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Sample records for identify biochemical pathways

  1. Metabolomic profiling identifies biochemical pathways associated with castration-resistant prostate cancer.

    PubMed

    Kaushik, Akash K; Vareed, Shaiju K; Basu, Sumanta; Putluri, Vasanta; Putluri, Nagireddy; Panzitt, Katrin; Brennan, Christine A; Chinnaiyan, Arul M; Vergara, Ismael A; Erho, Nicholas; Weigel, Nancy L; Mitsiades, Nicholas; Shojaie, Ali; Palapattu, Ganesh; Michailidis, George; Sreekumar, Arun

    2014-02-01

    Despite recent developments in treatment strategies, castration-resistant prostate cancer (CRPC) is still the second leading cause of cancer-associated mortality among American men, the biological underpinnings of which are not well understood. To this end, we measured levels of 150 metabolites and examined the rate of utilization of 184 metabolites in metastatic androgen-dependent prostate cancer (AD) and CRPC cell lines using a combination of targeted mass spectrometry and metabolic phenotyping. Metabolic data were used to derive biochemical pathways that were enriched in CRPC, using Oncomine concept maps (OCM). The enriched pathways were then examined in-silico for their association with treatment failure (i.e., prostate specific antigen (PSA) recurrence or biochemical recurrence) using published clinically annotated gene expression data sets. Our results indicate that a total of 19 metabolites were altered in CRPC compared to AD cell lines. These altered metabolites mapped to a highly interconnected network of biochemical pathways that describe UDP glucuronosyltransferase (UGT) activity. We observed an association with time to treatment failure in an analysis employing genes restricted to this pathway in three independent gene expression data sets. In summary, our studies highlight the value of employing metabolomic strategies in cell lines to derive potentially clinically useful predictive tools. PMID:24359151

  2. Metabolomic profiling and genomic analysis of wheat aneuploid lines to identify genes controlling biochemical pathways in mature grain.

    PubMed

    Francki, Michael G; Hayton, Sarah; Gummer, Joel P A; Rawlinson, Catherine; Trengove, Robert D

    2016-02-01

    Metabolomics is becoming an increasingly important tool in plant genomics to decipher the function of genes controlling biochemical pathways responsible for trait variation. Although theoretical models can integrate genes and metabolites for trait variation, biological networks require validation using appropriate experimental genetic systems. In this study, we applied an untargeted metabolite analysis to mature grain of wheat homoeologous group 3 ditelosomic lines, selected compounds that showed significant variation between wheat lines Chinese Spring and at least one ditelosomic line, tracked the genes encoding enzymes of their biochemical pathway using the wheat genome survey sequence and determined the genetic components underlying metabolite variation. A total of 412 analytes were resolved in the wheat grain metabolome, and principal component analysis indicated significant differences in metabolite profiles between Chinese Spring and each ditelosomic lines. The grain metabolome identified 55 compounds positively matched against a mass spectral library where the majority showed significant differences between Chinese Spring and at least one ditelosomic line. Trehalose and branched-chain amino acids were selected for detailed investigation, and it was expected that if genes encoding enzymes directly related to their biochemical pathways were located on homoeologous group 3 chromosomes, then corresponding ditelosomic lines would have a significant reduction in metabolites compared with Chinese Spring. Although a proportion showed a reduction, some lines showed significant increases in metabolites, indicating that genes directly and indirectly involved in biosynthetic pathways likely regulate the metabolome. Therefore, this study demonstrated that wheat aneuploid lines are suitable experimental genetic system to validate metabolomics-genomics networks. PMID:26032167

  3. Reconstructing biochemical pathways from time course data.

    PubMed

    Srividhya, Jeyaraman; Crampin, Edmund J; McSharry, Patrick E; Schnell, Santiago

    2007-03-01

    Time series data on biochemical reactions reveal transient behavior, away from chemical equilibrium, and contain information on the dynamic interactions among reacting components. However, this information can be difficult to extract using conventional analysis techniques. We present a new method to infer biochemical pathway mechanisms from time course data using a global nonlinear modeling technique to identify the elementary reaction steps which constitute the pathway. The method involves the generation of a complete dictionary of polynomial basis functions based on the law of mass action. Using these basis functions, there are two approaches to model construction, namely the general to specific and the specific to general approach. We demonstrate that our new methodology reconstructs the chemical reaction steps and connectivity of the glycolytic pathway of Lactococcus lactis from time course experimental data. PMID:17370261

  4. "Which Pathway Am I?" Using a Game Approach to Teach Students about Biochemical Pathways

    ERIC Educational Resources Information Center

    Ooi, Beng Guat; Sanger, Michael J.

    2009-01-01

    This game was designed to provide students with an alternative way to learn biochemical pathways through an interactive approach. In this game, students worked in pairs to help each other identify pathways taped to each other's backs by asking simple "yes or no" questions related to these pathways. This exercise was conducted after the traditional

  5. "Which Pathway Am I?" Using a Game Approach to Teach Students about Biochemical Pathways

    ERIC Educational Resources Information Center

    Ooi, Beng Guat; Sanger, Michael J.

    2009-01-01

    This game was designed to provide students with an alternative way to learn biochemical pathways through an interactive approach. In this game, students worked in pairs to help each other identify pathways taped to each other's backs by asking simple "yes or no" questions related to these pathways. This exercise was conducted after the traditional…

  6. "Which Pathway Am I?" Using a Game Approach To Teach Students about Biochemical Pathways

    NASA Astrophysics Data System (ADS)

    Guat Ooi, Beng; Sanger, Michael J.

    2009-04-01

    This game was designed to provide students with an alternative way to learn biochemical pathways through an interactive approach. In this game, students worked in pairs to help each other identify pathways taped to each other's backs by asking simple "yes or no" questions related to these pathways. This exercise was conducted after the traditional biochemistry lectures on pathways and was designed to complement the lectures and reinforce key information about pathways. The advantage of this approach is that the pair of students was able to explore and experience two mechanisms of learning biochemical pathways. The Student t test performed on the students' pre-test and post-test scores indicated that students showed significant improvement on their abilities to identify pathways after participating in this game.

  7. Network representations and methods for the analysis of chemical and biochemical pathways

    PubMed Central

    Sandefur, Conner I.; Mincheva, Maya; Schnell, Santiago

    2013-01-01

    Systems biologists increasingly use network representations to investigate biochemical pathways and their dynamic behaviours. In this critical review, we discuss four commonly used network representations of chemical and biochemical pathways. We illustrate how some of these representations reduce network complexity but result in the ambiguous representation of biochemical pathways. We also examine the current theoretical approaches available to investigate the dynamic behaviour of chemical and biochemical networks. Finally, we describe how the critical chemical and biochemical pathways responsible for emergent dynamic behaviour can be identified using network mining and functional mapping approaches. PMID:23857078

  8. Biochemical Pathway of Stress-induced Ethylene

    PubMed Central

    Abeles, A. L.; Abeles, F. B.

    1972-01-01

    Ethylene production from beam and tobacco leaves increased rapidly following the application of toxic compounds such as CuSO4, Endothal, and ozone. Treatments which increased ethylene evolution also increased the conversion of U-14C-methionine into ethylene. Cycloheximide inhibited the production of chemical stress-induced ethylene. These results suggest that ethylene is produced by the same biochemical pathway forming basal ethylene, auxin-induced ethylene, or that produced during the ripening of climacteric fruit. PMID:16658203

  9. Biochemical pathways in seed oil synthesis.

    PubMed

    Bates, Philip D; Stymne, Sten; Ohlrogge, John

    2013-06-01

    Oil produced in plant seeds is utilized as a major source of calories for human nutrition, as feedstocks for non-food uses such as soaps and polymers, and can serve as a high-energy biofuel. The biochemical pathways leading to oil (triacylglycerol) synthesis in seeds involve multiple subcellular organelles, requiring extensive lipid trafficking. Phosphatidylcholine plays a central role in these pathways as a substrate for acyl modifications and likely as a carrier for the trafficking of acyl groups between organelles and membrane subdomains. Although much has been clarified regarding the enzymes and pathways responsible for acyl-group flux, there are still major gaps in our understanding. These include the identity of several key enzymes, how flux between alternative pathways is controlled and the specialized cell biology leading to biogenesis of oil bodies that store up to 80% of carbon in seeds. PMID:23529069

  10. Identifying biochemical phenotypic differences between cryptic species

    PubMed Central

    Liebeke, Manuel; Bruford, Michael W.; Donnelly, Robert K.; Ebbels, Timothy M. D.; Hao, Jie; Kille, Peter; Lahive, Elma; Madison, Rachael M.; Morgan, A. John; Pinto-Juma, Gabriela A.; Spurgeon, David J.; Svendsen, Claus; Bundy, Jacob G.

    2014-01-01

    Molecular genetic methods can distinguish divergent evolutionary lineages in what previously appeared to be single species, but it is not always clear what functional differences exist between such cryptic species. We used a metabolomic approach to profile biochemical phenotype (metabotype) differences between two putative cryptic species of the earthworm Lumbricus rubellus. There were no straightforward metabolite biomarkers of lineage, i.e. no metabolites that were always at higher concentration in one lineage. Multivariate methods, however, identified a small number of metabolites that together helped distinguish the lineages, including uncommon metabolites such as N?-trimethyllysine, which is not usually found at high concentrations. This approach could be useful for characterizing functional trait differences, especially as it is applicable to essentially any species group, irrespective of its genome sequencing status. PMID:25252836

  11. Biochemical Genetic Pathways that Modulate Aging in Multiple Species

    PubMed Central

    Bitto, Alessandro; Wang, Adrienne M.; Bennett, Christopher F.; Kaeberlein, Matt

    2016-01-01

    The mechanisms underlying biological aging have been extensively studied in the past 20 years with the avail of mainly four model organisms: the budding yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, the fruitfly Drosophila melanogaster, and the domestic mouse Mus musculus. Extensive research in these four model organisms has identified a few conserved genetic pathways that affect longevity as well as metabolism and development. Here, we review how the mechanistic target of rapamycin (mTOR), sirtuins, adenosine monophosphate-activated protein kinase (AMPK), growth hormone/insulin-like growth factor 1 (IGF-1), and mitochondrial stress-signaling pathways influence aging and life span in the aforementioned models and their possible implications for delaying aging in humans. We also draw some connections between these biochemical pathways and comment on what new developments aging research will likely bring in the near future. PMID:26525455

  12. The Saccharomyces Genome Database: Exploring Biochemical Pathways and Mutant Phenotypes.

    PubMed

    Cherry, J Michael

    2015-01-01

    Many biochemical processes, and the proteins and cofactors involved, have been defined for the eukaryote Saccharomyces cerevisiae. This understanding has been largely derived through the awesome power of yeast genetics. The proteins responsible for the reactions that build complex molecules and generate energy for the cell have been integrated into web-based tools that provide classical views of pathways. The Yeast Pathways in the Saccharomyces Genome Database (SGD) is, however, the only database created from manually curated literature annotations. In this protocol, gene function is explored using phenotype annotations to enable hypotheses to be formulated about a gene's action. A common use of the SGD is to understand more about a gene that was identified via a phenotypic screen or found to interact with a gene/protein of interest. There are still many genes that do not yet have an experimentally defined function and so the information currently available can be used to speculate about their potential function. Typically, computational annotations based on sequence similarity are used to predict gene function. In addition, annotations are sometimes available for phenotypes of mutations in the gene of interest. Integrated results for a few example genes will be explored in this protocol. This will be instructive for the exploration of details that aid the analysis of experimental results and the establishment of connections within the yeast literature. PMID:26631123

  13. Cellular models to investigate biochemical pathways in Parkinson's disease.

    TOXLINE Toxicology Bibliographic Information

    Alberio T; Lopiano L; Fasano M

    2012-04-01

    Cellular models are instrumental in dissecting a complex pathological process into simpler molecular events. Parkinson's disease is multifactorial and clinically heterogeneous; the aetiology of the sporadic (and most common) form is still unclear and only a few molecular mechanisms have been clarified so far in the neurodegenerative cascade. In such a multifaceted picture, it is particularly important to identify experimental models that simplify the study of the different networks of proteins/genes involved. Cellular models that reproduce some of the features of the neurons that degenerate in Parkinson's disease have contributed to many advances in our comprehension of the pathogenic flow of the disease. In particular, the pivotal biochemical pathways (i.e. apoptosis and oxidative stress, mitochondrial impairment and dysfunctional mitophagy, unfolded protein stress and improper removal of misfolded proteins) have been widely explored in cell lines, challenged with toxic insults or genetically modified. The central role of α-synuclein has generated many models aiming to elucidate its contribution to the dysregulation of various cellular processes. In conclusion, classical cellular models appear to be the correct choice for preliminary studies on the molecular action of new drugs or potential toxins and for understanding the role of single genetic factors. Moreover, the availability of novel cellular systems, such as cybrids or induced pluripotent stem cells, offers the chance to exploit the advantages of an in vitro investigation, although mirroring more closely the cell population being affected.

  14. Identifying proteins controlling key disease signaling pathways

    PubMed Central

    Gitter, Anthony; Bar-Joseph, Ziv

    2013-01-01

    Motivation: Several types of studies, including genome-wide association studies and RNA interference screens, strive to link genes to diseases. Although these approaches have had some success, genetic variants are often only present in a small subset of the population, and screens are noisy with low overlap between experiments in different labs. Neither provides a mechanistic model explaining how identified genes impact the disease of interest or the dynamics of the pathways those genes regulate. Such mechanistic models could be used to accurately predict downstream effects of knocking down pathway members and allow comprehensive exploration of the effects of targeting pairs or higher-order combinations of genes. Results: We developed methods to model the activation of signaling and dynamic regulatory networks involved in disease progression. Our model, SDREM, integrates static and time series data to link proteins and the pathways they regulate in these networks. SDREM uses prior information about proteins likelihood of involvement in a disease (e.g. from screens) to improve the quality of the predicted signaling pathways. We used our algorithms to study the human immune response to H1N1 influenza infection. The resulting networks correctly identified many of the known pathways and transcriptional regulators of this disease. Furthermore, they accurately predict RNA interference effects and can be used to infer genetic interactions, greatly improving over other methods suggested for this task. Applying our method to the more pathogenic H5N1 influenza allowed us to identify several strain-specific targets of this infection. Availability: SDREM is available from http://sb.cs.cmu.edu/sdrem Contact: zivbj@cs.cmu.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23812988

  15. Characterizing autism spectrum disorders by key biochemical pathways

    PubMed Central

    Subramanian, Megha; Timmerman, Christina K.; Schwartz, Joshua L.; Pham, Daniel L.; Meffert, Mollie K.

    2015-01-01

    The genetic and phenotypic heterogeneity of autism spectrum disorders (ASD) presents a substantial challenge for diagnosis, classification, research, and treatment. Investigations into the underlying molecular etiology of ASD have often yielded mixed and at times opposing findings. Defining the molecular and biochemical underpinnings of heterogeneity in ASD is crucial to our understanding of the pathophysiological development of the disorder, and has the potential to assist in diagnosis and the rational design of clinical trials. In this review, we propose that genetically diverse forms of ASD may be usefully parsed into entities resulting from converse patterns of growth regulation at the molecular level, which lead to the correlates of general synaptic and neural overgrowth or undergrowth. Abnormal brain growth during development is a characteristic feature that has been observed both in children with autism and in mouse models of autism. We review evidence from syndromic and non-syndromic ASD to suggest that entities currently classified as autism may fundamentally differ by underlying pro- or anti-growth abnormalities in key biochemical pathways, giving rise to either excessive or reduced synaptic connectivity in affected brain regions. We posit that this classification strategy has the potential not only to aid research efforts, but also to ultimately facilitate early diagnosis and direct appropriate therapeutic interventions. PMID:26483618

  16. Characterizing autism spectrum disorders by key biochemical pathways.

    PubMed

    Subramanian, Megha; Timmerman, Christina K; Schwartz, Joshua L; Pham, Daniel L; Meffert, Mollie K

    2015-01-01

    The genetic and phenotypic heterogeneity of autism spectrum disorders (ASD) presents a substantial challenge for diagnosis, classification, research, and treatment. Investigations into the underlying molecular etiology of ASD have often yielded mixed and at times opposing findings. Defining the molecular and biochemical underpinnings of heterogeneity in ASD is crucial to our understanding of the pathophysiological development of the disorder, and has the potential to assist in diagnosis and the rational design of clinical trials. In this review, we propose that genetically diverse forms of ASD may be usefully parsed into entities resulting from converse patterns of growth regulation at the molecular level, which lead to the correlates of general synaptic and neural overgrowth or undergrowth. Abnormal brain growth during development is a characteristic feature that has been observed both in children with autism and in mouse models of autism. We review evidence from syndromic and non-syndromic ASD to suggest that entities currently classified as autism may fundamentally differ by underlying pro- or anti-growth abnormalities in key biochemical pathways, giving rise to either excessive or reduced synaptic connectivity in affected brain regions. We posit that this classification strategy has the potential not only to aid research efforts, but also to ultimately facilitate early diagnosis and direct appropriate therapeutic interventions. PMID:26483618

  17. Using the Biochemical Pathway Model To Teach the Concepts of Gene Interaction and Epistasis.

    ERIC Educational Resources Information Center

    Chinnici, Joseph P.

    1999-01-01

    Describes the successful use of giving examples of genes affecting various steps in biochemical pathways to teach gene interaction. Finds that once students grasp the notion that genes can interact because they control different steps within biochemical pathways, the reason why phenotypic ratios appear to be non-Mendelian becomes more obvious and

  18. Hierarchical modularization of biochemical pathways using fuzzy-c means clustering.

    PubMed

    de Luis Balaguer, Maria A; Williams, Cranos M

    2014-08-01

    Biological systems that are representative of regulatory, metabolic, or signaling pathways can be highly complex. Mathematical models that describe such systems inherit this complexity. As a result, these models can often fail to provide a path toward the intuitive comprehension of these systems. More coarse information that allows a perceptive insight of the system is sometimes needed in combination with the model to understand control hierarchies or lower level functional relationships. In this paper, we present a method to identify relationships between components of dynamic models of biochemical pathways that reside in different functional groups. We find primary relationships and secondary relationships. The secondary relationships reveal connections that are present in the system, which current techniques that only identify primary relationships are unable to show. We also identify how relationships between components dynamically change over time. This results in a method that provides the hierarchy of the relationships among components, which can help us to understand the low level functional structure of the system and to elucidate potential hierarchical control. As a proof of concept, we apply the algorithm to the epidermal growth factor signal transduction pathway, and to the C3 photosynthesis pathway. We identify primary relationships among components that are in agreement with previous computational decomposition studies, and identify secondary relationships that uncover connections among components that current computational approaches were unable to reveal. PMID:24196983

  19. Identifying Determinants of EGFR-Targeted Therapeutic Biochemical Efficacy Using Computational Modeling

    PubMed Central

    Monast, C S; Lazzara, M J

    2014-01-01

    We modeled cellular epidermal growth factor receptor (EGFR) tyrosine phosphorylation dynamics in the presence of receptor-targeting kinase inhibitors (e.g., gefitinib) or antibodies (e.g., cetuximab) to identify systematically the factors that contribute most to the ability of the therapeutics to antagonize EGFR phosphorylation, an effect we define here as biochemical efficacy. Our model identifies distinct processes as controlling gefitinib or cetuximab biochemical efficacy, suggests biochemical efficacy is favored in the presence of certain EGFR ligands, and suggests new drug design principles. For example, the model predicts that gefitinib biochemical efficacy is preferentially sensitive to perturbations in the activity of tyrosine phosphatases regulating EGFR, but that cetuximab biochemical efficacy is preferentially sensitive to perturbations in ligand binding. Our results highlight numerous other considerations that determine biochemical efficacy beyond those reflected by equilibrium affinities. By integrating these considerations, our model also predicts minimum therapeutic combination concentrations to maximally reduce receptor phosphorylation. PMID:25317724

  20. Characterization of Changes in Gene Expression and Biochemical Pathways at Low Levels of Benzene Exposure

    PubMed Central

    Thomas, Reuben; Hubbard, Alan E.; McHale, Cliona M.; Zhang, Luoping; Rappaport, Stephen M.; Lan, Qing; Rothman, Nathaniel; Vermeulen, Roel; Guyton, Kathryn Z.; Jinot, Jennifer; Sonawane, Babasaheb R.; Smith, Martyn T.

    2014-01-01

    Benzene, a ubiquitous environmental pollutant, causes acute myeloid leukemia (AML). Recently, through transcriptome profiling of peripheral blood mononuclear cells (PBMC), we reported dose-dependent effects of benzene exposure on gene expression and biochemical pathways in 83 workers exposed across four airborne concentration ranges (from <1 ppm to >10 ppm) compared with 42 subjects with non-workplace ambient exposure levels. Here, we further characterize these dose-dependent effects with continuous benzene exposure in all 125 study subjects. We estimated air benzene exposure levels in the 42 environmentally-exposed subjects from their unmetabolized urinary benzene levels. We used a novel non-parametric, data-adaptive model selection method to estimate the change with dose in the expression of each gene. We describe non-parametric approaches to model pathway responses and used these to estimate the dose responses of the AML pathway and 4 other pathways of interest. The response patterns of majority of genes as captured by mean estimates of the first and second principal components of the dose-response for the five pathways and the profiles of 6 AML pathway response-representative genes (identified by clustering) exhibited similar apparent supra-linear responses. Responses at or below 0.1 ppm benzene were observed for altered expression of AML pathway genes and CYP2E1. Together, these data show that benzene alters disease-relevant pathways and genes in a dose-dependent manner, with effects apparent at doses as low as 100 ppb in air. Studies with extensive exposure assessment of subjects exposed in the low-dose range between 10 ppb and 1 ppm are needed to confirm these findings. PMID:24786086

  1. Unraveling Biochemical Pathways Affected by Mitochondrial Dysfunctions Using Metabolomic Approaches

    PubMed Central

    Demine, Stéphane; Reddy, Nagabushana; Renard, Patricia; Raes, Martine; Arnould, Thierry

    2014-01-01

    Mitochondrial dysfunction(s) (MDs) can be defined as alterations in the mitochondria, including mitochondrial uncoupling, mitochondrial depolarization, inhibition of the mitochondrial respiratory chain, mitochondrial network fragmentation, mitochondrial or nuclear DNA mutations and the mitochondrial accumulation of protein aggregates. All these MDs are known to alter the capacity of ATP production and are observed in several pathological states/diseases, including cancer, obesity, muscle and neurological disorders. The induction of MDs can also alter the secretion of several metabolites, reactive oxygen species production and modify several cell-signalling pathways to resolve the mitochondrial dysfunction or ultimately trigger cell death. Many metabolites, such as fatty acids and derived compounds, could be secreted into the blood stream by cells suffering from mitochondrial alterations. In this review, we summarize how a mitochondrial uncoupling can modify metabolites, the signalling pathways and transcription factors involved in this process. We describe how to identify the causes or consequences of mitochondrial dysfunction using metabolomics (liquid and gas chromatography associated with mass spectrometry analysis, NMR spectroscopy) in the obesity and insulin resistance thematic. PMID:25257998

  2. A Computational Model for the Identification of Biochemical Pathways in the Krebs Cycle

    SciTech Connect

    Oliveira, Joseph S.; Bailey, Colin G.; Jones-Oliveira, Janet B.; Dixon, David A.; Gull, Dean W.; Chandler, Mary L.

    2003-03-01

    We have applied an algorithmic methodology which provably decomposes any complex network into a complete family of principal subcircuits to study the minimal circuits that describe the Krebs cycle. Every operational behavior that the network is capable of exhibiting can be represented by some combination of these principal subcircuits and this computational decomposition is linearly efficient. We have developed a computational model that can be applied to biochemical reaction systems which accurately renders pathways of such reactions via directed hypergraphs (Petri nets). We have applied the model to the citric acid cycle (Krebs cycle). The Krebs cycle, which oxidizes the acetyl group of acetyl CoA to CO2 and reduces NAD and FAD to NADH and FADH2 is a complex interacting set of nine subreaction networks. The Krebs cycle was selected because of its familiarity to the biological community and because it exhibits enough complexity to be interesting in order to introduce this novel analytic approach. This study validates the algorithmic methodology for the identification of significant biochemical signaling subcircuits, based solely upon the mathematical model and not upon prior biological knowledge. The utility of the algebraic-combinatorial model for identifying the complete set of biochemical subcircuits as a data set is demonstrated for this important metabolic process.

  3. A Biochemical Screen for Identification of Small-Molecule Regulators of the Wnt Pathway Using Xenopus Egg Extracts

    PubMed Central

    Thorne, Curtis A.; Lafleur, Bonnie; Lewis, Michelle; Hanson, Alison J.; Jernigan, Kristin K.; Weaver, David C.; Huppert, Kari A.; Chen, Tony W.; Wichadiit, Chonlarat; Cselenyi, Christopher S.; Tahinci, Emilios; Meyers, Kelly C.; Waskow, Emily; Orton, Darren; Salic, Adrian; Lee, Laura A.; Robbins, David J.; Huppert, Stacey S.; Lee, Ethan

    2013-01-01

    Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (?-catenin and Axin) in opposing fashion. We have now fused ?-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts. PMID:21859680

  4. Widespread flower color convergence in Solanaceae via alternate biochemical pathways.

    PubMed

    Ng, Julienne; Smith, Stacey D

    2016-01-01

    Phenotypic convergence is rampant throughout the tree of life. While recent studies have made significant progress in ascertaining the proximate mechanisms underlying convergent phenotypes, less is known about the frequency and predictability with which convergent phenotypes arise via the same or multiple pathways at the macroevolutionary scale. We investigated the proximate causes and evolutionary patterns of red flower color in the tomato family, Solanaceae, using large-scale data mining and new sequence data to reconstruct a megaphylogeny of 1341 species. We then combined spectral and anatomical data to assess how many times red flowers have evolved, the relative contribution of different pathways to independent origins of red, and whether the underlying pathway is predicted by phylogenetic relatedness. We estimated at least 30 relatively recent origins of red flowers using anthocyanins, carotenoids, or a dual production of both pigments, with significant phylogenetic signal in the use of anthocyanins and dual production, indicating that closely related red-flowered species tend to employ the same mechanism for coloration. Our study is the first to test whether developmental pathways exhibit phylogenetic signal and implies that historical contingency strongly influences the evolution of new phenotypes. PMID:26224118

  5. IDENTIFYING ESCHERICHIA SPECIES WITH BIOCHEMICAL TEST KITS AND STANDARD BACTERIOLOGICAL TESTS

    EPA Science Inventory

    Two commercially available biochemical test systems were evaluated for their ability to accurately identify speies of the genus Escherichia. Three laboratories participated in the study. The test kits did not always correctly identify species of Escherichia, but only once was a...

  6. Biochemical pathways analysis of microarray results: regulation of myogenesis in pigs

    PubMed Central

    te Pas, Marinus FW; Hulsegge, Ina; Coster, Albart; Pool, Marco H; Heuven, Henri H; Janss, Luc LG

    2007-01-01

    Background Combining microarray results and biological pathway information will add insight into biological processes. Pathway information is widely available in databases through the internet. Mammalian muscle formation has been previously studied using microarray technology in pigs because these animals are an interesting animal model for muscle formation due to selection for increased muscle mass. Results indicated regulation of the expression of genes involved in proliferation and differentiation of myoblasts, and energy metabolism. The aim of the present study was to analyse microarrays studying myogenesis in pigs. It was necessary to develop methods to search biochemical pathways databases. Results PERL scripts were developed that used the names of the genes on the microarray to search databases. Synonyms of gene names were added to the list by searching the Gene Ontology database. The KEGG database was searched for pathway information using this updated gene list. The KEGG database returned 88 pathways. Most genes were found in a single pathway, but others were found in up to seven pathways. Combining the pathways and the microarray information 21 pathways showed sufficient information content for further analysis. These pathways were related to regulation of several steps in myogenesis and energy metabolism. Pathways regulating myoblast proliferation and muscle fibre formation were described. Furthermore, two networks of pathways describing the formation of the myoblast cytoskeleton and regulation of the energy metabolism during myogenesis were presented. Conclusion Combining microarray results and pathways information available through the internet provide biological insight in how the process of porcine myogenesis is regulated. PMID:17567520

  7. Extending double modulation: combinatorial rules for identifying the modulations necessary for determining elasticities in metabolic pathways.

    PubMed

    Giersch, C; Cornish-Bowden, A

    1996-10-01

    The double modulation method for determining the elasticities of pathway enzymes, originally devised by Kacser & Burns (Biochem. Soc. Trans. 7, 1149-1160, 1979), is extended to pathways of complex topological structure, including branching and feedback loops. An explicit system of linear equations for the unknown elasticities is derived. The constraints imposed on this linear system imply that modulations of more than one enzyme are not necessarily independent. Simple combinatorial rules are described for identifying without using any algebra the set of independent modulations that allow the determination of the elasticities of any enzyme. By repeated application, the minimum numbers of modulations required to determine the elasticities of all enzymes of a given pathway can be determined. The procedure is illustrated with numerous examples. PMID:8944169

  8. The biochemical pathways of central nervous system neural degeneration in niacin deficiency

    PubMed Central

    Fu, Linshan; Doreswamy, Venkatesh; Prakash, Ravi

    2014-01-01

    Neural degeneration is a very complicated process. In spite of all the advancements in the molecular chemistry, there are many unknown aspects of the phenomena of neurodegeneration which need to be put together. It is a common sequela of the conditions of niacin deficiency. Neural degeneration in Pellagra manifests as chromatolysis mainly in pyramidal followed by other neurons and glial cells. However, there is a gross lack of understanding of biochemical mechanisms of neurodegeneration in niacin deficiency states. Because of the necessity of niacin or its amide derivative NAD in a number of biochemical pathways, it is understandable that several of these pathways may be involved in the common outcome of neural degeneration. Here, we highlight five pathways that could be involved in the neuraldegeneration for which evidence has accumulated through several studies. These pathways are: 1) the tryptophan-kyneurenic acid pathway, 2) the mitochondrial ATP generation related pathways, 3) the poly (ADP-ibose) polymerase (PARP) pathway, 4) the BDNF-TRKB Axis abnormalities, 5) the genetic influences of niacin deficiency. PMID:25317166

  9. Identifying Causal Genes and Dysregulated Pathways in Complex Diseases

    PubMed Central

    Kim, Yoo-Ah; Wuchty, Stefan; Przytycka, Teresa M.

    2011-01-01

    In complex diseases, various combinations of genomic perturbations often lead to the same phenotype. On a molecular level, combinations of genomic perturbations are assumed to dys-regulate the same cellular pathways. Such a pathway-centric perspective is fundamental to understanding the mechanisms of complex diseases and the identification of potential drug targets. In order to provide an integrated perspective on complex disease mechanisms, we developed a novel computational method to simultaneously identify causal genes and dys-regulated pathways. First, we identified a representative set of genes that are differentially expressed in cancer compared to non-tumor control cases. Assuming that disease-associated gene expression changes are caused by genomic alterations, we determined potential paths from such genomic causes to target genes through a network of molecular interactions. Applying our method to sets of genomic alterations and gene expression profiles of 158 Glioblastoma multiforme (GBM) patients we uncovered candidate causal genes and causal paths that are potentially responsible for the altered expression of disease genes. We discovered a set of putative causal genes that potentially play a role in the disease. Combining an expression Quantitative Trait Loci (eQTL) analysis with pathway information, our approach allowed us not only to identify potential causal genes but also to find intermediate nodes and pathways mediating the information flow between causal and target genes. Our results indicate that different genomic perturbations indeed dys-regulate the same functional pathways, supporting a pathway-centric perspective of cancer. While copy number alterations and gene expression data of glioblastoma patients provided opportunities to test our approach, our method can be applied to any disease system where genetic variations play a fundamental causal role. PMID:21390271

  10. Identifying Differentially Abundant Metabolic Pathways in Metagenomic Datasets

    NASA Astrophysics Data System (ADS)

    Liu, Bo; Pop, Mihai

    Enabled by rapid advances in sequencing technology, metagenomic studies aim to characterize entire communities of microbes bypassing the need for culturing individual bacterial members. One major goal of such studies is to identify specific functional adaptations of microbial communities to their habitats. Here we describe a powerful analytical method (MetaPath) that can identify differentially abundant pathways in metagenomic data-sets, relying on a combination of metagenomic sequence data and prior metabolic pathway knowledge. We show that MetaPath outperforms other common approaches when evaluated on simulated datasets. We also demonstrate the power of our methods in analyzing two, publicly available, metagenomic datasets: a comparison of the gut microbiome of obese and lean twins; and a comparison of the gut microbiome of infant and adult subjects. We demonstrate that the subpathways identified by our method provide valuable insights into the biological activities of the microbiome.

  11. A systems biology approach identifies the biochemical mechanisms regulating monoterpenoid essential oil composition in peppermint

    PubMed Central

    Rios-Estepa, Rigoberto; Turner, Glenn W.; Lee, James M.; Croteau, Rodney B.; Lange, B. Markus

    2008-01-01

    The integration of mathematical modeling and experimental testing is emerging as a powerful approach for improving our understanding of the regulation of metabolic pathways. In this study, we report on the development of a kinetic mathematical model that accurately simulates the developmental patterns of monoterpenoid essential oil accumulation in peppermint (Mentha piperita). This model was then used to evaluate the biochemical processes underlying experimentally determined changes in the monoterpene pathway under low ambient-light intensities, which led to an accumulation of the branchpoint intermediate (+)-pulegone and the side product (+)-menthofuran. Our simulations indicated that the environmentally regulated changes in monoterpene profiles could only be explained when, in addition to effects on biosynthetic enzyme activities, as yet unidentified inhibitory effects of (+)-menthofuran on the branchpoint enzyme pulegone reductase (PR) were assumed. Subsequent in vitro analyses with recombinant protein confirmed that (+)-menthofuran acts as a weak competitive inhibitor of PR (Ki = 300 ?M). To evaluate whether the intracellular concentration of (+)-menthofuran was high enough for PR inhibition in vivo, we isolated essential oil-synthesizing secretory cells from peppermint leaves and subjected them to steam distillations. When peppermint plants were grown under low-light conditions, (+)-menthofuran was selectively retained in secretory cells and accumulated to very high levels (up to 20 mM), whereas under regular growth conditions, (+)-menthofuran levels remained very low (<400 ?M). These results illustrate the utility of iterative cycles of mathematical modeling and experimental testing to elucidate the mechanisms controlling flux through metabolic pathways. PMID:18287058

  12. Chemical Shifts to Metabolic Pathways: Identifying Metabolic Pathways Directly from a Single 2D NMR Spectrum.

    PubMed

    Dubey, Abhinav; Rangarajan, Annapoorni; Pal, Debnath; Atreya, Hanudatta S

    2015-12-15

    Identifying cellular processes in terms of metabolic pathways is one of the avowed goals of metabolomics studies. Currently, this is done after relevant metabolites are identified to allow their mapping onto specific pathways. This task is daunting due to the complex nature of cellular processes and the difficulty in establishing the identity of individual metabolites. We propose here a new method: ChemSMP (Chemical Shifts to Metabolic Pathways), which facilitates rapid analysis by identifying the active metabolic pathways directly from chemical shifts obtained from a single two-dimensional (2D) [(13)C-(1)H] correlation NMR spectrum without the need for identification and assignment of individual metabolites. ChemSMP uses a novel indexing and scoring system comprised of a "uniqueness score" and a "coverage score". Our method is demonstrated on metabolic pathways data from the Small Molecule Pathway Database (SMPDB) and chemical shifts from the Human Metabolome Database (HMDB). Benchmarks show that ChemSMP has a positive prediction rate of >90% in the presence of decluttered data and can sustain the same at 60-70% even in the presence of noise, such as deletions of peaks and chemical shift deviations. The method tested on NMR data acquired for a mixture of 20 amino acids shows a success rate of 93% in correct recovery of pathways. When used on data obtained from the cell lysate of an unexplored oncogenic cell line, it revealed active metabolic pathways responsible for regulating energy homeostasis of cancer cells. Our unique tool is thus expected to significantly enhance analysis of NMR-based metabolomics data by reducing existing impediments. PMID:26556218

  13. Galactosemia: A strategy to identify new biochemical phenotypes and molecular genotypes

    SciTech Connect

    Elsas, L.J.; Langley, S.; Steele, E.; Evinger, J.; Brown, A.; Singh, R.; Fernhoff, P.; Hjelm, L.N.; Dembure, P.P.; Fridovich-Keil, J.L.

