabolic networks.
DNA transcription is a process that involves transcribing genetic information from DNA to RNA. The transcribed DNA message, or RNA transcript, is used to produce proteins. DNA is housed within the nucleus of our cells. It controls cellular activity by coding for the production of proteins. The information in DNA is not directly converted into proteins, but must first be copied into RNA. This ensures that the information contained within the DNA does not become tainted.
This document discusses multithreading and multicore processors. It begins by explaining that instruction level parallelism is difficult to achieve for a single program, but that thread level parallelism exists when running multiple threads or programs simultaneously. It then covers different multithreading paradigms including coarse-grained and fine-grained multithreading as well as challenges with context switching. The document also discusses techniques for multicore processors including cache sharing and instruction fetching policies. It provides examples of commercial multicore chips and research prototypes.
1) The document describes a new method for generating randomized metabolic networks using Markov chain Monte Carlo sampling and flux balance analysis. This allows imposing biological constraints to sample networks with the same functionality as real networks.
2) Analysis of structural properties of these randomized networks showed they have similar scale-free, small-world and bow-tie architecture as real metabolic networks. This suggests global network structure arises from simple biochemical and functional constraints.
3) The method provides a computational framework to study evolutionary questions in systems biology by uniformly sampling the vast space of genetically diverse yet functionally equivalent metabolic networks.
A Novel CMOS Integrated Amplifier for Sensing Single Ion-Channel Current in B...Iowa State University
Here, we present a novel integrated CMOS transimpedance amplifier that permits the recording of low-level, single-channel, fast transient ion-channel currents over a wide dynamic range (1–5 kHz). The
amplifier consists of an initial integrator stage, a post integrating differential stage with gain, a
clamp stage, a discrete-time differentiator stage, a pre-filter differential amplifier stage with gain, and a low pass filtering stage.
KDM5 epigenetic modifiers as a focus for drug discoveryChristopher Wynder
A summary presentation of my scientific work.
My laboratory focused on an enzyme KDM5b (aka PLU-1, JARID1b) that was widely expressed during development and played a key role in progression of breast cancer through HER-2.
My lab focused on understanding the key biochemical activity of the enzyme through dissecting the proteomic and genomic interactors.
Our results were confirmed through the use of ES cells, adult stem cells and mouse models.
Much of this work remains unpublished, please contact me for more information and/or access to any reagents that I still have as part of this work.
crwynder@gmail.com
- Cell signaling pathways regulate nearly all cellular functions through cascades of signaling events involving receptors, signal transducers, and effector proteins. Receptors include G-protein coupled receptors, receptor tyrosine kinases, cytokine receptors, and intracellular receptors.
- Signaling proteins that act as transducers include kinases, GTPases, adaptor proteins, and second messengers like cyclic nucleotides, calcium, lipids, and nitric oxide. These relay, integrate, and distribute signals within cells.
- Feedback loops allow cells to adapt their sensitivity to signaling and respond appropriately to their environment. Understanding cell signaling pathways is challenging due to their complexity, branching, and convergence.
Reducing Silicon Real Estate and Switching Activity Using Low Power Test Patt...ijsrd.com
Power dissipation is a challenging problem for today's system-on-chip design and test. This paper presents a novel architecture which generates the test patterns with reduced switching activities; it has the advantage of low test power and low hardware overhead. The proposed LP-TPG (test pattern generator) structure consists of modified low power linear feedback shift register (LP-LFSR), m-bit counter, gray counter, NOR-gate structure and XOR-array. The seed generated from LP-LFSR is EXCLUSIVE-OR ed with the data generated from gray code generator. The XOR result of the sequence is single input changing (SIC) sequence, in turn reduces the switching activity and so power dissipation will be very less. The proposed architecture is simulated using Modelsim and synthesized using Xilinx ISE9.2.The Xilinx chip scope tool will be used to test the logic running on FPGA.
1) Dr. Ramez Daniel of the Laboratory of Synthetic Biology & Bioelectronics discusses how analog electronics can mimic genetic and biochemical reactions in living cells.
2) Biology uses analog-digital feedback loop hybrid circuits for processes like sensing, gene expression, and signal amplification in a similar way that electronic circuits function.
