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1.  Genotypic and phenotypic analyses of a Pseudomonas aeruginosa chronic bronchiectasis isolate reveal differences from cystic fibrosis and laboratory strains 
BMC Genomics  2015;16:883.
Background
Pseudomonas aeruginosa is an environmentally ubiquitous Gram-negative bacterium and important opportunistic human pathogen, causing severe chronic respiratory infections in patients with underlying conditions such as cystic fibrosis (CF) or bronchiectasis. In order to identify mechanisms responsible for adaptation during bronchiectasis infections, a bronchiectasis isolate, PAHM4, was phenotypically and genotypically characterized.
Results
This strain displays phenotypes that have been associated with chronic respiratory infections in CF including alginate over-production, rough lipopolysaccharide, quorum-sensing deficiency, loss of motility, decreased protease secretion, and hypermutation. Hypermutation is a key adaptation of this bacterium during the course of chronic respiratory infections and analysis indicates that PAHM4 encodes a mutated mutS gene responsible for a ~1,000-fold increase in mutation rate compared to wild-type laboratory strain P. aeruginosa PAO1. Antibiotic resistance profiles and sequence data indicate that this strain acquired numerous mutations associated with increased resistance levels to β-lactams, aminoglycosides, and fluoroquinolones when compared to PAO1. Sequencing of PAHM4 revealed a 6.38 Mbp genome, 5.9 % of which were unrecognized in previously reported P. aeruginosa genome sequences. Transcriptome analysis suggests a general down-regulation of virulence factors, while metabolism of amino acids and lipids is up-regulated when compared to PAO1 and metabolic modeling identified further potential differences between PAO1 and PAHM4.
Conclusions
This work provides insights into the potential differential adaptation of this bacterium to the lung of patients with bronchiectasis compared to other clinical settings such as cystic fibrosis, findings that should aid the development of disease-appropriate treatment strategies for P. aeruginosa infections.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-2069-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-2069-0
PMCID: PMC4628258  PMID: 26519161
Pseudomonas aeruginosa; Metabolic model; Transcriptome; Comparative genomics; Cystic fibrosis; Bronchiectasis
3.  From “What Is?” to “What Isn't?” Computational Biology 
PLoS Computational Biology  2015;11(7):e1004318.
doi:10.1371/journal.pcbi.1004318
PMCID: PMC4489810  PMID: 26134043
4.  Inference of Network Dynamics and Metabolic Interactions in the Gut Microbiome 
PLoS Computational Biology  2015;11(6):e1004338.
We present a novel methodology to construct a Boolean dynamic model from time series metagenomic information and integrate this modeling with genome-scale metabolic network reconstructions to identify metabolic underpinnings for microbial interactions. We apply this in the context of a critical health issue: clindamycin antibiotic treatment and opportunistic Clostridium difficile infection. Our model recapitulates known dynamics of clindamycin antibiotic treatment and C. difficile infection and predicts therapeutic probiotic interventions to suppress C. difficile infection. Genome-scale metabolic network reconstructions reveal metabolic differences between community members and are used to explore the role of metabolism in the observed microbial interactions. In vitro experimental data validate a key result of our computational model, that B. intestinihominis can in fact slow C. difficile growth.
Author Summary
The community of bacteria that live in our intestines (called the “gut microbiome”) is important to normal intestinal function, and destruction of this community has a causative role in diseases including obesity, diabetes, and even neurological disorders. Clostridum difficile is an opportunistic pathogenic bacterium that causes potentially life-threatening intestinal inflammation and diarrhea and frequently occurs after antibiotic treatment, which wipes out the normal intestinal bacterial community. We use a mathematical model to identify how the normal bacterial community interacts and how this community changes with antibiotic treatment and C. difficile infection. We use this model to identify bacteria that may inhibit C. difficile growth. Our model and subsequent experiments indicate that Barnesiella intestinihominis inhibits C. difficile growth. This result suggests that B. intestinihominis could potentially be used as a probiotic to treat or prevent C. difficile infection.
doi:10.1371/journal.pcbi.1004338
PMCID: PMC4478025  PMID: 26102287
5.  MetDraw: automated visualization of genome-scale metabolic network reconstructions and high-throughput data 
Bioinformatics  2014;30(9):1327-1328.
