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1.  Mechanistic Exploration of Phthalimide Neovascular Factor 1 Using Network Analysis Tools 
Tissue engineering  2007;13(10):2561-2575.
Neovascularization is essential for the survival and successful integration of most engineering tissues after implantation in vivo. The objective of this study was to elucidate possible mechanisms of phthalimide neovascular factor 1 (PNF1), a new synthetic small molecule proposed for therapeutic induction of angiogenesis. Complementary deoxyribonucleic acid microarray analysis was used to identify 568 transcripts in human microvascular endothelial cells (HMVECs) that were significantly regulated after 24-h stimulation with 30 μM of PNF1, previously known as SC-3–149. Network analysis tools were used to identify genetic networks of the global biological processes involved in PNF1 stimulation and to describe known molecular and cellular functions that the drug regulated most highly. Examination of the most significantly perturbed networks identified gene products associated with transforming growth factor-beta (TGF-β), which has many known effects on angiogenesis, and related signal transduction pathways. These include molecules integral to the thrombospondin, plasminogen, fibroblast growth factor, epidermal growth factor, ephrin, Rho, and Ras signaling pathways that are essential to endothelial function. Moreover, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) of select genes showed significant increases in TGF-β-associated receptors endoglin and beta glycan. These experiments provide important insight into the pro-angiogenic mechanism of PNF1, namely, TGF-β-associated signaling pathways, and may ultimately offer new molecular targets for directed drug discovery.
doi:10.1089/ten.2007.0023
PMCID: PMC3124853  PMID: 17723106
2.  Systems Analysis of Small Signaling Modules Relevant to Eight Human Diseases 
Annals of Biomedical Engineering  2010;39(2):621-635.
Using eight newly generated models relevant to addiction, Alzheimer’s disease, cancer, diabetes, HIV, heart disease, malaria, and tuberculosis, we show that systems analysis of small (4–25 species), bounded protein signaling modules rapidly generates new quantitative knowledge from published experimental research. For example, our models show that tumor sclerosis complex (TSC) inhibitors may be more effective than the rapamycin (mTOR) inhibitors currently used to treat cancer, that HIV infection could be more effectively blocked by increasing production of the human innate immune response protein APOBEC3G, rather than targeting HIV’s viral infectivity factor (Vif), and how peroxisome proliferator-activated receptor alpha (PPARα) agonists used to treat dyslipidemia would most effectively stimulate PPARα signaling if drug design were to increase agonist nucleoplasmic concentration, as opposed to increasing agonist binding affinity for PPARα. Comparative analysis of system-level properties for all eight modules showed that a significantly higher proportion of concentration parameters fall in the top 15th percentile sensitivity ranking than binding affinity parameters. In infectious disease modules, host networks were significantly more sensitive to virulence factor concentration parameters compared to all other concentration parameters. This work supports the future use of this approach for informing the next generation of experimental roadmaps for known diseases.
Electronic supplementary material
The online version of this article (doi:10.1007/s10439-010-0208-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s10439-010-0208-y
PMCID: PMC3033523  PMID: 21132372
Systems biology; Human disease; Protein signaling; Comparative meta-analysis; Sensitivity analysis
3.  Phthalimide neovascular factor 1 (PNF1) modulates MT1-MMP activity in human microvascular endothelial cells 
Biotechnology and bioengineering  2009;103(4):796-807.
We are creating synthetic pharmaceuticals with angiogenic activity and potential to promote vascular invasion. We previously demonstrated that one of these molecules, phthalimide neovascular factor 1 (PNF1), significantly expands microvascular networks in vivo following sustained release from poly(lactic-co-glycolic acid) (PLAGA) films. In addition, to probe PNF1 mode-of-action, we recently applied a novel pathway-based compendium analysis to a multi-timepoint, controlled microarray dataset of PNF1-treated (versus control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-α) and, subsequently, transforming growth factor-beta (TGF-β) signaling networks by PNF1. Here we validate this microarray data-set with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Subsequently, we probe this dataset and identify three specific TGF-β-induced genes with regulation by PNF1 conserved over multiple timepoints—amyloid beta (A4) precursor protein (APP), early growth response 1 (EGR-1), and matrix metalloproteinase 14 (MMP14 or MT1-MMP)—that are also implicated in angiogenesis. We further focus on MMP14 given its unique role in angiogenesis, and we validate MT1-MMP modulation by PNF1 with an in vitro fluorescence assay that demonstrates the direct effects that PNF1 exerts on functional metalloproteinase activity. We also utilize endothelial cord formation in collagen gels to show that PNF1-induced stimulation of endothelial cord network formation in vitro is in some way MT1-MMP-dependent. Ultimately, this new network analysis of our transcriptional footprint characterizing PNF1 activity 1–48 h post-supplementation in HMVECs coupled with corresponding validating experiments suggests a key set of a few specific targets that are involved in PNF1 mode-of-action and important for successful promotion of the neovascularization that we have observed by the drug in vivo.
doi:10.1002/bit.22310
PMCID: PMC2711776  PMID: 19326468
Network analysis; transcriptional profiling; angiogenesis; matrix metalloproteinase; small molecule; drug discovery
4.  Novel pathway compendium analysis elucidates mechanism of pro-angiogenic synthetic small molecule 
Bioinformatics  2008;24(20):2384-2390.