3) The document maps similarities between biochemical reactions and analog electronics components like transistors, showing how principles like mass action, feedback, and noise can apply to both living and synthetic systems.
4) Engineering challenges remain in scaling synthetic biology from simple logic gates to complex multi-component systems that more accurately mimic the analog, probabilistic behaviors of natural biology.
The document describes the development of a high-throughput single-molecule imaging assay to identify small molecules that alter transcription or splicing kinetics. A reporter cell line was optimized and used to screen over 60 compounds. Hits were validated with live-cell single-molecule imaging, which revealed that SGC-CBP30, PFI-1, and JQ1 slow elongation rates, while Tenovin-1 may affect co-transcriptional splicing. The assay provides a method to characterize drug effects on transcription at a single-molecule level.
Rho GTPases as regulators of morphological neuroplasticityFatih University
Rho GTPases play a key role in regulating neuronal morphology by mediating interactions between cell adhesion molecules and the cytoskeleton. Specifically, Rho inhibits neurite extension while Rac and Cdc42 promote neurite outgrowth and dendritic spine formation. In glial cells, Rho GTPases are involved in myelination by oligodendrocytes and Schwann cells. The role of RhoA in particular contributes to the inhibitory environment of the CNS that prevents axon regeneration.
The document discusses various clock systems, interrupts, and operating modes for microcontrollers:
- It describes clock sources like crystal oscillators and a digitally controlled oscillator that provides the main system clock.
- When an interrupt occurs, the program counter and status register are pushed to the stack before jumping to the interrupt service routine, which must end with a reti instruction.
- Low power modes like LPM3 disable parts of the clock system to reduce power consumption while still allowing interrupts to wake the device.
- Other topics covered include watchdog timers, interrupt prioritization, and entering/waking from low power modes.
Principles of DNA isolation, PCR and LAMPPerez Eric
1. The document discusses principles of DNA isolation and purification as well as polymerase chain reaction (PCR). It describes how cells are broken to release DNA and the components of extraction buffers used to isolate DNA.
2. The three main steps of PCR - denaturation, annealing, and elongation - are explained. Denaturation separates DNA strands, annealing attaches primers, and elongation duplicates the DNA. Required PCR reagents and their roles are also outlined.
3. Loop-mediated isothermal amplification (LAMP) is introduced as an alternative to PCR that amplifies DNA at a constant temperature. LAMP uses multiple primers and has advantages like lower cost and faster results. The mechanism and applications of LAMP are summarized.
The document describes ongoing efforts to optimize a computational model of a CA1 pyramidal neuron. The model integrates synaptic and cellular interactions using the EONS/NEURON framework. The optimization aims to accurately reproduce various experimental data through tuning of channel conductances and other biophysical parameters. Several approaches are explored, including varying temperature effects, comparing different CA1 models, and deconvolving EPSCs to model presynaptic mechanisms. Future plans include using evolutionary optimization techniques and additional experimental objectives to further refine the model.
This document provides an introduction to single-cell RNA-seq (scRNA-seq) analysis. It discusses different scRNA-seq assays such as Smart-Seq2, Drop-seq, and 10X, and how their protocols and sequencing outputs differ. It also covers scRNA-seq data characteristics like zero inflation and overdispersion. The document outlines common analysis steps like filtering, dimensionality reduction, clustering, and differential expression. It emphasizes that scRNA-seq data requires specialized analysis due to its noisy and sparse nature compared to bulk RNA-seq data.
Synthetic gene networks were constructed in cells that function as digital counters. Two different counting mechanisms were implemented and tested experimentally. The first uses a riboregulated transcriptional cascade to count arabinose pulses, with fluorescence increasing only after the defined number of pulses. The second method uses site-specific DNA recombinases to invert promoter orientations and count pulses. Mathematical models were able to predict experimental results. While these synthetic gene networks demonstrate counting in cells, their reliability may be limited due to inherent variability in cellular mechanisms.