Motivation: Metabolic reaction maps allow visualization of genome-scale models and high-throughput data in a format familiar to many biologists. However, creating a map of a large metabolic model is a difficult and time-consuming process. MetDraw fully automates the map-drawing process for metabolic models containing hundreds to thousands of reactions. MetDraw can also overlay high-throughput ‘omics’ data directly on the generated maps.
Availability and implementation: Web interface and source code are freely available at http://www.metdraw.com.
Contact: papin@virginia.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btt758
PMCID: PMC3998126  PMID: 24413519
6.  Miniaturized Plate Readers for Low-Cost, High-Throughput Phenotypic Screening 
Jala (Charlottesville, Va.)  2015;20(1):51-55.
We present a miniaturized plate reader for measuring optical density in 96-well plates. Our standalone reader fits in most incubators, environmental chambers, or biological containment suites, allowing users to leverage their existing laboratory infrastructure. The device contains no moving parts, allowing an entire 96-well plate to be read several times per second. We demonstrate how the fast sampling rate allows our reader to detect small changes in optical density, even when the device is placed in a shaking incubator. A wireless communication module allows remote monitoring of multiple devices in real time. These features allow easy assembly of multiple readers to create a scalable, accurate solution for high-throughput phenotypic screening.
doi:10.1177/2211068214555414
PMCID: PMC4359207  PMID: 25366331
phenotypic screening; plate reader; growth curve; multiwell plate assays
7.  High temporal resolution of glucosyltransferase dependent and independent effects of Clostridium difficile toxins across multiple cell types 
BMC Microbiology  2015;15(1):7.
Background
Clostridium difficile toxins A and B (TcdA and TcdB), considered to be essential for C. difficile infection, affect the morphology of several cell types with different potencies and timing. However, morphological changes over various time scales are poorly characterized. The toxins’ glucosyltransferase domains are critical to their deleterious effects, and cell responses to glucosyltransferase-independent activities are incompletely understood. By tracking morphological changes of multiple cell types to C. difficile toxins with high temporal resolution, cellular responses to TcdA, TcdB, and a glucosyltransferase-deficient TcdB (gdTcdB) are elucidated.
Results
Human umbilical vein endothelial cells, J774 macrophage-like cells, and four epithelial cell lines (HCT8, T84, CHO, and immortalized mouse cecal epithelial cells) were treated with TcdA, TcdB, gdTcdB. Impedance across cell cultures was measured to track changes in cell morphology. Metrics from impedance data, developed to quantify rapid and long-lasting responses, produced standard curves with wide dynamic ranges that defined cell line sensitivities. Except for T84 cells, all cell lines were most sensitive to TcdB. J774 macrophages stretched and increased in size in response to TcdA and TcdB but not gdTcdB. High concentrations of TcdB and gdTcdB (>10 ng/ml) greatly reduced macrophage viability. In HCT8 cells, gdTcdB did not induce a rapid cytopathic effect, yet it delayed TcdA and TcdB’s rapid effects. gdTcdB did not clearly delay TcdA or TcdB’s toxin-induced effects on macrophages.
Conclusions
Epithelial and endothelial cells have similar responses to toxins yet differ in timing and degree. Relative potencies of TcdA and TcdB in mouse epithelial cells in vitro do not correlate with potencies in vivo. TcdB requires glucosyltransferase activity to cause macrophages to spread, but cell death from high TcdB concentrations is glucosyltransferase-independent. Competition experiments with gdTcdB in epithelial cells confirm common TcdA and TcdB mechanisms, yet different responses of macrophages to TcdA and TcdB suggest different, additional mechanisms or targets in these cells. This first-time, precise quantification of the response of multiple cell lines to TcdA and TcdB provides a comparative framework for delineating the roles of different cell types and toxin-host interactions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0361-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12866-015-0361-4
PMCID: PMC4323251  PMID: 25648517
Clostridium difficile; Toxin A; Toxin B; Glucosyltransferase; Epithelial; Endothelial
8.  A Thirteen-Gene Expression Signature Predicts Survival of Patients with Pancreatic Cancer and Identifies New Genes of Interest 
PLoS ONE  2014;9(9):e105631.