Motivation: Computational techniques have been applied to experimental datasets to identify drug mode-of-action. A shortcoming of existing approaches is the requirement of large reference databases of compound expression profiles. Here, we developed a new pathway-based compendium analysis that couples multi-timepoint, controlled microarray data for a single compound with systems-based network analysis to elucidate drug mechanism more efficiently.
Results: We applied this approach to a transcriptional regulatory footprint of phthalimide neovascular factor 1 (PNF1)—a novel synthetic small molecule that exhibits significant in vitro endothelial potency—spanning 1–48 h post-supplementation in human micro-vascular endothelial cells (HMVEC) to comprehensively interrogate PNF1 effects. We concluded that PNF1 first induces tumor necrosis factor-alpha (TNF-α) signaling pathway function which in turn affects transforming growth factor-beta (TGF-β) signaling. These results are consistent with our previous observations of PNF1-directed TGF-β signaling at 24 h, including differential regulation of TGF-β-induced matrix metalloproteinase 14 (MMP14/MT1-MMP) which is implicated in angiogenesis. Ultimately, we illustrate how our pathway-based compendium analysis more efficiently generates hypotheses for compound mechanism than existing techniques.
Availability: The microarray data generated as part of this study are available in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/).
Contact: botchwey@virginia.edu; papin@virginia.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btn451
PMCID: PMC2562016  PMID: 18718940
5.  Functional States of the Genome-Scale Escherichia Coli Transcriptional Regulatory System 
PLoS Computational Biology  2009;5(6):e1000403.
A transcriptional regulatory network (TRN) constitutes the collection of regulatory rules that link environmental cues to the transcription state of a cell's genome. We recently proposed a matrix formalism that quantitatively represents a system of such rules (a transcriptional regulatory system [TRS]) and allows systemic characterization of TRS properties. The matrix formalism not only allows the computation of the transcription state of the genome but also the fundamental characterization of the input-output mapping that it represents. Furthermore, a key advantage of this “pseudo-stoichiometric” matrix formalism is its ability to easily integrate with existing stoichiometric matrix representations of signaling and metabolic networks. Here we demonstrate for the first time how this matrix formalism is extendable to large-scale systems by applying it to the genome-scale Escherichia coli TRS. We analyze the fundamental subspaces of the regulatory network matrix (R) to describe intrinsic properties of the TRS. We further use Monte Carlo sampling to evaluate the E. coli transcription state across a subset of all possible environments, comparing our results to published gene expression data as validation. Finally, we present novel in silico findings for the E. coli TRS, including (1) a gene expression correlation matrix delineating functional motifs; (2) sets of gene ontologies for which regulatory rules governing gene transcription are poorly understood and which may direct further experimental characterization; and (3) the appearance of a distributed TRN structure, which is in stark contrast to the more hierarchical organization of metabolic networks.
Author Summary
Cells are comprised of genomic information that encodes for proteins, the basic building blocks underlying all biological processes. A transcriptional regulatory system (TRS) connects a cell's environmental cues to its genome and in turn determines which genes are turned “on” in response to these cues. Consequently, TRSs control which proteins of an intracellular biochemical reaction network are present. These systems have been mathematically described, often through Boolean expressions that represent the activation or inhibition of gene transcription in response to various inputs. We recently developed a matrix formalism that extends these approaches and facilitates a quantitative representation of the Boolean logic underlying a TRS. We demonstrated on small-scale TRSs that this matrix representation is advantageous in that it facilitates the calculation of unique properties of a given TRS. Here we apply this matrix formalism to the genome-scale Escherichia coli TRS, demonstrating for the first time the predictive power of the approach at a large scale. We use the matrix-based model of E. coli transcriptional regulation to generate novel findings about the system, including new functional motifs; sets of genes whose regulation is poorly understood; and features of the TRS structure.
doi:10.1371/journal.pcbi.1000403
PMCID: PMC2685017  PMID: 19503608
6.  Correction: Dynamic Analysis of Integrated Signaling, Metabolic, and Regulatory Networks 
PLoS Computational Biology  2008;4(6):10.1371/annotation/5594348b-de00-446a-bdd0-ec56e70b3553.
doi:10.1371/annotation/5594348b-de00-446a-bdd0-ec56e70b3553
PMCID: PMC2645273
7.  Dynamic Analysis of Integrated Signaling, Metabolic, and Regulatory Networks 
PLoS Computational Biology  2008;4(5):e1000086.