This document presents a novel approach to emulate power islands in FPGA platforms to enable earlier validation of low power features. It proposes corrupting logic in power-gated domains by connecting power gating enables to asynchronous set/clear signals on flip-flops. This approach more realistically mimics silicon behavior during power-off and can find issues like incorrect power sequencing bugs. The methodology was implemented using a custom EDF library and randomization to corrupt a parameterized percentage of logic. Challenges around FPGA architecture and optimization were addressed. Results showed the approach introduced minimal overhead while enabling important power management validation earlier in the design flow.
Review of Cell Press Article titled:
"Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module."
Jared Toettcher, Orion Weiner, Wendell Lim
Cell. 2013 Dec 5;155(6):1422-34. doi: 10.1016/j.cell.2013.11.004.
ESS-Bilbao Initiative Workshop. RF structure comparison for low energy accele...ESS BILBAO
This document compares different radio frequency (RF) structures that can be used for particle acceleration in the low energy range of 3 MeV to 200 MeV. It discusses figures of merit like shunt impedance and beam dynamics constraints for different structure types, including drift tube linacs (DTLs), coupled cavity drift tube linacs (CCDTLs), separated drift tube linacs (SDTLs), spoke cavities, and traveling wave structures. A comparison performed by the HIPPI collaboration assessed the performance of these structures based on factors like shunt impedance, beam dynamics acceptance, and mechanical complexity. Overall, no consensus structure exists for the low energy range, and the optimal design depends on project-specific parameters and future technology developments.
Randomizing genome-scale metabolic networksAreejit Samal
The document proposes a new Markov Chain Monte Carlo (MCMC) based method to generate randomized metabolic networks that impose biochemical and functional constraints. The method successively constrains the networks by (1) fixing the number of reactions, (2) fixing the number of metabolites, (3) excluding blocked reactions, and (4) requiring growth on a specified environment. Imposing these constraints causes the randomized networks to more closely match properties of real metabolic networks like E. coli. The approach generates an ensemble of diverse yet meaningful randomized networks to help identify design principles in metabolic networks.
DNA transcription is a process that involves transcribing genetic information from DNA to RNA. The transcribed DNA message, or RNA transcript, is used to produce proteins. DNA is housed within the nucleus of our cells. It controls cellular activity by coding for the production of proteins. The information in DNA is not directly converted into proteins, but must first be copied into RNA. This ensures that the information contained within the DNA does not become tainted.
This document discusses multithreading and multicore processors. It begins by explaining that instruction level parallelism is difficult to achieve for a single program, but that thread level parallelism exists when running multiple threads or programs simultaneously. It then covers different multithreading paradigms including coarse-grained and fine-grained multithreading as well as challenges with context switching. The document also discusses techniques for multicore processors including cache sharing and instruction fetching policies. It provides examples of commercial multicore chips and research prototypes.
1) The document describes a new method for generating randomized metabolic networks using Markov chain Monte Carlo sampling and flux balance analysis. This allows imposing biological constraints to sample networks with the same functionality as real networks.
2) Analysis of structural properties of these randomized networks showed they have similar scale-free, small-world and bow-tie architecture as real metabolic networks. This suggests global network structure arises from simple biochemical and functional constraints.
3) The method provides a computational framework to study evolutionary questions in systems biology by uniformly sampling the vast space of genetically diverse yet functionally equivalent metabolic networks.
A Novel CMOS Integrated Amplifier for Sensing Single Ion-Channel Current in B...Iowa State University
Here, we present a novel integrated CMOS transimpedance amplifier that permits the recording of low-level, single-channel, fast transient ion-channel currents over a wide dynamic range (1–5 kHz). The
amplifier consists of an initial integrator stage, a post integrating differential stage with gain, a
clamp stage, a discrete-time differentiator stage, a pre-filter differential amplifier stage with gain, and a low pass filtering stage.
KDM5 epigenetic modifiers as a focus for drug discoveryChristopher Wynder
A summary presentation of my scientific work.
My laboratory focused on an enzyme KDM5b (aka PLU-1, JARID1b) that was widely expressed during development and played a key role in progression of breast cancer through HER-2.
My lab focused on understanding the key biochemical activity of the enzyme through dissecting the proteomic and genomic interactors.
Our results were confirmed through the use of ES cells, adult stem cells and mouse models.