Background
Currently, prognostication for pancreatic ductal adenocarcinoma (PDAC) is based upon a coarse clinical staging system. Thus, more accurate prognostic tests are needed for PDAC patients to aid treatment decisions.
Methods and Findings
Affymetrix gene expression profiling was carried out on 15 human PDAC tumors and from the data we identified a 13-gene expression signature (risk score) that correlated with patient survival. The gene expression risk score was then independently validated using published gene expression data and survival data for an additional 101 patients with pancreatic cancer. Patients with high-risk scores had significantly higher risk of death compared to patients with low-risk scores (HR 2.27, p = 0.002). When the 13-gene score was combined with lymph node status the risk-score further discriminated the length of patient survival time (p<0.001). Patients with a high-risk score had poor survival independent of nodal status; however, nodal status increased predictability for survival in patients with a low-risk gene signature score (low-risk N1 vs. low-risk N0: HR = 2.0, p = 0.002). While AJCC stage correlated with patient survival (p = 0.03), the 13-gene score was superior at predicting survival. Of the 13 genes comprising the predictive model, four have been shown to be important in PDAC, six are unreported in PDAC but important in other cancers, and three are unreported in any cancer.
Conclusions
We identified a 13-gene expression signature that predicts survival of PDAC patients and could prove useful for making treatment decisions. This risk score should be evaluated prospectively in clinical trials for prognostication and for predicting response to chemotherapy. Investigation of new genes identified in our model may lead to novel therapeutic targets.
doi:10.1371/journal.pone.0105631
PMCID: PMC4152146  PMID: 25180633
9.  Linking genome-scale metabolic modeling and genome annotation 
Summary
Genome-scale metabolic network reconstructions, assembled from annotated genomes, serve as a platform for integrating data from heterogeneous sources and generating hypotheses for further experimental validation. Implementing constraint-based modeling techniques such as Flux Balance Analysis (FBA) on network reconstructions allow for interrogating metabolism at a systems-level, which aids in identifying and rectifying gaps in knowledge. With genome sequences for various organisms from prokaryotes to eukaryotes becoming increasingly available, a significant bottleneck lies in the structural and functional annotation of these sequences. Using topologically-based and biologically-inspired metabolic network refinement, we can better characterize enzymatic functions present in an organism and link annotation of these functions to candidate transcripts, both steps that can be experimentally validated.
doi:10.1007/978-1-62703-299-5_4
PMCID: PMC4079539  PMID: 23417799
metabolic network; gap filling; orphan reactions; flux balance analysis
10.  Comparative Metabolic Systems Analysis of Pathogenic Burkholderia 
Journal of Bacteriology  2014;196(2):210-226.
Burkholderia cenocepacia and Burkholderia multivorans are opportunistic drug-resistant pathogens that account for the majority of Burkholderia cepacia complex infections in cystic fibrosis patients and also infect other immunocompromised individuals. While they share similar genetic compositions, B. cenocepacia and B. multivorans exhibit important differences in pathogenesis. We have developed reconciled genome-scale metabolic network reconstructions of B. cenocepacia J2315 and B. multivorans ATCC 17616 in parallel (designated iPY1537 and iJB1411, respectively) to compare metabolic abilities and contextualize genetic differences between species. The reconstructions capture the metabolic functions of the two species and give insight into similarities and differences in their virulence and growth capabilities. The two reconstructions have 1,437 reactions in common, and iPY1537 and iJB1411 have 67 and 36 metabolic reactions unique to each, respectively. After curating the extensive reservoir of metabolic genes in Burkholderia, we identified 6 genes essential to growth that are unique to iPY1513 and 13 genes uniquely essential to iJB1411. The reconstructions were refined and validated by comparing in silico growth predictions to in vitro growth capabilities of B. cenocepacia J2315, B. cenocepacia K56-2, and B. multivorans ATCC 17616 on 104 carbon sources. Overall, we identified functional pathways that indicate B. cenocepacia can produce a wider array of virulence factors compared to B. multivorans, which supports the clinical observation that B. cenocepacia is more virulent than B. multivorans. The reconciled reconstructions provide a framework for generating and testing hypotheses on the metabolic and virulence capabilities of these two related emerging pathogens.