    1995-03-01

    We describe a stratagem for identifying new mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. GALT enzyme activity and isoforms were defined in erythrocytes from probands and their first-degree relatives. If the biochemical phenotypes segregated in an autosomal recesssive pattern, we screened for common mutations by using multiplex PCR and restriction endonuclease digestions. If common mutant alleles were not present, the 11 exons of the GALT gene were amplified by PCR, and variations from the normal nucleotide sequences were identified by SSCP. The suspected region(s) was then analyzed by direct DNA sequencing. We identified 86 mutant GALT alleles that reduced erythrocyte GALT activity. Seventy-five of these GALT genomes had abnormal SSCP patterns, of which 41 were sequenced, yielding 12 new and 21 previously reported, rare mutations. Among the novel group of 12 new mutations, an unusual biochemical phenotype was found in a family whose newborn proband has classical galactosemia. He had inherited two mutations in cis (N314D-E204K) from his father, whose GALT activity was near normal, and an additional GALT mutation in the splice-acceptor site of intron C (IVSC) from his mother. The substitution of a positively charged E204K mutation created a unique isoform-banding pattern. An asymptomatic sister`s GALT genes carries three mutations (E203K-N314D/N314D) with eight distinct isoform bands. Surprisingly, her erythrocytes have normal GALT activity. We conclude that the synergism of pedigree, biochemical, SSCP, and direct GALT gene analyses is an efficient protocol for identifying new mutations and speculate that E203K and N314D codon changes produce intra-allelic complementation when in cis. 40 refs., 4 figs., 3 tabs.

  14. Diversity of Bile Salts in Fish and Amphibians: Evolution of a Complex Biochemical Pathway

    PubMed Central

    Hagey, Lee R.; Møller, Peter R.; Hofmann, Alan F.; Krasowski, Matthew D.

    2010-01-01

    Bile salts are the major end-metabolites of cholesterol and are also important in lipid and protein digestion, as well as shaping of the gut microflora. Previous studies had demonstrated variation of bile salt structures across vertebrate species. We greatly extend prior surveys of bile salt variation in fish and amphibians, particularly in analysis of the biliary bile salts of Agnatha and Chondrichthyes. While there is significant structural variation of bile salts across all fish orders, bile salt profiles are generally stable within orders of fish and do not correlate with differences in diet. This large data set allowed us to infer evolutionary changes in the bile salt synthetic pathway. The hypothesized ancestral bile salt synthetic pathway, likely exemplified in extant hagfish, is simpler and much shorter than the pathway of most teleost fish and terrestrial vertebrates. Thus, the bile salt synthetic pathway has become longer and more complex throughout vertebrate evolution. Analysis of the evolution of bile salt synthetic pathways provides a rich model system for the molecular evolution of a complex biochemical pathway in vertebrates. PMID:20113173

  15. Invariant features of metabolic networks: a data analysis application on scaling properties of biochemical pathways

    NASA Astrophysics Data System (ADS)

    Giuliani, Alessandro; Zbilut, Joseph P.; Conti, Filippo; Manetti, Cesare; Miccheli, Alfredo

    2004-06-01

    The network metaphor is currently one of the most common general paradigms in biological sciences: this paradigm spans different scales of definition going from gene regulation to protein-protein interaction studies and metabolic regulation networks. Generally, the networks are defined by the nature of the connected elements (nodes) and their relative relations (edges). In this paper we demonstrate how the same biochemical regulation network can assume different shapes in terms of both constituting elements and intervening relations while remaining recognizable as a specific entity. This behaviour can be explained by the general scaling properties of biological networks and points to regulation pathways as emergent features of biochemical systems posited at a different hierarchical level with respect to the intervening metabolites.

  16. Fourier transform infrared spectroscopic imaging identifies early biochemical markers of tissue damage

    NASA Astrophysics Data System (ADS)

    Varma, Vishal K.; Ohlander, Samuel; Nguyen, Peter; Vendryes, Christopher; Parthiban, Sujeeth; Hamilton, Blake; Wallis, M. Chad; Kajdacsy-Balla, Andre; Hannaford, Blake; Lendvay, Thomas; Hotaling, James M.; Walsh, Michael J.

    2014-03-01

    Fourier Transform Infrared (FT-IR) spectroscopic imaging can allow for the rapid imaging of tissue biochemistry in a label-free and non-perturbing fashion. With the rapid adoption of new minimally invasive surgery (MIS) technologies over the last 20 years, adequate skill to safely and effectively use these technologies may not be achieved and risk of undue physical pressure being placed on tissues is a concern. Previous work has demonstrated that a number of histological stains can detect tissue damage, however, this process requires the initiation and progression of a signaling cascade that results in the epitope of interest being expressed. We proposed to identify the early biochemical markers associated with physical tissue damage from applied forces, thus not requiring transcriptional and translational protein synthesis as traditional immunohistochemistry does. To demonstrate that FT-IR can measure biochemical changes in tissues that have undergone physical force, we took ex-vivo lamb's liver that had been freshly excised and applied varying levels of physical pressure (0kPa to 30kPa). Tissues were then formalin-fixed, paraffin-embedded, and sectioned on to glass for H and E staining to identify damage and on to an IR slide for FT-IR imaging. Regions of interest containing hepatocytes were identified and average FT-IR spectra were extracted from the damaged and undamaged livers. FT-IR spectra showed clear biochemical changes associated with tissue damage. In addition, chemical changes could be observed proceeding histological changes observed when using conventional staining approaches.

  17. Molecular Factors and Biochemical Pathways Induced by Febrile Temperature in Intraerythrocytic Plasmodium falciparum Parasites▿ †

    PubMed Central

    Oakley, Miranda S. M.; Kumar, Sanjai; Anantharaman, Vivek; Zheng, Hong; Mahajan, Babita; Haynes, J. David; Moch, J. Kathleen; Fairhurst, Rick; McCutchan, Thomas F.; Aravind, L.

    2007-01-01

    Intermittent episodes of febrile illness are the most benign and recognized symptom of infection with malaria parasites, although the effects on parasite survival and virulence remain unclear. In this study, we identified the molecular factors altered in response to febrile temperature by measuring differential expression levels of individual genes using high-density oligonucleotide microarray technology and by performing biological assays in asexual-stage Plasmodium falciparum parasite cultures incubated at 37°C and 41°C (an elevated temperature that is equivalent to malaria-induced febrile illness in the host). Elevated temperature had a profound influence on expression of individual genes; 336 of approximately 5,300 genes (6.3% of the genome) had altered expression profiles. Of these, 163 genes (49%) were upregulated by twofold or greater, and 173 genes (51%) were downregulated by twofold or greater. In-depth sensitive sequence profile analysis revealed that febrile temperature-induced responses caused significant alterations in the major parasite biologic networks and pathways and that these changes are well coordinated and intricately linked. One of the most notable transcriptional changes occurs in genes encoding proteins containing the predicted Pexel motifs that are exported into the host cytoplasm or inserted into the host cell membrane and are likely to be associated with erythrocyte remodeling and parasite sequestration functions. Using our sensitive computational analysis, we were also able to assign biochemical or biologic functional predictions for at least 100 distinct genes previously annotated as “hypothetical.” We find that cultivation of P. falciparum parasites at 41°C leads to parasite death in a time-dependent manner. The presence of the “crisis forms” and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive parasites following heat treatment strongly support the notion that an apoptosis-like cell death mechanism might be induced in response to febrile temperatures. These studies enhance the possibility of designing vaccines and drugs on the basis of disruption in molecules and pathways of parasite survival and virulence activated in response to febrile temperatures. PMID:17283083

  18. Genome-Wide Pathway Analysis Identifies Genetic Pathways Associated with Psoriasis.

    PubMed

    Aterido, Adrià; Julià, Antonio; Ferrándiz, Carlos; Puig, Lluís; Fonseca, Eduardo; Fernández-López, Emilia; Dauden, Esteban; Sánchez-Carazo, José Luís; López-Estebaranz, José Luís; Moreno-Ramírez, David; Vanaclocha, Francisco; Herrera, Enrique; de la Cueva, Pablo; Dand, Nick; Palau, Núria; Alonso, Arnald; López-Lasanta, María; Tortosa, Raül; García-Montero, Andrés; Codó, Laia; Gelpí, Josep Lluís; Bertranpetit, Jaume; Absher, Devin; Capon, Francesca; Myers, Richard M; Barker, Jonathan N; Marsal, Sara

    2016-03-01

    Psoriasis is a chronic inflammatory disease with a complex genetic architecture. To date, the psoriasis heritability is only partially explained. However, there is increasing evidence that the missing heritability in psoriasis could be explained by multiple genetic variants of low effect size from common genetic pathways. The objective of this study was to identify new genetic variation associated with psoriasis risk at the pathway level. We genotyped 598,258 single nucleotide polymorphisms in a discovery cohort of 2,281 case-control individuals from Spain. We performed a genome-wide pathway analysis using 1,053 reference biological pathways. A total of 14 genetic pathways (PFDR ≤ 2.55 × 10(-2)) were found to be significantly associated with psoriasis risk. Using an independent validation cohort of 7,353 individuals from the UK, a total of 6 genetic pathways were significantly replicated (PFDR ≤ 3.46 × 10(-2)). We found genetic pathways that had not been previously associated with psoriasis risk such as retinol metabolism (Pcombined = 1.84 × 10(-4)), the transport of inorganic ions and amino acids (Pcombined = 1.57 × 10(-7)), and post-translational protein modification (Pcombined = 1.57 × 10(-7)). In the latter pathway, MGAT5 showed a strong network centrality, and its association with psoriasis risk was further validated in an additional case-control cohort of 3,429 individuals (P < 0.05). These findings provide insights into the biological mechanisms associated with psoriasis susceptibility. PMID:26743605

  19. Small RNA pathway genes identified by patterns of phylogenetic conservation and divergence

    PubMed Central

    Tabach, Yuval; Billi, Allison C.; Hayes, Gabriel D.; Newman, Martin A.; Zuk, Or; Gabel, Harrison; Kamath, Ravi; Yacoby, Keren; Chapman, Brad; Garcia, Susana M.; Borowsky, Mark; Kim, John K.; Ruvkun, Gary

    2013-01-01

    Genetic and biochemical analyses of RNA interference (RNAi) and microRNA (miRNA) pathways have revealed proteins such as Argonaute/PIWI and Dicer that process and present small RNAs to their targets. Well validated small RNA pathway cofactors, such as the Argonaute/PIWI proteins show distinctive patterns of conservation or divergence in particular animal, plant, fungal, and protist species. We compared 86 divergent eukaryotic genome sequences to discern sets of proteins that show similar phylogenetic profiles with known small RNA cofactors. A large set of additional candidate small RNA cofactors have emerged from functional genomic screens for defects in miRNA- or siRNA-mediated repression in C. elegans and D. melanogaster1,2 and from proteomic analyses of proteins co-purifying with validated small RNA pathway proteins3,4. The phylogenetic profiles of many of these candidate small RNA pathway proteins are similar to those of known small RNA cofactor proteins. We used a Bayesian approach to integrate the phylogenetic profile analysis with predictions from diverse transcriptional coregulation and proteome interaction datasets to assign a probability for each protein for a role in a small RNA pathway. Testing high-confidence candidates from this analysis for defects in RNAi silencing, we found that about half of the predicted small RNA cofactors are required for RNAi silencing. Many of the newly identified small RNA pathway proteins are orthologues of proteins implicated in RNA splicing. In support of a deep connection between the mechanism of RNA splicing and small RNA-mediated gene silencing, the presence of the Argonaute proteins and other small RNA components in the many species analysed strongly correlates with the number of introns in that species. PMID:23364702

  20. Extended CADLIVE: a novel graphical notation for design of biochemical network maps and computational pathway analysis.

    PubMed

    Kurata, Hiroyuki; Inoue, Kentaro; Maeda, Kazuhiro; Masaki, Koichi; Shimokawa, Yuki; Zhao, Quanyu

    2007-01-01

    Biochemical network maps are helpful for understanding the mechanism of how a collection of biochemical reactions generate particular functions within a cell. We developed a new and computationally feasible notation that enables drawing a wide resolution map from the domain-level reactions to phenomenological events and implemented it as the extended GUI network constructor of CADLIVE (Computer-Aided Design of LIVing systEms). The new notation presents 'Domain expansion' for proteins and RNAs, 'Virtual reaction and nodes' that are responsible for illustrating domain-based interaction and 'InnerLink' that links real complex nodes to virtual nodes to illustrate the exact components of the real complex. A modular box is also presented that packs related reactions as a module or a subnetwork, which gives CADLIVE a capability to draw biochemical maps in a hierarchical modular architecture. Furthermore, we developed a pathway search module for virtual knockout mutants as a built-in application of CADLIVE. This module analyzes gene function in the same way as molecular genetics, which simulates a change in mutant phenotypes or confirms the validity of the network map. The extended CADLIVE with the newly proposed notation is demonstrated to be feasible for computational simulation and analysis. PMID:17940089

  1. Signal duration and the time scale dependence of signal integration in biochemical pathways

    PubMed Central

    Locasale, Jason W

    2008-01-01

    Background Signal duration (e.g. the time over which an active signaling intermediate persists) is a key regulator of biological decisions in myriad contexts such as cell growth, proliferation, and developmental lineage commitments. Accompanying differences in signal duration are numerous downstream biological processes that require multiple steps of biochemical regulation. Results Here we present an analysis that investigates how simple biochemical motifs that involve multiple stages of regulation can be constructed to differentially process signals that persist at different time scales. We compute the dynamic, frequency dependent gain within these networks and resulting power spectra to better understand how biochemical networks can integrate signals at different time scales. We identify topological features of these networks that allow for different frequency dependent signal processing properties. Conclusion We show that multi-staged cascades are effective in integrating signals of long duration whereas multi-staged cascades that operate in the presence of negative feedback are effective in integrating signals of short duration. Our studies suggest principles for why signal duration in connection with multiple steps of downstream regulation is a ubiquitous motif in biochemical systems. PMID:19091071

  2. KNOWLEDGE-ASSISTED APPROACH TO IDENTIFY PATHWAYS WITH DIFFERENTIAL DEPENDENCIES*

    PubMed Central

    Speyer, Gil; Kiefer, Jeff; Dhruv, Harshil; Berens, Michael; Kim, Seungchan

    2015-01-01

    We have previously developed a statistical method to identify gene sets enriched with condition-specific genetic dependencies. The method constructs gene dependency networks from bootstrapped samples in one condition and computes the divergence between distributions of network likelihood scores from different conditions. It was shown to be capable of sensitive and specific identification of pathways with phenotype-specific dysregulation, i.e., rewiring of dependencies between genes in different conditions. We now present an extension of the method by incorporating prior knowledge into the inference of networks. The degree of prior knowledge incorporation has substantial effect on the sensitivity of the method, as the data is the source of condition specificity while prior knowledge incorporation can provide additional support for dependencies that are only partially supported by the data. Use of prior knowledge also significantly improved the interpretability of the results. Further analysis of topological characteristics of gene differential dependency networks provides a new approach to identify genes that could play important roles in biological signaling in a specific condition, hence, promising targets customized to a specific condition. Through analysis of TCGA glioblastoma multiforme data, we demonstrate the method can identify not only potentially promising targets but also underlying biology for new targets. PMID:26776171

  3. Pathway Analysis of Smoking Quantity in Multiple GWAS Identifies Cholinergic and Sensory Pathways

    PubMed Central

    Harari, Oscar; Wang, Jen-Chyong; Bucholz, Kathleen; Edenberg, Howard J.; Heath, Andrew; Martin, Nicholas G.; Pergadia, Michele L.; Montgomery, Grant; Schrage, Andrew; Bierut, Laura J.; Madden, Pamela F.; Goate, Alison M.

    2012-01-01

    Cigarette smoking is a common addiction that increases the risk for many diseases, including lung cancer and chronic obstructive pulmonary disease. Genome-wide association studies (GWAS) have successfully identified and validated several susceptibility loci for nicotine consumption and dependence. However, the trait variance explained by these genes is only a small fraction of the estimated genetic risk. Pathway analysis complements single marker methods by including biological knowledge into the evaluation of GWAS, under the assumption that causal variants lie in functionally related genes, enabling the evaluation of a broad range of signals. Our approach to the identification of pathways enriched for multiple genes associated with smoking quantity includes the analysis of two studies and the replication of common findings in a third dataset. This study identified pathways for the cholinergic receptors, which included SNPs known to be genome-wide significant; as well as novel pathways, such as genes involved in the sensory perception of smell, that do not contain any single SNP that achieves that stringent threshold. PMID:23227220

  4. Oxidation state, bioavailability & biochemical pathway define the fate of carbon in soil

    NASA Astrophysics Data System (ADS)

    Kuzyakov, Yakov; Apostel, Carolin; Gunina, Anna; Herrmann, Anke M.; Dippold, Michaela

    2015-04-01

    Numerous experiments under laboratory and field conditions analyzed microbial utilization and mean residence time (MRT) of carbon (C) from plant and microbial residues as well as root exudates in soil. Most of these studies tested the effects of various environmental factors, such as temperature, soil moisture, texture etc. on these parameters. However, only a few studies compared the properties of the substances themselves and there is no conceptual framework based on biochemical pathways. We hypothesize that the fate of C from organic substances in soil strongly depends on the first step of their microbial utilization, specifically, on biochemical pathway and initial C oxidation state, as well as its bioavailability in soils, defined by its hydrophobicity and molecular weight. Here we introduce and evaluate a new conceptual framework based on the following parameters: 1) C oxidation state, 2) molecular weight and hydrophobicity, 3) initial biochemical pathway of a substance class in microbial cells. To assess these parameters, two databases were prepared based on the literature and own studies. The first database included only the studies with 14C or 13C position specific labeled sugars, amino acids, carboxylic acids, phenols and lipids in soil. This database allowed us to analyze microbial utilization and mineralization of organics to CO2 depending on their C oxidation state (OS) and on functional groups. Additionally, we calculated data on the bond electronegativity of all compounds investigated in these studies. The second data base included the results of 14C and 13C studies with uniformly labeled substances of various classes. This database considered the free enthalpie (Delta H) per C unit from a variety of substrates differing in their aromaticity, hydrophobicity/electronegativity and location of the substance on the van Krevelen diagram. In addition, we calculated the hydrophobicity from the electronegativity of the individual bonds and recorded their molecular weight in our databases. For both data bases the decomposition rates and the MRT of C remaining in soil were calculated by the double first-order kinetics and related to the four parameter groups. The first database showed high correlation of mineralization rates to CO2 with the C oxidation state and biochemical pathway. Carboxyl group (OS = +3) was split at first from the skeleton of nearly all substances. In contrast, the methyl group (OS = -3) was mineralized as the slowest and after incorporation into microbial cells remained the longest period in soil. This general pattern reflects a clear preferential oxidation of already highly oxidized, polar functional groups. The initial use of substances within glucolysis (e.g. sugars) lead to a higher portion of remaining C in soil compared to C introduced via citric acid cycle (e.g. carboxylic acids). Concerning substance groups, the mineralization rates were the fastest for amino acids and sugars and the slowest for of the lipids - corresponding to their molecular weight and hydropobicity. This corresponded well with localization of the substance classes on the van Krevelen diagram. Generally, high oxidation state of the initial substance and consequently its low free enthalpy content lead to faster decomposition. In contrast, low oxidation state (e.g. lipids, aromatics) corresponds to high hydrophobicity and so, slow uptake from soil solution and utilization within microbial cells. Consequently, the optimum for microbial biomass utilization in soil and use for anabolic processes is common for sugars that have the oxidation state close to 0, have medium energy content and are hydrophilic. We conclude that from the tested substance properties, the oxidation state and biochemical pathway explained well the initial fate of C in soil, i.e. its mineralization to CO2 and incorporation into microbial biomass. Because the first step and microbial cycling are crucial for its further transformations, the same criteria are pivotal for C stabilization in soil.

  5. Identification of Genetic Bases of Vibrio fluvialis Species-Specific Biochemical Pathways and Potential Virulence Factors by Comparative Genomic Analysis

    PubMed Central

    Lu, Xin; Liang, Weili; Wang, Yunduan; Xu, Jialiang

    2014-01-01

    Vibrio fluvialis is an important food-borne pathogen that causes diarrheal illness and sometimes extraintestinal infections in humans. In this study, we sequenced the genome of a clinical V. fluvialis strain and determined its phylogenetic relationships with other Vibrio species by comparative genomic analysis. We found that the closest relationship was between V. fluvialis and V. furnissii, followed by those with V. cholerae and V. mimicus. Moreover, based on genome comparisons and gene complementation experiments, we revealed genetic mechanisms of the biochemical tests that differentiate V. fluvialis from closely related species. Importantly, we identified a variety of genes encoding potential virulence factors, including multiple hemolysins, transcriptional regulators, and environmental survival and adaptation apparatuses, and the type VI secretion system, which is indicative of complex regulatory pathways modulating pathogenesis in this organism. The availability of V. fluvialis genome sequences may promote our understanding of pathogenic mechanisms for this emerging pathogen. PMID:24441165

  6. MetaMapp: mapping and visualizing metabolomic data by integrating information from biochemical pathways and chemical and mass spectral similarity

    PubMed Central

    2012-01-01

    Background Exposure to environmental tobacco smoke (ETS) leads to higher rates of pulmonary diseases and infections in children. To study the biochemical changes that may precede lung diseases, metabolomic effects on fetal and maternal lungs and plasma from rats exposed to ETS were compared to filtered air control animals. Genome- reconstructed metabolic pathways may be used to map and interpret dysregulation in metabolic networks. However, mass spectrometry-based non-targeted metabolomics datasets often comprise many metabolites for which links to enzymatic reactions have not yet been reported. Hence, network visualizations that rely on current biochemical databases are incomplete and also fail to visualize novel, structurally unidentified metabolites. Results We present a novel approach to integrate biochemical pathway and chemical relationships to map all detected metabolites in network graphs (MetaMapp) using KEGG reactant pair database, Tanimoto chemical and NIST mass spectral similarity scores. In fetal and maternal lungs, and in maternal blood plasma from pregnant rats exposed to environmental tobacco smoke (ETS), 459 unique metabolites comprising 179 structurally identified compounds were detected by gas chromatography time of flight mass spectrometry (GC-TOF MS) and BinBase data processing. MetaMapp graphs in Cytoscape showed much clearer metabolic modularity and complete content visualization compared to conventional biochemical mapping approaches. Cytoscape visualization of differential statistics results using these graphs showed that overall, fetal lung metabolism was more impaired than lungs and blood metabolism in dams. Fetuses from ETS-exposed dams expressed lower lipid and nucleotide levels and higher amounts of energy metabolism intermediates than control animals, indicating lower biosynthetic rates of metabolites for cell division, structural proteins and lipids that are critical for in lung development. Conclusions MetaMapp graphs efficiently visualizes mass spectrometry based metabolomics datasets as network graphs in Cytoscape, and highlights metabolic alterations that can be associated with higher rate of pulmonary diseases and infections in children prenatally exposed to ETS. The MetaMapp scripts can be accessed at http://metamapp.fiehnlab.ucdavis.edu. PMID:22591066

  7. Least-squares methods for identifying biochemical regulatory networks from noisy measurements

    PubMed Central

    Kim, Jongrae; Bates, Declan G; Postlethwaite, Ian; Heslop-Harrison, Pat; Cho, Kwang-Hyun

    2007-01-01

    Background We consider the problem of identifying the dynamic interactions in biochemical networks from noisy experimental data. Typically, approaches for solving this problem make use of an estimation algorithm such as the well-known linear Least-Squares (LS) estimation technique. We demonstrate that when time-series measurements are corrupted by white noise and/or drift noise, more accurate and reliable identification of network interactions can be achieved by employing an estimation algorithm known as Constrained Total Least Squares (CTLS). The Total Least Squares (TLS) technique is a generalised least squares method to solve an overdetermined set of equations whose coefficients are noisy. The CTLS is a natural extension of TLS to the case where the noise components of the coefficients are correlated, as is usually the case with time-series measurements of concentrations and expression profiles in gene networks. Results The superior performance of the CTLS method in identifying network interactions is demonstrated on three examples: a genetic network containing four genes, a network describing p53 activity and mdm2 messenger RNA interactions, and a recently proposed kinetic model for interleukin (IL)-6 and (IL)-12b messenger RNA expression as a function of ATF3 and NF-?B promoter binding. For the first example, the CTLS significantly reduces the errors in the estimation of the Jacobian for the gene network. For the second, the CTLS reduces the errors from the measurements that are corrupted by white noise and the effect of neglected kinetics. For the third, it allows the correct identification, from noisy data, of the negative regulation of (IL)-6 and (IL)-12b by ATF3. Conclusion The significant improvements in performance demonstrated by the CTLS method under the wide range of conditions tested here, including different levels and types of measurement noise and different numbers of data points, suggests that its application will enable more accurate and reliable identification and modelling of biochemical networks. PMID:17212835

  8. Genetic and biochemical analysis of the twin-arginine translocation pathway in halophilic archaea.

    PubMed

    Dilks, Kieran; Gimnez, Mara Ins; Pohlschrder, Mechthild

    2005-12-01

    The twin-arginine translocation (Tat) pathway is present in a wide variety of prokaryotes and is capable of exporting partially or fully folded proteins from the cytoplasm. Although diverse classes of proteins are transported via the Tat pathway, in most organisms it facilitates the secretion of a relatively small number of substrates compared to the Sec pathway. However, computational evidence suggests that haloarchaea route nearly all secreted proteins to the Tat pathway. We have expanded previous computational analyses of the haloarchaeal Tat pathway and initiated in vivo characterization of the Tat machinery in a model haloarchaeon, Haloferax volcanii. Consistent with the predicted usage of the this pathway in the haloarchaea, we determined that three of the four identified tat genes in Haloferax volcanii are essential for viability when grown aerobically in complex medium. This represents the first report of an organism that requires the Tat pathway for viability when grown under such conditions. Deletion of the nonessential gene had no effect on the secretion of a verified substrate of the Tat pathway. The two TatA paralogs TatAo and TatAt were detected in both the membrane and cytoplasm and could be copurified from the latter fraction. Using size exclusion chromatography to further characterize cytoplasmic and membrane TatA proteins, we find these proteins present in high-molecular-weight complexes in both cellular fractions. PMID:16291683

  9. Path2Models: large-scale generation of computational models from biochemical pathway maps

    PubMed Central

    2013-01-01

    Background Systems biology projects and omics technologies have led to a growing number of biochemical pathway models and reconstructions. However, the majority of these models are still created de novo, based on literature mining and the manual processing of pathway data. Results To increase the efficiency of model creation, the Path2Models project has automatically generated mathematical models from pathway representations using a suite of freely available software. Data sources include KEGG, BioCarta, MetaCyc and SABIO-RK. Depending on the source data, three types of models are provided: kinetic, logical and constraint-based. Models from over 2 600 organisms are encoded consistently in SBML, and are made freely available through BioModels Database at http://www.ebi.ac.uk/biomodels-main/path2models. Each model contains the list of participants, their interactions, the relevant mathematical constructs, and initial parameter values. Most models are also available as easy-to-understand graphical SBGN maps. Conclusions To date, the project has resulted in more than 140 000 freely available models. Such a resource can tremendously accelerate the development of mathematical models by providing initial starting models for simulation and analysis, which can be subsequently curated and further parameterized. PMID:24180668

  10. Harnessing Intracellular Biochemical Pathways for In Vitro Synthesis of Designer Tellurium Nanorods.

    PubMed

    Xiong, Ling-Hong; Cui, Ran; Zhang, Zhi-Ling; Tu, Jia-Wei; Shi, Yun-Bo; Pang, Dai-Wen

    2015-10-28

    Synthesizing nanomaterials of desired properties is a big challenge, which requires extremely harsh conditions and/or use of toxic materials. More recently developed in vivo methods have brought a different set of problems such as separation and purification of nanomaterials made in vivo. Here, a novel approach that harnesses cellular pathways for in vitro synthesis of high-quality tellurium nanorods with tunable lengths and optical properties is reported. It is first demonstrated that in vivo biochemical pathways could be used to synthesize Te nanorods via the intracellular reduction of TeO3(2-) in living Staphylococcus aureus cells. The pathways to set up a quasi-biological system for Te precursor formation are then utilized, which could further synthesize Te nanorods in vitro. This allows to successfully synthesize in vitro, under routine laboratory conditions, Te nanorods with uniform and tunable lengths, ranging from about 10 to 200 nm, and controllable optical properties with high molar extinction coefficients. The approach here should open new avenues for controllable, facile, and efficient synthesis of designer nanomaterials for diverse industrial and biomedical applications. PMID:26313741

  11. Using String Metrics to Identify Patient Journeys through Care Pathways

    PubMed Central

    Williams, Richard; Buchan, Iain E.; Prosperi, Mattia; Ainsworth, John

    2014-01-01

    Given a computerized representation of a care pathway and an electronic record of a patients clinical journey, with potential omissions, insertions, discontinuities and reordering, we show that we can accurately match the journey to a particular route through the pathway by converting the problem into a string matching one. We discover that normalized string metrics lead to more unique pathway matches than non-normalized string metrics and should therefore be given preference when using these techniques. PMID:25954432

  12. Prediction and Biochemical Demonstration of a Catabolic Pathway for the Osmoprotectant Proline Betaine

    PubMed Central

    Kumar, Ritesh; Zhao, Suwen; Vetting, Matthew W.; Wood, B. McKay; Sakai, Ayano; Cho, Kyuil; Solbiati, Jos; Almo, Steven C.; Sweedler, Jonathan V.; Jacobson, Matthew P.; Gerlt, John A.; Cronan, John E.

    2014-01-01

    ABSTRACT Through the use of genetic, enzymatic, metabolomic, and structural analyses, we have discovered the catabolic pathway for proline betaine, an osmoprotectant, in Paracoccus denitrificans and Rhodobacter sphaeroides. Genetic and enzymatic analyses showed that several of the key enzymes of the hydroxyproline betaine degradation pathway also function in proline betaine degradation. Metabolomic analyses detected each of the metabolic intermediates of the pathway. The proline betaine catabolic pathway was repressed by osmotic stress and cold stress, and a regulatory transcription factor was identified. We also report crystal structure complexes of the P. denitrificans HpbD hydroxyproline betaine epimerase/proline betaine racemase with l-proline betaine and cis-hydroxyproline betaine. PMID:24520058

  13. Serum Metabolomic Profiling in Acute Alcoholic Hepatitis Identifies Multiple Dysregulated Pathways

    PubMed Central

    Rachakonda, Vikrant; Gabbert, Charles; Raina, Amit; Bell, Lauren N.; Cooper, Sara; Malik, Shahid; Behari, Jaideep

    2014-01-01

    Background and Objectives While animal studies have implicated derangements of global energy homeostasis in the pathogenesis of acute alcoholic hepatitis (AAH), the relevance of these findings to the development of human AAH remains unclear. Using global, unbiased serum metabolomics analysis, we sought to characterize alterations in metabolic pathways associated with severe AAH and identify potential biomarkers for disease prognosis. Methods This prospective, case-control study design included 25 patients with severe AAH and 25 ambulatory patients with alcoholic cirrhosis. Serum samples were collected within 24 hours of the index clinical encounter. Global, unbiased metabolomics profiling was performed. Patients were followed for 180 days after enrollment to determine survival. Results Levels of 234 biochemicals were altered in subjects with severe AAH. Random-forest analysis, principal component analysis, and integrated hierarchical clustering methods demonstrated that metabolomics profiles separated the two cohorts with 100% accuracy. Severe AAH was associated with enhanced triglyceride lipolysis, impaired mitochondrial fatty acid beta oxidation, and upregulated omega oxidation. Low levels of multiple lysolipids and related metabolites suggested decreased plasma membrane remodeling in severe AAH. While most measured bile acids were increased in severe AAH, low deoxycholate and glycodeoxycholate levels indicated intestinal dysbiosis. Several changes in substrate utilization for energy homeostasis were identified in severe AAH, including increased glucose consumption by the pentose phosphate pathway, altered tricarboxylic acid (TCA) cycle activity, and enhanced peptide catabolism. Finally, altered levels of small molecules related to glutathione metabolism and antioxidant vitamin depletion were observed in patients with severe AAH. Univariable logistic regression revealed 15 metabolites associated with 180-day survival in severe AAH. Conclusion Severe AAH is characterized by a distinct metabolic phenotype spanning multiple pathways. Metabolomics profiling revealed a panel of biomarkers for disease prognosis, and future studies are planned to validate these findings in larger cohorts of patients with severe AAH. PMID:25461442

  14. Identifying Breeding Priorities for Blueberry Flavor Using Biochemical, Sensory, and Genotype by Environment Analyses

    PubMed Central

    Gilbert, Jessica L.; Guthart, Matthew J.; Gezan, Salvador A.; Pisaroglo de Carvalho, Melissa; Schwieterman, Michael L.; Colquhoun, Thomas A.; Bartoshuk, Linda M.; Sims, Charles A.; Clark, David G.; Olmstead, James W.