Much of this work remains unpublished, please contact me for more information and/or access to any reagents that I still have as part of this work.
crwynder@gmail.com
- Cell signaling pathways regulate nearly all cellular functions through cascades of signaling events involving receptors, signal transducers, and effector proteins. Receptors include G-protein coupled receptors, receptor tyrosine kinases, cytokine receptors, and intracellular receptors.
- Signaling proteins that act as transducers include kinases, GTPases, adaptor proteins, and second messengers like cyclic nucleotides, calcium, lipids, and nitric oxide. These relay, integrate, and distribute signals within cells.
- Feedback loops allow cells to adapt their sensitivity to signaling and respond appropriately to their environment. Understanding cell signaling pathways is challenging due to their complexity, branching, and convergence.
Reducing Silicon Real Estate and Switching Activity Using Low Power Test Patt...ijsrd.com
Power dissipation is a challenging problem for today's system-on-chip design and test. This paper presents a novel architecture which generates the test patterns with reduced switching activities; it has the advantage of low test power and low hardware overhead. The proposed LP-TPG (test pattern generator) structure consists of modified low power linear feedback shift register (LP-LFSR), m-bit counter, gray counter, NOR-gate structure and XOR-array. The seed generated from LP-LFSR is EXCLUSIVE-OR ed with the data generated from gray code generator. The XOR result of the sequence is single input changing (SIC) sequence, in turn reduces the switching activity and so power dissipation will be very less. The proposed architecture is simulated using Modelsim and synthesized using Xilinx ISE9.2.The Xilinx chip scope tool will be used to test the logic running on FPGA.
1) Dr. Ramez Daniel of the Laboratory of Synthetic Biology & Bioelectronics discusses how analog electronics can mimic genetic and biochemical reactions in living cells.
2) Biology uses analog-digital feedback loop hybrid circuits for processes like sensing, gene expression, and signal amplification in a similar way that electronic circuits function.
3) The document maps similarities between biochemical reactions and analog electronics components like transistors, showing how principles like mass action, feedback, and noise can apply to both living and synthetic systems.
4) Engineering challenges remain in scaling synthetic biology from simple logic gates to complex multi-component systems that more accurately mimic the analog, probabilistic behaviors of natural biology.
The document describes the development of a high-throughput single-molecule imaging assay to identify small molecules that alter transcription or splicing kinetics. A reporter cell line was optimized and used to screen over 60 compounds. Hits were validated with live-cell single-molecule imaging, which revealed that SGC-CBP30, PFI-1, and JQ1 slow elongation rates, while Tenovin-1 may affect co-transcriptional splicing. The assay provides a method to characterize drug effects on transcription at a single-molecule level.
Rho GTPases as regulators of morphological neuroplasticityFatih University
Rho GTPases play a key role in regulating neuronal morphology by mediating interactions between cell adhesion molecules and the cytoskeleton. Specifically, Rho inhibits neurite extension while Rac and Cdc42 promote neurite outgrowth and dendritic spine formation. In glial cells, Rho GTPases are involved in myelination by oligodendrocytes and Schwann cells. The role of RhoA in particular contributes to the inhibitory environment of the CNS that prevents axon regeneration.
The document discusses various clock systems, interrupts, and operating modes for microcontrollers:
- It describes clock sources like crystal oscillators and a digitally controlled oscillator that provides the main system clock.
- When an interrupt occurs, the program counter and status register are pushed to the stack before jumping to the interrupt service routine, which must end with a reti instruction.
- Low power modes like LPM3 disable parts of the clock system to reduce power consumption while still allowing interrupts to wake the device.
- Other topics covered include watchdog timers, interrupt prioritization, and entering/waking from low power modes.
Principles of DNA isolation, PCR and LAMPPerez Eric
1. The document discusses principles of DNA isolation and purification as well as polymerase chain reaction (PCR). It describes how cells are broken to release DNA and the components of extraction buffers used to isolate DNA.
2. The three main steps of PCR - denaturation, annealing, and elongation - are explained. Denaturation separates DNA strands, annealing attaches primers, and elongation duplicates the DNA. Required PCR reagents and their roles are also outlined.