doi:10.1128/JB.00997-13
PMCID: PMC3911241  PMID: 24163337
11.  Multiscale Computational Models of Complex Biological Systems 
Integration of data across spatial, temporal, and functional scales is a primary focus of biomedical engineering efforts. The advent of powerful computing platforms, coupled with quantitative data from high-throughput experimental platforms, has allowed multiscale modeling to expand as a means to more comprehensively investigate biological phenomena in experimentally relevant ways. This review aims to highlight recently published multiscale models of biological systems while using their successes to propose the best practices for future model development. We demonstrate that coupling continuous and discrete systems best captures biological information across spatial scales by selecting modeling techniques that are suited to the task. Further, we suggest how to best leverage these multiscale models to gain insight into biological systems using quantitative, biomedical engineering methods to analyze data in non-intuitive ways. These topics are discussed with a focus on the future of the field, the current challenges encountered, and opportunities yet to be realized.
doi:10.1146/annurev-bioeng-071811-150104
PMCID: PMC3970111  PMID: 23642247
data integration; model validation; systems biology; bioinformatics; biochemical networks; model design
12.  In Vivo Physiological and Transcriptional Profiling Reveals Host Responses to Clostridium difficile Toxin A and Toxin B 
Infection and Immunity  2013;81(10):3814-3824.
Toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile cause gross pathological changes (e.g., inflammation, secretion, and diarrhea) in the infected host, yet the molecular and cellular pathways leading to observed host responses are poorly understood. To address this gap, we evaluated the effects of single doses of TcdA and/or TcdB injected into the ceca of mice, and several endpoints were analyzed, including tissue pathology, neutrophil infiltration, epithelial-layer gene expression, chemokine levels, and blood cell counts, 2, 6, and 16 h after injection. In addition to confirming TcdA's gross pathological effects, we found that both TcdA and TcdB resulted in neutrophil infiltration. Bioinformatics analyses identified altered expression of genes associated with the metabolism of lipids, fatty acids, and detoxification; small GTPase activity; and immune function and inflammation. Further analysis revealed transient expression of several chemokines (e.g., Cxcl1 and Cxcl2). Antibody neutralization of CXCL1 and CXCL2 did not affect TcdA-induced local pathology or neutrophil infiltration, but it did decrease the peripheral blood neutrophil count. Additionally, low serum levels of CXCL1 and CXCL2 corresponded with greater survival. Although TcdA induced more pronounced transcriptional changes than TcdB and the upregulated chemokine expression was unique to TcdA, the overall transcriptional responses to TcdA and TcdB were strongly correlated, supporting differences primarily in timing and potency rather than differences in the type of intracellular host response. In addition, the transcriptional data revealed novel toxin effects (e.g., altered expression of GTPase-associated and metabolic genes) underlying observed physiological responses to C. difficile toxins.
doi:10.1128/IAI.00869-13
PMCID: PMC3811747  PMID: 23897615
13.  Mechanistic systems modeling to guide drug discovery and development 
Drug discovery today  2012;18(0):116-127.
A crucial question that must be addressed in the drug development process is whether the proposed therapeutic target will yield the desired effect in the clinical population. Pharmaceutical and biotechnology companies place a large investment on research and development, long before confirmatory data are available from human trials. Basic science has greatly expanded the computable knowledge of disease processes, both through the generation of large omics data sets and a compendium of studies assessing cellular and systemic responses to physiologic and pathophysiologic stimuli. Given inherent uncertainties in drug development, mechanistic systems models can better inform target selection and the decision process for advancing compounds through preclinical and clinical research.
doi:10.1016/j.drudis.2012.09.003
PMCID: PMC3644584  PMID: 22999913
14.  A community-driven global reconstruction of human metabolism 
Nature biotechnology  2013;31(5):10.1038/nbt.2488.