Extracellular cues affect signaling, metabolic, and regulatory processes to elicit cellular responses. Although intracellular signaling, metabolic, and regulatory networks are highly integrated, previous analyses have largely focused on independent processes (e.g., metabolism) without considering the interplay that exists among them. However, there is evidence that many diseases arise from multifunctional components with roles throughout signaling, metabolic, and regulatory networks. Therefore, in this study, we propose a flux balance analysis (FBA)–based strategy, referred to as integrated dynamic FBA (idFBA), that dynamically simulates cellular phenotypes arising from integrated networks. The idFBA framework requires an integrated stoichiometric reconstruction of signaling, metabolic, and regulatory processes. It assumes quasi-steady-state conditions for “fast” reactions and incorporates “slow” reactions into the stoichiometric formalism in a time-delayed manner. To assess the efficacy of idFBA, we developed a prototypic integrated system comprising signaling, metabolic, and regulatory processes with network features characteristic of actual systems and incorporating kinetic parameters based on typical time scales observed in literature. idFBA was applied to the prototypic system, which was evaluated for different environments and gene regulatory rules. In addition, we applied the idFBA framework in a similar manner to a representative module of the single-cell eukaryotic organism Saccharomyces cerevisiae. Ultimately, idFBA facilitated quantitative, dynamic analysis of systemic effects of extracellular cues on cellular phenotypes and generated comparable time-course predictions when contrasted with an equivalent kinetic model. Since idFBA solves a linear programming problem and does not require an exhaustive list of detailed kinetic parameters, it may be efficiently scaled to integrated intracellular systems that incorporate signaling, metabolic, and regulatory processes at the genome scale, such as the S. cerevisiae system presented here.
Author Summary
Cellular systems comprise many diverse components and component interactions spanning signal transduction, transcriptional regulation, and metabolism. Although signaling, metabolic, and regulatory activities are often investigated independently of one another, there is growing evidence that considerable interplay occurs among them, and that the malfunctioning of this interplay is associated with disease. The computational analysis of integrated networks has been challenging because of the varying time scales involved as well as the sheer magnitude of such systems (e.g., the numbers of rate constants involved). To this end, we developed a novel computational framework called integrated dynamic flux balance analysis (idFBA) that generates quantitative, dynamic predictions of species concentrations spanning signaling, regulatory, and metabolic processes. idFBA extends an existing approach called flux balance analysis (FBA) in that it couples “fast” and “slow” reactions, thereby facilitating the study of whole-cell phenotypes and not just sub-cellular network properties. We applied this framework to a prototypic integrated system derived from literature as well as a representative integrated yeast module (the high-osmolarity glycerol [HOG] pathway) and generated time-course predictions that matched with available experimental data. By extending this framework to larger-scale systems, phenotypic profiles of whole-cell systems could be attained expeditiously.
doi:10.1371/journal.pcbi.1000086
PMCID: PMC2377155  PMID: 18483615
8.  Predicting biological system objectives de novo from internal state measurements 
BMC Bioinformatics  2008;9:43.
Background
Optimization theory has been applied to complex biological systems to interrogate network properties and develop and refine metabolic engineering strategies. For example, methods are emerging to engineer cells to optimally produce byproducts of commercial value, such as bioethanol, as well as molecular compounds for disease therapy. Flux balance analysis (FBA) is an optimization framework that aids in this interrogation by generating predictions of optimal flux distributions in cellular networks. Critical features of FBA are the definition of a biologically relevant objective function (e.g., maximizing the rate of synthesis of biomass, a unit of measurement of cellular growth) and the subsequent application of linear programming (LP) to identify fluxes through a reaction network. Despite the success of FBA, a central remaining challenge is the definition of a network objective with biological meaning.
Results
We present a novel method called Biological Objective Solution Search (BOSS) for the inference of an objective function of a biological system from its underlying network stoichiometry as well as experimentally-measured state variables. Specifically, BOSS identifies a system objective by defining a putative stoichiometric "objective reaction," adding this reaction to the existing set of stoichiometric constraints arising from known interactions within a network, and maximizing the putative objective reaction via LP, all the while minimizing the difference between the resultant in silico flux distribution and available experimental (e.g., isotopomer) flux data. This new approach allows for discovery of objectives with previously unknown stoichiometry, thus extending the biological relevance from earlier methods. We verify our approach on the well-characterized central metabolic network of Saccharomyces cerevisiae.
