Progress Report Summary
A. SPECIFIC AIMS
Computational. The specific aims continue to be the development of
computational tools and methodologies at molecular and supramolecular (Aim
1A), microphysiological or subcellular (Aim 1B) and cellular systems (Aim 1C).
Specific aims 2 (integration of these methods) and 3 (development and
implementation of tools for dissemination & visualization) remain unchanged.
Biomedical. The Biomedical Aims of the 3 developmental projects (DP1-3)
remain unchanged, as dynamics of apoptosis and effect of NO (DP1), DNA damage
recognition and signaling (DP2), and ligand interactions with signaling
molecules (DP3). A number of new collaborative efforts have also been
launched, on neurotransmitter release as described in section D.
B. STUDIES AND RESULTS
Specific Aim 1A.
We have made significant
progresses in developing and implementing methods based on elastic network
models (or Gaussian Network Model, GNM) for examining the structure and
dynamics of biomolecular systems as recently reviewed (Bahar & Rader, 2005).
Notable extensions of the methodology to supramolecular systems include the
examination of the dynamics of HK97 bacteriophage capsid (Rader et al., 2005);
the elucidation of ribosomal machinery (Wang et al., 2004). We have also
developed a new algorithm for switching between different levels of resolution
in protein models (Lyman & Zuckerman, 2005). The approach, termed “Resolution
Exchange,” generalizes the widely-used replica exchange approach by allowing
configuration swaps between simulations at different levels of resolution.
Specific Aim 1B.
MCell and DReAMM (www.mcell.psc.edu)
were originally designed to simulate and visualize the 3-D reaction/diffusion
aspects of neurotransmission, and a continuing aim is expansion of both to
encompass more general signaling pathways such as those of the DPs. Since
early 2005, new versions (v.3.0) of both have been in heavy alpha testing. As
originally proposed, new MCell testing and use focuses on reaction network
motifs such as single and coupled autocatalytic feedback loops (e.g., the
Lotka-Volterra reaction, the Repressilator circuit, and the Oregonator version
of the Belousov-Zhabotinski reaction). Our recent studies have illustrated
surprising quantitative and qualitative differences between MCell simulations
and ODE-based simulations of the same networks. Reports on spatially realistic
modeling (Stiles et al, 2004) and Grid computation with MCell 2.5 (Casanova et
al, 2004) have also appeared.
Specific Aim 1C.
We have made major developments in our software for a logical network model
of apoptosis (Ta’asan lab, CMU) so as to include stochastic modeling and ODEs,
on top of agent based architecture that we have. We have attempted to connect
between existing modeling approaches such as ODEs and Stochastic differential
equations and the Logical Modeling approach. This has led us to examine
different implementations which were derived from the law of mass action with
certain assumptions of discreteness. Our recent implementation of logical
models can be viewed as a coarse graining of stochastic models. Our current
BioLogic software platform can simulate arbitrary hierarchy of objects with
arbitrary compartment structure. A new version of XPPAUT (version 5.91) has
been made available on the internet by the lab of Dr. Ermentrout (Pitt, Math),
which also includes an SBML to ODE file converter.
Specific Aim 2.
With recent developments in the elastic network models and methods, it is now
possible to examine the collective dynamics of large structures at
residue-level. The extension of molecular models by Bahar’s lab to systems
comprised of 105 residues or 106 atoms (e.g. Rader et
al., 2005) and the elucidation of the functional dynamics of supramolecular
systems by efficient computational methods is an important advance in the
field towards filling the gap between molecular and microphysiological
simulations. Another effort launched towards bridging across these two scales
is the collaboration between molecular and subcellular simulation groups for
exchange of output/input, e.g. the use of on- and off- rates for ligand
binding, or associated free energy changes, or the diffusion coefficients
predicted by molecular simulations, as input parameters for microphysiological
simulations.
Specific Aim 3.