Multiple models of human metabolism have been reconstructed, but each represents only a subset of our knowledge. Here we describe Recon 2, a community-driven, consensus ‘metabolic reconstruction’, which is the most comprehensive representation of human metabolism that is applicable to computational modeling. Compared with its predecessors, the reconstruction has improved topological and functional features, including ~2× more reactions and ~1.7× more unique metabolites. Using Recon 2 we predicted changes in metabolite biomarkers for 49 inborn errors of metabolism with 77% accuracy when compared to experimental data. Mapping metabolomic data and drug information onto Recon 2 demonstrates its potential for integrating and analyzing diverse data types. Using protein expression data, we automatically generated a compendium of 65 cell type–specific models, providing a basis for manual curation or investigation of cell-specific metabolic properties. Recon 2 will facilitate many future biomedical studies and is freely available at http://humanmetabolism.org/.
doi:10.1038/nbt.2488
PMCID: PMC3856361  PMID: 23455439
15.  Clinical, Molecular and Genetic Validation of a Murine Orthotopic Xenograft Model of Pancreatic Adenocarcinoma Using Fresh Human Specimens 
PLoS ONE  2013;8(10):e77065.
Background
Relevant preclinical models that recapitulate the key features of human pancreatic ductal adenocarcinoma (PDAC) are needed in order to provide biologically tractable models to probe disease progression and therapeutic responses and ultimately improve patient outcomes for this disease. Here, we describe the establishment and clinical, pathological, molecular and genetic validation of a murine, orthotopic xenograft model of PDAC.
Methods
Human PDACs were resected and orthotopically implanted and propagated in immunocompromised mice. Patient survival was correlated with xenograft growth and metastatic rate in mice. Human and mouse tumor pathology were compared. Tumors were analyzed for genetic mutations, gene expression, receptor tyrosine kinase activation, and cytokine expression.
Results
Fifteen human PDACs were propagated orthotopically in mice. Xenograft-bearing mice developed peritoneal and liver metastases. Time to tumor growth and metastatic efficiency in mice each correlated with patient survival. Tumor architecture, nuclear grade and stromal content were similar in patient and xenografted tumors. Propagated tumors closely exhibited the genetic and molecular features known to characterize pancreatic cancer (e.g. high rate of KRAS, P53, SMAD4 mutation and EGFR activation). The correlation coefficient of gene expression between patient tumors and xenografts propagated through multiple generations was 93 to 99%. Analysis of gene expression demonstrated distinct differences between xenografts from fresh patient tumors versus commercially available PDAC cell lines.
Conclusions
The orthotopic xenograft model derived from fresh human PDACs closely recapitulates the clinical, pathologic, genetic and molecular aspects of human disease. This model has resulted in the identification of rational therapeutic strategies to be tested in clinical trials and will permit additional therapeutic approaches and identification of biomarkers of response to therapy.
doi:10.1371/journal.pone.0077065
PMCID: PMC3799939  PMID: 24204737
16.  Novel Multiscale Modeling Tool Applied to Pseudomonas aeruginosa Biofilm Formation 
PLoS ONE  2013;8(10):e78011.
Multiscale modeling is used to represent biological systems with increasing frequency and success. Multiscale models are often hybrids of different modeling frameworks and programming languages. We present the MATLAB-NetLogo extension (MatNet) as a novel tool for multiscale modeling. We demonstrate the utility of the tool with a multiscale model of Pseudomonas aeruginosa biofilm formation that incorporates both an agent-based model (ABM) and constraint-based metabolic modeling. The hybrid model correctly recapitulates oxygen-limited biofilm metabolic activity and predicts increased growth rate via anaerobic respiration with the addition of nitrate to the growth media. In addition, a genome-wide survey of metabolic mutants and biofilm formation exemplifies the powerful analyses that are enabled by this computational modeling tool.