    2015-01-01

    Breeding for a subjective goal such as flavor is challenging, as many blueberry cultivars are grown worldwide, and identifying breeding targets relating to blueberry flavor biochemistry that have a high degree of genetic control and low environmental variability are priorities. A variety of biochemical compounds and physical characters induce the sensory responses of taste, olfaction, and somatosensation, all of which interact to create what is perceived flavor. The goal of this study was to identify the flavor compounds with a larger genetic versus environmental component regulating their expression over an array of cultivars, locations, and years. Over the course of three years, consumer panelists rated overall liking, texture, sweetness, sourness, and flavor intensity of 19 southern highbush blueberry (Vaccinium corymbosum hybrids) genotypes in 30 sensory panels. Significant positive correlations to overall liking of blueberry fruit (P<0.001) were found with sweetness (R2 = 0.70), texture (R2 = 0.68), and flavor (R2 = 0.63). Sourness had a significantly negative relationship with overall liking (R2 = 0.55). The relationship between flavor and texture liking was also linear (R2 = 0.73, P<0.0001) demonstrating interaction between olfaction and somatosensation. Partial least squares analysis was used to identify sugars, acids, and volatile compounds contributing to liking and sensory intensities, and revealed strong effects of fructose, pH, and several volatile compounds upon all sensory parameters measured. To assess the feasibility of breeding for flavor components, a three year study was conducted to compare genetic and environmental influences on flavor biochemistry. Panelists could discern genotypic variation in blueberry sensory components, and many of the compounds affecting consumer favor of blueberries, such as fructose, pH, β-caryophyllene oxide and 2-heptanone, were sufficiently genetically controlled that allocating resources for their breeding is worthwhile. PMID:26378911

  15. Identifying Breeding Priorities for Blueberry Flavor Using Biochemical, Sensory, and Genotype by Environment Analyses.

    PubMed

    Gilbert, Jessica L; Guthart, Matthew J; Gezan, Salvador A; Pisaroglo de Carvalho, Melissa; Schwieterman, Michael L; Colquhoun, Thomas A; Bartoshuk, Linda M; Sims, Charles A; Clark, David G; Olmstead, James W

    2015-01-01

    Breeding for a subjective goal such as flavor is challenging, as many blueberry cultivars are grown worldwide, and identifying breeding targets relating to blueberry flavor biochemistry that have a high degree of genetic control and low environmental variability are priorities. A variety of biochemical compounds and physical characters induce the sensory responses of taste, olfaction, and somatosensation, all of which interact to create what is perceived flavor. The goal of this study was to identify the flavor compounds with a larger genetic versus environmental component regulating their expression over an array of cultivars, locations, and years. Over the course of three years, consumer panelists rated overall liking, texture, sweetness, sourness, and flavor intensity of 19 southern highbush blueberry (Vaccinium corymbosum hybrids) genotypes in 30 sensory panels. Significant positive correlations to overall liking of blueberry fruit (P<0.001) were found with sweetness (R2 = 0.70), texture (R2 = 0.68), and flavor (R2 = 0.63). Sourness had a significantly negative relationship with overall liking (R2 = 0.55). The relationship between flavor and texture liking was also linear (R2 = 0.73, P<0.0001) demonstrating interaction between olfaction and somatosensation. Partial least squares analysis was used to identify sugars, acids, and volatile compounds contributing to liking and sensory intensities, and revealed strong effects of fructose, pH, and several volatile compounds upon all sensory parameters measured. To assess the feasibility of breeding for flavor components, a three year study was conducted to compare genetic and environmental influences on flavor biochemistry. Panelists could discern genotypic variation in blueberry sensory components, and many of the compounds affecting consumer favor of blueberries, such as fructose, pH, ?-caryophyllene oxide and 2-heptanone, were sufficiently genetically controlled that allocating resources for their breeding is worthwhile. PMID:26378911

  16. Resveratrol suppresses interferon-gamma-induced biochemical pathways in human peripheral blood mononuclear cells in vitro.

    PubMed

    Wirleitner, Barbara; Schroecksnadel, Katharina; Winkler, Christiana; Schennach, Harald; Fuchs, Dietmar

    2005-09-15

    A wide range of biological activities of resveratrol (3,5,4'-trihydroxystilbene) in vitro and in vivo has been proved, including antioxidant, antitumor, and also anti-inflammatory effects. Resveratrol found in, e.g., grapes and red wine has been suggested to counteract the progression of coronary heart disease by lowering serum lipid concentrations and inhibiting platelet aggregation. Cellular immune activation is known to be involved crucially in the pathogenesis of coronary heart diseases. In this in vitro study, the modulatory effect of resveratrol on two interferon-gamma-mediated pathways, the degradation of tryptophan by the enzyme indoleamine 2,3-dioxygenase, and the production of neopterin by activation of the GTP-cyclohydrolase I, was tested. Cultures of human peripheral blood mononuclear cells were exposed to resveratrol, in combination with mitogenic stimulation. A significant down-regulatory effect of resveratrol on both biochemical pathways was found, and also the production of Th1-type cytokine interferon-gamma was significantly suppressed. If these results can be verified in vivo, an explanation is provided how resveratrol may interfere with immune activation and cytokine cascades, which are important in the development and progression of cardiovascular disorders and also other diseases. PMID:16154495

  17. Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology (edited by Gerhard Michal)

    NASA Astrophysics Data System (ADS)

    Voige, Reviewed By William H.

    2000-02-01

    For decades, a wall chart detailing living organisms' metabolic pathways has been a fixture in many classrooms and laboratories where biochemistry is taught. One of the most popular of those charts first appeared 30 years ago. Now its editor, Gerhard Michal, has produced a book that summarizes metabolism (broadly defined) in graphical and textual formats. The book retains the elegance of the chart. Names of molecules are printed in a crisp, easy-to-read font, and structural formulas are shown with exemplary clarity. Color coding serves multiple purposes: to differentiate enzymes, substrates, cofactors, and effector molecules; to indicate in which group or groups of organisms a reaction has been observed; and to distinguish enzymatic reactions from regulatory effects. The primary advantage of presenting this information in book format is immediately apparent. A typical metabolic chart covers about 2 m2; the book has a total surface area nearly 10 times greater. The extra space is used to add explanatory text to the figures and to include many topics not covered by the traditional definition of metabolism. Examples include replication, transcription, translation, reaction mechanisms for proteolytic enzymes, and the role of chaperones in protein folding. Illustrating these topics is not as straightforward as delineating a metabolic pathway, but the author has done an admirable job of designing figures that clarify these and other aspects of biochemistry and complement the accompanying text. A potential deficiency of book format is the inability to clearly show links between different realms of metabolism: carbohydrate and amino acid pathways, for example. The book overcomes this problem in two ways. A diagrammatic overview of metabolism (with references to applicable sections of the book) is printed inside its front cover, and key compounds (pyruvate, for example) have a distinctive green background to provide a visual link between pathways. (The author compares this feature to the hyperlinks in an electronic document.) The book's index is comprehensive and useful. Entries for "phenylketonuria" and "sickle cell anemia", for example, lead to commendably concise summaries of these hereditary diseases (and the relevant metabolic pathway, in the former case). Looking up a specific molecule, however, is less helpful. The listing for fumarate hydratase, a citric acid cycle enzyme, directs the reader to the chapter on special bacterial metabolism but not to the section on the citric acid cycle itself. Literature references are included at the end of each section and are mainly from the 1990s, but they could be more useful. A long section on heme proteins, for example, concludes with eight citations, but their titles are not included, so it is impossible to determine what topic each one addresses. This book will be most useful to those with a good understanding of the fundamentals of biochemistry. Some of the information it presents could easily confuse less experienced readers. For example, it classifies selenocysteine as a standard amino acid in a figure but not in the accompanying text. In the diagram of anaerobic glycolysis, a double-headed arrow for the hexokinase reaction reinforces the frustratingly common student misperception that the phosphoryl group of glucose-6-phosphate can be used to phosphorylate ADP. Biochemical Pathways compiles a large amount of information in a single source. Its good index and clear, concise text and diagrams should make it a reliable way of gaining insight into many biochemical topics. With a price similar to that of most textbooks, it merits a place in the libraries of individuals and academic departments that teach biochemistry.

  18. A method for integrating and ranking the evidence for biochemical pathways by mining reactions from text

    PubMed Central

    Miwa, Makoto; Ohta, Tomoko; Rak, Rafal; Rowley, Andrew; Kell, Douglas B.; Pyysalo, Sampo; Ananiadou, Sophia

    2013-01-01

    Motivation: To create, verify and maintain pathway models, curators must discover and assess knowledge distributed over the vast body of biological literature. Methods supporting these tasks must understand both the pathway model representations and the natural language in the literature. These methods should identify and order documents by relevance to any given pathway reaction. No existing system has addressed all aspects of this challenge. Method: We present novel methods for associating pathway model reactions with relevant publications. Our approach extracts the reactions directly from the models and then turns them into queries for three text mining-based MEDLINE literature search systems. These queries are executed, and the resulting documents are combined and ranked according to their relevance to the reactions of interest. We manually annotate document-reaction pairs with the relevance of the document to the reaction and use this annotation to study several ranking methods, using various heuristic and machine-learning approaches. Results: Our evaluation shows that the annotated document-reaction pairs can be used to create a rule-based document ranking system, and that machine learning can be used to rank documents by their relevance to pathway reactions. We find that a Support Vector Machine-based system outperforms several baselines and matches the performance of the rule-based system. The success of the query extraction and ranking methods are used to update our existing pathway search system, PathText. Availability: An online demonstration of PathText 2 and the annotated corpus are available for research purposes at http://www.nactem.ac.uk/pathtext2/. Contact: makoto.miwa@manchester.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23813008

  19. Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in Flavonoid Biosynthetic Pathway

    PubMed Central

    Sun, Wei; Meng, Xiangyu; Liang, Lingjie; Jiang, Wangshu; Huang, Yafei; He, Jing; Hu, Haiyan; Almqvist, Jonas; Gao, Xiang; Wang, Li

    2015-01-01

    Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants. PMID:25742495

  20. Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

    PubMed

    Sun, Wei; Meng, Xiangyu; Liang, Lingjie; Jiang, Wangshu; Huang, Yafei; He, Jing; Hu, Haiyan; Almqvist, Jonas; Gao, Xiang; Wang, Li

    2015-01-01

    Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants. PMID:25742495

  1. Biochemical Modulation of Lipid Pathway in Microalgae Dunaliella sp. for Biodiesel Production

    PubMed Central

    Talebi, Ahmad Farhad; Tohidfar, Masoud; Mousavi Derazmahalleh, Seyedeh Mahsa; Sulaiman, Alawi; Baharuddin, Azhari Samsu; Tabatabaei, Meisam

    2015-01-01

    Exploitation of renewable sources of energy such as algal biodiesel could turn energy supplies problem around. Studies on a locally isolated strain of Dunaliella sp. showed that the mean lipid content in cultures enriched by 200 mg L−1 myoinositol was raised by around 33% (1.5 times higher than the control). Similarly, higher lipid productivity values were achieved in cultures treated by 100 and 200 mg L−1 myoinositol. Fluorometry analyses (microplate fluorescence and flow cytometry) revealed increased oil accumulation in the Nile red-stained algal samples. Moreover, it was predicted that biodiesel produced from myoinositol-treated cells possessed improved oxidative stability, cetane number, and cloud point values. From the genomic point of view, real-time analyses revealed that myoinositol negatively influenced transcript abundance of AccD gene (one of the key genes involved in lipid production pathway) due to feedback inhibition and that its positive effect must have been exerted through other genes. The findings of the current research are not to interprete that myoinositol supplementation could answer all the challenges faced in microalgal biodiesel production but instead to show that “there is a there there” for biochemical modulation strategies, which we achieved, increased algal oil quantity and enhanced resultant biodiesel quality. PMID:26146623

  2. Characterization of the biochemical-pathway of uranium (VI) reduction in facultative anaerobic bacteria.

    PubMed

    Mtimunye, Phalazane J; Chirwa, Evans M N

    2014-10-01

    Cultures of U(VI) reducing bacteria sourced from abandoned uranium mine tailing dam were evaluated for their ability to reduce U(VI) to U(IV). The species in the cultures reduced U(VI) in solutions with initial U(VI) concentration up to 400mgL(-)(1) under a near neutral pH of 6.5. The electron flow pathway and fate of reduced species was also analysed in the individual species in order to evaluate the potential for control and optimisation of the reduction potential at the biochemical level. The results showed that U(VI) reduction in live cells was completely blocked by the NADH-dehydrogenase inhibitor, rotenone (C23H22O6), and thioredoxin inhibitor, cadmium chloride (CdCl2), showing that U(VI) reduction involves the electron flow through NADH-dehydrogenase, a primary electron donor to the electron transport respiratory (ETR) system. Mass balance analysis of uranium species aided by visual and electron microscopy suggest that most U(VI) reduction occurred on the cell surface of the isolated species. This finding indicates the possibility of easy uranium recovery for beneficial use through biological remediation. Should the U(VI) be reduced inside the cell, recovery would require complete disruption of the cells and therefore would be difficult. The study contributes new knowledge on the underlying mechanisms in the U(VI) reduction in facultative anaerobes. PMID:25065785

  3. Biochemical Modulation of Lipid Pathway in Microalgae Dunaliella sp. for Biodiesel Production.

    PubMed

    Talebi, Ahmad Farhad; Tohidfar, Masoud; Mousavi Derazmahalleh, Seyedeh Mahsa; Sulaiman, Alawi; Baharuddin, Azhari Samsu; Tabatabaei, Meisam

    2015-01-01

    Exploitation of renewable sources of energy such as algal biodiesel could turn energy supplies problem around. Studies on a locally isolated strain of Dunaliella sp. showed that the mean lipid content in cultures enriched by 200?mg?L(-1) myoinositol was raised by around 33% (1.5 times higher than the control). Similarly, higher lipid productivity values were achieved in cultures treated by 100 and 200?mg?L(-1) myoinositol. Fluorometry analyses (microplate fluorescence and flow cytometry) revealed increased oil accumulation in the Nile red-stained algal samples. Moreover, it was predicted that biodiesel produced from myoinositol-treated cells possessed improved oxidative stability, cetane number, and cloud point values. From the genomic point of view, real-time analyses revealed that myoinositol negatively influenced transcript abundance of AccD gene (one of the key genes involved in lipid production pathway) due to feedback inhibition and that its positive effect must have been exerted through other genes. The findings of the current research are not to interprete that myoinositol supplementation could answer all the challenges faced in microalgal biodiesel production but instead to show that "there is a there there" for biochemical modulation strategies, which we achieved, increased algal oil quantity and enhanced resultant biodiesel quality. PMID:26146623

  4. Identifiability and inference of pathway motifs by epistasis analysis.

    PubMed

    Phenix, Hilary; Perkins, Theodore; Krn, Mads

    2013-06-01

    The accuracy of genetic network inference is limited by the assumptions used to determine if one hypothetical model is better than another in explaining experimental observations. Most previous work on epistasis analysis-in which one attempts to infer pathway relationships by determining equivalences among traits following mutations-has been based on Boolean or linear models. Here, we delineate the ultimate limits of epistasis-based inference by systematically surveying all two-gene network motifs and use symbolic algebra with arbitrary regulation functions to examine trait equivalences. Our analysis divides the motifs into equivalence classes, where different genetic perturbations result in indistinguishable experimental outcomes. We demonstrate that this partitioning can reveal important information about network architecture, and show, using simulated data, that it greatly improves the accuracy of genetic network inference methods. Because of the minimal assumptions involved, equivalence partitioning has broad applicability for gene network inference. PMID:23822501

  5. Identifiability and inference of pathway motifs by epistasis analysis

    NASA Astrophysics Data System (ADS)

    Phenix, Hilary; Perkins, Theodore; Krn, Mads

    2013-06-01

    The accuracy of genetic network inference is limited by the assumptions used to determine if one hypothetical model is better than another in explaining experimental observations. Most previous work on epistasis analysisin which one attempts to infer pathway relationships by determining equivalences among traits following mutationshas been based on Boolean or linear models. Here, we delineate the ultimate limits of epistasis-based inference by systematically surveying all two-gene network motifs and use symbolic algebra with arbitrary regulation functions to examine trait equivalences. Our analysis divides the motifs into equivalence classes, where different genetic perturbations result in indistinguishable experimental outcomes. We demonstrate that this partitioning can reveal important information about network architecture, and show, using simulated data, that it greatly improves the accuracy of genetic network inference methods. Because of the minimal assumptions involved, equivalence partitioning has broad applicability for gene network inference.

  6. Receptor-Drug Interaction: Europium Employment for Studying the Biochemical Pathway of G-Protein-Coupled Receptor Activation

    PubMed Central

    Antonio, Colabufo Nicola; Grazia, Perrone Maria; Marialessandra, Contino; Francesco, Berardi; Roberto, Perrone

    2007-01-01

    In medicinal chemistry field, the biochemical pathways, involved in 7-transmembrane domains G-protein coupled receptors (GPCRs) activation, are commonly studied to establish the activity of ligands towards GPCRs. The most studied steps are the measurement of activated GTP-? subunit and stimulated intracellular cAMP. At the present, many researchers defined agonist or antagonist activity of potential GPCRs drugs employing [35S]GTP?S or [3H]cAMP as probes. Recently, the corresponding lanthanide labels Eu-GTP and Eu-cAMP as alternative to radiochemicals have been developed because they are highly sensitive, easy to automate, easily synthesized, they display a much longer shelf-life and they can be used in multilabel experiments. In the present review, the receptor-drug interaction by europium employment for studying the biochemical pathway of GPCR activation has been focused. Moreover, comparative studies between lanthanide label probes and the corresponding radiolabeled compounds have been carried out. PMID:18350113

  7. Biological Pathway-Based Genome-Wide Association Analysis Identified the Vasoactive Intestinal Peptide (VIP) Pathway Important for Obesity

    PubMed Central

    Liu, Yong-Jun; Guo, Yan-Fang; Zhang, Li-Shu; Pei, Yu-Fang; Yu, Na; Yu, Ping; Papasian, Christopher J; Deng, Hong-Wen

    2010-01-01

    Recent genome-wide association (GWA) studies have identified a number of novel genes/variants predisposing to obesity. However, most GWA studies have focused on individual single-nucleotide polymorphism (SNPs)/genes with a strong statistical association with a phenotypic trait without considering potential biological interplay of the tested genes. In this study, we performed biological pathway-based GWA analysis for BMI and body fat mass. We used individual level genotype data generated from 1,000 unrelated US whites that were genotyped for ~500,000 SNPs. Statistical analysis of pathways was performed using a modification of the Gene Set Enrichment Algorithm. A total of 963 pathways extracted from the BioCarta, Kyoto Encyclopedia of Genes and Genomes (KEGG), Ambion GeneAssist, and Gene Ontology (GO) databases were analyzed. Among all of the pathways analyzed, the vasoactive intestinal peptide (VIP) pathway was most strongly associated with fat mass (nominal P = 0.0009) and was the third most strongly associated pathway with BMI (nominal P = 0.0006). After multiple testing correction, the VIP pathway achieved false-discovery rate (FDR) q values of 0.042 and 0.120 for fat mass and BMI, respectively. Our study is the first to demonstrate that the VIP pathway may play an important role in development of obesity. The study also highlights the importance of pathway-based GWA analysis in identification of additional genes/variants for complex human diseases. PMID:20379146

  8. Genomic, Proteomic, and Biochemical Analysis of the Organohalide Respiratory Pathway in Desulfitobacterium dehalogenans

    PubMed Central

    van de Pas, Bram A.; Atteia, Ariane; Krab, Klaas; Hagen, Wilfred R.; Goodwin, Lynne; Chain, Patrick; Boeren, Sjef; Maphosa, Farai; Schraa, Gosse; de Vos, Willem M.; van der Oost, John; Smidt, Hauke

    2014-01-01

    Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1T consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA. PMID:25512312

  9. Genes and biochemical pathways in human skeletal muscle affecting resting energy expenditure and fuel partitioning

    PubMed Central

    Wu, Xuxia; Patki, Amit; Lara-Castro, Cristina; Cui, Xiangqin; Zhang, Kui; Walton, R. Grace; Osier, Michael V.; Gadbury, Gary L.; Allison, David B.; Martin, Mitchell

    2011-01-01

    Genes influencing resting energy expenditure (REE) and respiratory quotient (RQ) represent candidate genes for obesity and the metabolic syndrome because of the involvement of these traits in energy balance and substrate oxidation. We aim to explore the molecular basis for individual variation in REE and fuel partitioning as reflected by RQ. We performed microarray studies in human vastus lateralis muscle biopsies from 40 healthy subjects with measured REE and RQ values. We identified 2,392 and 1,115 genes significantly correlated with REE and RQ, respectively. Genes correlated with REE and RQ encompass a broad array of functions, including carbohydrate and lipid metabolism, gene expression, mitochondrial processes, and membrane transport. Microarray pathway analysis revealed that REE was positively correlated with upregulation of G protein-coupled receptor signaling (meet criteria/total genes: 65 of 283) involved in autonomic nervous system functions, including those receptors mediating adrenergic, dopamine, ?-aminobutyric acid (GABA), neuropeptide Y (NPY), and serotonin action (meet criteria/total genes: 46 of 176). Reduced REE was associated with an increase in genes participating in ubiquitin-proteasome-dependent proteolytic pathways (58 of 232). Serine-type peptidase activity (9 of 76) was positively correlated with RQ, while genes involved in the protein phosphatase type 2A complex (4 of 9), mitochondrial function and cellular respiration (38 of 315), and unfolded protein binding (19 of 97) were associated with reduced RQ values and a preference for lipid fuel metabolism. Individual variations in whole body REE and RQ are regulated by differential expressions of specific genes and pathways intrinsic to skeletal muscle. PMID:21109598

  10. Hypericin biosynthesis in Hypericum hookerianum Wight and Arn: investigation on biochemical pathways using metabolite inhibitors and suppression subtractive hybridization.

    PubMed

    Pillai, Padmesh P; Nair, Aswati R

    2014-10-01

    The biochemical pathway to hypericin biosynthesis is presumed to be polyketide synthase (PKS) mediated, but it has not been experimentally validated, and no alternate route (chorismate/o-succinylbenzoate pathway) has been analyzed. We report here our earlier developed auxin inducible culture systems of Hypericum hookerianum as a model, to study the metabolic pathway to hypericin synthesis. Inhibitors of the alternate pathway at varying concentrations showed steady synthesis of total hypericins with means of 2.800.22, 18.750.01; 16.393.75, 29.601.90 (mevinolin) 2.530.10, 18.120.56; 0.140.01, 14.281.11 (fosmidomycin) and 2.70.35, 18.750.61; 0.140.01, 12.801.09 mg g(-1) DW (glyphosate) in the control and auxin-induced shoot and shoot-forming callus cultures, respectively. SSH analysis classified the differentially expressed sequences into protein synthesis (38%), modification (20%), electron transport (9%) and remaining as unclassified (11%) and unknown proteins (22%). Functional annotation of sequences indicates the presence of additional protein components besides PKS activity. Our results demonstrate direct biochemical and molecular evidence of PKS hypothesis of hypericin biosynthesis for the first time. PMID:25282172

  11. Biochemical and structural characterization of mouse mitochondrial aspartate aminotransferase, a newly identified kynurenine aminotransferase-IV

    SciTech Connect

    Han, Q.; Robinson, H.; Cai, T.; Tagle, D. A.; Li, J.

    2011-10-01

    Mammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine aminotransferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14 of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of other KATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

  12. Identifying Differences Between Biochemical Failure and Cure: Incidence Rates and Predictors

    SciTech Connect

    Vicini, Frank A.; Shah, Chirag; Kestin, Larry; Ghilezan, Mihai; Krauss, Daniel; Ye Hong; Brabbins, Donald; Martinez, Alvaro A.

    2011-11-15

    Background: Patients treated with radiation therapy (RT) for prostate cancer were evaluated to estimate the length of time required to document biochemical cure (BC) after treatment and the variables associated with long-term treatment efficacy. Patients and Methods: 2,100 patients received RT alone for localized prostate carcinoma (external-beam RT, n = 1,504; brachytherapy alone, n = 241; or brachytherapy + pelvic radiation, n = 355). The median external-beam dose was 68.4 Gy, and the median follow-up time was 8.6 years. Biochemical failure (BF) was defined according to the Phoenix definition. Results: Biochemical failure was experienced by 685 patients (32.6%). The median times to BF for low-, intermediate-, and high-risk groups were 6.0, 5.6, and 4.5 years respectively (p < 0.001). The average annual incidence rates of BF for years 1-5, 5-10,11-15, and 16-20 in low-risk patients were 2.0%, 2.0%, 0.3%, and 0.06% (p < 0.001); for intermediate-risk patients, 4%, 3%, 0.3%, and 0% (p < 0.001); and for high-risk patients, 10.0%, 5.0%, 0.3%, and 0.3% (p < 0.001). After 5 years of treatment, 36.9% of all patients experienced BF. The percentage of total failures occurring during years 1-5, 5-10, 11-15, and 16-20 were 48.7%, 43.5%, 6.5%, and 1.3% for low-risk patients; 64.0%, 32.2%, 3.8%, and 0% for intermediate-risk patients; and 71.9%, 25.9%, 1.1%, and 1.1% for high-risk patients, respectively. Increasing time to nadir was associated with increased time to BF. On multivariate analysis, factors significantly associated with 10-year BC included prostate-specific antigen nadir and time to nadir. Conclusions: The incidence rates for BF did not plateau until later than 10 years after treatment, suggesting that extended follow-up time is required to monitor patients after treatment. Prostate-specific antigen nadir and time to nadir have the strongest association with long-term BC.

  13. Identifying Distinct Healthcare Pathways During Episodes of Chronic Obstructive Pulmonary Disease Exacerbations

    PubMed Central

    Kuwornu, John P.; Lix, Lisa M.; Quail, Jacqueline M.; Forget, Evelyn; Muthukumarana, Saman; Wang, Xiaoyun E.; Osman, Meric; Teare, Gary F.

    2016-01-01

    Abstract Healthcare pathways are important to measure because they are expected to affect outcomes. However, they are challenging to define because patients exhibit heterogeneity in their use of healthcare services. The objective of this study was to identify and describe healthcare pathways during episodes of chronic obstructive pulmonary disease (COPD) exacerbations. Linked administrative databases from Saskatchewan, Canada were used to identify a cohort of newly diagnosed COPD patients and their episodes of healthcare use for disease exacerbations. Latent class analysis (LCA) was used to classify the cohort into homogeneous pathways using indicators of respiratory-related hospitalizations, emergency department (ED) visits, general and specialist physician visits, and outpatient prescription drug dispensations. Multinomial logistic regression models tested patients’ demographic and disease characteristics associated with pathway group membership. The most frequent healthcare contact sequences in each pathway were described. Tests of mean costs across groups were conducted using a model-based approach with χ2 statistics. LCA identified 3 distinct pathways for patients with hospital- (n = 963) and ED-initiated (n = 364) episodes. For the former, pathway group 1 members followed complex pathways in which multiple healthcare services were repeatedly used and incurred substantially higher costs than patients in the other pathway groups. For patients with an ED-initiated episode, pathway group 1 members also had higher costs than other groups. Pathway groups differed with respect to patient demographic and disease characteristics. A minority of patients were discharged from ED or hospital, but did not have any follow-up care during the remainder of their episode. Patients who followed complex pathways could benefit from case management interventions to streamline their journeys through the healthcare system. The minority of patients whose pathways were not consistent with recommended follow-up care should be further investigated to fully align COPD treatment in the province with recommended care practices. PMID:26945376

  14. Identifying Distinct Healthcare Pathways During Episodes of Chronic Obstructive Pulmonary Disease Exacerbations.

    PubMed

    Kuwornu, John P; Lix, Lisa M; Quail, Jacqueline M; Forget, Evelyn; Muthukumarana, Saman; Wang, Xiaoyun E; Osman, Meric; Teare, Gary F

    2016-03-01

    Healthcare pathways are important to measure because they are expected to affect outcomes. However, they are challenging to define because patients exhibit heterogeneity in their use of healthcare services. The objective of this study was to identify and describe healthcare pathways during episodes of chronic obstructive pulmonary disease (COPD) exacerbations.Linked administrative databases from Saskatchewan, Canada were used to identify a cohort of newly diagnosed COPD patients and their episodes of healthcare use for disease exacerbations. Latent class analysis (LCA) was used to classify the cohort into homogeneous pathways using indicators of respiratory-related hospitalizations, emergency department (ED) visits, general and specialist physician visits, and outpatient prescription drug dispensations. Multinomial logistic regression models tested patients' demographic and disease characteristics associated with pathway group membership. The most frequent healthcare contact sequences in each pathway were described. Tests of mean costs across groups were conducted using a model-based approach with χ statistics.LCA identified 3 distinct pathways for patients with hospital- (n = 963) and ED-initiated (n = 364) episodes. For the former, pathway group 1 members followed complex pathways in which multiple healthcare services were repeatedly used and incurred substantially higher costs than patients in the other pathway groups. For patients with an ED-initiated episode, pathway group 1 members also had higher costs than other groups. Pathway groups differed with respect to patient demographic and disease characteristics. A minority of patients were discharged from ED or hospital, but did not have any follow-up care during the remainder of their episode.Patients who followed complex pathways could benefit from case management interventions to streamline their journeys through the healthcare system. The minority of patients whose pathways were not consistent with recommended follow-up care should be further investigated to fully align COPD treatment in the province with recommended care practices. PMID:26945376

  15. An Effective Method to Identify Shared Pathways and Common Factors among Neurodegenerative Diseases

    PubMed Central

    Li, Ping; Nie, Yaling; Yu, Jingkai

    2015-01-01

    Groups of distinct but related diseases often share common symptoms, which suggest likely overlaps in underlying pathogenic mechanisms. Identifying the shared pathways and common factors among those disorders can be expected to deepen our understanding for them and help designing new treatment strategies effected on those diseases. Neurodegeneration diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD), were taken as a case study in this research. Reported susceptibility genes for AD, PD and HD were collected and human protein-protein interaction network (hPPIN) was used to identify biological pathways related to neurodegeneration. 81 KEGG pathways were found to be correlated with neurodegenerative disorders. 36 out of the 81 are human disease pathways, and the remaining ones are involved in miscellaneous human functional pathways. Cancers and infectious diseases are two major subclasses within the disease group. Apoptosis is one of the most significant functional pathways. Most of those pathways found here are actually consistent with prior knowledge of neurodegenerative diseases except two cell communication pathways: adherens and tight junctions. Gene expression analysis showed a high probability that the two pathways were related to neurodegenerative diseases. A combination of common susceptibility genes and hPPIN is an effective method to study shared pathways involved in a group of closely related disorders. Common modules, which might play a bridging role in linking neurodegenerative disorders and the enriched pathways, were identified by clustering analysis. The identified shared pathways and common modules can be expected to yield clues for effective target discovery efforts on neurodegeneration. PMID:26575483

  16. Identifying novel prostate cancer associated pathways based on integrative microarray data analysis.

    PubMed

    Wang, Ying; Chen, Jiajia; Li, Qinghui; Wang, Haiyun; Liu, Ganqiang; Jing, Qing; Shen, Bairong

    2011-06-01

    The development and diverse application of microarray and next generation sequencing technologies has made the meta-analysis widely used in expression data analysis. Although it is commonly accepted that pathway, network and systemic level approaches are more reproducible than reductionism analyses, the meta-analysis of prostate cancer associated molecular signatures at the pathway level remains unexplored. In this article, we performed a meta-analysis of 10 prostate cancer microarray expression datasets to identify the common signatures at both the gene and pathway levels. As the enrichment analysis result of GeneGo's database and KEGG database, 97.8% and 66.7% of the signatures show higher similarity at pathway level than that at gene level, respectively. Analysis by using gene set enrichment analysis (GSEA) method also supported the hypothesis. Further analysis of PubMed citations verified that 207 out of 490 (42%) pathways from GeneGo and 48 out of 74 (65%) pathways from KEGG were related to prostate cancer. An overlap of 15 enriched pathways was observed in at least eight datasets. Eight of these pathways were first described as being associated with prostate cancer. In particular, endothelin-1/EDNRA transactivation of the EGFR pathway was found to be overlapped in nine datasets. The putative novel prostate cancer related pathways identified in this paper were indirectly supported by PubMed citations and would provide essential information for further development of network biomarkers and individualized therapy strategy for prostate cancer. PMID:21704261

  17. Transcriptomics profiling of Indian mustard (Brassica juncea) under arsenate stress identifies key candidate genes and regulatory pathways

    PubMed Central

    Srivastava, Sudhakar; Srivastava, Ashish K.; Sablok, Gaurav; Deshpande, Tejaswini U.; Suprasanna, Penna

    2015-01-01

    Arsenic (As) is a non-essential element, a groundwater pollutant, whose uptake by plants produces toxic effects. The use of As-contaminated groundwater for irrigation can affect the crop productivity. Realizing the importance of the Brassica juncea as a crop plant in terms of oil-yield, there is a need to unravel mechanistic details of response to As stress and identify key functional genes and pathways. In this research, we studied time-dependent (4–96 h) transcriptome changes in roots and shoots of B. juncea under arsenate [As(V)] stress using Agilent platform. Among the whole transcriptome profiled genes, a total of 1,285 genes showed significant change in expression pattern upon As(V) exposure. The differentially expressed genes were categorized to various signaling pathways including hormones (jasmonate, abscisic acid, auxin, and ethylene) and kinases. Significant effects were also noticed on genes related to sulfur, nitrogen, CHO, and lipid metabolisms along with photosynthesis. Biochemical assays were conducted using specific inhibitors of glutathione and jasmonate biosynthesis, and kinases. The inhibitor studies revealed interconnection among sulfur metabolism, jasmonate, and kinase signaling pathways. In addition, various transposons also constituted a part of the altered transcriptome. Lastly, we profiled a set of key functional up- and down-regulated genes using real-time RT-PCR, which could act as an early indicators of the As stress. PMID:26347763

  18. Transcriptomics profiling of Indian mustard (Brassica juncea) under arsenate stress identifies key candidate genes and regulatory pathways.