3. Loop-mediated isothermal amplification (LAMP) is introduced as an alternative to PCR that amplifies DNA at a constant temperature. LAMP uses multiple primers and has advantages like lower cost and faster results. The mechanism and applications of LAMP are summarized.
The document describes ongoing efforts to optimize a computational model of a CA1 pyramidal neuron. The model integrates synaptic and cellular interactions using the EONS/NEURON framework. The optimization aims to accurately reproduce various experimental data through tuning of channel conductances and other biophysical parameters. Several approaches are explored, including varying temperature effects, comparing different CA1 models, and deconvolving EPSCs to model presynaptic mechanisms. Future plans include using evolutionary optimization techniques and additional experimental objectives to further refine the model.
This document provides an introduction to single-cell RNA-seq (scRNA-seq) analysis. It discusses different scRNA-seq assays such as Smart-Seq2, Drop-seq, and 10X, and how their protocols and sequencing outputs differ. It also covers scRNA-seq data characteristics like zero inflation and overdispersion. The document outlines common analysis steps like filtering, dimensionality reduction, clustering, and differential expression. It emphasizes that scRNA-seq data requires specialized analysis due to its noisy and sparse nature compared to bulk RNA-seq data.
Synthetic gene networks were constructed in cells that function as digital counters. Two different counting mechanisms were implemented and tested experimentally. The first uses a riboregulated transcriptional cascade to count arabinose pulses, with fluorescence increasing only after the defined number of pulses. The second method uses site-specific DNA recombinases to invert promoter orientations and count pulses. Mathematical models were able to predict experimental results. While these synthetic gene networks demonstrate counting in cells, their reliability may be limited due to inherent variability in cellular mechanisms.
This document presents a novel approach to emulate power islands in FPGA platforms to enable earlier validation of low power features. It proposes corrupting logic in power-gated domains by connecting power gating enables to asynchronous set/clear signals on flip-flops. This approach more realistically mimics silicon behavior during power-off and can find issues like incorrect power sequencing bugs. The methodology was implemented using a custom EDF library and randomization to corrupt a parameterized percentage of logic. Challenges around FPGA architecture and optimization were addressed. Results showed the approach introduced minimal overhead while enabling important power management validation earlier in the design flow.
1. Signaling model for IFN-γ
•Step 0: (Optional) pre-equilibration of JAK1 with
R1, JAK2 with R2, R1 with R2, R1 with itself
(dimerization), R2 with itself, L with itself to make
functional IFN-γ, and STAT1 with itself.
•Parameters are taken largely from Yamada’s.
ligand
L
R1
L
R1
L
R1
R2
J1
S1
Y440
L
R1
R2
J1
S1
Y440
R2
R1
J2
R2
R1
J2
R2
Y
R1
Y
R2
Y R1
Y
R1 chain
JAK1
JAK2
R2 chain
Step 1: Ligand binds to
receptor.
+
ligand
L
R1
L
R1
L
R1
R2
J1
S1
Y440
L
R1
R2
J1
S1
Y440
R2
R1
J2
R2
R1
J2
R2
Y
R1
Y
R2
Y R1
Y
R1 chain
JAK1
JAK2
R2 chain
Ligand binding opens up the
receptor complex, thereby
allowing STAT1 dimer to bind.
A model of IFN-γ mediated JAK-STAT signaling that accounts for the combinatorial complexity
James S. Cavenaugh, James R. Faeder, Michael L. Blinov,
Jeremy E. Kozdon, William S. Hlavacek
Theoretical Biology and Biophysics Group
Los Alamos National Laboratory
Los Alamos, NM 87545
Acknowledgements: We
thank Emi Shudo, David
Torney, Amitabh Trehan,
Satoshi Yamada, Jin Yang,
and Zhike Zi for helpful
discussions.
Background
• IFN-γ is an inflammatory cytokine with
broad effects, especially important in
intracellular infections.
• IFN-γ is a compact homodimer:
fro m T. Kisseleva et al. Gene 285 (2002) 1-24
primary input
presumed
primary output
Assumptions
• Cell type: “liver cells”
• R1 = R2 = R
• JAK1 = JAK2 = JAK
• ligand-induced receptor
dimerization
• Combinatorial complexity
not fully addressed, e.g.:
– STAT1 dimerizesonly
after phosphorylation
– IFN-γ doesn’t bindIFN-
γ receptor without JAK.