doi:10.1371/journal.pone.0078011
PMCID: PMC3798466  PMID: 24147108
17.  MicroRNAs induced in melanoma treated with combination targeted therapy of Temsirolimus and Bevacizumab 
Background
Targeted therapies directed at commonly overexpressed pathways in melanoma have clinical activity in numerous trials. Little is known about how these therapies influence microRNA (miRNA) expression, particularly with combination regimens. Knowledge of miRNAs altered with treatment may contribute to understanding mechanisms of therapeutic effects, as well as mechanisms of tumor escape from therapy. We analyzed miRNA expression in metastatic melanoma tissue samples treated with a novel combination regimen of Temsirolimus and Bevacizumab. Given the preliminary clinical activity observed with this combination regimen, we hypothesized that we would see significant changes in miRNA expression with combination treatment.
Methods
Using microarray analysis we analyzed miRNA expression levels in melanoma samples from a Cancer Therapy Evaluation Program-sponsored phase II trial of combination Temsirolimus and Bevacizumab in advanced melanoma, which elicited clinical benefit in a subset of patients. Pre-treatment and post-treatment miRNA levels were compared using paired t-tests between sample groups (patients), using a p-value < 0.01 for significance.
Results
microRNA expression remained unchanged with Temsirolimus alone; however, expression of 15 microRNAs was significantly upregulated (1.4 to 2.5-fold) with combination treatment, compared to pre-treatment levels. Interestingly, twelve of these fifteen miRNAs possess tumor suppressor capabilities. We identified 15 putative oncogenes as potential targets of the 12 tumor suppressor miRNAs, based on published experimental evidence. For 15 of 25 miRNA-target mRNA pairings, changes in gene expression from pre-treatment to post-combination treatment samples were inversely correlated with changes in miRNA expression, supporting a functional effect of those miRNA changes. Clustering analyses based on selected miRNAs suggest preliminary signatures characteristic of clinical response to combination treatment and of tumor BRAF mutational status.
Conclusions
To our knowledge, this is the first study analyzing miRNA expression in pre-treatment and post-treatment human metastatic melanoma tissue samples. This preliminary investigation suggests miRNAs that may be involved in the mechanism of action of combination Temsirolimus and Bevacizumab in metastatic melanoma, possibly through inhibition of oncogenic pathways, and provides the preliminary basis for further functional studies of these miRNAs.
doi:10.1186/1479-5876-11-218
PMCID: PMC3853033  PMID: 24047116
18.  A metabolic network approach for the identification and prioritization of antimicrobial drug targets 
Trends in Microbiology  2012;20(3):113-123.
For many infectious diseases, novel treatment options are needed to address problems with cost, toxicity and resistance to current drugs. Systems biology tools can be used to gain valuable insight into pathogenic processes and aid in expediting drug discovery. In the past decade, constraint-based modeling of genome-scale metabolic networks has become widely used. Focusing on pathogen metabolic networks, we review in silico strategies to identify effective drug targets, and we highlight recent successes as well as limitations associated with such computational analyses. We further discuss how accounting for the host environment and even targeting the host may offer new therapeutic options. These systems-level approaches are beginning to provide novel avenues for drug targeting against infectious agents.
doi:10.1016/j.tim.2011.12.004
PMCID: PMC3299924  PMID: 22300758
systems biology; metabolic network reconstruction; flux balance analysis; drug targeting; computational biology; microbial pathogens
19.  Integration of expression data in genome-scale metabolic network reconstructions 
With the advent of high-throughput technologies, the field of systems biology has amassed an abundance of “omics” data, quantifying thousands of cellular components across a variety of scales, ranging from mRNA transcript levels to metabolite quantities. Methods are needed to not only integrate this omics data but to also use this data to heighten the predictive capabilities of computational models. Several recent studies have successfully demonstrated how flux balance analysis (FBA), a constraint-based modeling approach, can be used to integrate transcriptomic data into genome-scale metabolic network reconstructions to generate predictive computational models. In this review, we summarize such FBA-based methods for integrating expression data into genome-scale metabolic network reconstructions, highlighting their advantages as well as their limitations.
doi:10.3389/fphys.2012.00299
PMCID: PMC3429070  PMID: 22934050
flux balance analysis; data integration; transcriptomics; expression data; metabolic networks
20.  Comprehensive analysis of RTK activation in human melanomas reveals autocrine signaling through IGF-1R 
Melanoma research  2011;21(4):274-284.