    PubMed

    Srivastava, Sudhakar; Srivastava, Ashish K; Sablok, Gaurav; Deshpande, Tejaswini U; Suprasanna, Penna

    2015-01-01

    Arsenic (As) is a non-essential element, a groundwater pollutant, whose uptake by plants produces toxic effects. The use of As-contaminated groundwater for irrigation can affect the crop productivity. Realizing the importance of the Brassica juncea as a crop plant in terms of oil-yield, there is a need to unravel mechanistic details of response to As stress and identify key functional genes and pathways. In this research, we studied time-dependent (4-96 h) transcriptome changes in roots and shoots of B. juncea under arsenate [As(V)] stress using Agilent platform. Among the whole transcriptome profiled genes, a total of 1,285 genes showed significant change in expression pattern upon As(V) exposure. The differentially expressed genes were categorized to various signaling pathways including hormones (jasmonate, abscisic acid, auxin, and ethylene) and kinases. Significant effects were also noticed on genes related to sulfur, nitrogen, CHO, and lipid metabolisms along with photosynthesis. Biochemical assays were conducted using specific inhibitors of glutathione and jasmonate biosynthesis, and kinases. The inhibitor studies revealed interconnection among sulfur metabolism, jasmonate, and kinase signaling pathways. In addition, various transposons also constituted a part of the altered transcriptome. Lastly, we profiled a set of key functional up- and down-regulated genes using real-time RT-PCR, which could act as an early indicators of the As stress. PMID:26347763

  19. Systematic Tracking of Disrupted Modules Identifies Altered Pathways Associated with Congenital Heart Defects in Down Syndrome

    PubMed Central

    Chen, Denghong; Zhang, Zhenhua; Meng, Yuxiu

    2015-01-01

    Background This work aimed to identify altered pathways in congenital heart defects (CHD) in Down syndrome (DS) by systematically tracking the dysregulated modules of reweighted protein-protein interaction (PPI) networks. Material/Methods We performed systematic identification and comparison of modules across normal and disease conditions by integrating PPI and gene-expression data. Based on Pearson correlation coefficient (PCC), normal and disease PPI networks were inferred and reweighted. Then, modules in the PPI network were explored by clique-merging algorithm; altered modules were identified via maximum weight bipartite matching and ranked in non-increasing order. Finally, pathways enrichment analysis of genes in altered modules was carried out based on Database for Annotation, Visualization, and Integrated Discovery (DAVID) to study the biological pathways in CHD in DS. Results Our analyses revealed that 348 altered modules were identified by comparing modules in normal and disease PPI networks. Pathway functional enrichment analysis of disrupted module genes showed that the 4 most significantly altered pathways were: ECM-receptor interaction, purine metabolism, focal adhesion, and dilated cardiomyopathy. Conclusions We successfully identified 4 altered pathways and we predicted that these pathways would be good indicators for CHD in DS. PMID:26524729

  20. Understanding the Biochemical Basis of Temperature-Induced Lipid Pathway Adjustments in Plants

    PubMed Central

    Li, Qiang; Zheng, Qian; Shen, Wenyun; Cram, Dustin; Fowler, D. Brian; Wei, Yangdou; Zou, Jitao

    2015-01-01

    Glycerolipid biosynthesis in plants proceeds through two major pathways compartmentalized in the chloroplast and the endoplasmic reticulum (ER). The involvement of glycerolipid pathway interactions in modulating membrane desaturation under temperature stress has been suggested but not fully explored. We profiled glycerolipid changes as well as transcript dynamics under suboptimal temperature conditions in three plant species that are distinctively different in the mode of lipid pathway interactions. In Arabidopsis thaliana, a 16:3 plant, the chloroplast pathway is upregulated in response to low temperature, whereas high temperature promotes the eukaryotic pathway. Operating under a similar mechanistic framework, Atriplex lentiformis at high temperature drastically increases the contribution of the eukaryotic pathway and correspondingly suppresses the prokaryotic pathway, resulting in the switch of lipid profile from 16:3 to 18:3. In wheat (Triticum aestivum), an 18:3 plant, low temperature also influences the channeling of glycerolipids from the ER to chloroplast. Evidence of differential trafficking of diacylglycerol moieties from the ER to chloroplast was uncovered in three plant species as another layer of metabolic adaptation under temperature stress. We propose a model that highlights the predominance and prevalence of lipid pathway interactions in temperature-induced lipid compositional changes. PMID:25564555

  1. How Can Men Destined for Biochemical Failure After Androgen Deprivation and Radiotherapy be Identified Earlier?

    PubMed Central

    D'Ambrosio, David J.; Ruth, Karen; Horwitz, Eric M.; Uzzo, Robert G.; Pollack, Alan; Buyyounouski, Mark K.

    2009-01-01

    Purpose The significance of prostate-specific antigen (PSA) increases during the recovery of androgen after androgen deprivation therapy (ADT) and radiotherapy for prostate cancer is not well understood. This study sought to determine whether the initial PSA increase from undetectable after completion of all treatment predicts for eventual biochemical failure (BF). Methods and Materials Between July 1992 and March 2004, 163 men with a Gleason score of 810 or initial PSA level >20 ng/mL, or Stage T3 prostate cancer were treated with radiotherapy (median dose, 76 Gy) and ADT and achieved an undetectable PSA level. The first detectable PSA level after the cessation of ADT was defined as the PSA sentinel rise (SR). A PSA-SR of >0.25, >0.5, >0.75, and >1.0 ng/mL was studied as predictors of BF (nadir plus 2 ng/mL). Cox proportional hazards models were used for univariate and multivariate analyses for BF adjusting for pretreatment differences in Gleason score, stage, PSA level (continuous), dose (continuous), and ADT duration (<12 vs. ?12 months). Results Of the 163 men, 41 had BF after therapy. The median time to BF was 25 months (range, 496). The 5-year BF rate stratified by a PSA-SR of ?0.25 vs. >0.25 ng/mL was 28% vs. 43% (p = 0.02), ?0.5 vs. >0.5 ng/mL was 30% vs. 56% (p = 0.0003), ?0.75 vs. >0.75 ng/mL was 29% vs. 66% (p < 0.0001), and ?1.0 vs. >1.0 ng/mL was 29% vs. 75% (p < 0.0001). All four PSA-SRs were independently predictive of BF on multivariate analysis. Conclusion The PSA-SR predicts for BF. A PSA-SR of >0.5 ng/mL can be used for early identification of men at greater risk of BF. PMID:18164854

  2. How Can Men Destined for Biochemical Failure After Androgen Deprivation and Radiotherapy Be Identified Earlier?

    SciTech Connect

    D'Ambrosio, David J.; Ruth, Karen; Horwitz, Eric M.; Uzzo, Robert G.; Pollack, Alan; Buyyounouski, Mark K.

    2008-04-01

    Purpose: The significance of prostate-specific antigen (PSA) increases during the recovery of androgen after androgen deprivation therapy (ADT) and radiotherapy for prostate cancer is not well understood. This study sought to determine whether the initial PSA increase from undetectable after completion of all treatment predicts for eventual biochemical failure (BF). Methods and Materials: Between July 1992 and March 2004, 163 men with a Gleason score of 8-10 or initial PSA level >20 ng/mL, or Stage T3 prostate cancer were treated with radiotherapy (median dose, 76 Gy) and ADT and achieved an undetectable PSA level. The first detectable PSA level after the cessation of ADT was defined as the PSA sentinel rise (SR). A PSA-SR of >0.25, >0.5, >0.75, and >1.0 ng/mL was studied as predictors of BF (nadir plus 2 ng/mL). Cox proportional hazards models were used for univariate and multivariate analyses for BF adjusting for pretreatment differences in Gleason score, stage, PSA level (continuous), dose (continuous), and ADT duration (<12 vs. {>=}12 months). Results: Of the 163 men, 41 had BF after therapy. The median time to BF was 25 months (range, 4-96). The 5-year BF rate stratified by a PSA-SR of {<=}0.25 vs. >0.25 ng/mL was 28% vs. 43% (p = 0.02), {<=}0.5 vs. >0.5 ng/mL was 30% vs. 56% (p = 0.0003), {<=}0.75 vs. >0.75 ng/mL was 29% vs. 66% (p < 0.0001), and {<=}1.0 vs. >1.0 ng/mL was 29% vs. 75% (p < 0.0001). All four PSA-SRs were independently predictive of BF on multivariate analysis. Conclusion: The PSA-SR predicts for BF. A PSA-SR of >0.5 ng/mL can be used for early identification of men at greater risk of BF.

  3. Identifying the biological pathways underlying human focal epilepsy: from complexity to coherence to centrality.

    PubMed

    Mirza, Nasir; Appleton, Richard; Burn, Sasha; Carr, Daniel; Crooks, Daniel; du Plessis, Daniel; Duncan, Roderick; Farah, Jibril Osman; Josan, Vivek; Miyajima, Fabio; Mohanraj, Rajiv; Shukralla, Arif; Sills, Graeme J; Marson, Anthony G; Pirmohamed, Munir

    2015-08-01

    Numerous diverse biological pathways are dysregulated in the epileptic focus. Which of these pathways are most critical in producing the biological abnormalities that lead to epilepsy? Answering this question is key to identifying the primary causes of epilepsy and for discovering new therapeutic strategies with greater efficacy than currently available antiepileptics (AEDs). We have performed the largest genome-wide transcriptomic analysis to date comparing epileptic with normal human hippocampi. We have identified 118 differentially expressed and, for the first time, differentially connected pathways in the epileptic focus. Using network mapping techniques, we have shown that these dysregulated pathways, though seemingly disparate, form a coherent interconnected central network. Using closeness centrality analysis, we have identified that the most influential hub pathways in this network are signalling through G protein-coupled receptors, in particular opioid receptors, and their downstream effectors PKA/CREB and DAG/IP3. Next, we have objectively demonstrated that genetic association of gene sets in independent genome-wide association studies (GWASs) can be used to identify causally relevant gene sets: we show that proven causal epilepsy genes, which cause familial Mendelian epilepsy syndromes, are associated in published sporadic epilepsy GWAS results. Using the same technique, we have shown that central pathways identified (opioid receptor and PKA/CREB and DAG/IP3 signalling pathways) are genetically associated with focal epilepsy and, hence, likely causal. Published functional studies in animal models provide evidence of a role for these pathways in epilepsy. Our work shows that these pathways play a central role in human focal epilepsy and that they are important currently unexploited antiepileptic drug targets. PMID:25941323

  4. Using soil moisture and spatial yield patterns to identify subsurface flow pathways.

    PubMed

    Gish, T J; Walthall, C L; Daughtry, C S T; Kung, K-J S

    2005-01-01

    Subsurface soil water dynamics can influence crop growth and the fate of surface-applied fertilizers and pesticides. Recently, a method was proposed using only ground-penetrating radar (GPR) and digital elevation maps (DEMs) to identify locations where subsurface water converged into discrete pathways. For this study, the GPR protocol for identifying horizontal subsurface flow pathways was extended to a 3.2-ha field, uncertainty is discussed, and soil moisture and yield patterns are presented as confirming evidence of the extent of the subsurface flow pathways. Observed soil water contents supported the existence of discrete preferential funnel flow processes occurring near the GPR-identified preferential flow pathways. Soil moisture also played a critical role in the formation of corn (Zea mays L.) grain yield patterns with yield spatial patterns being similar for mild and severe drought conditions. A buffer zone protocol was introduced that allowed the impact of subsurface flow pathways on corn grain yield to be quantified. Results indicate that when a GPR-identified subsurface clay layer was within 2 m of the soil surface, there was a beneficial impact on yield during a drought year. Furthermore, the buffer zone analysis demonstrated that corn grain yields decreased as the horizontal distance from the GPR-identified subsurface flow pathways increased during a drought year. Averaged real-time soil moisture contents at 0.1 m also decreased with increasing distance from the GPR-identified flow pathways. This research suggests that subsurface flow pathways exist and influence soil moisture and corn grain yield patterns. PMID:15647558

  5. Method for identifying biochemical and chemical reactions and micromechanical processes using nanomechanical and electronic signal identification

    DOEpatents

    Holzrichter, J.F.; Siekhaus, W.J.

    1997-04-15

    A scanning probe microscope, such as an atomic force microscope (AFM) or a scanning tunneling microscope (STM), is operated in a stationary mode on a site where an activity of interest occurs to measure and identify characteristic time-varying micromotions caused by biological, chemical, mechanical, electrical, optical, or physical processes. The tip and cantilever assembly of an AFM is used as a micromechanical detector of characteristic micromotions transmitted either directly by a site of interest or indirectly through the surrounding medium. Alternatively, the exponential dependence of the tunneling current on the size of the gap in the STM is used to detect micromechanical movement. The stationary mode of operation can be used to observe dynamic biological processes in real time and in a natural environment, such as polymerase processing of DNA for determining the sequence of a DNA molecule. 6 figs.

  6. Method for identifying biochemical and chemical reactions and micromechanical processes using nanomechanical and electronic signal identification

    DOEpatents

    Holzrichter, John F. (Berkeley, CA); Siekhaus, Wigbert J. (Berkeley, CA)

    1997-01-01

    A scanning probe microscope, such as an atomic force microscope (AFM) or a scanning tunneling microscope (STM), is operated in a stationary mode on a site where an activity of interest occurs to measure and identify characteristic time-varying micromotions caused by biological, chemical, mechanical, electrical, optical, or physical processes. The tip and cantilever assembly of an AFM is used as a micromechanical detector of characteristic micromotions transmitted either directly by a site of interest or indirectly through the surrounding medium. Alternatively, the exponential dependence of the tunneling current on the size of the gap in the STM is used to detect micromechanical movement. The stationary mode of operation can be used to observe dynamic biological processes in real time and in a natural environment, such as polymerase processing of DNA for determining the sequence of a DNA molecule.

  7. The use of an imagery mnemonic to teach the porphyrin biochemical pathway.

    PubMed

    De Moll, Ellen H; Routt, Ethan; Heinecke, Gillian; Tsui, Cindy; Levitt, Jacob

    2015-04-01

    We designed an imagery mnemonic to help medical students and residents learn the porphyrin pathway and associated diseases. Fourth year medical students at the Icahn School of Medicine at Mount Sinai in the spring of 2014 participated. One group (n=11) received the porphyrin pathway in a lecture explaining a mnemonic, whereas a second group (n=11) was simply taught the steps of the pathway. A pre-intervention assessment before the lectures was given to assess baseline differences in knowledge of the porphyrin pathway between the groups. Immediately following the lecture, 1 week after the lecture, and 3 weeks after the lecture, the students were given quizzes to assess their knowledge. Students were aware of the week 1 quiz and were asked not to study for it. The week 3 quiz was a surprise. There were no statistically significant differences in knowledge of the pathway at baseline (p=.45), at the immediate post-intervention (p=.22), or one week post-intervention (p=.40). Three weeks after the lecture, students in the mnemonic group scored 20% higher than controls (p=.02). Students who had learned the mnemonic demonstrated better long-term retention of information than students learning by the control method. This mnemonic minimizes study time while improving long-term retention. PMID:25933070

  8. AN INTEGRATED NETWORK APPROACH TO IDENTIFYING BIOLOGICAL PATHWAYS AND ENVIRONMENTAL EXPOSURE INTERACTIONS IN COMPLEX DISEASES.

    PubMed

    Darabos, Christian; Qiu, Jingya; Moore, Jason H

    2016-01-01

    Complex diseases are the result of intricate interactions between genetic, epigenetic and environmental factors. In previous studies, we used epidemiological and genetic data linking environmental exposure or genetic variants to phenotypic disease to construct Human Phenotype Networks and separately analyze the effects of both environment and genetic factors on disease interactions. To better capture the intricacies of the interactions between environmental exposure and the biological pathways in complex disorders, we integrate both aspects into a single "tripartite" network. Despite extensive research, the mechanisms by which chemical agents disrupt biological pathways are still poorly understood. In this study, we use our integrated network model to identify specific biological pathway candidates possibly disrupted by environmental agents. We conjecture that a higher number of co-occurrences between an environmental substance and biological pathway pair can be associated with a higher likelihood that the substance is involved in disrupting that pathway. We validate our model by demonstrating its ability to detect known arsenic and signal transduction pathway interactions and speculate on candidate cell-cell junction organization pathways disrupted by cadmium. The validation was supported by distinct publications of cell biology and genetic studies that associated environmental exposure to pathway disruption. The integrated network approach is a novel method for detecting the biological effects of environmental exposures. A better understanding of the molecular processes associated with specific environmental exposures will help in developing targeted molecular therapies for patients who have been exposed to the toxicity of environmental chemicals. PMID:26776169

  9. AN INTEGRATED NETWORK APPROACH TO IDENTIFYING BIOLOGICAL PATHWAYS AND ENVIRONMENTAL EXPOSURE INTERACTIONS IN COMPLEX DISEASES

    PubMed Central

    DARABOS, CHRISTIAN; QIU, JINGYA; MOORE, JASON H.

    2015-01-01

    Complex diseases are the result of intricate interactions between genetic, epigenetic and environmental factors. In previous studies, we used epidemiological and genetic data linking environmental exposure or genetic variants to phenotypic disease to construct Human Phenotype Networks and separately analyze the effects of both environment and genetic factors on disease interactions. To better capture the intricacies of the interactions between environmental exposure and the biological pathways in complex disorders, we integrate both aspects into a single “tripartite” network. Despite extensive research, the mechanisms by which chemical agents disrupt biological pathways are still poorly understood. In this study, we use our integrated network model to identify specific biological pathway candidates possibly disrupted by environmental agents. We conjecture that a higher number of co-occurrences between an environmental substance and biological pathway pair can be associated with a higher likelihood that the substance is involved in disrupting that pathway. We validate our model by demonstrating its ability to detect known arsenic and signal transduction pathway interactions and speculate on candidate cell-cell junction organization pathways disrupted by cadmium. The validation was supported by distinct publications of cell biology and genetic studies that associated environmental exposure to pathway disruption. The integrated network approach is a novel method for detecting the biological effects of environmental exposures. A better understanding of the molecular processes associated with specific environmental exposures will help in developing targeted molecular therapies for patients who have been exposed to the toxicity of environmental chemicals. PMID:26776169

  10. Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins

    PubMed Central

    Hassan, Karl A.; Jackson, Scott M.; Penesyan, Anahit; Patching, Simon G.; Tetu, Sasha G.; Eijkelkamp, Bart A.; Brown, Melissa H.; Henderson, Peter J. F.; Paulsen, Ian. T.

    2013-01-01

    Chlorhexidine is widely used as an antiseptic or disinfectant in both hospital and community settings. A number of bacterial species display resistance to this membrane-active biocide. We examined the transcriptomic response of a representative nosocomial human pathogen, Acinetobacter baumannii, to chlorhexidine to identify the primary chlorhexidine resistance elements. The most highly up-regulated genes encoded components of a major multidrug efflux system, AdeAB. The next most highly overexpressed gene under chlorhexidine stress was annotated as encoding a hypothetical protein, named here as AceI. Orthologs of the aceI gene are conserved within the genomes of a broad range of proteobacterial species. Expression of aceI or its orthologs from several other ?- or ?-proteobacterial species in Escherichia coli resulted in significant increases in resistance to chlorhexidine. Additionally, disruption of the aceI ortholog in Acinetobacter baylyi rendered it more susceptible to chlorhexidine. The AceI protein was localized to the membrane after overexpression in E. coli. This protein was purified, and binding assays demonstrated direct and specific interactions between AceI and chlorhexidine. Transport assays using [14C]-chlorhexidine determined that AceI was able to mediate the energy-dependent efflux of chlorhexidine. An E15Q AceI mutant with a mutation in a conserved acidic residue, although unable to mediate chlorhexidine resistance and transport, was still able to bind chlorhexidine. Taken together, these data are consistent with AceI being an active chlorhexidine efflux protein and the founding member of a family of bacterial drug efflux transporters. PMID:24277845

  11. Evidence from Biochemical Pathways in Favor of Unfinished Evolution Rather than Intelligent Design

    ERIC Educational Resources Information Center

    Behrman, Edward J.; Marzluf, George A.

    2004-01-01

    An argument is made in favor of imperfect or unfinished evolution based on some metabolic pathways in which it seems that intelligent design would have done better. The case studies noted indicate the absence of highly intelligent design and are not intended as comprehensive collection but as a limited sample of inefficient situations in

  12. Evidence from Biochemical Pathways in Favor of Unfinished Evolution Rather than Intelligent Design

    ERIC Educational Resources Information Center

    Behrman, Edward J.; Marzluf, George A.

    2004-01-01

    An argument is made in favor of imperfect or unfinished evolution based on some metabolic pathways in which it seems that intelligent design would have done better. The case studies noted indicate the absence of highly intelligent design and are not intended as comprehensive collection but as a limited sample of inefficient situations in…

  13. Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway

    PubMed Central

    Miller, Bryan W; Lau, Garnet; Grouios, Chris; Mollica, Emanuela; Barrios-Rodiles, Miriam; Liu, Yongmei; Datti, Alessandro; Morris, Quaid; Wrana, Jeffrey L; Attisano, Liliana

    2009-01-01

    Large-scale proteomic approaches have been used to study signaling pathways. However, identification of biologically relevant hits from a single screen remains challenging due to limitations inherent in each individual approach. To overcome these limitations, we implemented an integrated, multi-dimensional approach and used it to identify Wnt pathway modulators. The LUMIER proteinprotein interaction mapping method was used in conjunction with two functional screens that examined the effect of overexpression and siRNA-mediated gene knockdown on Wnt signaling. Meta-analysis of the three data sets yielded a combined pathway score (CPS) for each tested component, a value reflecting the likelihood that an individual protein is a Wnt pathway regulator. We characterized the role of two proteins with high CPSs, Ube2m and Nkd1. We show that Ube2m interacts with and modulates ?-catenin stability, and that the antagonistic effect of Nkd1 on Wnt signaling requires interaction with Axin, itself a negative pathway regulator. Thus, integrated physical and functional mapping in mammalian cells can identify signaling components with high confidence and provides unanticipated insights into pathway regulators. PMID:19888210

  14. Steady and transient fluid shear stress stimulate NO release in osteoblasts through distinct biochemical pathways

    NASA Technical Reports Server (NTRS)

    McAllister, T. N.; Frangos, J. A.

    1999-01-01

    Fluid flow has been shown to be a potent stimulus in osteoblasts and osteocytes and may therefore play an important role in load-induced bone remodeling. The objective of this study was to investigate the characteristics of flow-activated pathways. Previously we reported that fluid flow stimulates rapid and continuous release of nitric oxide (NO) in primary rat calvarial osteoblasts. Here we demonstrate that flow-induced NO release is mediated by shear stress and that this response is distinctly biphasic. Transients in shear stress associated with the onset of flow stimulated a burst in NO production (8.2 nmol/mg of protein/h), while steady flow stimulated sustained NO production (2.2 nmol/mg of protein/h). Both G-protein inhibition and calcium chelation abolished the burst phase but had no effect on sustained production. Activation of G-proteins stimulated dose-dependent NO release in static cultures of both calvarial osteoblasts and UMR-106 osteoblast-like cells. Pertussis toxin had no effect on NO release. Calcium ionophore stimulated low levels of NO production within 15 minutes but had no effect on sustained production. Taken together, these data suggest that fluid shear stress stimulates NO release by two distinct pathways: a G-protein and calcium-dependent phase sensitive to flow transients, and a G-protein and calcium-independent pathway stimulated by sustained flow.

  15. Flexible and Versatile Strategy for the Construction of Large Biochemical Pathways.

    PubMed

    Yuan, Yongbo; Andersen, Erik; Zhao, Huimin

    2016-01-15

    Synthetic pathways and circuits have been increasingly used for microbial production of fuels and chemicals. Here, we report a flexible and versatile DNA assembly strategy that allows rapid, modular, and reliable construction of biological pathways and circuits from basic genetic parts. This strategy combines the automation-friendly ligase cycling reaction (LCR) method and the high-fidelity in vivo yeast-based DNA assembly method, DNA assembler. Briefly, LCR is used to preassemble basic genetic parts into gene expression cassettes or to preassemble small parts into larger parts to reduce the number of parts, in which many basic genetic parts can be reused. With the help of specially designed unique linkers, all preassembled parts will then be directly assembled using DNA assembler to build the target constructs. As proof of concept, three plasmids with varying sizes of 13.4, 24, and 44 kb were rapidly constructed with fidelities of 100, 88, and 71%, respectively. The yeast strain harboring the constructed 44 kb plasmid was confirmed to be capable of utilizing xylose, cellobiose, and glucose to produce zeaxanthin. This strategy should be generally applicable to any custom-designed pathways, circuits, or plasmids. PMID:26332374

  16. Genetic Modifier Screens to Identify Components of a Redox-Regulated Cell Adhesion and Migration Pathway

    PubMed Central

    Hurd, Thomas Ryan; Leblanc, Michelle Gail; Jones, Leonard Nathaniel; DeGennaro, Matthew; Lehmann, Ruth

    2016-01-01

    Under normal physiological conditions, cells use oxidants, particularly H2O2, for signal transduction during processes such as proliferation and migration. Though recent progress has been made in determining the precise role H2O2 plays in these processes, many gaps still remain. To further understand this, we describe the use of a dominant enhancer screen to identify novel components of a redox-regulated cell migration and adhesion pathway in Drosophila melanogaster. Here, we discuss our methodology and progress as well as the benefits and limitations of applying such an approach to study redox-regulated pathways. Depending on the nature of these pathways, unbiased genetic modifier screens may prove a productive way to identify novel redox-regulated signaling components. PMID:23849867

  17. A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae.

    PubMed

    Shi, Shuobo; Liang, Youyun; Zhang, Mingzi M; Ang, Ee Lui; Zhao, Huimin

    2016-01-01

    Despite recent advances in genome editing capabilities for the model organism Saccharomyces cerevisiae, the chromosomal integration of large biochemical pathways for stable industrial production remains challenging. In this work, we developed a simple platform for high-efficiency, single-step, markerless, multi-copy chromosomal integration of full biochemical pathways in Saccharomyces cerevisiae. In this Di-CRISPR (delta integration CRISPR-Cas) platform based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas), we specifically designed guide RNA sequences to target multiple delta sites in the yeast genome. The generation of double stranded breaks at the delta sites allowed simultaneous integration of multiple copies of linearized donor DNA containing large biochemical pathways. With our newly developed Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways and achieve an unprecedented 18-copy genomic integration of a 24kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, thus generating a strain that was able to produce BDO directly from xylose. The simplicity and high efficiency of the Di-CRISPR platform could provide a superior alternative to high copy plasmids and would render this platform an invaluable tool for genome editing and metabolic engineering in S. cerevisiae. PMID:26546089

  18. Genetic network identifies novel pathways contributing to atherosclerosis susceptibility in the innominate artery

    PubMed Central

    2014-01-01

    Background Atherosclerosis, the underlying cause of cardiovascular disease, results from both genetic and environmental factors. Methods In the current study we take a systems-based approach using weighted gene co-expression analysis to identify a candidate pathway of genes related to atherosclerosis. Bioinformatic analyses are performed to identify candidate genes and interactions and several novel genes are characterized using in-vitro studies. Results We identify 1 coexpression module associated with innominate artery atherosclerosis that is also enriched for inflammatory and macrophage gene signatures. Using a series of bioinformatics analysis, we further prioritize the genes in this pathway and identify Cd44 as a critical mediator of the atherosclerosis. We validate our predictions generated by the network analysis using Cd44 knockout mice. Conclusion These results indicate that alterations in Cd44 expression mediate inflammation through a complex transcriptional network involving a number of previously uncharacterized genes. PMID:25115202

  19. IDENTIFYING DISEASE RESISTANCE GENES AND PATHWAYS THROUGH HOST-PATHOGEN PROTEIN INTERACTIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major objective of both animal and plant genomics research is to identify disease resistance genes and pathways. Popular approaches to achieve this goal include candidate gene testing, genome-wide QTL screens, and DNA microarrays. We argue that the two-hybrid assay, which detects protein-protein...

  20. Biochemical and Structural Characterization of a Ureidoglycine Aminotransferase in the Klebsiella pneumoniae Uric Acid Catabolic Pathway

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2010-09-03

    Many plants, fungi, and bacteria catabolize allantoin as a mechanism for nitrogen assimilation. Recent reports have shown that in plants and some bacteria the product of hydrolysis of allantoin by allantoinase is the unstable intermediate ureidoglycine. While this molecule can spontaneously decay, genetic analysis of some bacterial genomes indicates that an aminotransferase may be present in the pathway. Here we present evidence that Klebsiella pneumoniae HpxJ is an aminotransferase that preferentially converts ureidoglycine and an {alpha}-keto acid into oxalurate and the corresponding amino acid. We determined the crystal structure of HpxJ, allowing us to present an explanation for substrate specificity.

  1. Transcriptome and Biochemical Analyses Revealed a Detailed Proanthocyanidin Biosynthesis Pathway in Brown Cotton Fiber

    PubMed Central

    Xiao, Yue-Hua; Yan, Qian; Ding, Hui; Luo, Ming; Hou, Lei; Zhang, Mi; Yao, Dan; Liu, Hou-Sheng; Li, Xin; Zhao, Jia; Pei, Yan

    2014-01-01

    Brown cotton fiber is the major raw material for colored cotton industry. Previous studies have showed that the brown pigments in cotton fiber belong to proanthocyanidins (PAs). To clarify the details of PA biosynthesis pathway in brown cotton fiber, gene expression profiles in developing brown and white fibers were compared via digital gene expression profiling and qRT-PCR. Compared to white cotton fiber, all steps from phenylalanine to PA monomers (flavan-3-ols) were significantly up-regulated in brown fiber. Liquid chromatography mass spectrometry analyses showed that most of free flavan-3-ols in brown fiber were in 2, 3-trans form (gallocatechin and catechin), and the main units of polymeric PAs were trihydroxylated on B ring. Consistent with monomeric composition, the transcript levels of flavonoid 3?, 5?-hydroxylase and leucoanthocyanidin reductase in cotton fiber were much higher than their competing enzymes acting on the same substrates (dihydroflavonol 4-reductase and anthocyanidin synthase, respectively). Taken together, our data revealed a detailed PA biosynthesis pathway wholly activated in brown cotton fiber, and demonstrated that flavonoid 3?, 5?-hydroxylase and leucoanthocyanidin reductase represented the primary flow of PA biosynthesis in cotton fiber. PMID:24466041

  2. Integrated genomic approaches identify major pathways and upstream regulators in late onset Alzheimers disease

    PubMed Central

    Li, Xinzhong; Long, Jintao; He, Taigang; Belshaw, Robert; Scott, James

    2015-01-01

    Previous studies have evaluated gene expression in Alzheimers disease (AD) brains to identify mechanistic processes, but have been limited by the size of the datasets studied. Here we have implemented a novel meta-analysis approach to identify differentially expressed genes (DEGs) in published datasets comprising 450 late onset AD (LOAD) brains and 212 controls. We found 3124 DEGs, many of which were highly correlated with Braak stage and cerebral atrophy. Pathway Analysis revealed the most perturbed pathways to be (a) nitric oxide and reactive oxygen species in macrophages (NOROS), (b) NFkB and (c) mitochondrial dysfunction. NOROS was also up-regulated, and mitochondrial dysfunction down-regulated, in healthy ageing subjects. Upstream regulator analysis predicted the TLR4 ligands, STAT3 and NFKBIA, for activated pathways and RICTOR for mitochondrial genes. Protein-protein interaction network analysis emphasised the role of NFKB; identified a key interaction of CLU with complement; and linked TYROBP, TREM2 and DOK3 to modulation of LPS signalling through TLR4 and to phosphatidylinositol metabolism. We suggest that NEUROD6, ZCCHC17, PPEF1 and MANBAL are potentially implicated in LOAD, with predicted links to calcium signalling and protein mannosylation. Our study demonstrates a highly injurious combination of TLR4-mediated NFKB signalling, NOROS inflammatory pathway activation, and mitochondrial dysfunction in LOAD. PMID:26202100

  3. Integrated genomic approaches identify major pathways and upstream regulators in late onset Alzheimer's disease.

    PubMed

    Li, Xinzhong; Long, Jintao; He, Taigang; Belshaw, Robert; Scott, James

    2015-01-01

    Previous studies have evaluated gene expression in Alzheimer's disease (AD) brains to identify mechanistic processes, but have been limited by the size of the datasets studied. Here we have implemented a novel meta-analysis approach to identify differentially expressed genes (DEGs) in published datasets comprising 450 late onset AD (LOAD) brains and 212 controls. We found 3124 DEGs, many of which were highly correlated with Braak stage and cerebral atrophy. Pathway Analysis revealed the most perturbed pathways to be (a) nitric oxide and reactive oxygen species in macrophages (NOROS), (b) NFkB and (c) mitochondrial dysfunction. NOROS was also up-regulated, and mitochondrial dysfunction down-regulated, in healthy ageing subjects. Upstream regulator analysis predicted the TLR4 ligands, STAT3 and NFKBIA, for activated pathways and RICTOR for mitochondrial genes. Protein-protein interaction network analysis emphasised the role of NFKB; identified a key interaction of CLU with complement; and linked TYROBP, TREM2 and DOK3 to modulation of LPS signalling through TLR4 and to phosphatidylinositol metabolism. We suggest that NEUROD6, ZCCHC17, PPEF1 and MANBAL are potentially implicated in LOAD, with predicted links to calcium signalling and protein mannosylation. Our study demonstrates a highly injurious combination of TLR4-mediated NFKB signalling, NOROS inflammatory pathway activation, and mitochondrial dysfunction in LOAD. PMID:26202100

  4. Hydrograph Separations can Identify Contaminant-Specific Pathways for Conservation Targeting in a Tile-Drained Watershed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Water quality issues continue to vex agriculture. Understanding contaminant-specific pathways could help clarify effective water quality management strategies in watersheds. Hypothesis: If conducted at nested scales, hydrograph separation techniques can identify contaminant-specific pathways that co...

  5. Physiological and biochemical characteristics of adrenergic receptors and pathways in brown adipocytes

    NASA Technical Reports Server (NTRS)

    Horwitz, B. A.

    1975-01-01

    Mechanisms involved in the thermogenic response of brown adipose tissue (BAT) to sympathetic nervous stimulation (e.g., by cold exposure) and to norepinephrine (NE) release are investigated. Three effects appear to play a role in the increased oxygen consumption (and heat production) of the adipocytes: increased membrane permeability, activation of the beta-adrenergic pathway, and enhancement of Na(+)/K(+) membrane pump activity. Increased passive influx of Na(+) and efflux of K(+) due to greater permeability raise the energy demands of the Na/K pump; the pump is also stimulated by increased cyclic AMP synthesis resulting from activation by NE of membrane-bound adenyl cyclase. Studies with inhibitors such as propanolol, phentolamine, and ouabain support this hypothesis.

  6. Biochemical characterization of GDP-L-fucose de novo synthesis pathway in fungus Mortierella alpina

    SciTech Connect

    Ren, Yan; Perepelov, Andrei V.; Wang, Haiyan; Zhang, Hao; Knirel, Yuriy A.; Wang, Lei; Chen, Wei

    2010-01-22

    Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. L-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-L-fucose (the nucleotide-activated form of L-fucose) in M. alpina. Genes encoding GDP-D-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-L-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k{sub m} values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 {sup o}C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-L-fucose biosynthetic pathway in fungi.

  7. An Arabidopsis thaliana gene for methylsalicylate biosynthesis, identified by a biochemical genomics approach, has a role in defense.