For continued improvements to microphysiological model building and
visualization, DReAMM v.3 (see Aim 1B) has been expanded dramatically. We
have made extensive improvements to OpenDX (www.opendx.org,
originally from IBM) hardware and software rendering routines (corrected
shading and lighting so that images match), as well as user interface design
modules. Another database/server recently implemented by Bahar’s lab is the
iGNM server (Yang et al., 2005). The mathematical modeling site XPPAUT
maintained by Ermentrout has continued to be extensively used for cell
modeling and simulations.
Developmental Project 1
(DP1). Previously,
we increased the realism of the model of apoptosis originally reported by
Fussenegger et al. (2000) by including Bid/tBid interactions as proposed in
the DP1. We also created the first-generation model of the reaction pathways
of NO, and coupled it to our apoptosis model. This mathematical model
reproduced our previous experimental results on the differential effect of NO
on apoptosis observed in different cellular environment (Vodovotz et al.,
2004). We have shown that Hill-type cooperativity in the formation of
apoptosome complex is instrumental in leading to a bistable response (Bagci et
al., 2005, submitted). Bistability could explain the effects of excessive
mitochondrial transmembrane pore (MPTP) formation and excessive iron-nitrosyl
nitric oxide species on cellular fate. Excessive MPTP formation may cause
pathological cell death (Green and Kromer, 2004) and excessive
iron-nitrosyl NO species produced in hepatocytes may render these cells very
resistant to apoptosis (Kim et al., 2000). We showed that if the modulators
exceed some critical values, the bistable responses may convert to a
monostable response (either cell survival or cell death). The model suggests
that a passage from bistable to monostable response may be induced by changes
in Bax and Bcl-2 synthesis and degradation rates (Bagci et al., submitted).
Developmental Project 2
(DP2). We
modeled the structure of the DNA polymerase domain of POLQ, as described in
the collaborative manuscript published by Dr. Wood and Dr. Bahar’s labs (Seki
et al, 2004). Importantly, the collaborative efforts between the labs of Drs.
Wood, Bahar and Stiles within the scope of the pre-NPEBC DP2 have led to an
NIH Nanomedicine Center proposal. A concept development memo was
submitted to NIH in an open competition, in July 2004. Of 86 such memos
submitted, ours was one of 20 approved for planning for a nanomedicine center
application. A “concept development plan” was submitted in February, 2005.
This project stemmed from discussions of DP2, initiated by the pre-NPEBC
grant. Thus, the pre-NPEBC funding has been valuable in stimulating a
collaborative effort for specific applications for future funding from the
NIH.
Developmental Project 3
(DP3).
Substantial progress has been made in free energy methods of potential use in
computing protein-ligand affinities (Ytreberg & Zuckerman, 2005) and
protein-protein docked conformations (Tobi & Bahar). The new approaches allow
free energy differences to be computed between configurational ensembles.
Following a general strategy championed by H. Meirovitch, a new protocol
accurately calculates absolute free energies in molecular systems, and has
been successfully applied to peptides. Another noteworthy development is the
productive collaboration between Bahar’s lab and Lazo’s lab in the prediction
of the binding affinity and geometries of lead compounds that potentially
inhibit dual specificity phosphatases (Brisson et al., 2004). Two other
important publications appeared from Bahar’s lab, which aim at improving our
understanding of the mechanism of enzyme inhibition (Sluis-Kremer et al.,
2004; Yang & Bahar, 2005).
C. SIGNIFICANCE. As
mentioned above, supramolecular systems have been modeled for the first time
at the residue-level resolution, and advanced models and methods have been
implemented in iGNM and MCell for improving the ability and extending the
applicability of both software.
The
collaboration with experimental groups started to be extremely productive, as
evidenced by the large number and high quality of collaborative publications
described above. Many joint publications appeared for the first time between
team members (e.g. co-authored by Bahar & Wood, by Bahar & Lazo, by Billiar,
Ermentrout, Vodovotz and Bahar, by Sluis-Cremer & Bahar) indicative of the
utility and effectiveness of multidisciplinary collaborations launched within
the scope of the pre-NPEBC between experimental and computational groups.