Melanomas depend on autocrine signals for proliferation and survival; however, no systematic screen of known RTKs has been performed to identify which autocrine signaling pathways are activated in melanoma. Here we performed a comprehensive analysis of 42 receptor tyrosine kinases (RTKs) in 6 individual human melanoma tumor specimens as well as 17 melanoma cell lines, some of which were derived from the tumor specimens. We identified 5 RTKs that were active in almost every one of the melanoma tissue specimens and cell lines, including two previously unreported receptors, IGF1R and MSPR, in addition to three receptors (VEGFR, FGFR and HGFR) known to be autocrine activated in melanoma. We show by real time quantitative PCR that all melanoma cell lines expressed genes for the RTK ligands HGF, IGF1 and MSP. Addition of antibodies to either IGF1 or HGF, but not to MSP, to the culture medium blocked melanoma cell proliferation, and even caused net loss of melanoma cells. Antibody addition deactivated IGF1R and HGFR receptors, as well as MAPK signaling. Thus, IGF1 is a new growth factor for autocrine driven proliferation of human melanoma in vitro. Our results suggest that IGF1-IGF1R autocrine pathway in melanoma is a possible target for therapy in human melanomas.
doi:10.1097/CMR.0b013e328343a1d6
PMCID: PMC3131461  PMID: 21654344
IGF1; IGF1R; HGF; HGFR; c-Met; melanoma; Receptor Tyrosine Kinases
21.  Whole-genome metabolic network reconstruction and constraint-based modeling 
Methods in Enzymology  2011;500:411-433.
With the advent of modern high throughput genomics, there is a significant need for genome-scale analysis techniques that can assist in complex systems analysis. Metabolic genome-scale network reconstructions (GENREs) paired with constraint-based modeling are an efficient method to integrate genomics, transcriptomics, and proteomics to conduct organism-specific analysis. This text explains key steps in the GENRE construction process and several methods of constraint-based modeling that can help elucidate basic life processes and development of disease treatment, bioenergy solutions, and industrial bioproduction applications.
doi:10.1016/B978-0-12-385118-5.00021-9
PMCID: PMC3361743  PMID: 21943909
22.  Matrix Rigidity Regulates Cancer Cell Growth by Modulating Cellular Metabolism and Protein Synthesis 
PLoS ONE  2012;7(5):e37231.
Background
Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy.
Methodology/Principal Findings
This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa), cells on soft substrates (150–300 Pa) exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC) and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins) and glycolysis (e.g., phosphofructokinase-1), whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway.
Conclusions/Significance
The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under conditions that reflect the different mechanical environments encountered by cancer cells upon metastasis to distant sites.
doi:10.1371/journal.pone.0037231
PMCID: PMC3356407  PMID: 22623999
23.  Metabolic network analysis predicts efficacy of FDA-approved drugs targeting the causative agent of a neglected tropical disease 
BMC Systems Biology  2012;6:27.
Background
Systems biology holds promise as a new approach to drug target identification and drug discovery against neglected tropical diseases. Genome-scale metabolic reconstructions, assembled from annotated genomes and a vast array of bioinformatics/biochemical resources, provide a framework for the interrogation of human pathogens and serve as a platform for generation of future experimental hypotheses. In this article, with the application of selection criteria for both Leishmania major targets (e.g. in silico gene lethality) and drugs (e.g. toxicity), a method (MetDP) to rationally focus on a subset of low-toxic Food and Drug Administration (FDA)-approved drugs is introduced.
Results
This metabolic network-driven approach identified 15 L. major genes as high-priority targets, 8 high-priority synthetic lethal targets, and 254 FDA-approved drugs. Results were compared to previous literature findings and existing high-throughput screens. Halofantrine, an antimalarial agent that was prioritized using MetDP, showed noticeable antileishmanial activity when experimentally evaluated in vitro against L. major promastigotes. Furthermore, synthetic lethality predictions also aided in the prediction of superadditive drug combinations. For proof-of-concept, double-drug combinations were evaluated in vitro against L. major and four combinations involving the drug disulfiram that showed superadditivity are presented.