    PubMed

    Chen, Feng; D'Auria, John C; Tholl, Dorothea; Ross, Jeannine R; Gershenzon, Jonathan; Noel, Joseph P; Pichersky, Eran

    2003-12-01

    Emission of methylsalicylate (MeSA), and occasionally of methylbenzoate (MeBA), from Arabidopsis thaliana leaves was detected following the application of some forms of both biotic and abiotic stresses to the plant. Maximal emission of MeSA was observed following alamethicin treatment of leaves. A gene (AtBSMT1) encoding a protein with both benzoic acid (BA) and salicylic acid (SA) carboxyl methyltransferase activities was identified using a biochemical genomics approach. Its ortholog (AlBSMT1) in A. lyrata, a close relative of A. thaliana, was also isolated. The AtBSMT1 protein utilizes SA more efficiently than BA, whereas AlBSMT1 catalyzes the methylation of SA less effectively than that of BA. The AtBSMT1 and AlBSMT1 genes showed expression in leaves under normal growth conditions and were more highly expressed in the flowers. In A. thaliana leaves, the expression of AtBSMT1 was induced by alamethicin, Plutella xylostella herbivory, uprooting, physical wounding, and methyl jasmonate. SA was not an effective inducer. Using a beta-glucuronidase (GUS) reporter approach, the promoter activity of AtBSMT1 was localized to the sepals of flowers, and also to leaf trichomes and hydathodes. Upon thrip damage to leaves, AtBSMT1 promoter activity was induced specifically around the lesions. PMID:14617060

  8. Combined Analysis of SNP Array Data Identifies Novel CNV Candidates and Pathways in Ependymoma and Mesothelioma

    PubMed Central

    Wajnberg, Gabriel; Carvalho, Benilton S.; Ferreira, Carlos G.; Passetti, Fabio

    2015-01-01

    Copy number variation is a class of structural genomic modifications that includes the gain and loss of a specific genomic region, which may include an entire gene. Many studies have used low-resolution techniques to identify regions that are frequently lost or amplified in cancer. Usually, researchers choose to use proprietary or non-open-source software to detect these regions because the graphical interface tends to be easier to use. In this study, we combined two different open-source packages into an innovative strategy to identify novel copy number variations and pathways associated with cancer. We used a mesothelioma and ependymoma published datasets to assess our tool. We detected previously described and novel copy number variations that are associated with cancer chemotherapy resistance. We also identified altered pathways associated with these diseases, like cell adhesion in patients with mesothelioma and negative regulation of glutamatergic synaptic transmission in ependymoma patients. In conclusion, we present a novel strategy using open-source software to identify copy number variations and altered pathways associated with cancer. PMID:26185765

  9. Identifying and exploiting defects in the Fanconi anemia/BRCA pathway in oncology.

    PubMed

    Stecklein, Shane R; Jensen, Roy A

    2012-09-01

    Defects in components of DNA repair pathways are responsible for numerous hereditary cancer syndromes and are also common in many sporadic malignancies. Inherited mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 or components of the Fanconi anemia (FA) complex incite genomic instability and predispose to malignancy. The products of the BRCA and FA genes participate in a conserved DNA damage repair pathway that is responsible for repairing interstrand crosslinks and double-strand DNA breaks by homologous recombination. While the genetic instability resulting from FA/BRCA dysfunction contributes to cancer pathogenesis, deficiency of these genes also lends to therapeutic exploitation. Crosslinking agents and ionizing radiation induce damage in cancer cells that requires the FA/BRCA pathway to be resolved; thus cancers that are deficient in BRCA1, BRCA2, or any other component of the FA/BRCA pathway are hypersensitive to these agents. Moreover, emerging synthetic lethal strategies offer opportunities to selectively target cancer cells with defects in homologous recombination. Conversely, enhanced activity of the FA/BRCA pathway is responsible for acquired resistance to specific therapeutic agents, suggesting that both dysfunction and hyperfunction of the FA/BRCA repair machinery are rational targets for cancer therapy. Selection of specific cytotoxic agents based on repair capacity may improve responses and enable personalized cytotoxic chemotherapy. This article reviews the FA/BRCA pathway and current approaches to identify deficiencies within it, discusses synthetic lethality and enhanced repair capacity as causes of therapeutic hypersensitivity and resistance, respectively, and highlights recent studies that have linked FA/BRCA pathway function with therapeutic efficacy. PMID:22683426

  10. Formation of soil organic matter via biochemical and physical pathways of litter mass loss

    NASA Astrophysics Data System (ADS)

    Cotrufo, M. Francesca; Soong, Jennifer L.; Horton, Andrew J.; Campbell, Eleanor E.; Haddix, Michelle L.; Wall, Diana H.; Parton, William J.

    2015-10-01

    Soil organic matter is the largest terrestrial carbon pool. The pool size depends on the balance between formation of soil organic matter from decomposition of plant litter and its mineralization to inorganic carbon. Knowledge of soil organic matter formation remains limited and current C numerical models assume that stable soil organic matter is formed primarily from recalcitrant plant litter. However, labile components of plant litter could also form mineral-stabilized soil organic matter. Here we followed the decomposition of isotopically labelled above-ground litter and its incorporation into soil organic matter over three years in a grassland in Kansas, USA, and used laboratory incubations to determine the decay rates and pool structure of litter-derived organic matter. Early in decomposition, soil organic matter formed when non-structural compounds were lost from litter. Soil organic matter also formed at the end of decomposition, when both non-structural and structural compounds were lost at similar rates. We conclude that two pathways yield soil organic matter efficiently. A dissolved organic matter-microbial path occurs early in decomposition when litter loses mostly non-structural compounds, which are incorporated into microbial biomass at high rates, resulting in efficient soil organic matter formation. An equally efficient physical-transfer path occurs when litter fragments move into soil.

  11. Identifying drug effects via pathway alterations using an integer linear programming optimization formulation on phosphoproteomic data.

    PubMed

    Mitsos, Alexander; Melas, Ioannis N; Siminelakis, Paraskeuas; Chairakaki, Aikaterini D; Saez-Rodriguez, Julio; Alexopoulos, Leonidas G

    2009-12-01

    Understanding the mechanisms of cell function and drug action is a major endeavor in the pharmaceutical industry. Drug effects are governed by the intrinsic properties of the drug (i.e., selectivity and potency) and the specific signaling transduction network of the host (i.e., normal vs. diseased cells). Here, we describe an unbiased, phosphoproteomic-based approach to identify drug effects by monitoring drug-induced topology alterations. With our proposed method, drug effects are investigated under diverse stimulations of the signaling network. Starting with a generic pathway made of logical gates, we build a cell-type specific map by constraining it to fit 13 key phopshoprotein signals under 55 experimental conditions. Fitting is performed via an Integer Linear Program (ILP) formulation and solution by standard ILP solvers; a procedure that drastically outperforms previous fitting schemes. Then, knowing the cell's topology, we monitor the same key phosphoprotein signals under the presence of drug and we re-optimize the specific map to reveal drug-induced topology alterations. To prove our case, we make a topology for the hepatocytic cell-line HepG2 and we evaluate the effects of 4 drugs: 3 selective inhibitors for the Epidermal Growth Factor Receptor (EGFR) and a non-selective drug. We confirm effects easily predictable from the drugs' main target (i.e., EGFR inhibitors blocks the EGFR pathway) but we also uncover unanticipated effects due to either drug promiscuity or the cell's specific topology. An interesting finding is that the selective EGFR inhibitor Gefitinib inhibits signaling downstream the Interleukin-1alpha (IL1alpha) pathway; an effect that cannot be extracted from binding affinity-based approaches. Our method represents an unbiased approach to identify drug effects on small to medium size pathways which is scalable to larger topologies with any type of signaling interventions (small molecules, RNAi, etc). The method can reveal drug effects on pathways, the cornerstone for identifying mechanisms of drug's efficacy. PMID:19997482

  12. Meta-Analysis of Placental Transcriptome Data Identifies a Novel Molecular Pathway Related to Preeclampsia

    PubMed Central

    van Uitert, Miranda; Moerland, Perry D.; Enquobahrie, Daniel A.; Laivuori, Hannele; van der Post, Joris A. M.; Ris-Stalpers, Carrie; Afink, Gijs B.

    2015-01-01

    Studies using the placental transcriptome to identify key molecules relevant for preeclampsia are hampered by a relatively small sample size. In addition, they use a variety of bioinformatics and statistical methods, making comparison of findings challenging. To generate a more robust preeclampsia gene expression signature, we performed a meta-analysis on the original data of 11 placenta RNA microarray experiments, representing 139 normotensive and 116 preeclamptic pregnancies. Microarray data were pre-processed and analyzed using standardized bioinformatics and statistical procedures and the effect sizes were combined using an inverse-variance random-effects model. Interactions between genes in the resulting gene expression signature were identified by pathway analysis (Ingenuity Pathway Analysis, Gene Set Enrichment Analysis, Graphite) and protein-protein associations (STRING). This approach has resulted in a comprehensive list of differentially expressed genes that led to a 388-gene meta-signature of preeclamptic placenta. Pathway analysis highlights the involvement of the previously identified hypoxia/HIF1A pathway in the establishment of the preeclamptic gene expression profile, while analysis of protein interaction networks indicates CREBBP/EP300 as a novel element central to the preeclamptic placental transcriptome. In addition, there is an apparent high incidence of preeclampsia in women carrying a child with a mutation in CREBBP/EP300 (Rubinstein-Taybi Syndrome). The 388-gene preeclampsia meta-signature offers a vital starting point for further studies into the relevance of these genes (in particular CREBBP/EP300) and their concomitant pathways as biomarkers or functional molecules in preeclampsia. This will result in a better understanding of the molecular basis of this disease and opens up the opportunity to develop rational therapies targeting the placental dysfunction causal to preeclampsia. PMID:26171964

  13. Identifiability and estimation of multiple transmission pathways in cholera and waterborne disease.

    PubMed

    Eisenberg, Marisa C; Robertson, Suzanne L; Tien, Joseph H

    2013-05-01

    Cholera and many waterborne diseases exhibit multiple characteristic timescales or pathways of infection, which can be modeled as direct and indirect transmission. A major public health issue for waterborne diseases involves understanding the modes of transmission in order to improve control and prevention strategies. An important epidemiological question is: given data for an outbreak, can we determine the role and relative importance of direct vs. environmental/waterborne routes of transmission? We examine whether parameters for a differential equation model of waterborne disease transmission dynamics can be identified, both in the ideal setting of noise-free data (structural identifiability) and in the more realistic setting in the presence of noise (practical identifiability). We used a differential algebra approach together with several numerical approaches, with a particular emphasis on identifiability of the transmission rates. To examine these issues in a practical public health context, we apply the model to a recent cholera outbreak in Angola (2006). Our results show that the model parameters-including both water and person-to-person transmission routes-are globally structurally identifiable, although they become unidentifiable when the environmental transmission timescale is fast. Even for water dynamics within the identifiable range, when noisy data are considered, only a combination of the water transmission parameters can practically be estimated. This makes the waterborne transmission parameters difficult to estimate, leading to inaccurate estimates of important epidemiological parameters such as the basic reproduction number (R0). However, measurements of pathogen persistence time in environmental water sources or measurements of pathogen concentration in the water can improve model identifiability and allow for more accurate estimation of waterborne transmission pathway parameters as well as R0. Parameter estimates for the Angola outbreak suggest that both transmission pathways are needed to explain the observed cholera dynamics. These results highlight the importance of incorporating environmental data when examining waterborne disease. PMID:23333764

  14. Cerebral Biochemical Pathways in Experimental Autoimmune Encephalomyelitis and Adjuvant Arthritis: A Comparative Metabolomic Study

    PubMed Central

    Lutz, Norbert W.; Fernandez, Carla; Pellissier, Jean-Franois; Cozzone, Patrick J.; Braud, Evelyne

    2013-01-01

    Many diseases, including brain disorders, are associated with perturbations of tissue metabolism. However, an often overlooked issue is the impact that inflammations outside the brain may have on brain metabolism. Our main goal was to study similarities and differences between brain metabolite profiles of animals suffering from experimental autoimmune encephalomyelitis (EAE) and adjuvant arthritis (AA) in Lewis rat models. Our principal objective was the determination of molecular protagonists involved in the metabolism underlying these diseases. EAE was induced by intraplantar injection of complete Freunds adjuvant (CFA) and spinal-cord homogenate (SC-H), whereas AA was induced by CFA only. Naive rats served as controls (n?=?9 for each group). Two weeks after inoculation, animals were sacrificed, and brains were removed and processed for metabolomic analysis by NMR spectroscopy or for immunohistochemistry. Interestingly, both inflammatory diseases caused similar, though not identical, changes in metabolites involved in regulation of brain cell size and membrane production: among the osmolytes, taurine and the neuronal marker, N-acetylaspartate, were decreased, and the astrocyte marker, myo-inositol, slightly increased in both inoculated groups compared with controls. Also ethanolamine-containing phospholipids, sources of inflammatory agents, and several glycolytic metabolites were increased in both inoculated groups. By contrast, the amino acids, aspartate and isoleucine, were less concentrated in CFA/SC-H and control vs. CFA rats. Our results suggest that inflammatory brain metabolite profiles may indicate the existence of either cerebral (EAE) or extra-cerebral (AA) inflammation. These inflammatory processes may act through distinct pathways that converge toward similar brain metabolic profiles. Our findings open new avenues for future studies aimed at demonstrating whether brain metabolic effects provoked by AA are pain/stress-mediated and/or due to the presence of systemic proinflammatory molecules. Regardless of the nature of these mechanisms, our findings may be of interest for future clinical studies, e.g. by in-vivo magnetic resonance spectroscopy. PMID:23457507

  15. Cerebral biochemical pathways in experimental autoimmune encephalomyelitis and adjuvant arthritis: a comparative metabolomic study.

    PubMed

    Lutz, Norbert W; Fernandez, Carla; Pellissier, Jean-Franois; Cozzone, Patrick J; Braud, Evelyne

    2013-01-01

    Many diseases, including brain disorders, are associated with perturbations of tissue metabolism. However, an often overlooked issue is the impact that inflammations outside the brain may have on brain metabolism. Our main goal was to study similarities and differences between brain metabolite profiles of animals suffering from experimental autoimmune encephalomyelitis (EAE) and adjuvant arthritis (AA) in Lewis rat models. Our principal objective was the determination of molecular protagonists involved in the metabolism underlying these diseases. EAE was induced by intraplantar injection of complete Freund's adjuvant (CFA) and spinal-cord homogenate (SC-H), whereas AA was induced by CFA only. Naive rats served as controls (n?=?9 for each group). Two weeks after inoculation, animals were sacrificed, and brains were removed and processed for metabolomic analysis by NMR spectroscopy or for immunohistochemistry. Interestingly, both inflammatory diseases caused similar, though not identical, changes in metabolites involved in regulation of brain cell size and membrane production: among the osmolytes, taurine and the neuronal marker, N-acetylaspartate, were decreased, and the astrocyte marker, myo-inositol, slightly increased in both inoculated groups compared with controls. Also ethanolamine-containing phospholipids, sources of inflammatory agents, and several glycolytic metabolites were increased in both inoculated groups. By contrast, the amino acids, aspartate and isoleucine, were less concentrated in CFA/SC-H and control vs. CFA rats. Our results suggest that inflammatory brain metabolite profiles may indicate the existence of either cerebral (EAE) or extra-cerebral (AA) inflammation. These inflammatory processes may act through distinct pathways that converge toward similar brain metabolic profiles. Our findings open new avenues for future studies aimed at demonstrating whether brain metabolic effects provoked by AA are pain/stress-mediated and/or due to the presence of systemic proinflammatory molecules. Regardless of the nature of these mechanisms, our findings may be of interest for future clinical studies, e.g. by in-vivo magnetic resonance spectroscopy. PMID:23457507

  16. Transcriptome analysis identifies pathways associated with enhanced maternal performance in QSi5 mice

    PubMed Central

    Ramanathan, Palaniappan; Martin, Ian C; Gardiner-Garden, Margaret; Thomson, Peter C; Taylor, Rosanne M; Ormandy, Christopher J; Moran, Christopher; Williamson, Peter

    2008-01-01

    Background Highly fecund mouse strains provide an ideal model to understand the factors affecting maternal performance. The QSi5 inbred strain of mice was selected for high fecundity and low inter-litter interval, and is very successful at weaning large numbers of offspring when compared to other inbred strains. Results Post-natal pup weight gain was used to estimate mammary gland output and to compare the performance of QSi5 mice to CBA mice. Cumulative litter weights and individual pup weight gain was significantly higher throughout the first eight days of lactation in QSi5 mice compared to CBA mice. Morphometric analysis of mammary glands during pregnancy in QSi5 mice revealed a 150 percent greater ductal side branching compared to CBA mice (P < 0.001). Ontology and pathway classification of transcript profiles from the two strains identified an enrichment of genes involved in a number of pathways, including the MAPK, tight junction, insulin signalling and Wnt signalling. Eleven of these genes, including six genes from the MAPK signalling pathway, were identified as associated with postnatal growth. Further, positive mediators of Wnt signalling, including Wnt4, Csnk2a1 and Smad4, were over-represented in the QSi5 strain profile, while negative regulators, including Dkkl1, Ppp2r1a and Nlk, were under-represented. These findings are consistent with the role of Wnt and MAPK signalling pathway in ductal morphogenesis and lobuloalveolar development suggesting enhanced activity in QSi5 mice. A similar pattern of phenotype concordance was seen amongst 12 genes from the tight junction pathway, but a pattern did not emerge from the insulin signalling genes. Amongst a group of differentially expressed imprinted genes, two maternal imprinted genes that suppress growth induced via the IGF signalling pathway, Grb10 and Igf2r, were under-represented in QSi5 mice. Whereas Peg3 and Plagl1, both paternally imprinted genes that enhance neonatal growth, were over-represented in QSi5 mice. Conclusion We propose that the combined action of at least three major signalling pathways involved in mammary gland development and milk secretion, namely Wnt, MAPK and tight junction pathways, contribute to the superior maternal performance phenotype in QSi5 mice. Additionally, favourable expression patterns of the imprinted genes Peg3, Plagl1, Grb10 and Igf2r may also contribute. PMID:18442417

  17. Knowledge-Assisted Approach to Identify Pathways with Differential Dependencies | Office of Cancer Genomics

    Cancer.gov

    We have previously developed a statistical method to identify gene sets enriched with condition-specific genetic dependencies. The method constructs gene dependency networks from bootstrapped samples in one condition and computes the divergence between distributions of network likelihood scores from different conditions. It was shown to be capable of sensitive and specific identification of pathways with phenotype-specific dysregulation, i.e., rewiring of dependencies between genes in different conditions.

  18. Identification and Biochemical Characterization of Mutants in the Proanthocyanidin Pathway in Arabidopsis1

    PubMed Central

    Abrahams, Sharon; Tanner, Gregory J.; Larkin, Philip J.; Ashton, Anthony R.

    2002-01-01

    Proanthocyanidin (PA), or condensed tannin, is a polymeric flavanol that accumulates in a number of tissues in a wide variety of plants. In Arabidopsis, we found that PA precursors (detected histochemically using OsO4) accumulate in the endothelial cell layer of the seed coat from the two-terminal cell stage of embryo development onwards. To understand how PA is made, we screened mature seed pools of T-DNA-tagged Arabidopsis lines to identify mutants defective in the synthesis of PA and found six tds (tannin-deficient seed) complementation groups defective in PA synthesis. Mutations in these loci disrupt the amount (tds1, tds2, tds3, tds5, and tds6) or location and amount of PA (tds4) in the endothelial cell layer. The PA intermediate epicatechin has been identified in wild type and mutants tds1, tds2, tds3, and tds5 (which do not produce PA) and tds6 (6% of wild-type PA), whereas tds4 (2% of wild-type PA) produces an unidentified dimethylaminocinnamaldehyde-reacting compound, indicating that the mutations may be acting on genes beyond leucoanthocyanidin reductase, the first enzymatic reduction step dedicated to PA synthesis. Two other mutants were identified, an allele of tt7, which has a spotted pattern of PA deposition and produces only 8% of the wild-type level of type PA as propelargonidin, and an allele of tt8 producing no PA. Spotted patterns of PA deposition observed in seed of mutants tds4 and tt7-3 result from altered PA composition and distribution in the cell. Our mutant screen, which was not exhaustive, suggests that the cooperation of many genes is required for successful PA accumulation. PMID:12376625

  19. Gene Set Enrichment in eQTL Data Identifies Novel Annotations and Pathway Regulators

    PubMed Central

    Wu, Chunlei; Delano, David L.; Mitro, Nico; Su, Stephen V.; Janes, Jeff; McClurg, Phillip; Batalov, Serge; Welch, Genevieve L.; Zhang, Jie; Orth, Anthony P.; Walker, John R.; Glynne, Richard J.; Cooke, Michael P.; Takahashi, Joseph S.; Shimomura, Kazuhiro; Kohsaka, Akira; Bass, Joseph; Saez, Enrique; Wiltshire, Tim; Su, Andrew I.

    2008-01-01

    Genome-wide gene expression profiling has been extensively used to generate biological hypotheses based on differential expression. Recently, many studies have used microarrays to measure gene expression levels across genetic mapping populations. These gene expression phenotypes have been used for genome-wide association analyses, an analysis referred to as expression QTL (eQTL) mapping. Here, eQTL analysis was performed in adipose tissue from 28 inbred strains of mice. We focused our analysis on trans-eQTL bands, defined as instances in which the expression patterns of many genes were all associated to a common genetic locus. Genes comprising trans-eQTL bands were screened for enrichments in functional gene sets representing known biological pathways, and genes located at associated trans-eQTL band loci were considered candidate transcriptional modulators. We demonstrate that these patterns were enriched for previously characterized relationships between known upstream transcriptional regulators and their downstream target genes. Moreover, we used this strategy to identify both novel regulators and novel members of known pathways. Finally, based on a putative regulatory relationship identified in our analysis, we identified and validated a previously uncharacterized role for cyclin H in the regulation of oxidative phosphorylation. We believe that the specific molecular hypotheses generated in this study will reveal many additional pathway members and regulators, and that the analysis approaches described herein will be broadly applicable to other eQTL data sets. PMID:18464898

  20. Identifying low variance pathways for free energy calculations of molecular transformations in solution phase

    NASA Astrophysics Data System (ADS)

    Pham, Tri T.; Shirts, Michael R.

    2011-07-01

    Improving the efficiency of free energy calculations is important for many biological and materials design applications, such as protein-ligand binding affinities in drug design, partitioning between immiscible liquids, and determining molecular association in soft materials. We show that for any pair potential, moderately accurate estimation of the radial distribution function for a solute molecule is sufficient to accurately estimate the statistical variance of a sampling along a free energy pathway. This allows inexpensive analytical identification of low statistical error free energy pathways. We employ a variety of methods to estimate the radial distribution function (RDF) and find that the computationally cheap two-body "dilute gas" limit performs as well or better than 3D-RISM theory and other approximations for identifying low variance free energy pathways. With a RDF estimate in hand, we can search for pairwise interaction potentials that produce low variance. We give an example of a search minimizing statistical variance of solvation free energy over the entire parameter space of a generalized "soft core" potential. The free energy pathway arising from this optimization procedure has lower curvature in the variance and reduces the total variance by at least 50% compared to the traditional soft core solvation pathway. We also demonstrate that this optimized pathway allows free energies to be estimated with fewer intermediate states due to its low curvature. This free energy variance optimization technique is generalizable to solvation in any homogeneous fluid and for any type of pairwise potential and can be performed in minutes to hours, depending on the method used to estimate g(r).

  1. PCOSKB: A KnowledgeBase on genes, diseases, ontology terms and biochemical pathways associated with PolyCystic Ovary Syndrome.

    PubMed

    Joseph, Shaini; Barai, Ram Shankar; Bhujbalrao, Rasika; Idicula-Thomas, Susan

    2016-01-01

    Polycystic ovary syndrome (PCOS) is one of the major causes of female subfertility worldwide and ?7-10% of women in reproductive age are affected by it. The affected individuals exhibit varying types and levels of comorbid conditions, along with the classical PCOS symptoms. Extensive studies on PCOS across diverse ethnic populations have resulted in a plethora of information on dysregulated genes, gene polymorphisms and diseases linked to PCOS. However, efforts have not been taken to collate and link these data. Our group, for the first time, has compiled PCOS-related information available through scientific literature; cross-linked it with molecular, biochemical and clinical databases and presented it as a user-friendly, web-based online knowledgebase for the benefit of the scientific and clinical community. Manually curated information on associated genes, single nucleotide polymorphisms, diseases, gene ontology terms and pathways along with supporting reference literature has been collated and included in PCOSKB (http://pcoskb.bicnirrh.res.in). PMID:26578565

  2. PCOSKB: A KnowledgeBase on genes, diseases, ontology terms and biochemical pathways associated with PolyCystic Ovary Syndrome

    PubMed Central

    Joseph, Shaini; Barai, Ram Shankar; Bhujbalrao, Rasika; Idicula-Thomas, Susan

    2016-01-01

    Polycystic ovary syndrome (PCOS) is one of the major causes of female subfertility worldwide and ≈7–10% of women in reproductive age are affected by it. The affected individuals exhibit varying types and levels of comorbid conditions, along with the classical PCOS symptoms. Extensive studies on PCOS across diverse ethnic populations have resulted in a plethora of information on dysregulated genes, gene polymorphisms and diseases linked to PCOS. However, efforts have not been taken to collate and link these data. Our group, for the first time, has compiled PCOS-related information available through scientific literature; cross-linked it with molecular, biochemical and clinical databases and presented it as a user-friendly, web-based online knowledgebase for the benefit of the scientific and clinical community. Manually curated information on associated genes, single nucleotide polymorphisms, diseases, gene ontology terms and pathways along with supporting reference literature has been collated and included in PCOSKB (http://pcoskb.bicnirrh.res.in). PMID:26578565

  3. Gene Expression in Relation to Exhaled Nitric Oxide Identifies Novel Asthma Phenotypes with Unique Biomolecular Pathways

    PubMed Central

    Modena, Brian D.; Tedrow, John R.; Milosevic, Jadranka; Bleecker, Eugene R.; Meyers, Deborah A.; Wu, Wei; Bar-Joseph, Ziv; Erzurum, Serpil C.; Gaston, Benjamin M.; Busse, William W.; Jarjour, Nizar N.; Kaminski, Naftali

    2014-01-01

    Rationale Although asthma is recognized as a heterogeneous disease associated with clinical phenotypes, the molecular basis of these phenotypes remains poorly understood. Although genomic studies have successfully broadened our understanding in diseases such as cancer, they have not been widely used in asthma studies. Objectives To link gene expression patterns to clinical asthma phenotypes. Methods We used a microarray platform to analyze bronchial airway epithelial cell gene expression in relation to the asthma biomarker fractional exhaled nitric oxide (FeNO) in 155 subjects with asthma and healthy control subjects from the Severe Asthma Research Program (SARP). Measurements and Main Results We first identified a diverse set of 549 genes whose expression correlated with FeNO. We used k-means to cluster the patient samples according to the expression of these genes, identifying five asthma clusters/phenotypes with distinct clinical, physiological, cellular, and gene transcription characteristicstermed subject clusters (SCs). To then investigate differences in gene expression between SCs, a total of 1,384 genes were identified that highly differentiated the SCs at an unadjusted P value?identified nine gene clusters or biclusters, whose coexpression suggested biological characteristics unique to each SC. Although genes related to type 2 inflammation were present, novel pathways, including those related to neuronal function, WNT pathways, and actin cytoskeleton, were noted. Conclusions These findings show that bronchial epithelial cell gene expression, as related to the asthma biomarker FeNO, can identify distinct asthma phenotypes, while also suggesting the presence of underlying novel gene pathways relevant to these phenotypes. PMID:25338189

  4. Biochemical Analysis of the Biosynthetic Pathway of an Anticancer Tetracycline SF2575

    PubMed Central

    Pickens, Lauren B.; Kim, Woncheol; Wang, Peng; Zhou, Hui; Watanabe, Kenji; Gomi, Shuichi; Tang, Yi

    2009-01-01

    SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity towards a broad range of cancer cell lines. The structure of SF2575 is characterized by a highly substituted tetracycline aglycon. The modifications include methylation of the C-6 and C-12a hydroxyl groups, acylation of the 4-(S)-hydroxyl with salicylic acid, C-glycosylation of the C-9 of the D-ring with d-olivose and further acylation of the C4?-hydroxyl of d-olivose with the unusual angelic acid. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogs. In this study, we identified, sequenced and functionally analyzed the ssf biosynthetic gene cluster which contains 40 putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as well as installing these unique structural features are found in the gene cluster. Biosynthetic intermediates were isolated from the SF2575 culture extract to suggest the order of pendant groups addition is C-9 glycosylation, C-4 salicylation and O-4? angelycylation. Using in vitro assays, two enzymes that are responsible for C-4 acylation of salicylic acid were identified. These enzymes include an ATP-dependent salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown to have relaxed substrate specificity towards substituted benzoic acids. Since the salicylic acid moiety is critically important for the anticancer properties of SF2575, verification of the activities of SsfL1 and SsfX3 sets the stage for biosynthetic modification of the C-4 group towards structural-activity relationship studies of SF2575. Using heterologous biosynthesis in Streptomyces lividans, we also determined that biosynthesis of the SF2575 tetracycline aglycon 8 parallels that of oxytetracycline 4 and diverges after the assembly of 4-keto-anhydrotetracycline 51. The minimal ssf polyketide synthase together with the amidotransferase SsfD produced the amidated decaketide backbone that is required for the formation of 2-naphthacenecarboxamide skeleton. Additional enzymes, such as cyclases, C-6 methyltransferase and C-4/C-12a dihydroxylase were functionally reconstituted. PMID:19908837

  5. Metabolic profiling of chickpea-Fusarium interaction identifies differential modulation of disease resistance pathways.

    PubMed

    Kumar, Yashwant; Dholakia, Bhushan B; Panigrahi, Priyabrata; Kadoo, Narendra Y; Giri, Ashok P; Gupta, Vidya S

    2015-08-01

    Chickpea is the third most widely grown legume in the world and mainly used as a vegetarian source of human dietary protein. Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceri (Foc), is one of the major threats to global chickpea production. Host resistance is the best way to protect crops from diseases; however, in spite of using various approaches, the mechanism of Foc resistance in chickpea remains largely obscure. In the present study, non-targeted metabolic profiling at several time points of resistant and susceptible chickpea cultivars using high-resolution liquid chromatography-mass spectrometry was applied to better understand the mechanistic basis of wilt resistance or susceptibility. Multivariate analysis of the data (OPLS-DA) revealed discriminating metabolites in chickpea root tissue after Foc inoculation such as flavonoids, isoflavonoids, alkaloids, amino acids and sugars. Foc inoculated resistant plants had more flavonoids and isoflavonoids along with their malonyl conjugates. Many antifungal metabolites that were induced after Foc infection viz., aurantion-obstine ?-glucosides and querecitin were elevated in resistant cultivar. Overall, diverse genetic and biochemical mechanisms were operational in the resistant cultivar for Foc defense as compared to the susceptible plant. The resistant chickpea plants employed the above-mentioned metabolic pathways as potential defense strategy against Foc. PMID:25935544

  6. Insertional Mutagenesis Identifies a STAT3/Arid1b/β-catenin Pathway Driving Neurofibroma Initiation.

    PubMed

    Wu, Jianqiang; Keng, Vincent W; Patmore, Deanna M; Kendall, Jed J; Patel, Ami V; Jousma, Edwin; Jessen, Walter J; Choi, Kwangmin; Tschida, Barbara R; Silverstein, Kevin A T; Fan, Danhua; Schwartz, Eric B; Fuchs, James R; Zou, Yuanshu; Kim, Mi-Ok; Dombi, Eva; Levy, David E; Huang, Gang; Cancelas, Jose A; Stemmer-Rachamimov, Anat O; Spinner, Robert J; Largaespada, David A; Ratner, Nancy

    2016-03-01

    To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/β-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and β-catenin activity. β-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and β-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3β and the SWI/SNF gene Arid1b to increase β-catenin. Knockdown of Arid1b or Gsk3β in Stat3(fl/fl);Nf1(fl/fl);DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/β-catenin pathway inhibitors in neurofibroma therapeutic trials. PMID:26904939

  7. A cellular genetics approach identifies gene-drug interactions and pinpoints drug toxicity pathway nodes

    PubMed Central

    Suzuki, Oscar T.; Frick, Amber; Parks, Bethany B.; Trask, O. Joseph; Butz, Natasha; Steffy, Brian; Chan, Emmanuel; Scoville, David K.; Healy, Eric; Benton, Cristina; McQuaid, Patricia E.; Thomas, Russell S.; Wiltshire, Tim

    2014-01-01

    New approaches to toxicity testing have incorporated high-throughput screening across a broad-range of in vitro assays to identify potential key events in response to chemical or drug treatment. To date, these approaches have primarily utilized repurposed drug discovery assays. In this study, we describe an approach that combines in vitro screening with genetic approaches for the experimental identification of genes and pathways involved in chemical or drug toxicity. Primary embryonic fibroblasts isolated from 32 genetically-characterized inbred mouse strains were treated in concentration-response format with 65 compounds, including pharmaceutical drugs, environmental chemicals, and compounds with known modes-of-action. Integrated cellular responses were measured at 24 and 72 h using high-content imaging and included cell loss, membrane permeability, mitochondrial function, and apoptosis. Genetic association analysis of cross-strain differences in the cellular responses resulted in a collection of candidate loci potentially underlying the variable strain response to each chemical. As a demonstration of the approach, one candidate gene involved in rotenone sensitivity, Cybb, was experimentally validated in vitro and in vivo. Pathway analysis on the combined list of candidate loci across all chemicals identified a number of over-connected nodes that may serve as core regulatory points in toxicity pathways. PMID:25221565

  8. A new screening pathway for identifying asymptomatic patients using dental panoramic radiographs

    NASA Astrophysics Data System (ADS)

    Hayashi, Tatsuro; Matsumoto, Takuya; Sawagashira, Tsuyoshi; Tagami, Motoki; Katsumata, Akitoshi; Hayashi, Yoshinori; Muramatsu, Chisako; Zhou, Xiangrong; Iida, Yukihiro; Matsuoka, Masato; Katagi, Kiyoji; Fujita, Hiroshi

    2012-03-01

    To identify asymptomatic patients is the challenging task and the essential first step in diagnosis. Findings of dental panoramic radiographs include not only dental conditions but also radiographic signs that are suggestive of possible systemic diseases such as osteoporosis, arteriosclerosis, and maxillary sinusitis. Detection of such signs on panoramic radiographs has a potential to provide supplemental benefits for patients. However, it is not easy for general dental practitioners to pay careful attention to such signs. We addressed the development of a computer-aided detection (CAD) system that detects radiographic signs of pathology on panoramic images, and the design of the framework of new screening pathway by cooperation of dentists and our CAD system. The performance evaluation of our CAD system showed the sensitivity and specificity in the identification of osteoporotic patients were 92.6 % and 100 %, respectively, and those of the maxillary sinus abnormality were 89.6 % and 73.6 %, respectively. The detection rate of carotid artery calcifications that suggests the need for further medical evaluation was approximately 93.6 % with 4.4 false-positives per image. To validate the utility of the new screening pathway, preliminary clinical trials by using our CAD system were conducted. To date, 223 panoramic images were processed and 4 asymptomatic patients with suspected osteoporosis, 7 asymptomatic patients with suspected calcifications, and 40 asymptomatic patients with suspected maxillary sinusitis were detected in our initial trial. It was suggested that our new screening pathway could be useful to identify asymptomatic patients with systemic diseases.