D. PLANS
Given the large number of
involved labs and limited funds in the pre-NPEBC budget, and the considerable
progresses made in both the computationally driven and the developmental
(pilot) projects, it can be anticipated that efforts will now be focused on
seeking additional funds for pursuing the collaborative studies that have been
launched in the last two years. A large number of investigators have indeed
submitted proposals in small groups (to NIH, NSF or other agencies) and
succeeded in finding support for their lab members that have effectively
contributed to the research and educational goals of the pre-NPEBC. In line
with the overall goal of the pre-Center, our overarching goal in the coming
year will be to continue to conduct interdisciplinary research and integrate
the models and methods developed/used by different groups towards gaining a
deeper understanding of the molecular basis of cell signaling and regulation
processes. Below is a more specific summary of the planned activities, both
research and educational.
Specific Aim 1. We will continue to develop models and methods at multiple
scales. With regard to GNM/ANM, the goal is to extent the ability of the
methodology at both lower and higher scales, by incorporating amino acid
specificity, on the one hand, and adopting hierarchically coarse-grained
representations at higher scales, on the other. Hybrid models that explore a
system at different resolutions will be constructed, and simulations based on
the resolution exchange method described above will be conducted. At the
microphysiological level simulations, a preliminary report on MCell v.3 has
appeared (Kerr et al, 2004) and in the coming year, this version will be in
the final stages of testing for accuracy and optimized execution before more
generalized release to the Computational Biology and Neuroscience communities.
At the higher level (mathematical models), reactions will be implemented using
logical modeling, while extensions to include ODEs as well as stochastic
differential equations will be developed to allow us to perform multiscale
computations in the same framework. Notably, XPPAUT started to support SBML as
of May 2005, which will enable users to use/share tools/models more
efficiently.
Specific Aim 2. The integration of the different tools for multiscale
modeling remains a challenging task despite the progress made in exploring
intermediate scales (between molecular and cellular) that were beyond reach
using conventional molecular models or classical chemical kinetics-based
mathematical models. More efforts will be concentrated towards further
advancing the models and methods for exploring multiscale dynamics.
Specific Aim 3. The new codes/routines of
DReAMM v.3 will be released to users in source code form as PSCDX. DReAMM
presently supports direct import of surface and volume meshes from CAD and
VRML files as well as simulation output from MCell. We will also improve the
streamlined handling of MCell3 datasets that can include thousands of mesh
objects, mesh regions, and fixed and diffusing molecular species, and the mesh
editing pipeline with preliminary mesh region annotation that allows direct
export of MCell MDL (Model Description Language) files for simulations (Stiles
et al, 2004).
DP1-3.
With respect to DP1,
we are planning to couple
this model with pathways involving nitric oxide (NO) that we presented before
(Vodovotz et al., 2004). We will change the parameters of the model that are
obtained from the literature where possible to account for different
environments in different cell types. This will be a generic model that might
explain the dichotomous effects of NO. We will then examine the pivotal role
of superoxide anion in directing the effect of NO (Wink et al., 1999). These
results will be tested in vitro by incubating hepatocyte cultures with
different concentrations of NO and superoxide donors, as well as using NO
donors that release NO with different kinetics, to examine the response of
cells to apoptotic stimuli. These studies will greatly enhance our
understanding NO as a modulator of apoptosis.