• Only SOCS1 for negative
feedback
• No cross-talk considered
• No positive feedback
• No ligand internalization
or receptor recycling
BioNetGen modeling
Input: seed set,
reaction rules
Reaction network:
species and reactions
Time courses for all species and observables
by ODE or SSA
• Hlavaceket al., Biotechnol. Bioeng. 2003
• Blinov et al., Bioinformatics 2004
• Faeder et al., Complexity 2005
• Faeder et al., Proc. ACM 2005
• Blinov et al., BioConcur 2005
Version 2.0 (alpha) allows
bonds (edges in graphs).
ligand
L
R1
L
R1
L
R1
R2
J1
S1
Y440
L
R1
R2
J1
S1
Y440
R2
R1
J2
R2
R1
J2
R2
Y
R1
Y
R2
Y R1
Y
R1 chain
JAK1
JAK2
R2 chain
Step 2: JAK2 autophosphorylates itself.
Step 3: Phosphorylated JAK2 phosphorylates JAK1.
Step 4: Phosphorylated JAK1 phosphorylates R1.
Step 5: STAT1 dimer can bind to phosphorylated R1.
S1
R1
Y701
loc
S1
R1
Y701
loc
STAT1
Steps 6, 7, 8: STAT1
dimer gets phosphorylated
and dissociates, then
migrates to nucleus and
activates transcription of
various genes.
SHP-2
SOCS-1
TC-45
some inhibitors of
this pathway
Combinatorial complexity
• How many individual
reactions are possible?
• Current paradigm is to limit
possible rxns by assumptions.
– Are ad hoc assumptions the
origin of system behavior?
– Arbitrary: which to ignore?
• Rule based modeling
– Treats protein-protein
interactions as reaction rules
– Example: “R” notation in
organic chemistry
(e.g., R-X + Cl- → R-CL + X-)
– Can incorporate knowledge of
important domains
L
R1
R2
J1
S1
Y440
L
R1
R2
J1
S1
Y440
R2
R1
J2
R2
R1
J2
R2
Y
R2
Y R1
Y
R1 chain
JAK1
JAK2
R2 chain
ligand
L
R1
L
R1
+
3,646,350 possible species!
(without additional restrictions)
Goals
(1) To better account for the
consequences of combinatorial
complexity inherent in protein-
protein signaling interactions;
(2) To incorporate known
biochemistry in a more mechanistic
understanding of signaling events
Yamada’s model: The starting point
•Canonical model
–Based on earlier dogma for receptor, STAT1
dimerizations
•Extended by others but essentially unchanged
–Zi et al., FEBS Letters 579 (2005) 1101-1108
–Shudo et al., submitted.
Experimental benchmarks
• Four critical tyrosines (on JAK1, JAK2, R1,
STAT1) are phosphorylated within 1 min of
ligand binding.
• STAT1 begins entering nucleus after 15 min and
is complete by 30 min of ligand binding.
• 1st wave of transcription occurs very shortly
afterwards (within 15-30 min).
• STAT1 activation is inhibited within 1 h of
activation.
Results
• The total number of allowed species is 135,200.
• Achieved 53,836 species and 500,895 reactions in
13 iterations, then…
• Out of memory!
– Running on Dell Precision 670 Plus: Dual 3.60 GHz Intel
Xeon Processors, 2 MB L2 cache, 2 GB ECC RAM
– 40% completed
• Can the network be split into parts, i.e. separating
the signal transduction from the receptor binding?
YES (approximately)
Timecourses: without signal transduction
Timecourses: only signal transduction
Timecourses: only signal transductionConclusions
• Most extensive model to
date with BioNetGen.
– Major driving force for BNG
v.2
• Network generation
takes by far the longest
amount of time.
– Reaction generation took the
longest CPU time.
– ODE solver is very fast.
• OK to split the network
into subsections
• Unsplit network is likely
doable with optimized BNG
code
• “Full” complexity (with R1,
R2 dissociation) still too big.