  9. FamNet: A Framework to Identify Multiplied Modules Driving Pathway Expansion in Plants.

    PubMed

    Ruprecht, Colin; Mendrinna, Amelie; Tohge, Takayuki; Sampathkumar, Arun; Klie, Sebastian; Fernie, Alisdair R; Nikoloski, Zoran; Persson, Staffan; Mutwil, Marek

    2016-03-01

    Gene duplications generate new genes that can acquire similar but often diversified functions. Recent studies of gene coexpression networks have indicated that, not only genes, but also pathways can be multiplied and diversified to perform related functions in different parts of an organism. Identification of such diversified pathways, or modules, is needed to expand our knowledge of biological processes in plants and to understand how biological functions evolve. However, systematic explorations of modules remain scarce, and no user-friendly platform to identify them exists. We have established a statistical framework to identify modules and show that approximately one-third of the genes of a plant's genome participate in hundreds of multiplied modules. Using this framework as a basis, we implemented a platform that can explore and visualize multiplied modules in coexpression networks of eight plant species. To validate the usefulness of the platform, we identified and functionally characterized pollen- and root-specific cell wall modules that multiplied to confer tip growth in pollen tubes and root hairs, respectively. Furthermore, we identified multiplied modules involved in secondary metabolite synthesis and corroborated them by metabolite profiling of tobacco (Nicotiana tabacum) tissues. The interactive platform, referred to as FamNet, is available at http://www.gene2function.de/famnet.html. PMID:26754669

  10. A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation.

    PubMed

    Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

    2016-01-01

    Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGL?) and -beta (DAGL?) to neurons and microglia, respectively. Disruption of DAGL? perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function. PMID:26779719

  11. Identifying the potential extracellular electron transfer pathways from a c-type cytochrome network.

    PubMed

    Ding, De-Wu; Xu, Jun; Li, Ling; Xie, Jian-Ming; Sun, Xiao

    2014-12-01

    Extracellular electron transfer (EET) is the key feature of some bacteria, such as Geobacter sulfurreducens and Shewanella oneidensis. Via EET processes, these bacteria can grow on electrode surfaces and make current output of microbial fuel cells. c-Type cytochromes can be used as carriers to transfer electrons, which play an important role in EET processes. Typically, from the inner (cytoplasmic) membrane through the periplasm to the outer membrane, they could form EET pathways. Recent studies suggest that a group of c-type cytochromes could form a network which extended the well-known EET pathways. We obtained the protein interaction information for all 41 c-type cytochromes in Shewanella oneidensis MR-1, constructed a large-scale protein interaction network, and studied its structural characteristics and functional significance. Centrality analysis has identified the top 10 key proteins of the network, and 7 of them are associated with electricity production in the bacteria, which suggests that the ability of Shewanella oneidensis MR-1 to produce electricity might be derived from the unique structure of the c-type cytochrome network. By modularity analysis, we obtained 5 modules from the network. The subcellular localization study has shown that the proteins in these modules all have diversiform cellular compartments, which reflects their potential to form EET pathways. In particular, combination of protein subcellular localization and operon analysis, the well-known and new candidate EET pathways are obtained from the Mtr-like module, indicating that potential EET pathways could be obtained from such a c-type cytochrome network. PMID:25227320

  12. Carbon and chlorine isotope analysis to identify abiotic degradation pathways of 1,1,1-trichloroethane.

    PubMed

    Palau, Jordi; Shouakar-Stash, Orfan; Hunkeler, Daniel

    2014-12-16

    This study investigates dual C-Cl isotope fractionation during 1,1,1-TCA transformation by heat-activated persulfate (PS), hydrolysis/dehydrohalogenation (HY/DH) and Fe(0). Compound-specific chlorine isotope analysis of 1,1,1-TCA was performed for the first time, and transformation-associated isotope fractionation ? bulk C and ? bulk Cl values were -4.0 0.2 and no chlorine isotope fractionation with PS, -1.6 0.2 and -4.7 0.1 for HY/DH, -7.8 0.4 and -5.2 0.2 with Fe(0). Distinctly different dual isotope slopes (??13C/??37Cl): ? with PS, 0.33 0.04 for HY/DH and 1.5 0.1 with Fe(0) highlight the potential of this approach to identify abiotic degradation pathways of 1,1,1-TCA in the field. The trend observed with PS agreed with a C-H bond oxidation mechanism in the first reaction step. For HY/DH and Fe(0) pathways, different slopes were obtained although both pathways involve cleavage of a C-Cl bond in their initial reaction step. In contrast to the expected larger primary carbon isotope effects relative to chlorine for C-Cl bond cleavage, ? bulk C < ? bulk Cl was observed for HY/DH and in a similar range for reduction by Fe(0), suggesting the contribution of secondary chlorine isotope effects. Therefore, different magnitude of secondary chlorine isotope effects could at least be partly responsible for the distinct slopes between HY/DH and Fe(0) pathways. Following this dual isotope approach, abiotic transformation processes can unambiguously be identified and quantified. PMID:25379605

  13. Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation.

    PubMed

    Rovira, Meritxell; Huang, Wei; Yusuff, Shamila; Shim, Joong Sup; Ferrante, Anthony A; Liu, Jun O; Parsons, Michael J

    2011-11-29

    Pancreatic ?-cells are an essential source of insulin and their destruction because of autoimmunity causes type I diabetes. We conducted a chemical screen to identify compounds that would induce the differentiation of insulin-producing ?-cells in vivo. To do this screen, we brought together the use of transgenic zebrafish as a model of ?-cell differentiation, a unique multiwell plate that allows easy visualization of lateral views of swimming larval fish and a library of clinical drugs. We identified six hits that can induce precocious differentiation of secondary islets in larval zebrafish. Three of these six hits were known drugs with a considerable background of published data on mechanism of action. Using pharmacological approaches, we have identified and characterized two unique pathways in ?-cell differentiation in the zebrafish, including down-regulation of GTP production and retinoic acid biosynthesis. PMID:22084084

  14. Using Smoke Injection in Drains to Identify Potential Preferential Pathways in a Drained Arable Field

    NASA Astrophysics Data System (ADS)

    Nielsen, M. H.; Petersen, C. T.; Hansen, S.

    2014-12-01

    Macropores forming a continuous pathway between the soil surface and subsurface drains favour the transport of many contaminants from agricultural fields to surface waters. The smoke injection method presented by Shipitalo and Gibbs (2000) used for demonstrating and quantifying such pathways has been further developed and used on a drained Danish sandy loam. In order to identify the preferential pathways to drains, smoke was injected in three 1.15 m deep tile drains (total drain length 93 m), and smoke emitting macropores (SEMP) at the soil surface were counted and characterized as producing either strong or weak plumes compared to reference plumes from 3 and 6 mm wide tubes. In the two situations investigated in the present study - an early spring and an autumn situation, smoke only penetrated the soil surface layer via earthworm burrows located in a 1.0 m wide belt directly above the drain lines. However, it is known from previous studies that desiccation fractures in a dry summer situation also can contribute to the smoke pattern. The distance between SEMP measured along the drain lines was on average 0.46 m whereas the average spacing between SEMP with strong plumes was 2.3 m. Ponded water was applied in 6 cm wide rings placed above 52 burrows including 17 reference burrows which did not emit smoke. Thirteen pathways in the soil were examined using dye tracer and profile excavation. SEMP with strong plumes marked the entrance of highly efficient transport pathways conducting surface applied water and dye tracer into the drain. However, no single burrow was traced all the way from the surface into the drain, the dye patterns branched off in a network of other macropores. Water infiltration rates were significantly higher (P < 0.05) in SEMP with strong plumes (average rate: 247 mL min-1 n = 19) compared to SEMP with weak plumes (average rate: 87 mL min-1 n = 16) and no plumes (average rate: 56 mL min-1 n = 17). The results suggest that the smoke injection method is useful for identification of potentially efficient pathways for surface applied contaminants to drains and surface waters, pathways being associated primarily with unevenly distributed SEMP producing strong smoke plumes.

  15. Transcriptional profiling of GBM invasion genes identifies effective inhibitors of the LIM kinase-Cofilin pathway

    PubMed Central

    Golbourn, Brian; Bertrand, Kelsey C.; Luck, Amanda; Sabha, Nesrin; Smith, Christian A.; Byron, Sara; Zadeh, Gelareh; Croul, Sidney; Berens, Michael; Rutka, James T.

    2014-01-01

    Malignant gliomas are highly proliferative and invasive neoplasms where total surgical resection is often impossible and effective local radiation therapy difficult. Consequently, there is a need to develop a greater understanding of the molecular events driving invasion and to identify novel treatment targets. Using microarray analysis comparing normal brain samples and mesenchymal glioblastoma multiforme (GBM), we identified over 140 significant genes involved in cell migration and invasion. The cofilin (CFL) pathway, which disassembles actin filaments, was highly up-regulated compared to normal brain. Up-regulation of LIM domain kinase 1 and 2 (LIMK1/2), that phosphorylates and inactivates cofilin, was confirmed in an additional independent data set comparing normal brain to GBM. We identified and utilized two small molecule inhibitors BMS-5 and Cucurbitacin I directed against the cofilin regulating kinases, LIMK1 and LIMK2, to target this pathway. Significant decreases in cell viability were observed in glioma cells treated with BMS-5 and Cucurbitacin I, while no cytotoxic effects were seen in normal astrocytes that lack LIMK. BMS-5 and Cucurbitacin I promoted increased adhesion in GBM cells, and decreased migration and invasion. Collectively, these data suggest that use of LIMK inhibitors may provide a novel way to target the invasive machinery in GBM. PMID:25237832

  16. Anthropogenic Molecular Markers: Tools to Identify the Sources and Transport Pathways of Pollutants

    USGS Publications Warehouse

    Takada, H.; Satoh, F.; Bothner, Michael H.; Tripp, B.W.; Johnson, C.G.; Farrington, J.W.

    1997-01-01

    The activities of modern civilization have released to the oceans a wide variety of both mobilized natural compounds and synthetic compounds not found prior to modern times. Many of these compounds provide a means of identifying sources of inputs and pathways of movement of chemicals through oceanic ecosystems and serve as molecular markers of human activities. A coastal ocean (Tokyo Bay) and a deep ocean (Deep Water Dump Site 106 in the Western North Atlantic Ocean) example are presented. In the deep ocean study, the correlation between potential sewage marker, i.e. linear alkylbenzenes (LABs), and polychlorinated biphenyls (PCBs) concentrations indicates a contribution of sewage sludge PCBs to the dump site sediments.

  17. Mechanisms and consequences of the impaired trans-sulphuration pathway in liver disease: Part I. Biochemical implications.

    PubMed

    Mato, J M; Corrales, F; Martin-Duce, A; Ortiz, P; Pajares, M A; Cabrero, C

    1990-01-01

    The energy-dependent conversion of methionine to S-adenosyl-L-methionine (SAMe) is catalysed by S-adenosyl-L-methionine synthetase (SAMe-synthetase) in the liver. In the hepatocyte, an equilibrium exists between the high and low molecular weight forms of SAMe-synthetase, which consist of a tetramer and a dimer, respectively, of a 48.5 kilodalton subunit. The 2 enzymic forms differ in their affinity for methionine and sensitivity to inhibition by pyrophosphate; 2 of the sulfhydryl groups of SAMe-synthetase have been identified as essential for the normal functioning of the enzyme. In patients with liver cirrhosis, a marked reduction in the utilisation of the high molecular weight SAMe-synthetase and displacement of the equilibrium occur, the molecular mechanism of which has yet to be established. This loss of activity is associated with a delay in methionine clearance and impairment of the trans-sulphuration pathway, which normally eliminates excess methionine by oxidising homocysteine to sulphate anion. It is hypothesised that in normal liver function the essential sulfhydryl groups of SAMe-synthetase are protected from oxidation by glutathione, a by-product of the trans-sulphuration pathway. However, glutathione levels are reduced in liver cirrhosis, and this may result in increased oxidation of the essential sulfhydryl groups, and consequent inactivation of the enzyme. Thus, the trans-sulphuration pathway may play an important role in the maintenance of normal SAMe-synthetase activity. PMID:2081481

  18. Identifying Genetic Variants for Heart Rate Variability in the Acetylcholine Pathway

    PubMed Central

    Riese, Harritte; Muoz, Loretto M.; Hartman, Catharina A.; Ding, Xiuhua; Su, Shaoyong; Oldehinkel, Albertine J.; van Roon, Arie M.; van der Most, Peter J.; Lefrandt, Joop; Gansevoort, Ron T.; van der Harst, Pim; Verweij, Niek; Licht, Carmilla M. M.; Boomsma, Dorret I.; Hottenga, Jouke-Jan; Willemsen, Gonneke; Penninx, Brenda W. J. H.; Nolte, Ilja M.; de Geus, Eco J. C.; Wang, Xiaoling; Snieder, Harold

    2014-01-01

    Heart rate variability is an important risk factor for cardiovascular disease and all-cause mortality. The acetylcholine pathway plays a key role in explaining heart rate variability in humans. We assessed whether 443 genotyped and imputed common genetic variants in eight key genes (CHAT, SLC18A3, SLC5A7, CHRNB4, CHRNA3, CHRNA, CHRM2 and ACHE) of the acetylcholine pathway were associated with variation in an established measure of heart rate variability reflecting parasympathetic control of the heart rhythm, the root mean square of successive differences (RMSSD) of normal RR intervals. The association was studied in a two stage design in individuals of European descent. First, analyses were performed in a discovery sample of four cohorts (n?=?3429, discovery stage). Second, findings were replicated in three independent cohorts (n?=?3311, replication stage), and finally the two stages were combined in a meta-analysis (n?=?6740). RMSSD data were obtained under resting conditions. After correction for multiple testing, none of the SNPs showed an association with RMSSD. In conclusion, no common genetic variants for heart rate variability were identified in the largest and most comprehensive candidate gene study on the acetylcholine pathway to date. Future gene finding efforts for RMSSD may want to focus on hypothesis free approaches such as the genome-wide association study. PMID:25384021

  19. Pan-Cancer Network Analysis Identifies Combinations of Rare Somatic Mutations across Pathways and Protein Complexes

    PubMed Central

    Leiserson, Mark D.M.; Vandin, Fabio; Wu, Hsin-Ta; Dobson, Jason R.; Eldridge, Jonathan V.; Thomas, Jacob L.; Papoutsaki, Alexandra; Kim, Younhun; Niu, Beifang; McLellan, Michael; Lawrence, Michael S.; Gonzalez-Perez, Abel; Tamborero, David; Cheng, Yuwei; Ryslik, Gregory A.; Lopez-Bigas, Nuria; Getz, Gad; Ding, Li; Raphael, Benjamin J.

    2014-01-01

    Cancers exhibit extensive mutational heterogeneity and the resulting long tail phenomenon complicates the discovery of the genes and pathways that are significantly mutated in cancer. We perform a Pan-Cancer analysis of mutated networks in 3281 samples from 12 cancer types from The Cancer Genome Atlas (TCGA) using HotNet2, a novel algorithm to find mutated subnetworks that overcomes limitations of existing single gene and pathway/network approaches.. We identify 14 significantly mutated subnetworks that include well-known cancer signaling pathways as well as subnetworks with less characterized roles in cancer including cohesin, condensin, and others. Many of these subnetworks exhibit co-occurring mutations across samples. These subnetworks contain dozens of genes with rare somatic mutations across multiple cancers; many of these genes have additional evidence supporting a role in cancer. By illuminating these rare combinations of mutations, Pan-Cancer network analyses provide a roadmap to investigate new diagnostic and therapeutic opportunities across cancer types. PMID:25501392

  20. Inside the biochemical pathways of thymidylate synthase perturbed by anticancer drugs: Novel strategies to overcome cancer chemoresistance.

    PubMed

    Taddia, Laura; D'Arca, Domenico; Ferrari, Stefania; Marraccini, Chiara; Severi, Leda; Ponterini, Glauco; Assaraf, Yahuda G; Marverti, Gaetano; Costi, Maria Paola

    2015-11-01

    Our current understanding of the mechanisms of action of antitumor agents and the precise mechanisms underlying drug resistance is that these two processes are directly linked. Moreover, it is often possible to delineate chemoresistance mechanisms based on the specific mechanism of action of a given anticancer drug. A more holistic approach to the chemoresistance problem suggests that entire metabolic pathways, rather than single enzyme targets may better explain and educate us about the complexity of the cellular responses upon cytotoxic drug administration. Drugs, which target thymidylate synthase and folate-dependent enzymes, represent an important therapeutic arm in the treatment of various human malignancies. However, prolonged patient treatment often provokes drug resistance phenomena that render the chemotherapeutic treatment highly ineffective. Hence, strategies to overcome drug resistance are primarily designed to achieve either enhanced intracellular drug accumulation, to avoid the upregulation of folate-dependent enzymes, and to circumvent the impairment of DNA repair enzymes which are also responsible for cross-resistance to various anticancer drugs. The current clinical practice based on drug combination therapeutic regimens represents the most effective approach to counteract drug resistance. In the current paper, we review the molecular aspects of the activity of TS-targeting drugs and describe how such mechanisms are related to the emergence of clinical drug resistance. We also discuss the current possibilities to overcome drug resistance by using a molecular mechanistic approach based on medicinal chemistry methods focusing on rational structural modifications of novel antitumor agents. This paper also focuses on the importance of the modulation of metabolic pathways upon drug administration, their analysis and the assessment of their putative roles in the networks involved using a meta-analysis approach. The present review describes the main pathways that are modulated by TS-targeting anticancer drugs starting from the description of the normal functioning of the folate metabolic pathway, through the protein modulation occurring upon drug delivery to cultured tumor cells as well as cancer patients, finally describing how the pathways are modulated by drug resistance development. The data collected are then analyzed using network/netwire connecting methods in order to provide a wider view of the pathways involved and of the importance of such information in identifying additional proteins that could serve as novel druggable targets for efficacious cancer therapy. PMID:26690339

  1. Gene Expression Profiling Identifies Molecular Pathways Associated with Collagen VI Deficiency and Provides Novel Therapeutic Targets

    PubMed Central

    Paco, Sonia; Kalko, Susana G.; Jou, Cristina; Rodrguez, Mara A.; Corbera, Joan; Muntoni, Francesco; Feng, Lucy; Rivas, Eloy; Torner, Ferran; Gualandi, Francesca; Gomez-Foix, Anna M.; Ferrer, Anna; Ortez, Carlos; Nascimento, Andrs; Colomer, Jaume; Jimenez-Mallebrera, Cecilia

    2013-01-01

    Ullrich congenital muscular dystrophy (UCMD), caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered. PMID:24223098

  2. Gene expression profiling identifies molecular pathways associated with collagen VI deficiency and provides novel therapeutic targets.

    PubMed

    Paco, Sonia; Kalko, Susana G; Jou, Cristina; Rodrguez, Mara A; Corbera, Joan; Muntoni, Francesco; Feng, Lucy; Rivas, Eloy; Torner, Ferran; Gualandi, Francesca; Gomez-Foix, Anna M; Ferrer, Anna; Ortez, Carlos; Nascimento, Andrs; Colomer, Jaume; Jimenez-Mallebrera, Cecilia

    2013-01-01

    Ullrich congenital muscular dystrophy (UCMD), caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered. PMID:24223098

  3. GS2PATH: A web-based integrated analysis tool for finding functional relationships using gene ontology and biochemical pathway data

    PubMed Central

    Yang, Jin Ok; Charny, Park; Lee, Byungwook; Kim, Sangsoo; Bhak, Jong; Woo, Hyun Goo

    2007-01-01

    GS2PATH is a Web-based pipeline tool to permit functional enrichment of a given gene set from prior knowledge databases, including gene ontology (GO) database and biological pathway databases. The tool also provides an estimation of gene set enrichment, in GO terms, from the databases of the KEGG and BioCarta pathways, which may allow users to compute and compare functional over-representations. This is especially useful in the perspective of biological pathways such as metabolic, signal transduction, genetic information processing, environmental information processing, cellular process, disease, and drug development. It provides relevant images of biochemical pathways with highlighting of the gene set by customized colors, which can directly assist in the visualization of functional alteration. Availability The GS2PATH system is freely available at http://array.kobic.re.kr:8080/arrayport/gs2path/. PMID:18305828

  4. Ubiquitin Ligase Trapping Identifies an SCFSaf1 Pathway Targeting Unprocessed Vacuolar/Lysosomal Proteins

    PubMed Central

    Mark, Kevin G.; Simonetta, Marco; Maiolica, Alessio; Seller, Charles A.

    2014-01-01

    SUMMARY We have developed a technique, called Ubiquitin Ligase Substrate Trapping, for the isolation of ubiquitinated substrates in complex with their ubiquitin ligase (E3). By fusing a ubiquitin associated (UBA) domain to an E3 ligase, we were able to selectively purify the polyubiquitinated forms of E3 substrates. Using Ligase Traps of eight different F-box proteins (SCF specificity factors) coupled with mass spectrometry, we identified known, as well as previously uncharacterized substrates. Polyubiquitinated forms of candidate substrates associated with their cognate F-box partner, but not other Ligase Traps. Interestingly, the four most abundant candidate substrates identified for the F-box protein Saf1 were all vacuolar/lysosomal proteins. Analysis of one of these substrates, Prb1, showed that Saf1 selectively promotes ubiquitination of the unprocessed form of the zymogen. This suggests that Saf1 is part of a pathway that targets protein precursors for proteasomal degradation. PMID:24389104

  5. Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways

    PubMed Central

    Cirulli, Elizabeth T.; Lasseigne, Brittany N.; Petrovski, Slav; Sapp, Peter C.; Dion, Patrick A.; Leblond, Claire S.; Couthouis, Julien; Lu, Yi-Fan; Wang, Quanli; Krueger, Brian J.; Ren, Zhong; Keebler, Jonathan; Han, Yujun; Levy, Shawn E.; Boone, Braden E.; Wimbish, Jack R.; Waite, Lindsay L.; Jones, Angela L.; Carulli, John P.; Day-Williams, Aaron G.; Staropoli, John F.; Xin, Winnie W.; Chesi, Alessandra; Raphael, Alya R.; McKenna-Yasek, Diane; Cady, Janet; de Jong, J.M.B. Vianney; Kenna, Kevin P.; Smith, Bradley N.; Topp, Simon; Miller, Jack; Gkazi, Athina; Al-Chalabi, Ammar; van den Berg, Leonard H.; Veldink, Jan; Silani, Vincenzo; Ticozzi, Nicola; Shaw, Christopher E.; Baloh, Robert H.; Appel, Stanley; Simpson, Ericka; Lagier-Tourenne, Clotilde; Pulst, Stefan M.; Gibson, Summer; Trojanowski, John Q.; Elman, Lauren; McCluskey, Leo; Grossman, Murray; Shneider, Neil A.; Chung, Wendy K.; Ravits, John M.; Glass, Jonathan D.; Sims, Katherine B.; Van Deerlin, Vivianna M.; Maniatis, Tom; Hayes, Sebastian D.; Ordureau, Alban; Swarup, Sharan; Landers, John; Baas, Frank; Allen, Andrew S.; Bedlack, Richard S.; Harper, J. Wade; Gitler, Aaron D.; Rouleau, Guy A.; Brown, Robert; Harms, Matthew B.; Cooper, Gregory M.; Harris, Tim; Myers, Richard M.; Goldstein, David B.

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. Here we report the results of a moderate-scale sequencing study aimed at identifying new genes contributing to predisposition for ALS. We performed whole exome sequencing of 2,874 ALS patients and compared them to 6,405 controls. Several known ALS genes were found to be associated, and the non-canonical I?B kinase family TANK-Binding Kinase 1 (TBK1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention. PMID:25700176

  6. A Sleeping Beauty forward genetic screen identifies new genes and pathways driving osteosarcoma development and metastasis.

    PubMed

    Moriarity, Branden S; Otto, George M; Rahrmann, Eric P; Rathe, Susan K; Wolf, Natalie K; Weg, Madison T; Manlove, Luke A; LaRue, Rebecca S; Temiz, Nuri A; Molyneux, Sam D; Choi, Kwangmin; Holly, Kevin J; Sarver, Aaron L; Scott, Milcah C; Forster, Colleen L; Modiano, Jaime F; Khanna, Chand; Hewitt, Stephen M; Khokha, Rama; Yang, Yi; Gorlick, Richard; Dyer, Michael A; Largaespada, David A

    2015-06-01

    Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma. PMID:25961939

  7. A Sleeping Beauty forward genetic screen identifies new genes and pathways driving osteosarcoma development and metastasis

    PubMed Central

    Moriarity, Branden S; Otto, George M; Rahrmann, Eric P; Rathe, Susan K; Wolf, Natalie K; Weg, Madison T; Manlove, Luke A; LaRue, Rebecca S; Temiz, Nuri A; Molyneux, Sam D; Choi, Kwangmin; Holly, Kevin J; Sarver, Aaron L; Scott, Milcah C; Forster, Colleen L; Modiano, Jaime F; Khanna, Chand; Hewitt, Stephen M; Khokha, Rama; Yang, Yi; Gorlick, Richard; Dyer, Michael A; Largaespada, David A

    2016-01-01

    Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma. PMID:25961939

  8. Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells.

    PubMed

    Villar, Margarita; Ayllón, Nieves; Alberdi, Pilar; Moreno, Andrés; Moreno, María; Tobes, Raquel; Mateos-Hernández, Lourdes; Weisheit, Sabine; Bell-Sakyi, Lesley; de la Fuente, José

    2015-12-01

    Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human granulocytic anaplasmosis. These intracellular bacteria establish infection by affecting cell function in both the vertebrate host and the tick vector, Ixodes scapularis. Previous studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection. However, in the postgenomic era, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host-pathogen interactions and the discovery of new targets for prevention and control of infectious diseases. This study reports the first systems biology integration of metabolomics, transcriptomics, and proteomics data to characterize essential metabolic pathways involved in the tick response to A. phagocytophilum infection. The ISE6 tick cells used in this study constitute a model for hemocytes involved in pathogen infection and immune response. The results showed that infection affected protein processing in endoplasmic reticulum and glucose metabolic pathways in tick cells. These results supported tick-Anaplasma co-evolution by providing new evidence of how tick cells limit pathogen infection, while the pathogen benefits from the tick cell response to establish infection. Additionally, ticks benefit from A. phagocytophilum infection by increasing survival while pathogens guarantee transmission. The results suggested that A. phagocytophilum induces protein misfolding to limit the tick cell response and facilitate infection but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells. Additionally, A. phagocytophilum may benefit from the tick cell's ability to limit bacterial infection through PEPCK inhibition leading to decreased glucose metabolism, which also results in the inhibition of cell apoptosis that increases infection of tick cells. These results support the use of this experimental approach to systematically identify cell pathways and molecular mechanisms involved in tick-pathogen interactions. Data are available via ProteomeXchange with identifier PXD002181. PMID:26424601

  9. Suppressors of systemin signaling identify genes in the tomato wound response pathway.

    PubMed Central

    Howe, G A; Ryan, C A

    1999-01-01

    In tomato plants, systemic induction of defense genes in response to herbivory or mechanical wounding is regulated by an 18-amino-acid peptide signal called systemin. Transgenic plants that overexpress prosystemin, the systemin precursor, from a 35S::prosystemin (35S::prosys) transgene exhibit constitutive expression of wound-inducible defense proteins including proteinase inhibitors and polyphenol oxidase. To study further the role of (pro)systemin in the wound response pathway, we isolated and characterized mutations that suppress 35S::prosys-mediated phenotypes. Ten recessive, extragenic suppressors were identified. Two of these define new alleles of def-1, a previously identified mutation that blocks both wound- and systemin-induced gene expression and renders plants susceptible to herbivory. The remaining mutants defined four loci designated Spr-1, Spr-2, Spr-3, and Spr-4 (for Suppressed in 35S::prosystemin-mediated responses). spr-3 and spr-4 mutants were not significantly affected in their response to either systemin or mechanical wounding. In contrast, spr-1 and spr-2 plants lacked systemic wound responses and were insensitive to systemin. These results confirm the function of (pro)systemin in the transduction of systemic wound signals and further establish that wounding, systemin, and 35S::prosys induce defensive gene expression through a common signaling pathway defined by at least three genes (Def-1, Spr-1, and Spr-2). PMID:10545469

  10. Integrated genomics identifies convergence of ankylosing spondylitis with global immune mediated disease pathways.

    PubMed

    Uddin, Mohammed; Codner, Dianne; Hasan, S M Mahmud; Scherer, Stephen W; O'Rielly, Darren D; Rahman, Proton

    2015-01-01

    Ankylosing spondylitis(AS), a highly heritable complex inflammatory arthritis. Although, a handful of non-HLA risk loci have been identified, capturing the unexplained genetic contribution to AS pathogenesis remains a challenge attributed to additive, pleiotropic and epistatic-interactions at the molecular level. Here, we developed multiple integrated genomic approaches to quantify molecular convergence of non-HLA loci with global immune mediated diseases. We show that non-HLA genes are significantly sensitive to deleterious mutation accumulation in the general population compared with tolerant genes. Human developmental proteomics (prenatal to adult) analysis revealed that proteins encoded by non-HLA AS risk loci are 2-fold more expressed in adult hematopoietic cells.Enrichment analysis revealed AS risk genes overlap with a significant number of immune related pathways (p < 0.0001 to 9.8 × 0(-12)). Protein-protein interaction analysis revealed non-shared AS risk genes are highly clustered seeds that significantly converge (empirical; p < 0.01 to 1.6 × 10(-4)) into networks of global immune mediated disease risk loci. We have also provided initial evidence for the involvement of STAT2/3 in AS pathogenesis. Collectively, these findings highlight molecular insight on non-HLA AS risk loci that are not exclusively connected with overlapping immune mediated diseases; rather a component of common pathophysiological pathways with other immune mediated diseases. This information will be pivotal to fully explain AS pathogenesis and identify new therapeutic targets. PMID:25980808

  11. Simulated Annealing Based Algorithm for Identifying Mutated Driver Pathways in Cancer

    PubMed Central

    Li, Hai-Tao; Zhang, Yu-Lang; Zheng, Chun-Hou; Wang, Hong-Qiang

    2014-01-01

    With the development of next-generation DNA sequencing technologies, large-scale cancer genomics projects can be implemented to help researchers to identify driver genes, driver mutations, and driver pathways, which promote cancer proliferation in large numbers of cancer patients. Hence, one of the remaining challenges is to distinguish functional mutations vital for cancer development, and filter out the unfunctional and random “passenger mutations.” In this study, we introduce a modified method to solve the so-called maximum weight submatrix problem which is used to identify mutated driver pathways in cancer. The problem is based on two combinatorial properties, that is, coverage and exclusivity. Particularly, we enhance an integrative model which combines gene mutation and expression data. The experimental results on simulated data show that, compared with the other methods, our method is more efficient. Finally, we apply the proposed method on two real biological datasets. The results show that our proposed method is also applicable in real practice. PMID:24982873

  12. Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma

    PubMed Central

    Weiser, Keith C.; Liu, Bin; Hansen, Gwenn M.; Skapura, Darlene; Hentges, Kathryn E.; Yarlagadda, Sujatha; Morse III, Herbert C.

    2007-01-01

    AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NF?B, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at http://www.mouse-genome.bcm.tmc.edu/vision. PMID:17926094

  13. 2D NMR-based metabolomics uncovers interactions between conserved biochemical pathways in the model organism Caenorhabditis elegans

    PubMed Central

    Izrayelit, Yevgeniy; Robinette, Steven L.; Bose, Neelanjan; von Reuss, Stephan H.; Schroeder, Frank C.

    2012-01-01

    Ascarosides are small-molecule signals that play a central role in C. elegans biology, including dauer formation, aging, and social behaviors, but many aspects of their biosynthesis remain unknown. Using automated 2D NMR-based comparative metabolomics, we identified ascaroside ethanolamides as shunt metabolites in C. elegans mutants of daf-22, a gene with homology to mammalian 3-ketoacyl-CoA thiolases predicted to function in conserved peroxisomal lipid ?-oxidation. Two groups of ethanolamides feature ?-keto functionalization confirming the predicted role of daf-22 in ascaroside biosynthesis, whereas ?-methyl substitution points to unexpected inclusion of methylmalonte at a late stage in the biosynthesis of long-chain fatty acids in C. elegans. We show that ascaroside ethanolamide formation in response to defects in daf-22 and other peroxisomal genes is associated with severe depletion of endocannabinoid pools. These results indicate unexpected interaction between peroxisomal lipid ?-oxidation and the biosynthesis of endocannabinoids, which are major regulators of lifespan in C. elegans. Our study demonstrates the utility of unbiased comparative metabolomics for investigating biochemical networks in metazoans. PMID:23163760

  14. 2D NMR-based metabolomics uncovers interactions between conserved biochemical pathways in the model organism Caenorhabditis elegans.

    PubMed

    Izrayelit, Yevgeniy; Robinette, Steven L; Bose, Neelanjan; von Reuss, Stephan H; Schroeder, Frank C

    2013-02-15

    Ascarosides are small-molecule signals that play a central role in C. elegans biology, including dauer formation, aging, and social behaviors, but many aspects of their biosynthesis remain unknown. Using automated 2D NMR-based comparative metabolomics, we identified ascaroside ethanolamides as shunt metabolites in C. elegans mutants of daf-22, a gene with homology to mammalian 3-ketoacyl-CoA thiolases predicted to function in conserved peroxisomal lipid β-oxidation. Two groups of ethanolamides feature β-keto functionalization confirming the predicted role of daf-22 in ascaroside biosynthesis, whereas α-methyl substitution points to unexpected inclusion of methylmalonate at a late stage in the biosynthesis of long-chain fatty acids in C. elegans. We show that ascaroside ethanolamide formation in response to defects in daf-22 and other peroxisomal genes is associated with severe depletion of endocannabinoid pools. These results indicate unexpected interaction between peroxisomal lipid β-oxidation and the biosynthesis of endocannabinoids, which are major regulators of lifespan in C. elegans. Our study demonstrates the utility of unbiased comparative metabolomics for investigating biochemical networks in metazoans. PMID:23163760

  15. Large-scale integrative network-based analysis identifies common pathways disrupted by copy number alterations across cancers

    PubMed Central

    2013-01-01

    Background Many large-scale studies analyzed high-throughput genomic data to identify altered pathways essential to the development and progression of specific types of cancer. However, no previous study has been extended to provide a comprehensive analysis of pathways disrupted by copy number alterations across different human cancers. Towards this goal, we propose a network-based method to integrate copy number alteration data with human protein-protein interaction networks and pathway databases to identify pathways that are commonly disrupted in many different types of cancer. Results We applied our approach to a data set of 2,172 cancer patients across 16 different types of cancers, and discovered a set of commonly disrupted pathways, which are likely essential for tumor formation in majority of the cancers. We also identified pathways that are only disrupted in specific cancer types, providing molecular markers for different human cancers. Analysis with independent microarray gene expression datasets confirms that the commonly disrupted pathways can be used to identify patient subgroups with significantly different survival outcomes. We also provide a network view of disrupted pathways to explain how copy number alterations affect pathways that regulate cell growth, cycle, and differentiation for tumorigenesis. Conclusions In this work, we demonstrated that the network-based integrative analysis can help to identify pathways disrupted by copy number alterations across 16 types of human cancers, which are not readily identifiable by conventional overrepresentation-based and other pathway-based methods. All the results and source code are available at http://compbio.cs.umn.edu/NetPathID/. PMID:23822816

  16. A Genome-Wide Screen with Nicotinamide to Identify Sirtuin-Dependent Pathways in Saccharomyces cerevisiae

    PubMed Central

    Choy, John S.; Qadri, Bayan; Henry, Leah; Shroff, Kunal; Bifarin, Olatomiwa; Basrai, Munira A.