As an extension of the
research studies initiated within the scope of DP2, we are currently
organizing a concept development application for the nanomedicine center, to
be submitted in July 2005. The concept development centers around the
examination of the mechanism of nucleotide excision repair of DNA and its
future manipulation in nanomedicine. In the coming year, our efforts in this
field will be intensified, with the addition of new team members (Drs. Camacho
and Benos at the Dept of Comp Bio, SOM, Pitt and Dr. Sanford Leuba at Cell
Biol & Physiology, SOM, Pitt). Finally, the efforts on understanding
protein-protein and protein-inhibitor interactions within the scope of DP3 are
expected to be conducted more efficiently in the coming term, building on the
computational accumulation in Bahar’s and Madura’s labs, and on the productive
collaboration that has already started between experimental and theoretical
labs.
Other DPs. (1) Calcium imaging and neurotransmitter release.
These ongoing experimental and applied mathematical studies,
undertaken by J. Stiles in collaboration with S. Meriney (Neuroscience, Pitt),
focus on determination of the number and opening probability of voltage-gated
calcium channels in active zones of the frog neuromuscular junction. A
publication from the previous grant period (Wachman et al., 2004, J. Neurosci.
24:2877) reported values averaged across multiple entire active zones, and
current work focuses on subregions of single active zones (Luo et al, 2005, in
press). (2) Monte Carlo simulations of presynaptic calcium dynamics
and neurotransmitter release, directed by J. Stiles and carried out by
John Pattillo, a post-doc in Stiles’s lab. These computational studies are
directly coupled to experimental inputs and tests from the preceding project.
Based on those and other experimental constraints, our model directly predicts
recent evidence for up to 8 SNARE complexes and 40 Ca2+ binding
sites per synaptic vesicle, and possible cooperativity of synaptotagmin
binding to trigger fusion. It also makes novel predictions for the spatial
relationships and stoichiometry of channels and vesicles during exocytosis
(Pattillo et al, 2004; ibid 2005, submitted). These presynaptic
simulations are complemented by postsynaptic simulations of quantal
variability arising from normal or ectopic sites of neurotransmitter release
at a reconstructed central synapse, carried out by Stiles and collaborators at
the Salk Institute (Coggan et al, 2005, in press).
Other Significant
Collaborations:
One outcome of the collaborative research efforts of the Pre-NPEBC has been
the newly established Center for Inflammation and Regenerative Modeling (CIRM)
within the McGowan Institute for Regenerative Medicine. The CIRM, directed by
Y. Vodovotz (Surgery, Pitt) with G. Bard Ermentrout (Math, Pitt) as the
co-director of the Simulation Core, aims at understanding the molecular basis
of inflammation in the initial stages of injury, healing and eventually tissue
regeneration. Details can be found at www.mirm.pitt.edu/cirm.
Another outcome, as mentioned earlier, is the NIH
Nanomedicine Center (section B, DP2). This is a collaborative effort between
the experimental group of R. Wood supported by the computational research
group of I. Bahar, and focuses on the mechanism of nucleotide excision repair
of DNA and its future manipulation in nanomedicine. A “concept development
application” is planned for submission in July 2005.
E. PUBLICATIONS.
Publications related to
this grant are presented in Appendix I. A total of 23 publications (21 papers
+ 2 book chapter) are listed, of which
16 are published/accepted,
5 submitted, and 2 are currently in preparation. Electronic copies of most of
the published/accepted papers are made accessible in the pre-NPEBC website
(Appendix
I).
F. SPECIAL REQUIREMENTS
F.1. Description of
activities.
Research activities have been described above. Educational activities are
described in F.4.
F.2. Organizational
activities.
1. External Advisory Committee. Douglas Lauffenberger
(MIT), who accepted our invitation to serve on the EAC, visited Pittsburgh on
October 5, 2004. During his visit, he presented a seminar and also discussed
the current and future plans of the Pre-NPEBC with Dr. Bahar and other members
involved with research and organizational activities of the award.