Conclusions
A direct metabolic network-driven method that incorporates single gene essentiality and synthetic lethality predictions is proposed that generates a set of high-priority L. major targets, which are in turn associated with a select number of FDA-approved drugs that are candidate antileishmanials. Additionally, selection of high-priority double-drug combinations might provide for an attractive and alternative avenue for drug discovery against leishmaniasis.
doi:10.1186/1752-0509-6-27
PMCID: PMC3388006  PMID: 22540944
24.  Systems analysis of the transcriptional response of human ileocecal epithelial cells to Clostridium difficile toxins and effects on cell cycle control 
Background
Toxins A and B (TcdA and TcdB) are Clostridium difficile's principal virulence factors, yet the pathways by which they lead to inflammation and severe diarrhea remain unclear. Also, the relative role of either toxin during infection and the differences in their effects across cell lines is still poorly understood. To better understand their effects in a susceptible cell line, we analyzed the transciptome-wide gene expression response of human ileocecal epithelial cells (HCT-8) after 2, 6, and 24 hr of toxin exposure.
Results
We show that toxins elicit very similar changes in the gene expression of HCT-8 cells, with the TcdB response occurring sooner. The high similarity suggests differences between toxins are due to events beyond transcription of a single cell-type and that their relative potencies during infection may depend on differential effects across cell types within the intestine. We next performed an enrichment analysis to determine biological functions associated with changes in transcription. Differentially expressed genes were associated with response to external stimuli and apoptotic mechanisms and, at 24 hr, were predominately associated with cell-cycle control and DNA replication. To validate our systems approach, we subsequently verified a novel G1/S and known G2/M cell-cycle block and increased apoptosis as predicted from our enrichment analysis.
Conclusions
This study shows a successful example of a workflow deriving novel biological insight from transcriptome-wide gene expression. Importantly, we do not find any significant difference between TcdA and TcdB besides potency or kinetics. The role of each toxin in the inhibition of cell growth and proliferation, an important function of cells in the intestinal epithelium, is characterized.
doi:10.1186/1752-0509-6-2
PMCID: PMC3266197  PMID: 22225989
Clostridium difficile; Toxin A; Toxin B; gene expression; epithelial cell; cell-cycle
25.  TIGER: Toolbox for integrating genome-scale metabolic models, expression data, and transcriptional regulatory networks 
BMC Systems Biology  2011;5:147.
Background
Several methods have been developed for analyzing genome-scale models of metabolism and transcriptional regulation. Many of these methods, such as Flux Balance Analysis, use constrained optimization to predict relationships between metabolic flux and the genes that encode and regulate enzyme activity. Recently, mixed integer programming has been used to encode these gene-protein-reaction (GPR) relationships into a single optimization problem, but these techniques are often of limited generality and lack a tool for automating the conversion of rules to a coupled regulatory/metabolic model.
Results
We present TIGER, a Toolbox for Integrating Genome-scale Metabolism, Expression, and Regulation. TIGER converts a series of generalized, Boolean or multilevel rules into a set of mixed integer inequalities. The package also includes implementations of existing algorithms to integrate high-throughput expression data with genome-scale models of metabolism and transcriptional regulation. We demonstrate how TIGER automates the coupling of a genome-scale metabolic model with GPR logic and models of transcriptional regulation, thereby serving as a platform for algorithm development and large-scale metabolic analysis. Additionally, we demonstrate how TIGER's algorithms can be used to identify inconsistencies and improve existing models of transcriptional regulation with examples from the reconstructed transcriptional regulatory network of Saccharomyces cerevisiae.
Conclusion
The TIGER package provides a consistent platform for algorithm development and extending existing genome-scale metabolic models with regulatory networks and high-throughput data.
doi:10.1186/1752-0509-5-147
PMCID: PMC3224351  PMID: 21943338

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