    2015-01-01

    Sirtuins are evolutionarily conserved NAD-dependent deacetylases that catalyze the cleavage of NAD+ into nicotinamide (NAM), which can act as a pan-sirtuin inhibitor in unicellular and multicellular organisms. Sirtuins regulate processes such as transcription, DNA damage repair, chromosome segregation, and longevity extension in yeast and metazoans. The founding member of the evolutionarily conserved sirtuin family, SIR2, was first identified in budding yeast. Subsequent studies led to the identification of four yeast SIR2 homologs HST1, HST2, HST3, and HST4. Understanding the downstream physiological consequences of inhibiting sirtuins can be challenging since most studies focus on single or double deletions of sirtuins, and mating defects in SIR2 deletions hamper genome-wide screens. This represents an important gap in our knowledge of how sirtuins function in highly complex biological processes such as aging, metabolism, and chromosome segregation. In this report, we used a genome-wide screen to explore sirtuin-dependent processes in Saccharomyces cerevisiae by identifying deletion mutants that are sensitive to NAM. We identified 55 genes in total, 36 of which have not been previously reported to be dependent on sirtuins. We find that genome stability pathways are particularly vulnerable to loss of sirtuin activity. Here, we provide evidence that defects in sister chromatid cohesion renders cells sensitive to growth in the presence of NAM. The results of our screen provide a broad view of the biological pathways sensitive to inhibition of sirtuins, and advance our understanding of the function of sirtuins and NAD+ biology. PMID:26646153

  17. A genome-wide deletion mutant screen identifies pathways affected by nickel sulfate in Saccharomyces cerevisiae

    PubMed Central

    2009-01-01

    Background The understanding of the biological function, regulation, and cellular interactions of the yeast genome and proteome, along with the high conservation in gene function found between yeast genes and their human homologues, has allowed for Saccharomyces cerevisiae to be used as a model organism to deduce biological processes in human cells. Here, we have completed a systematic screen of the entire set of 4,733 haploid S. cerevisiae gene deletion strains (the entire set of nonessential genes for this organism) to identify gene products that modulate cellular toxicity to nickel sulfate (NiSO4). Results We have identified 149 genes whose gene deletion causes sensitivity to NiSO4 and 119 genes whose gene deletion confers resistance. Pathways analysis with proteins whose absence renders cells sensitive and resistant to nickel identified a wide range of cellular processes engaged in the toxicity of S. cerevisiae to NiSO4. Functional categories overrepresented with proteins whose absence renders cells sensitive to NiSO4 include homeostasis of protons, cation transport, transport ATPases, endocytosis, siderophore-iron transport, homeostasis of metal ions, and the diphthamide biosynthesis pathway. Functional categories overrepresented with proteins whose absence renders cells resistant to nickel include functioning and transport of the vacuole and lysosome, protein targeting, sorting, and translocation, intra-Golgi transport, regulation of C-compound and carbohydrate metabolism, transcriptional repression, and chromosome segregation/division. Interactome analysis mapped seven nickel toxicity modulating and ten nickel-resistance networks. Additionally, we studied the degree of sensitivity or resistance of the 111 nickel-sensitive and 72 -resistant strains whose gene deletion product has a similar protein in human cells. Conclusion We have undertaken a whole genome approach in order to further understand the mechanism(s) regulating the cell's toxicity to nickel compounds. We have used computational methods to integrate the data and generate global models of the yeast's cellular response to NiSO4. The results of our study shed light on molecular pathways associated with the cellular response of eukaryotic cells to nickel compounds and provide potential implications for further understanding the toxic effects of nickel compounds to human cells. PMID:19917080

  18. Network analysis identifies a putative role for the PPAR and type 1 interferon pathways in glucocorticoid actions in asthmatics

    PubMed Central

    2012-01-01

    Background Asthma is a chronic inflammatory airway disease influenced by genetic and environmental factors that affects ~300 million people worldwide, leading to ~250,000 deaths annually. Glucocorticoids (GCs) are well-known therapeutics that are used extensively to suppress airway inflammation in asthmatics. The airway epithelium plays an important role in the initiation and modulation of the inflammatory response. While the role of GCs in disease management is well understood, few studies have examined the holistic effects on the airway epithelium. Methods Gene expression data were used to generate a co-transcriptional network, which was interrogated to identify modules of functionally related genes. In parallel, expression data were mapped to the human protein-protein interaction (PPI) network in order to identify modules with differentially expressed genes. A common pathways approach was applied to highlight genes and pathways functionally relevant and significantly altered following GC treatment. Results Co-transcriptional network analysis identified pathways involved in inflammatory processes in the epithelium of asthmatics, including the Toll-like receptor (TLR) and PPAR signaling pathways. Analysis of the PPI network identified RXRA, PPARGC1A, STAT1 and IRF9, among others genes, as differentially expressed. Common pathways analysis highlighted TLR and PPAR signaling pathways, providing a link between general inflammatory processes and the actions of GCs. Promoter analysis identified genes regulated by the glucocorticoid receptor (GCR) and PPAR pathways as well as highlighted the interferon pathway as a target of GCs. Conclusions Network analyses identified known genes and pathways associated with inflammatory processes in the airway epithelium of asthmatics. This workflow illustrated a hypothesis generating experimental design that integrated multiple analysis methods to produce a weight-of-evidence based approach upon which future focused studies can be designed. In this case, results suggested a mechanism whereby GCs repress TLR-mediated interferon production via upregulation of the PPAR signaling pathway. These results highlight the role of interferons in asthma and their potential as targets of future therapeutic efforts. PMID:22713245

  19. A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation

    PubMed Central

    Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

    2016-01-01

    Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGL?) and beta (DAGL?) to neurons and microglia, respectively. Disruption of DAGL? perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function. DOI: http://dx.doi.org/10.7554/eLife.12345.001 PMID:26779719

  20. Transcriptional profiles of drought-responsive genes in modulating transcription signal transduction, and biochemical pathways in tomato

    PubMed Central

    Gong, Pengjuan; Zhang, Junhong; Li, Hanxia; Yang, Changxian; Zhang, Chanjuan; Zhang, Xiaohui; Khurram, Ziaf; Zhang, Yuyang; Wang, Taotao; Fei, Zhangjun; Ye, Zhibiao

    2010-01-01

    To unravel the molecular mechanisms of drought responses in tomato, gene expression profiles of two drought-tolerant lines identified from a population of Solanum pennellii introgression lines, and the recurrent parent S. lycopersicum cv. M82, a drought-sensitive cultivar, were investigated under drought stress using tomato microarrays. Around 400 genes identified were responsive to drought stress only in the drought-tolerant lines. These changes in genes expression are most likely caused by the two inserted chromosome segments of S. pennellii, which possibly contain drought-tolerance quantitative trait loci (QTLs). Among these genes are a number of transcription factors and signalling proteins which could be global regulators involved in the tomato responses to drought stress. Genes involved in organism growth and development processes were also specifically regulated by drought stress, including those controlling cell wall structure, wax biosynthesis, and plant height. Moreover, key enzymes in the pathways of gluconeogenesis (fructose-bisphosphate aldolase), purine and pyrimidine nucleotide biosynthesis (adenylate kinase), tryptophan degradation (aldehyde oxidase), starch degradation (?-amylase), methionine biosynthesis (cystathionine ?-lyase), and the removal of superoxide radicals (catalase) were also specifically affected by drought stress. These results indicated that tomato plants could adapt to water-deficit conditions through decreasing energy dissipation, increasing ATP energy provision, and reducing oxidative damage. The drought-responsive genes identified in this study could provide further information for understanding the mechanisms of drought tolerance in tomato. PMID:20643807

  1. Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways.

    PubMed

    Cirulli, Elizabeth T; Lasseigne, Brittany N; Petrovski, Slavé; Sapp, Peter C; Dion, Patrick A; Leblond, Claire S; Couthouis, Julien; Lu, Yi-Fan; Wang, Quanli; Krueger, Brian J; Ren, Zhong; Keebler, Jonathan; Han, Yujun; Levy, Shawn E; Boone, Braden E; Wimbish, Jack R; Waite, Lindsay L; Jones, Angela L; Carulli, John P; Day-Williams, Aaron G; Staropoli, John F; Xin, Winnie W; Chesi, Alessandra; Raphael, Alya R; McKenna-Yasek, Diane; Cady, Janet; Vianney de Jong, J M B; Kenna, Kevin P; Smith, Bradley N; Topp, Simon; Miller, Jack; Gkazi, Athina; Al-Chalabi, Ammar; van den Berg, Leonard H; Veldink, Jan; Silani, Vincenzo; Ticozzi, Nicola; Shaw, Christopher E; Baloh, Robert H; Appel, Stanley; Simpson, Ericka; Lagier-Tourenne, Clotilde; Pulst, Stefan M; Gibson, Summer; Trojanowski, John Q; Elman, Lauren; McCluskey, Leo; Grossman, Murray; Shneider, Neil A; Chung, Wendy K; Ravits, John M; Glass, Jonathan D; Sims, Katherine B; Van Deerlin, Vivianna M; Maniatis, Tom; Hayes, Sebastian D; Ordureau, Alban; Swarup, Sharan; Landers, John; Baas, Frank; Allen, Andrew S; Bedlack, Richard S; Harper, J Wade; Gitler, Aaron D; Rouleau, Guy A; Brown, Robert; Harms, Matthew B; Cooper, Gregory M; Harris, Tim; Myers, Richard M; Goldstein, David B

    2015-03-27

    Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. We report the results of a moderate-scale sequencing study aimed at increasing the number of genes known to contribute to predisposition for ALS. We performed whole-exome sequencing of 2869 ALS patients and 6405 controls. Several known ALS genes were found to be associated, and TBK1 (the gene encoding TANK-binding kinase 1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention. PMID:25700176

  2. Whole-Genome Pathway Analysis on 132,497 Individuals Identifies Novel Gene-Sets Associated with Body Mass Index

    PubMed Central

    Simonson, Matthew A.; McQueen, Matthew B.; Keller, Matthew C.

    2014-01-01

    Whole genome pathway analysis is a powerful tool for the exploration of the combined effects of gene-sets within biological pathways. This study applied Interval Based Enrichment Analysis (INRICH) to perform whole-genome pathway analysis of body-mass index (BMI). We used a discovery set composed of summary statistics from a meta-analysis of 123,865 subjects performed by the GIANT Consortium, and an independent sample of 8,632 subjects to assess replication of significant pathways. We examined SNPs within nominally significant pathways using linear mixed models to estimate their contribution to overall BMI heritability. Six pathways replicated as having significant enrichment for association after correcting for multiple testing, including the previously unknown relationships between BMI and the Reactome regulation of ornithine decarboxylase pathway, the KEGG lysosome pathway, and the Reactome stabilization of P53 pathway. Two non-overlapping sets of genes emerged from the six significant pathways. The clustering of shared genes based on previously identified protein-protein interactions listed in PubMed and OMIM supported the relatively independent biological effects of these two gene-sets. We estimate that the SNPs located in examined pathways explain ?20% of the heritability for BMI that is tagged by common SNPs (3.35% of the 16.93% total). PMID:24497910

  3. Integrated Genomics Identifies Convergence of Ankylosing Spondylitis with Global Immune Mediated Disease Pathways

    PubMed Central

    Uddin, Mohammed; Codner, Dianne; Mahmud Hasan, S M; Scherer, Stephen W; O’Rielly, Darren D; Rahman, Proton

    2015-01-01

    Ankylosing spondylitis(AS), a highly heritable complex inflammatory arthritis. Although, a handful of non-HLA risk loci have been identified, capturing the unexplained genetic contribution to AS pathogenesis remains a challenge attributed to additive, pleiotropic and epistatic-interactions at the molecular level. Here, we developed multiple integrated genomic approaches to quantify molecular convergence of non-HLA loci with global immune mediated diseases. We show that non-HLA genes are significantly sensitive to deleterious mutation accumulation in the general population compared with tolerant genes. Human developmental proteomics (prenatal to adult) analysis revealed that proteins encoded by non-HLA AS risk loci are 2-fold more expressed in adult hematopoietic cells.Enrichment analysis revealed AS risk genes overlap with a significant number of immune related pathways (p < 0.0001 to 9.8 × 10-12). Protein-protein interaction analysis revealed non-shared AS risk genes are highly clustered seeds that significantly converge (empirical; p < 0.01 to 1.6 × 10-4) into networks of global immune mediated disease risk loci. We have also provided initial evidence for the involvement of STAT2/3 in AS pathogenesis. Collectively, these findings highlight molecular insight on non-HLA AS risk loci that are not exclusively connected with overlapping immune mediated diseases; rather a component of common pathophysiological pathways with other immune mediated diseases. This information will be pivotal to fully explain AS pathogenesis and identify new therapeutic targets. PMID:25980808

  4. Metabolomics Identifies Novel Hnf1α-Dependent Physiological Pathways in Vivo

    PubMed Central

    Bonzo, Jessica A.; Patterson, Andrew D.; Krausz, Kristopher W.; Gonzalez, Frank J.

    2010-01-01

    Mutations in the HNF1A gene cause maturity-onset diabetes of the young type 3, one of the most common genetic causes of non-insulin-dependent (type 2) diabetes mellitus. Although the whole-body Hnf1a-null mouse recapitulates the low insulin levels and high blood glucose observed in human maturity-onset diabetes of the young type 3 patients, these mice also suffer from Laron dwarfism and aminoaciduria, suggesting a role for hepatocyte nuclear factor 1α (Hnf1α) in pathophysiologies distinct from non-insulin-dependent (type 2) diabetes mellitus. In an effort to identify pathways associated with inactivation of Hnf1α, an ultraperformance liquid chromatography coupled to mass spectrometry-based metabolomics study was conducted on urine samples from wild-type and Hnf1a-null mice. An increase in phenylalanine metabolites is in agreement with the known regulation of the phenylalanine hydroxylase gene by Hnf1α. This metabolomic approach also identified urinary biomarkers for three tissue-specific dysfunctions previously unassociated with Hnf1α function. 1) Elevated indolelactate coupled to decreased xanthurenic acid also indicated defects in the indole and kynurenine pathways of tryptophan metabolism, respectively. 2) An increase in the neutral amino acid proline in the urine of Hnf1a-null mice correlated with loss of renal apical membrane transporters of the Slc6a family. 3) Further investigation into the mechanism of aldosterone increase revealed an overactive adrenal gland in Hnf1a-null mice possibly due to inhibition of negative feedback regulation. Although the phenotype of the Hnf1a-null mouse is complex, metabolomics has opened the door to investigation of several physiological systems in which Hnf1α may be a critical regulatory component. PMID:20943816

  5. Long-term recovery from hippocampal-related behavioral and biochemical abnormalities induced by noise exposure during brain development. Evaluation of auditory pathway integrity.

    PubMed

    Uran, S L; Gmez-Casati, M E; Guelman, L R

    2014-10-01

    Sound is an important part of man's contact with the environment and has served as critical means for survival throughout his evolution. As a result of exposure to noise, physiological functions such as those involving structures of the auditory and non-auditory systems might be damaged. We have previously reported that noise-exposed developing rats elicited hippocampal-related histological, biochemical and behavioral changes. However, no data about the time lapse of these changes were reported. Moreover, measurements of auditory pathway function were not performed in exposed animals. Therefore, with the present work, we aim to test the onset and the persistence of the different extra-auditory abnormalities observed in noise-exposed rats and to evaluate auditory pathway integrity. Male Wistar rats of 15 days were exposed to moderate noise levels (95-97 dB SPL, 2 h a day) during one day (acute noise exposure, ANE) or during 15 days (sub-acute noise exposure, SANE). Hippocampal biochemical determinations as well as short (ST) and long term (LT) behavioral assessments were performed. In addition, histological and functional evaluations of the auditory pathway were carried out in exposed animals. Our results show that hippocampal-related behavioral and biochemical changes (impairments in habituation, recognition and associative memories as well as distortion of anxiety-related behavior, decreases in reactive oxygen species (ROS) levels and increases in antioxidant enzymes activities) induced by noise exposure were almost completely restored by PND 90. In addition, auditory evaluation shows that increased cochlear thresholds observed in exposed rats were re-established at PND 90, although with a remarkable supra-threshold amplitude reduction. These data suggest that noise-induced hippocampal and auditory-related alterations are mostly transient and that the effects of noise on the hippocampus might be, at least in part, mediated by the damage on the auditory pathway. However, we cannot exclude that a different mechanism might be responsible for the observed hippocampal-related changes. PMID:24911434

  6. Converging mediators from immune and trophic pathways to identify Parkinson disease dementia

    PubMed Central

    Schmitz, Christopher T.; Snyder, Noelle L.; Chen, Kewei; Walker, Douglas G.; Davis, Kathryn J.; Belden, Christine; Caviness, John N.; Driver-Dunckley, Erika; Adler, Charles H.; Sabbagh, Marwan N.; Shill, Holly A.

    2016-01-01

    Objective: To identify a panel of peripheral inflammatory/immune mediators that could discriminate Parkinson disease with dementia (PDD) from Parkinson disease (PD) without dementia. Methods: Plasma samples from 52 patients with PD and 22 patients with PDD were prepared from freshly collected blood following an institutional review board–approved protocol. A total of 160 proteins were measured using a multiplex antibody array. Plasma α-synuclein levels were analyzed by an electrochemiluminescence immunoassay. The main objective of the statistical analyses was to identify PDD discriminants using the plasma protein profile alone or in combination with age. Results: The PD and PDD groups differed significantly in cognitive measurements (Mini-Mental State Examination, Auditory Verbal Learning Test-A7, and Clinical Dementia Rating) and age. The age-adjusted levels of thymus and activation-regulated chemokine (TARC) and platelet-derived growth factor (PDGF)-AA were significantly different between disease groups. The levels of plasma α-synuclein significantly correlated with 26 proteins; among them, PDGF-BB, TARC, PDGF-AA, and epidermal growth factor were the highest. Linear discriminant analysis with leave-one-out cross-validation identified a 14-protein panel with age as discriminants of PDD (96% sensitivity, 89% specificity, area under the curve = 0.9615). Conclusions: We showed that multiple proteins that are mediators of growth/trophic and immune response-related pathways had discriminatory power for identifying PDD in patients with PD. Validation of this discovery-based study in longitudinal population-based studies is warranted. Classification of evidence: This study provides Class III evidence that a 14-protein panel plasma assay combined with age has a sensitivity of 96% and a specificity of 89% for PDD. PMID:26848485

  7. Characterization of the novel dimethyl sulfide-degrading bacterium Alcaligenes sp. SY1 and its biochemical degradation pathway.

    PubMed

    Sun, Yiming; Qiu, Jiguo; Chen, Dongzhi; Ye, Jiexu; Chen, Jianmeng

    2016-03-01

    Recently, the biodegradation of volatile organic sulfur compounds (VOSCs) has become a burgeoning field, with a growing focus on the reduction of VOSCs. The reduction of VOSCs encompasses both organic emission control and odor control. Herein, Alcaligenes sp. SY1 was isolated from active sludge and found to utilize dimethyl sulfide (DMS) as a growth substrate in a mineral salt medium. Response surface methodology (RSM) analysis was applied to optimize the incubation conditions. The following conditions for optimal degradation were identified: temperature 27.03°C; pH 7.80; inoculum salinity 0.84%; and initial DMS concentration 1585.39μM. Under these conditions, approximately 99% of the DMS was degraded within 30h of incubation. Two metabolic compounds were detected and identified by gas chromatography-mass spectrometry (GC-MS): dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS). The DMS degradation kinetics for different concentrations were evaluated using the Haldane-Andrews model and the pseudo first-order model. The maximum specific growth rate and degradation rate of Alcaligenes sp. SY1 were 0.17h(-1) and 0.63gsgx(-1)h(-1). A possible degradation pathway is proposed, and the results suggest that Alcaligenes sp. SY1 has the potential to control odor emissions under aerobic conditions. PMID:26623933

  8. Molecular mechanisms of the non-coenzyme action of thiamin in brain: biochemical, structural and pathway analysis

    PubMed Central

    Mkrtchyan, Garik; Aleshin, Vasily; Parkhomenko, Yulia; Kaehne, Thilo; Luigi Di Salvo, Martino; Parroni, Alessia; Contestabile, Roberto; Vovk, Andrey; Bettendorff, Lucien; Bunik, Victoria

    2015-01-01

    Thiamin (vitamin B1) is a pharmacological agent boosting central metabolism through the action of the coenzyme thiamin diphosphate (ThDP). However, positive effects, including improved cognition, of high thiamin doses in neurodegeneration may be observed without increased ThDP or ThDP-dependent enzymes in brain. Here, we determine protein partners and metabolic pathways where thiamin acts beyond its coenzyme role. Malate dehydrogenase, glutamate dehydrogenase and pyridoxal kinase were identified as abundant proteins binding to thiamin- or thiazolium-modified sorbents. Kinetic studies, supported by structural analysis, revealed allosteric regulation of these proteins by thiamin and/or its derivatives. Thiamin triphosphate and adenylated thiamin triphosphate activate glutamate dehydrogenase. Thiamin and ThDP regulate malate dehydrogenase isoforms and pyridoxal kinase. Thiamin regulation of enzymes related to malate-aspartate shuttle may impact on malate/citrate exchange, responsible for exporting acetyl residues from mitochondria. Indeed, bioinformatic analyses found an association between thiamin- and thiazolium-binding proteins and the term acetylation. Our interdisciplinary study shows that thiamin is not only a coenzyme for acetyl-CoA production, but also an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis. Moreover, thiamin action in neurodegeneration may also involve neurodegeneration-related 14-3-3, DJ-1 and β-amyloid precursor proteins identified among the thiamin- and/or thiazolium-binding proteins. PMID:26212886

  9. Molecular mechanisms of the non-coenzyme action of thiamin in brain: biochemical, structural and pathway analysis.

    PubMed

    Mkrtchyan, Garik; Aleshin, Vasily; Parkhomenko, Yulia; Kaehne, Thilo; Luigi Di Salvo, Martino; Parroni, Alessia; Contestabile, Roberto; Vovk, Andrey; Bettendorff, Lucien; Bunik, Victoria

    2015-01-01

    Thiamin (vitamin B1) is a pharmacological agent boosting central metabolism through the action of the coenzyme thiamin diphosphate (ThDP). However, positive effects, including improved cognition, of high thiamin doses in neurodegeneration may be observed without increased ThDP or ThDP-dependent enzymes in brain. Here, we determine protein partners and metabolic pathways where thiamin acts beyond its coenzyme role. Malate dehydrogenase, glutamate dehydrogenase and pyridoxal kinase were identified as abundant proteins binding to thiamin- or thiazolium-modified sorbents. Kinetic studies, supported by structural analysis, revealed allosteric regulation of these proteins by thiamin and/or its derivatives. Thiamin triphosphate and adenylated thiamin triphosphate activate glutamate dehydrogenase. Thiamin and ThDP regulate malate dehydrogenase isoforms and pyridoxal kinase. Thiamin regulation of enzymes related to malate-aspartate shuttle may impact on malate/citrate exchange, responsible for exporting acetyl residues from mitochondria. Indeed, bioinformatic analyses found an association between thiamin- and thiazolium-binding proteins and the term acetylation. Our interdisciplinary study shows that thiamin is not only a coenzyme for acetyl-CoA production, but also an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis. Moreover, thiamin action in neurodegeneration may also involve neurodegeneration-related 14-3-3, DJ-1 and ?-amyloid precursor proteins identified among the thiamin- and/or thiazolium-binding proteins. PMID:26212886

  10. Integrated Analysis of Mutation Data from Various Sources Identifies Key Genes and Signaling Pathways in Hepatocellular Carcinoma

    PubMed Central

    Wei, Lin; Tang, Ruqi; Lian, Baofeng; Zhao, Yingjun; He, Xianghuo; Xie, Lu

    2014-01-01

    Background Recently, a number of studies have performed genome or exome sequencing of hepatocellular carcinoma (HCC) and identified hundreds or even thousands of mutations in protein-coding genes. However, these studies have only focused on a limited number of candidate genes, and many important mutation resources remain to be explored. Principal Findings In this study, we integrated mutation data obtained from various sources and performed pathway and network analysis. We identified 113 pathways that were significantly mutated in HCC samples and found that the mutated genes included in these pathways contained high percentages of known cancer genes, and damaging genes and also demonstrated high conservation scores, indicating their important roles in liver tumorigenesis. Five classes of pathways that were mutated most frequently included (a) proliferation and apoptosis related pathways, (b) tumor microenvironment related pathways, (c) neural signaling related pathways, (d) metabolic related pathways, and (e) circadian related pathways. Network analysis further revealed that the mutated genes with the highest betweenness coefficients, such as the well-known cancer genes TP53, CTNNB1 and recently identified novel mutated genes GNAL and the ADCY family, may play key roles in these significantly mutated pathways. Finally, we highlight several key genes (e.g., RPS6KA3 and PCLO) and pathways (e.g., axon guidance) in which the mutations were associated with clinical features. Conclusions Our workflow illustrates the increased statistical power of integrating multiple studies of the same subject, which can provide biological insights that would otherwise be masked under individual sample sets. This type of bioinformatics approach is consistent with the necessity of making the best use of the ever increasing data provided in valuable databases, such as TCGA, to enhance the speed of deciphering human cancers. PMID:24988079

  11. Rare Copy Number Variants Identified Suggest the Regulating Pathways in Hypertension-Related Left Ventricular Hypertrophy

    PubMed Central

    Marshall, Christian R.; Majid, Fadhlina; Danuri, Norlaila; Basir, Fashieha; Thiruvahindrapuram, Bhooma; Scherer, Stephen W.; Yusoff, Khalid

    2016-01-01

    Left ventricular hypertrophy (LVH) is an independent risk factor for cardiovascular morbidity and mortality, and a powerful predictor of adverse cardiovascular outcomes in the hypertensive patients. It has complex multifactorial and polygenic basis for its pathogenesis. We hypothesized that rare copy number variants (CNVs) contribute to the LVH pathogenesis in hypertensive patients. Copy number variants (CNV) were identified in 258 hypertensive patients, 95 of whom had LVH, after genotyping with a high resolution SNP array. Following stringent filtering criteria, we identified 208 rare, or private CNVs that were only present in our patients with hypertension related LVH. Preliminary findings from Gene Ontology and pathway analysis of this study confirmed the involvement of the genes known to be functionally involved in cardiac development and phenotypes, in line with previously reported transcriptomic studies. Network enrichment analyses suggested that the gene-set was, directly or indirectly, involved in the transcription factors regulating the “foetal cardiac gene programme” which triggered the hypertrophic cascade, confirming previous reports. These findings suggest that multiple, individually rare copy number variants altering genes may contribute to the pathogenesis of hypertension-related LVH. In summary, we have provided further supporting evidence that rare CNV could potentially impact this common and complex disease susceptibility with lower heritability. PMID:26930585

  12. Rare Copy Number Variants Identified Suggest the Regulating Pathways in Hypertension-Related Left Ventricular Hypertrophy.

    PubMed

    Boon-Peng, Hoh; Mat Jusoh, Julia Ashazila; Marshall, Christian R; Majid, Fadhlina; Danuri, Norlaila; Basir, Fashieha; Thiruvahindrapuram, Bhooma; Scherer, Stephen W; Yusoff, Khalid

    2016-01-01

    Left ventricular hypertrophy (LVH) is an independent risk factor for cardiovascular morbidity and mortality, and a powerful predictor of adverse cardiovascular outcomes in the hypertensive patients. It has complex multifactorial and polygenic basis for its pathogenesis. We hypothesized that rare copy number variants (CNVs) contribute to the LVH pathogenesis in hypertensive patients. Copy number variants (CNV) were identified in 258 hypertensive patients, 95 of whom had LVH, after genotyping with a high resolution SNP array. Following stringent filtering criteria, we identified 208 rare, or private CNVs that were only present in our patients with hypertension related LVH. Preliminary findings from Gene Ontology and pathway analysis of this study confirmed the involvement of the genes known to be functionally involved in cardiac development and phenotypes, in line with previously reported transcriptomic studies. Network enrichment analyses suggested that the gene-set was, directly or indirectly, involved in the transcription factors regulating the "foetal cardiac gene programme" which triggered the hypertrophic cascade, confirming previous reports. These findings suggest that multiple, individually rare copy number variants altering genes may contribute to the pathogenesis of hypertension-related LVH. In summary, we have provided further supporting evidence that rare CNV could potentially impact this common and complex disease susceptibility with lower heritability. PMID:26930585

  13. Significant Deregulated Pathways in Diabetes Type II Complications Identified through Expression Based Network Biology

    NASA Astrophysics Data System (ADS)

    Ukil, Sanchaita; Sinha, Meenakshee; Varshney, Lavneesh; Agrawal, Shipra

    Type 2 Diabetes is a complex multifactorial disease, which alters several signaling cascades giving rise to serious complications. It is one of the major risk factors for cardiovascular diseases. The present research work describes an integrated functional network biology approach to identify pathways that get transcriptionally altered and lead to complex complications thereby amplifying the phenotypic effect of the impaired disease state. We have identified two sub-network modules, which could be activated under abnormal circumstances in diabetes. Present work describes key proteins such as P85A and SRC serving as important nodes to mediate alternate signaling routes during diseased condition. P85A has been shown to be an important link between stress responsive MAPK and CVD markers involved in fibrosis. MAPK8 has been shown to interact with P85A and further activate CTGF through VEGF signaling. We have traced a novel and unique route correlating inflammation and fibrosis by considering P85A as a key mediator of signals. The next sub-network module shows SRC as a junction for various signaling processes, which results in interaction between NF-kB and beta catenin to cause cell death. The powerful interaction between these important genes in response to transcriptionally altered lipid metabolism and impaired inflammatory response via SRC causes apoptosis of cells. The crosstalk between inflammation, lipid homeostasis and stress, and their serious effects downstream have been explained in the present analyses.

  14. Identifying specific profiles in patients with different degrees of painful knee osteoarthritis based on serological biochemical and mechanistic pain biomarkers: a diagnostic approach based on cluster analysis.

    PubMed

    Egsgaard, Line Lindhardt; Eskehave, Thomas Navndrup; Bay-Jensen, Anne C; Hoeck, Hans Christian; Arendt-Nielsen, Lars

    2015-01-01

    Biochemical and pain biomarkers can be applied to patients with painful osteoarthritis profiles and may provide more details compared with conventional clinical tools. The aim of this study was to identify an optimal combination of biochemical and pain biomarkers for classification of patients with different degrees of knee pain and joint damage. Such profiling may provide new diagnostic and therapeutic options. A total of 216 patients with different degrees of knee pain (maximal pain during the last 24 hours rated on a visual analog scale [VAS]) (VAS 0-100) and 64 controls (VAS 0-9) were recruited. Patients were separated into 3 groups: VAS 10 to 39 (N = 81), VAS 40 to 69 (N = 70), and VAS 70 to 100 (N = 65). Pressure pain thresholds, temporal summation to pressure stimuli, and conditioning pain modulation were measured from the peripatellar and extrasegmental sites. Biochemical markers indicative for autoinflammation and immunity (VICM, CRP, and CRPM), synovial inflammation (CIIIM), cartilage loss (CIIM), and bone degradation (CIM) were analyzed. WOMAC, Lequesne, and pain catastrophizing scores were collected. Principal component analysis was applied to select the optimal variable subset, and cluster analysis was applied to this subset to create distinctly different knee pain profiles. Four distinct knee pain profiles were identified: profile A (N = 27), profile B (N = 59), profile C (N = 85), and profile D (N = 41). Each knee pain profile had a unique combination of biochemical markers, pain biomarkers, physical impairments, and psychological factors that may provide the basis for mechanism-based diagnosis, individualized treatment, and selection of patients for clinical trials evaluating analgesic compounds. These results introduce a new profiling for knee OA and should be regarded as preliminary. PMID:25599306

  15. Functional signaling pathway analysis of lung adenocarcinomas identifies novel therapeutic targets for KRAS mutant tumors

    PubMed Central

    Baldelli, Elisa; Bellezza, Guido; Haura, Eric B.; Crinó, Lucio; Cress, W. Douglas; Deng, Jianghong; Ludovini, Vienna; Sidoni, Angelo; Schabath, Matthew B.; Puma, Francesco; Vannucci, Jacopo; Siggillino, Annamaria; Liotta, Lance A.; Petricoin, Emanuel F.; Pierobon, Mariaelena

    2015-01-01

    Little is known about the complex signaling architecture of KRAS and the interconnected RAS-driven protein-protein interactions, especially as it occurs in human clinical specimens. This study explored the activated and interconnected signaling network of KRAS mutant lung adenocarcinomas (AD) to identify novel therapeutic targets. Thirty-four KRAS mutant (MT) and twenty-four KRAS wild-type (WT) frozen biospecimens were obtained from surgically treated lung ADs. Samples were subjected to laser capture microdissection and reverse phase protein microarray analysis to explore the expression/activation levels of 150 signaling proteins along with co-activation concordance mapping. An independent set of 90 non-small cell lung cancers (NSCLC) was used to validate selected findings by immunohistochemistry (IHC). Compared to KRAS WT tumors, the signaling architecture of KRAS MT ADs revealed significant interactions between KRAS downstream substrates, the AKT/mTOR pathway, and a number of Receptor Tyrosine Kinases (RTK). Approximately one-third of the KRAS MT tumors had ERK activation greater than the WT counterpart (p<0.01). Notably 18% of the KRAS MT tumors had elevated activation of the Estrogen Receptor alpha (ER-α) (p=0.02). This finding was verified in an independent population by IHC (p=0.03). KRAS MT lung ADs appear to have a more intricate RAS linked signaling network than WT tumors with linkage to many RTKs and to the AKT-mTOR pathway. Combination therapy targeting different nodes of this network may be necessary to treat this group of patients. In addition, for patients with KRAS MT tumors and activation of the ER-α, anti-estrogen therapy may have important clinical implications. PMID:26468985

  16. An integrative framework for Bayesian variable selection with informative priors for identifying genes and pathways.

    PubMed

    Peng, Bin; Zhu, Dianwen; Ander, Bradley P; Zhang, Xiaoshuai; Xue, Fuzhong; Sharp, Frank R; Yang, Xiaowei

    2013-01-01

    The discovery of genetic or genomic markers plays a central role in the development of personalized medicine. A notable challenge exists when dealing with the high dimensionality of the data sets, as thousands of genes or millions of genetic variants are collected on a relatively small number of subjects. Traditional gene-wise selection methods using univariate analyses face difficulty to incorporate correlational, structural, or functional structures amongst the molecular measures. For microarray gene expression data, we first summarize solutions in dealing with 'large p, small n' problems, and then propose an integrative Bayesian variable selection (iBVS) framework for simultaneously identifying causal or marker genes and regulatory pathways. A novel partial least squares (PLS) g-prior for iBVS is developed to allow the incorporation of prior knowledge on gene-gene interactions or functional relationships. From the point view of systems biology, iBVS enables user to directly target the joint effects of multiple genes and pathways in a hierarchical modeling diagram to predict disease status or phenotype. The estimated posterior selection probabilities offer probabilitic and biological interpretations. Both simulated data and a set of microarray data in predicting stroke status are used in validating the performance of iBVS in a Probit model with binary outcomes. iBVS offers a general framework for effective discovery of various molecular biomarkers by combining data-based statistics and knowledge-based priors. Guidelines on making posterior inferences, determining Bayesian significance levels, and improving computational efficiencies are also discussed. PMID:23844055

  17. An Integrative Framework for Bayesian Variable Selection with Informative Priors for Identifying Genes and Pathways

    PubMed Central

    Ander, Bradley P.; Zhang, Xiaoshuai; Xue, Fuzhong; Sharp, Frank R.; Yang, Xiaowei

    2013-01-01

    The discovery of genetic or genomic markers plays a central role in the development of personalized medicine. A notable challenge exists when dealing with the high dimensionality of the data sets, as thousands of genes or millions of genetic variants are collected on a relatively small number of subjects. Traditional gene-wise selection methods using univariate analyses face difficulty to incorporate correlational, structural, or functional structures amongst the molecular measures. For microarray gene expression data, we first summarize solutions in dealing with large p, small n problems, and then propose an integrative Bayesian variable selection (iBVS) framework for simultaneously identifying causal or marker genes and regulatory pathways. A novel partial least squares (PLS) g-prior for iBVS is developed to allow the incorporation of prior knowledge on gene-gene interactions or functional relationships. From the point view of systems biology, iBVS enables user to directly target the joint effects of multiple genes and pathways in a hierarchical modeling diagram to predict disease status or phenotype. The estimated posterior selection probabilities offer probabilitic and biological interpretations. Both simulated data and a set of microarray data in predicting stroke status are used in validating the performance of iBVS in a Probit model with binary outcomes. iBVS offers a general framework for effective discovery of various molecular biomarkers by combining data-based statistics and knowledge-based priors. Guidelines on making posterior inferences, determining Bayesian significance levels, and improving computational efficiencies are also discussed. PMID:23844055

  18. Genome-Wide Chromatin Landscape Transitions Identify Novel Pathways in Early Commitment to Osteoblast Differentiation

    PubMed Central

    Thompson, Bethtrice; Varticovski, Lyuba; Baek, Songjoon; Hager, Gordon L.