• Meets initial experimental
benchmarks better than
Yamada’s model
• Sensitivity analysis and
parameter optimizations
needed for later events (too
slow, even more than
Yamada’s)
2. Results
• The total number of allowed species is 135,200.
• Achieved 53,836 species and 500,895 reactions in
13 iterations, then…
• Out of memory!
– Running on Dell Precision 670 Plus: Dual 3.60 GHz Intel
Xeon Processors, 2 MB L2 cache, 2 GB ECC RAM
– 40% completed
• Can the network be split into parts, i.e. separating
the signal transduction from the receptor binding?
YES (approximately)
3. Signaling model for IFN-γ
•Step 0: (Optional) pre-equilibration of JAK1 with
R1, JAK2 with R2, R1 with R2, R1 with itself
(dimerization), R2 with itself, L with itself to make
functional IFN-γ, and STAT1 with itself.
•Parameters are taken largely from Yamada’s.
ligand
L
R1
L
R1
L
R1
R2
J1
S1
Y440
L
R1
R2
J1
S1
Y440
R2
R1
J2
R2
R1
J2
R2
Y
R1
Y
R2
Y R1
Y
R1 chain
JAK1
JAK2
R2 chain
Step 1: Ligand binds to
receptor.
+
ligand
L
R1
L
R1
L
R1
R2
J1
S1
Y440
L
R1
R2
J1
S1
Y440
R2
R1
J2
R2
R1
J2
R2
Y
R1
Y
R2
Y R1
Y
R1 chain
JAK1
JAK2
R2 chain
Ligand binding opens up the
receptor complex, thereby
allowing STAT1 dimer to bind.
4. ligand
L
R1
L
R1
L
R1
R2
J1
S1
Y440
L
R1
R2
J1
S1
Y440
R2
R1
J2
R2
R1
J2
R2
Y
R1
Y
R2
Y R1
Y
R1 chain
JAK1
JAK2
R2 chain
Step 2: JAK2 autophosphorylates itself.
Step 3: Phosphorylated JAK2 phosphorylates JAK1.
Step 4: Phosphorylated JAK1 phosphorylates R1.
Step 5: STAT1 dimer can bind to phosphorylated R1.
S1
R1
Y701
loc
S1
R1
Y701
loc
STAT1
Steps 6, 7, 8: STAT1
dimer gets phosphorylated
and dissociates, then
migrates to nucleus and
activates transcription of
various genes.
SHP-2
SOCS-1
TC-45
some inhibitors of
this pathway
8. Experimental benchmarks
•Four critical tyrosines (on JAK1, JAK2, R1,
STAT1) are phosphorylated within 1 min of ligand
binding.
•STAT1 begins entering nucleus after 15 min and is
complete by 30 min of ligand binding.
•1st
wave of transcription occurs very shortly
afterwards (within 15-30 min).
•STAT1 activation is inhibited within 1 h of
activation.
9. Conclusions
• Most extensive model to date with BioNetGen.
– Major driving force for BNG v.2
• Splitting the network into subsections seems to work.
• Network generation takes by far the longest amount of
time.
– Reaction generation took the longest CPU time.
– ODE solver is very fast.
• Unsplit network is likely doable with optimized BNG
code:
– Better memory usage, parallelization, etc.
– Parallelization (to spread memory requirements)
– Stochastic simulation
– Improved logic in restricting reaction rules
• “Full” complexity (with R1, R2 dissociation) is too big.
10. Yamada’s model: The starting point
•Canonical model
–Based on earlier dogma for receptor, STAT1
dimerizations
•Extended by others but essentially unchanged
–Zi et al., FEBS Letters 579 (2005) 1101-1108
–Shudo et al., submitted.
11. Conclusions
• Most extensive model to
date with BioNetGen.
– Major driving force for BNG
v.2
• Network generation
takes by far the longest
amount of time.
– Reaction generation took the
longest CPU time.
– ODE solver is very fast.
• OK to split the network
into subsections
• Unsplit network is likely
doable with optimized BNG
code
• “Full” complexity (with R1,
R2 dissociation) still too big.
• Meets initial experimental
benchmarks better than
Yamada’s model
• Sensitivity analysis and
parameter optimizations
needed for later events (too
slow, even more than
Yamada’s)