2. The second Pre-NPEBC
workshop, titled “Computational Methodology in Modeling Complex Biological
Systems”, was held on October 13, 2004 at the Pittsburgh Supercomputing
Center, and was attended by a large number of researchers (PIs, postdoctoral
fellows, and students) from all the participating institutions (Pitt, CMU,
PSC, and Duquesne). The goal of this workshop was to focus on "computational
models and methods" developed and used by researchers in the Pittsburgh area
for investigating complex biological systems at molecular, supramolecular, and
subcellular/cellular levels, and the integration of these methods. An outline
of the workshop program can be found in Appendix III.
3. New pre-NPEBC Investigators. New Faculty that
joined pre-NPEBC activities are C. Camacho (Comp Bio, Pitt SOM), I. Maly (Comp
Bio, Pitt SOM), and D. Swigon (Math, Pitt). All three have already contributed
to this project using their start-up funds and we plan to provide partial
support from the pre-NPEBC award from the next granting period (7% for
Camacho, none for others).
4. Formation of a new
Department of Computational Biology at Pitt SOM. A new Department of
Computational Biology (CB), chaired by Dr. Bahar, was approved and established
at Pitt SOM in October 2004. The department is amongst the first in the
nation, and currently has 7 full-time Faculty with interests in computational
structural biology, systems biology, and genomics (bioinformatics), and we
anticipate recruiting
additional faculty to the department.
The Pre-NPEBC award is now administered by this new department allowing for
dedicated financial and administrative resources for the development and
implementation of computational tools and methodologies in collaboration with
experimental groups at the participating institutions. Furthermore, this
department is also the administrative center of a new PhD-granting program in
computational biology jointly offered by CMU (see F.4.1)
F.3. DPs that have been
initiated and progress made in each of them.
See the paragraphs DP1,
DP2 and DP3 in Section
B
(Studies and Results), and DP1-3 and Other DPs in Section D.
F.4. Progress toward
developing educational opportunities in biomedical computing.
1. CMU-Pitt Joint PhD
Program in Computational Biology (JPCB).
A cross-campus PhD program
in computational biology between Pitt and CMU has been newly proposed by Drs.
Ivet Bahar (Program Director, Comp Bio, Pitt) and Robert Murphy (Biol Sci,
CMU), and approved by both universities (www.compbio.cmu.edu).
Students have been admitted to the new program for the 2004-2005 academic
year, starting in the Fall 2005. The program aims to
provide intensive interdisciplinary education to enable outstanding
students to become leaders in identifying and solving tomorrow’s biological
problems using computational and/or mathematical methods and fundamental
principles of life and physical sciences. Currently, the program offers five
areas of specialization, consistent with the goals of the Pre-NPEBC:
computational genomics, comp structural biology, cellular and systems
modeling, bioimage informatics, and comp neurobiology. The program currently
includes 67 Faculty from physical, life, computer and mathematical sciences at
both universities.
2. Biomedical and
Bioinformatics Summer Institute (BBSI). The BBSI is an NSF/NIH funded
program offering a 10-week summer program to undergraduate (junior and senior)
and 1st/2nd year graduate students. The major theme of
the program is ‘Multiscale Modeling’ in line with the pre-NPEBC goals.
Six PIs playing a key role in the pre-NPEBC (Bahar, Stiles, Coalson, Madura,
Ermentrout and Meirovitch) also serve as instructors and co-PIs in the BBSI.
Additionally, 17 Faculty from Pitt, CMU, PSC and Duquesne serve as research
mentors. A manuscript on BBSI has been recently submitted to Biotechnology
Progress (Munshi et al., 2005; invited article).
3. Other programs and
courses. A PhD program in Molecular Biophysics, with a strong component in
Comp Bio, started in the Fall 2004. In addition to the molecular biophysics
basics, first-year classes introduce concepts of modeling at different scales.
CMU started a new course "Modeling and Simulation of Biological Systems" for
undergrad students (taught by Dr. Ta’asan).
4. Several postdoctoral
fellows and students funded by other (NIH or NSF) sources are involved in,
and benefit from, collaborative multidisciplinary activities conducted within
the scope of the pre-NPEBC (see Appendix IV).