Conclusion
We illustrate how BOSS offers insight into the functional organization of biochemical networks, facilitating the interrogation of cellular design principles and development of cellular engineering applications. Furthermore, we describe how growth is the best-fit objective function for the yeast metabolic network given experimentally-measured fluxes.
doi:10.1186/1471-2105-9-43
PMCID: PMC2258290  PMID: 18218092
9.  Matrix Formalism to Describe Functional States of Transcriptional Regulatory Systems 
PLoS Computational Biology  2006;2(8):e101.
Complex regulatory networks control the transcription state of a genome. These transcriptional regulatory networks (TRNs) have been mathematically described using a Boolean formalism, in which the state of a gene is represented as either transcribed or not transcribed in response to regulatory signals. The Boolean formalism results in a series of regulatory rules for the individual genes of a TRN that in turn can be used to link environmental cues to the transcription state of a genome, thereby forming a complete transcriptional regulatory system (TRS). Herein, we develop a formalism that represents such a set of regulatory rules in a matrix form. Matrix formalism allows for the systemic characterization of the properties of a TRS and facilitates the computation of the transcriptional state of the genome under any given set of environmental conditions. Additionally, it provides a means to incorporate mechanistic detail of a TRS as it becomes available. In this study, the regulatory network matrix, R, for a prototypic TRS is characterized and the fundamental subspaces of this matrix are described. We illustrate how the matrix representation of a TRS coupled with its environment (R*) allows for a sampling of all possible expression states of a given network, and furthermore, how the fundamental subspaces of the matrix provide a way to study key TRS features and may assist in experimental design.
Synopsis
Complex regulatory networks control the transcription state of a genome that defines the components of a biochemical network. These transcriptional regulatory networks have been mathematically described. The purpose of many such mathematical models is to allow for the prediction of gene expression under a variety of environmental conditions. However, to date, quantitative models have been limited in scope due to a paucity of relevant data, and models of larger networks have been limited in their quantitative predictive power. Herein, Gianchandani and colleagues present a formalism that represents regulatory rules in a matrix form which attempts to address these issues. This matrix formalism allows for the systemic characterization of the properties of a transcriptional regulatory system and facilitates the computation of the transcriptional state of the corresponding genome under any given set of environmental conditions. Additionally, it provides a means to incorporate mechanistic detail of a transcriptional regulatory system as it becomes available. The authors illustrate how this matrix representation allows for a sampling of all possible expression states of a given network and provides a way to study key features. They also present how it may assist in experimental design to interrogate genome-scale cellular networks.
doi:10.1371/journal.pcbi.0020101
PMCID: PMC1534074  PMID: 16895435
10.  Matrix Formalism to Describe Functional States of Transcriptional Regulatory Systems 
PLoS Computational Biology  2006;2(8):e101.
Complex regulatory networks control the transcription state of a genome. These transcriptional regulatory networks (TRNs) have been mathematically described using a Boolean formalism, in which the state of a gene is represented as either transcribed or not transcribed in response to regulatory signals. The Boolean formalism results in a series of regulatory rules for the individual genes of a TRN that in turn can be used to link environmental cues to the transcription state of a genome, thereby forming a complete transcriptional regulatory system (TRS). Herein, we develop a formalism that represents such a set of regulatory rules in a matrix form. Matrix formalism allows for the systemic characterization of the properties of a TRS and facilitates the computation of the transcriptional state of the genome under any given set of environmental conditions. Additionally, it provides a means to incorporate mechanistic detail of a TRS as it becomes available. In this study, the regulatory network matrix, R, for a prototypic TRS is characterized and the fundamental subspaces of this matrix are described. We illustrate how the matrix representation of a TRS coupled with its environment (R*) allows for a sampling of all possible expression states of a given network, and furthermore, how the fundamental subspaces of the matrix provide a way to study key TRS features and may assist in experimental design.
Synopsis
Complex regulatory networks control the transcription state of a genome that defines the components of a biochemical network. These transcriptional regulatory networks have been mathematically described. The purpose of many such mathematical models is to allow for the prediction of gene expression under a variety of environmental conditions. However, to date, quantitative models have been limited in scope due to a paucity of relevant data, and models of larger networks have been limited in their quantitative predictive power. Herein, Gianchandani and colleagues present a formalism that represents regulatory rules in a matrix form which attempts to address these issues. This matrix formalism allows for the systemic characterization of the properties of a transcriptional regulatory system and facilitates the computation of the transcriptional state of the corresponding genome under any given set of environmental conditions. Additionally, it provides a means to incorporate mechanistic detail of a transcriptional regulatory system as it becomes available. The authors illustrate how this matrix representation allows for a sampling of all possible expression states of a given network and provides a way to study key features. They also present how it may assist in experimental design to interrogate genome-scale cellular networks.
doi:10.1371/journal.pcbi.0020101
PMCID: PMC1534074  PMID: 16895435

Results 1-10 (10)