    2016-01-01

    Bone continuously undergoes remodeling by a tightly regulated process that involves osteoblast differentiation from Mesenchymal Stem Cells (MSC). However, commitment of MSC to osteoblastic lineage is a poorly understood process. Chromatin organization functions as a molecular gatekeeper of DNA functions. Detection of sites that are hypersensitive to Dnase I has been used for detailed examination of changes in response to hormones and differentiation cues. To investigate the early steps in commitment of MSC to osteoblasts, we used a model human temperature-sensitive cell line, hFOB. When shifted to non-permissive temperature, these cells undergo "spontaneous" differentiation that takes several weeks, a process that is greatly accelerated by osteogenic induction media. We performed Dnase I hypersensitivity assays combined with deep sequencing to identify genome-wide potential regulatory events in cells undergoing early steps of commitment to osteoblasts. Massive reorganization of chromatin occurred within hours of differentiation. Whereas ~30% of unique DHS sites were located in the promoters, the majority was outside of the promoters, designated as enhancers. Many of them were at novel genomic sites and need to be confirmed experimentally. We developed a novel method for identification of cellular networks based solely on DHS enhancers signature correlated to gene expression. The analysis of enhancers that were unique to differentiating cells led to identification of bone developmental program encompassing 147 genes that directly or indirectly participate in osteogenesis. Identification of these pathways provided an unprecedented view of genomic regulation during early steps of differentiation and changes related to WNT, AP-1 and other pathways may have therapeutic implications. PMID:26890492

  19. Functional signaling pathway analysis of lung adenocarcinomas identifies novel therapeutic targets for KRAS mutant tumors.

    PubMed

    Baldelli, Elisa; Bellezza, Guido; Haura, Eric B; Crin, Lucio; Cress, W Douglas; Deng, Jianghong; Ludovini, Vienna; Sidoni, Angelo; Schabath, Matthew B; Puma, Francesco; Vannucci, Jacopo; Siggillino, Annamaria; Liotta, Lance A; Petricoin, Emanuel F; Pierobon, Mariaelena

    2015-10-20

    Little is known about the complex signaling architecture of KRAS and the interconnected RAS-driven protein-protein interactions, especially as it occurs in human clinical specimens. This study explored the activated and interconnected signaling network of KRAS mutant lung adenocarcinomas (AD) to identify novel therapeutic targets.Thirty-four KRAS mutant (MT) and twenty-four KRAS wild-type (WT) frozen biospecimens were obtained from surgically treated lung ADs. Samples were subjected to Laser Capture Microdissection and Reverse Phase Protein Microarray analysis to explore the expression/activation levels of 150 signaling proteins along with co-activation concordance mapping. An independent set of 90 non-small cell lung cancers (NSCLC) was used to validate selected findings by immunohistochemistry (IHC).Compared to KRAS WT tumors, the signaling architecture of KRAS MT ADs revealed significant interactions between KRAS downstream substrates, the AKT/mTOR pathway, and a number of Receptor Tyrosine Kinases (RTK). Approximately one-third of the KRAS MT tumors had ERK activation greater than the WT counterpart (p<0.01). Notably 18% of the KRAS MT tumors had elevated activation of the Estrogen Receptor alpha (ER-?) (p=0.02).This finding was verified in an independent population by IHC (p=0.03).KRAS MT lung ADs appear to have a more intricate RAS linked signaling network than WT tumors with linkage to many RTKs and to the AKT-mTOR pathway. Combination therapy targeting different nodes of this network may be necessary to treat this group of patients. In addition, for patients with KRAS MT tumors and activation of the ER-?, anti-estrogen therapy may have important clinical implications. PMID:26468985

  20. Genetic and Biochemical Dissection of a HisKA Domain Identifies Residues Required Exclusively for Kinase and Phosphatase Activities

    PubMed Central

    Willett, Jonathan W.; Kirby, John R.

    2012-01-01

    Two-component signal transduction systems, composed of histidine kinases (HK) and response regulators (RR), allow bacteria to respond to diverse environmental stimuli. The HK can control both phosphorylation and subsequent dephosphorylation of its cognate RR. The majority of HKs utilize the HisKA subfamily of dimerization and histidine phosphotransfer (DHp) domains, which contain the phospho-accepting histidine and directly contact the RR. Extensive genetics, biochemistry, and structural biology on several prototypical TCS systems including NtrB-NtrC and EnvZ-OmpR have provided a solid basis for understanding the function of HKRR signaling. Recently, work on NarX, a HisKA_3 subfamily protein, indicated that two residues in the highly conserved region of the DHp domain are responsible for phosphatase activity. In this study we have carried out both genetic and biochemical analyses on Myxococcus xanthus CrdS, a member of the HisKA subfamily of bacterial HKs. CrdS is required for the regulation of spore formation in response to environmental stress. Following alanine-scanning mutagenesis of the ?1 helix of the DHp domain of CrdS, we determined the role for each mutant protein for both kinase and phosphatase activity. Our results indicate that the conserved acidic residue (E372) immediately adjacent to the site of autophosphorylation (H371) is specifically required for kinase activity but not for phosphatase activity. Conversely, we found that the conserved Thr/Asn residue (N375) was required for phosphatase activity but not for kinase activity. We extended our biochemical analyses to two CrdS homologs from M. xanthus, HK1190 and HK4262, as well as Thermotoga maritima HK853. The results were similar for each HisKA family protein where the conserved acidic residue is required for kinase activity while the conserved Thr/Asn residue is required for phosphatase activity. These data are consistent with conserved mechanisms for kinase and phosphatase activities in the broadly occurring HisKA family of sensor kinases in bacteria. PMID:23226719

  1. Characterization of a novel ?-cypermethrin-degrading Aspergillus niger YAT strain and the biochemical degradation pathway of ?-cypermethrin.

    PubMed

    Deng, Weiqin; Lin, Derong; Yao, Kai; Yuan, Huaiyu; Wang, Zhilong; Li, Jianlong; Zou, Likou; Han, Xinfeng; Zhou, Kang; He, Li; Hu, Xinjie; Liu, Shuliang

    2015-10-01

    Aspergillus niger YAT strain was obtained from Chinese brick tea (Collection number: CGMCC 10,568) and identified on the basis of morphological characteristics and internal transcribed spacer (ITS) sequence. The strain could degrade 54.83 % of ?-cypermethrin (?-CY; 50 mg L(-1)) in 7 days and 100 % of 3-phenoxybenzoic acid (3-PBA; 100 mg L(-1)) in 22 h. The half-lives of ?-CY and 3-PBA range from 3.573 to 11.748 days and from 5.635 to 12.160 h, respectively. The degradation of ?-CY and 3-PBA was further described using first-order kinetic models. The pathway and mechanism of ?-CY degraded by YAT were investigated by analyzing the degraded metabolites through high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Relevant enzymatic activities and substrate utilization were also investigated. ?-CY degradation products were analyzed. Results indicated that YAT strain transformed ?-CY into 3-PBA. 3-PBA was then gradually transformed into permethric acid, protocatechuic acid, 3-hydroxy-5-phenoxy benzoic acid, gallic acid, and phenol gradually. The YAT strain can also effectively degrade these metabolites. The results indicated that YAT strain has potential applications in bioremediation of pyrethroid insecticide (PI)-contaminated environments and fermented food. PMID:26022858

  2. Analysis of biochemical compounds and differentially expressed genes of the anthocyanin biosynthetic pathway in variegated peach flowers.

    PubMed

    Hassani, D; Liu, H L; Chen, Y N; Wan, Z B; Zhuge, Q; Li, S X

    2015-01-01

    Variegated plants are highly valuable in the floricultural market, yet the genetic mechanism underlying this attractive phenomenon has not been completely elucidated. In this study, we identified and measured different compounds in pink and white flower petals of peach (Prunus persica) by high-performance liquid chromatography and liquid chromatography/mass spectrometry analyses. No cyanidin-based or pelargonidin-based compounds were detected in white petals, but high levels of these compounds were found in pink petals. Additionally, we sequenced and analyzed the expression of six key structural genes in the anthocyanin biosynthesis pathway (CHI, CHS, DFR, F3'H, ANS, and UFGT) in both white and pink petals. Quantitative real-time polymerase chain reaction revealed all six genes to be expressed at greatly reduced levels in white flower petals, relative to pink. No allelic variations were found in the transcribed sequences. However, alignment of transcribed and genomic sequences of the ANS gene detected alternative splicing, resulting in transcripts of 1.071 and 942 bp. Only the longer transcript was observed in white flower petals. Since ANS is the key intermediate enzyme catalyzing the colorless leucopelargonidin and leucocyanidin to substrates required for completion of anthocyanin biosynthesis, the ANS gene is implicated in flower color variegation and should be explored in future studies. This article, together with a previous transcriptome study, elucidates the mechanism underlying peach flower color variegation in terms of the key structural genes involved in anthocyanin biosynthesis. PMID:26535657

  3. Network-based methods for identifying critical pathways of complex diseases: a survey.

    PubMed

    Zhang, Qiaosheng; Li, Jie; Xue, Hanqing; Kong, Leilei; Wang, Yadong

    2016-04-22

    Biological pathways play important roles in the development of complex diseases, such as cancers, which are multifactorial complex diseases that are generally caused by mutation of multiple genes or dysregulation of pathways. It has become one of the most important issues to analyze pathways through combining multiple types of high-throughput data, such as genomics and proteomics, to understand the mechanisms of complex diseases. Currently, several network-based pathway analysis methods have been proposed. In this overview, we review seven major network-based pathway analysis methods and enumerate their benefits and limitations from an algorithmic perspective to provide a reference for the next generation of pathway analysis methods. Finally, we discuss the challenges that the next generation of methods faces. PMID:26888073

  4. An evidence-based knowledgebase of metastasis suppressors to identify key pathways relevant to cancer metastasis

    PubMed Central

    Zhao, Min; Li, Zhe; Qu, Hong

    2015-01-01

    Metastasis suppressor genes (MS genes) are genes that play important roles in inhibiting the process of cancer metastasis without preventing growth of the primary tumor. Identification of these genes and understanding their functions are critical for investigation of cancer metastasis. Recent studies on cancer metastasis have identified many new susceptibility MS genes. However, the comprehensive illustration of diverse cellular processes regulated by metastasis suppressors during the metastasis cascade is lacking. Thus, the relationship between MS genes and cancer risk is still unclear. To unveil the cellular complexity of MS genes, we have constructed MSGene (http://MSGene.bioinfo-minzhao.org/), the first literature-based gene resource for exploring human MS genes. In total, we manually curated 194 experimentally verified MS genes and mapped to 1448 homologous genes from 17 model species. Follow-up functional analyses associated 194 human MS genes with epithelium/tissue morphogenesis and epithelia cell proliferation. In addition, pathway analysis highlights the prominent role of MS genes in activation of platelets and coagulation system in tumor metastatic cascade. Moreover, global mutation pattern of MS genes across multiple cancers may reveal common cancer metastasis mechanisms. All these results illustrate the importance of MSGene to our understanding on cell development and cancer metastasis. PMID:26486520

  5. A model for genetic and epigenetic regulatory networks identifies rare pathways for transcription factor induced pluripotency

    NASA Astrophysics Data System (ADS)

    Artyomov, Maxim; Meissner, Alex; Chakraborty, Arup

    2010-03-01

    Most cells in an organism have the same DNA. Yet, different cell types express different proteins and carry out different functions. This is because of epigenetic differences; i.e., DNA in different cell types is packaged distinctly, making it hard to express certain genes while facilitating the expression of others. During development, upon receipt of appropriate cues, pluripotent embryonic stem cells differentiate into diverse cell types that make up the organism (e.g., a human). There has long been an effort to make this process go backward -- i.e., reprogram a differentiated cell (e.g., a skin cell) to pluripotent status. Recently, this has been achieved by transfecting certain transcription factors into differentiated cells. This method does not use embryonic material and promises the development of patient-specific regenerative medicine, but it is inefficient. The mechanisms that make reprogramming rare, or even possible, are poorly understood. We have developed the first computational model of transcription factor-induced reprogramming. Results obtained from the model are consistent with diverse observations, and identify the rare pathways that allow reprogramming to occur. If validated, our model could be further developed to design optimal strategies for reprogramming and shed light on basic questions in biology.

  6. First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic ?-cell mass.

    PubMed

    Wang, Guangliang; Rajpurohit, Surendra K; Delaspre, Fabien; Walker, Steven L; White, David T; Ceasrine, Alexis; Kuruvilla, Rejji; Li, Ruo-Jing; Shim, Joong S; Liu, Jun O; Parsons, Michael J; Mumm, Jeff S

    2015-01-01

    Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing ? cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of ?-cell proliferation. Further, we discovered novel roles for NF-?B signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating ?-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling ?-cell mass, potential therapeutic targets for treating diabetes. PMID:26218223

  7. What is a clinical pathway? Refinement of an operational definition to identify clinical pathway studies for a Cochrane systematic review.

    PubMed

    Lawal, Adegboyega K; Rotter, Thomas; Kinsman, Leigh; Machotta, Andreas; Ronellenfitsch, Ulrich; Scott, Shannon D; Goodridge, Donna; Plishka, Christopher; Groot, Gary

    2016-01-01

    Clinical pathways (CPWs) are a common component in the quest to improve the quality of health. CPWs are used to reduce variation, improve quality of care, and maximize the outcomes for specific groups of patients. An ongoing challenge is the operationalization of a definition of CPW in healthcare. This may be attributable to both the differences in definition and a lack of conceptualization in the field of clinical pathways. This correspondence article describes a process of refinement of an operational definition for CPW research and proposes an operational definition for the future syntheses of CPWs literature. Following the approach proposed by Kinsman et al. (BMC Medicine 8(1):31, 2010) and Wieland et al. (Alternative Therapies in Health and Medicine 17(2):50, 2011), we used a four-stage process to generate a five criteria checklist for the definition of CPWs. We refined the operational definition, through consensus, merging two of the checklist's criteria, leading to a more inclusive criterion for accommodating CPW studies conducted in various healthcare settings. The following four criteria for CPW operational definition, derived from the refinement process described above, are (1) the intervention was a structured multidisciplinary plan of care; (2) the intervention was used to translate guidelines or evidence into local structures; (3) the intervention detailed the steps in a course of treatment or care in a plan, pathway, algorithm, guideline, protocol or other 'inventory of actions' (i.e. the intervention had time-frames or criteria-based progression); and (4) the intervention aimed to standardize care for a specific population. An intervention meeting all four criteria was considered to be a CPW. The development of operational definitions for complex interventions is a useful approach to appraise and synthesize evidence for policy development and quality improvement. PMID:26904977

  8. Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage

    PubMed Central

    Park, Jung; Gebhardt, Matthias; Golovchenko, Svitlana; Perez-Branguli, Francesc; Hattori, Takako; Hartmann, Christine; Zhou, Xin; deCrombrugghe, Benoit; Stock, Michael; Schneider, Holm; von der Mark, Klaus

    2015-01-01

    According to the general understanding, the chondrocyte lineage terminates with the elimination of late hypertrophic cells by apoptosis in the growth plate. However, recent cell tracking studies have shown that murine hypertrophic chondrocytes can survive beyond terminal differentiation and give rise to a progeny of osteoblasts participating in endochondral bone formation. The question how chondrocytes convert into osteoblasts, however, remained open. Following the cell fate of hypertrophic chondrocytes by genetic lineage tracing using BACCol10;Cre induced YFP-reporter gene expression we show that a progeny of Col10Cre-reporter labelled osteoprogenitor cells and osteoblasts appears in the primary spongiosa and participates depending on the developmental stage substantially in trabecular, endosteal, and cortical bone formation. YFP+ trabecular and endosteal cells isolated by FACS expressed Col1a1, osteocalcin and runx2, thus confirming their osteogenic phenotype. In searching for transitory cells between hypertrophic chondrocytes and trabecular osteoblasts we identified by confocal microscopy a novel, small YFP+Osx+ cell type with mitotic activity in the lower hypertrophic zone at the chondro-osseous junction. When isolated from growth plates by fractional enzymatic digestion, these cells termed CDOP (chondrocyte-derived osteoprogenitor) cells expressed bone typical genes and differentiated into osteoblasts in vitro. We propose the Col10Cre-labeled CDOP cells mark the initiation point of a second pathway giving rise to endochondral osteoblasts, alternative to perichondrium derived osteoprogenitor cells. These findings add to current concepts of chondrocyte-osteocyte lineages and give new insight into the complex cartilage-bone transition process in the growth plate. PMID:25882555

  9. Genome-wide expression profiling identifies type 1 interferon response pathways in active tuberculosis.

    PubMed

    Ottenhoff, Tom H M; Dass, Ranjeeta Hari; Yang, Ninghan; Zhang, Mingzi M; Wong, Hazel E E; Sahiratmadja, Edhyana; Khor, Chiea Chuen; Alisjahbana, Bachti; van Crevel, Reinout; Marzuki, Sangkot; Seielstad, Mark; van de Vosse, Esther; Hibberd, Martin L

    2012-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains the leading cause of mortality from a single infectious agent. Each year around 9 million individuals newly develop active TB disease, and over 2 billion individuals are latently infected with M.tb worldwide, thus being at risk of developing TB reactivation disease later in life. The underlying mechanisms and pathways of protection against TB in humans, as well as the dynamics of the host response to M.tb infection, are incompletely understood. We carried out whole-genome expression profiling on a cohort of TB patients longitudinally sampled along 3 time-points: during active infection, during treatment, and after completion of curative treatment. We identified molecular signatures involving the upregulation of type-1 interferon (α/β) mediated signaling and chronic inflammation during active TB disease in an Indonesian population, in line with results from two recent studies in ethnically and epidemiologically different populations in Europe and South Africa. Expression profiles were captured in neutrophil-depleted blood samples, indicating a major contribution of lymphocytes and myeloid cells. Expression of type-1 interferon (α/β) genes mediated was also upregulated in the lungs of M.tb infected mice and in infected human macrophages. In patients, the regulated gene expression-signature normalized during treatment, including the type-1 interferon mediated signaling and a concurrent opposite regulation of interferon-gamma. Further analysis revealed IL15RA, UBE2L6 and GBP4 as molecules involved in the type-I interferon response in all three experimental models. Our data is highly suggestive that the innate immune type-I interferon signaling cascade could be used as a quantitative tool for monitoring active TB disease, and provide evidence that components of the patient's blood gene expression signature bear similarities to the pulmonary and macrophage response to mycobacterial infection. PMID:23029268

  10. CSF Proteomics Identifies Specific and Shared Pathways for Multiple Sclerosis Clinical Subtypes

    PubMed Central

    Avsar, Timucin; Duras?, ?lknur Melis; Uyguno?lu, U?ur; Ttnc, Melih; Demirci, Nuri Onat; Saip, Sabahattin; Sezerman, O. U?ur; Siva, Aksel; Tahir Turanl?, Eda

    2015-01-01

    Multiple sclerosis (MS) is an immune-mediated, neuro-inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS) with a heterogeneous clinical presentation and course. There is a remarkable phenotypic heterogeneity in MS, and the molecular mechanisms underlying it remain unknown. We aimed to investigate further the etiopathogenesis related molecular pathways in subclinical types of MS using proteomic and bioinformatics approaches in cerebrospinal fluids of patients with clinically isolated syndrome, relapsing remitting MS and progressive MS (n=179). Comparison of disease groups with controls revealed a total of 151 proteins that are differentially expressed in clinically different MS subtypes. KEGG analysis using PANOGA tool revealed the disease related pathways including aldosterone-regulated sodium reabsorption (p=8.02x10-5) which is important in the immune cell migration, renin-angiotensin (p=6.88x10-5) system that induces Th17 dependent immunity, notch signaling (p=1.83x10-10) pathway indicating the activated remyelination and vitamin digestion and absorption pathways (p=1.73x10-5). An emerging theme from our studies is that whilst all MS clinical forms share common biological pathways, there are also clinical subtypes specific and pathophysiology related pathways which may have further therapeutic implications. PMID:25942430

  11. Combinatorial high-throughput experimental and bioinformatic approach identifies molecular pathways linked with the sensitivity to anticancer target drugs

    PubMed Central

    Venkova, Larisa; Aliper, Alexander; Suntsova, Maria; Kholodenko, Roman; Shepelin, Denis; Borisov, Nicolas; Malakhova, Galina; Vasilov, Raif; Roumiantsev, Sergey; Zhavoronkov, Alex; Buzdin, Anton

    2015-01-01

    Effective choice of anticancer drugs is important problem of modern medicine. We developed a method termed OncoFinder for the analysis of new type of biomarkers reflecting activation of intracellular signaling and metabolic molecular pathways. These biomarkers may be linked with the sensitivity to anticancer drugs. In this study, we compared the experimental data obtained in our laboratory and in the Genomics of Drug Sensitivity in Cancer (GDS) project for testing response to anticancer drugs and transcriptomes of various human cell lines. The microarray-based profiling of transcriptomes was performed for the cell lines before the addition of drugs to the medium, and experimental growth inhibition curves were built for each drug, featuring characteristic IC50 values. We assayed here four target drugs - Pazopanib, Sorafenib, Sunitinib and Temsirolimus, and 238 different cell lines, of which 11 were profiled in our laboratory and 227 - in GDS project. Using the OncoFinder-processed transcriptomic data on ?600 molecular pathways, we identified pathways showing significant correlation between pathway activation strength (PAS) and IC50 values for these drugs. Correlations reflect relationships between response to drug and pathway activation features. We intersected the results and found molecular pathways significantly correlated in both our assay and GDS project. For most of these pathways, we generated molecular models of their interaction with known molecular target(s) of the respective drugs. For the first time, our study uncovered mechanisms underlying cancer cell response to drugs at the high-throughput molecular interactomic level. PMID:26317900

  12. Combinatorial high-throughput experimental and bioinformatic approach identifies molecular pathways linked with the sensitivity to anticancer target drugs.

    PubMed

    Venkova, Larisa; Aliper, Alexander; Suntsova, Maria; Kholodenko, Roman; Shepelin, Denis; Borisov, Nicolas; Malakhova, Galina; Vasilov, Raif; Roumiantsev, Sergey; Zhavoronkov, Alex; Buzdin, Anton

    2015-09-29

    Effective choice of anticancer drugs is important problem of modern medicine. We developed a method termed OncoFinder for the analysis of new type of biomarkers reflecting activation of intracellular signaling and metabolic molecular pathways. These biomarkers may be linked with the sensitivity to anticancer drugs. In this study, we compared the experimental data obtained in our laboratory and in the Genomics of Drug Sensitivity in Cancer (GDS) project for testing response to anticancer drugs and transcriptomes of various human cell lines. The microarray-based profiling of transcriptomes was performed for the cell lines before the addition of drugs to the medium, and experimental growth inhibition curves were built for each drug, featuring characteristic IC50 values. We assayed here four target drugs - Pazopanib, Sorafenib, Sunitinib and Temsirolimus, and 238 different cell lines, of which 11 were profiled in our laboratory and 227 - in GDS project. Using the OncoFinder-processed transcriptomic data on ~600 molecular pathways, we identified pathways showing significant correlation between pathway activation strength (PAS) and IC50 values for these drugs. Correlations reflect relationships between response to drug and pathway activation features. We intersected the results and found molecular pathways significantly correlated in both our assay and GDS project. For most of these pathways, we generated molecular models of their interaction with known molecular target(s) of the respective drugs. For the first time, our study uncovered mechanisms underlying cancer cell response to drugs at the high-throughput molecular interactomic level. PMID:26317900

  13. Integrated Physiological, Biochemical, and Molecular Analysis Identifies Important Traits and Mechanisms Associated with Differential Response of Rice Genotypes to Elevated Temperature

    PubMed Central

    Sailaja, Boghireddy; Subrahmanyam, Desiraju; Neelamraju, Sarla; Vishnukiran, Turaga; Rao, Yadavalli Venkateswara; Vijayalakshmi, Pujarula; Voleti, Sitapati R.; Bhadana, Vijai P.; Mangrauthia, Satendra K.

    2015-01-01

    In changing climatic conditions, heat stress caused by high temperature poses a serious threat to rice cultivation. A multiple organizational analysis at physiological, biochemical, and molecular levels is required to fully understand the impact of elevated temperature in rice. This study was aimed at deciphering the elevated temperature response in 11 popular and mega rice cultivars widely grown in India. Physiological and biochemical traits specifically membrane thermostability (MTS), antioxidants, and photosynthesis were studied at vegetative and reproductive phases, which were used to establish a correlation with grain yield under stress. Several useful traits in different genotypes were identified, which will be an important resource to develop high temperature-tolerant rice cultivars. Interestingly, Nagina22 emerged as the best performer in terms of yield as well as expression of physiological and biochemical traits at elevated temperature. It showed lesser relative injury, lesser reduction in chlorophyll content, increased super oxide dismutase, catalase and peroxidase activities, lesser reduction in net photosynthetic rate (PN), high transpiration rate (E), and other photosynthetic/fluorescence parameters contributing to least reduction in spikelet fertility and grain yield at elevated temperature. Furthermore, expression of 14 genes including heat shock transcription factors and heat shock proteins was analyzed in Nagina22 (tolerant) and Vandana (susceptible) at flowering phase, strengthening the fact that N22 performed better at molecular level also during elevated temperature. This study shows that elevated temperature response is complex and involves multiple biological processes that are needed to be characterized to address the challenges of extreme conditions of future climate. PMID:26640473

  14. A phosphoproteomics approach to identify candidate kinase inhibitor pathway targets in lymphoma-like primary cell lines.

    PubMed

    Vojvodic, Miliana; Hansford, Loen M; Morozova, Olena; Blakely, Kim M; Taylor, Paul; Fathers, Kelly E; Moffat, Jason; Marra, Marco; Smith, Kristen M; Moran, Michael F; Kaplan, David R

    2013-12-01

    Mass spectrometry-based technologies are increasingly utilized in drug discovery. Phosphoproteomics in particular has allowed for the efficient surveying of phosphotyrosine signaling pathways involved in various diseases states, most prominently in cancer. We describe a phosphotyrosine-based proteomics screening approach to identify signaling pathways and tyrosine kinase inhibitor targets in highly tumorigenic human lymphoma-like primary cells. We identified several receptor tyrosine kinase pathways and validated SRC family kinases (SFKs) as potential drug targets for targeted selection of small molecule inhibitors. BMS-354825 (dasatinib) and SKI-606 (bosutinib), second and third generation clinical SFK/ABL inhibitors, were found to be potent cytotoxic agents against tumorigenic cells with low toxicity to normal pediatric stem cells. Both SFK inhibitors reduced ERK1/2 and AKT phosphorylation and induced apoptosis. This study supports the adaptation of high-end mass spectrometry techniques for the efficient identification of candidate tyrosine kinases as novel therapeutic targets in primary cancer cell lines. PMID:23701117

  15. Candidate Pathway-Based GWAS Identifies Novel Associations of Genomic Variants in the Complement System Associated with Coronary Artery Disease

    PubMed Central

    Xu, Chengqi; Yang, Qin; Xiong, Hongbo; Wang, Longfei; Cai, Jianping; Wang, Fan; Li, Sisi; Chen, Jing; Wang, Chuchu; Wang, Dan; Xiong, Xin; Wang, Pengyun; Zhao, Yuanyuan; Wang, Xiaojing; Huang, Yufeng; Chen, Shanshan; Yin, Dan; Li, Xiuchun; Liu, Ying; Liu, Jinqiu; Wang, Jingjing; Li, Hui; Ke, Tie; Ren, Xiang; Wu, Yanxia; Wu, Gang; Wan, Jing; Zhang, Rongfeng; Wu, Tangchun; Wang, Junhan; Xia, Yunlong; Yang, Yanzong; Cheng, Xiang; Liao, Yuhua; Chen, Qiuyun; Zhou, Yanhong; He, Qing; Tu, Xin; Wang, Qing K.

    2014-01-01

    Background Genomic variants identified by genome-wide association studies (GWAS) explain <20% of heritability of coronary artery disease (CAD), thus many risk variants remain missing for CAD. Identification of new variants may unravel new biological pathways and genetic mechanisms for CAD. To identify new variants associated with CAD, we developed a candidate pathway-based GWAS by integrating expression quantitative loci (eQTL) analysis and mining of GWAS data with variants in a candidate pathway. Methods and Results Mining of GWAS data was performed to analyze variants in 32 complement system genes for positive association with CAD. Functional variants in genes showing positive association were then identified by searching existing expression quantitative loci databases and validated by RT-PCR. A follow-up case control design was then used to determine whether the functional variants are associated with CAD in two independent GeneID Chinese populations. Candidate pathway-based GWAS identified positive association between variants in C3AR1 and C6 and CAD. Two functional variants, rs7842 in C3AR1 and rs4400166 in C6, were found to be associated with expression levels of C3AR1 and C6, respectively. Significant association was identified between rs7842 and CAD (P=3.99×10−6, OR=1.47) and between rs4400166 and CAD (P=9.30×10−3, OR=1.24) in the validation cohort. The significant findings were confirmed in the replication cohort (P=1.53×10−5, OR=1.37 for rs7842; P=8.41×10−3, OR=1.21 for rs4400166. Conclusions Integration of GWAS with biological pathways and eQTL is effective in identifying new risk variants for CAD. Functional variants increasing C3AR1 and C6 expression were shown to confer significant risk of CAD for the first time. PMID:25249547

  16. Integrated pathway-based approach identifies association between genomic regions at CTCF and CACNB2 and schizophrenia.

    PubMed

    Juraeva, Dilafruz; Haenisch, Britta; Zapatka, Marc; Frank, Josef; Witt, Stephanie H; Mhleisen, Thomas W; Treutlein, Jens; Strohmaier, Jana; Meier, Sandra; Degenhardt, Franziska; Giegling, Ina; Ripke, Stephan; Leber, Markus; Lange, Christoph; Schulze, Thomas G; Mssner, Rainald; Nenadic, Igor; Sauer, Heinrich; Rujescu, Dan; Maier, Wolfgang; Brglum, Anders; Ophoff, Roel; Cichon, Sven; Nthen, Markus M; Rietschel, Marcella; Mattheisen, Manuel; Brors, Benedikt

    2014-06-01

    In the present study, an integrated hierarchical approach was applied to: (1) identify pathways associated with susceptibility to schizophrenia; (2) detect genes that may be potentially affected in these pathways since they contain an associated polymorphism; and (3) annotate the functional consequences of such single-nucleotide polymorphisms (SNPs) in the affected genes or their regulatory regions. The Global Test was applied to detect schizophrenia-associated pathways using discovery and replication datasets comprising 5,040 and 5,082 individuals of European ancestry, respectively. Information concerning functional gene-sets was retrieved from the Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, and the Molecular Signatures Database. Fourteen of the gene-sets or pathways identified in the discovery dataset were confirmed in the replication dataset. These include functional processes involved in transcriptional regulation and gene expression, synapse organization, cell adhesion, and apoptosis. For two genes, i.e. CTCF and CACNB2, evidence for association with schizophrenia was available (at the gene-level) in both the discovery study and published data from the Psychiatric Genomics Consortium schizophrenia study. Furthermore, these genes mapped to four of the 14 presently identified pathways. Several of the SNPs assigned to CTCF and CACNB2 have potential functional consequences, and a gene in close proximity to CACNB2, i.e. ARL5B, was identified as a potential gene of interest. Application of the present hierarchical approach thus allowed: (1) identification of novel biological gene-sets or pathways with potential involvement in the etiology of schizophrenia, as well as replication of these findings in an independent cohort; (2) detection of genes of interest for future follow-up studies; and (3) the highlighting of novel genes in previously reported candidate regions for schizophrenia. PMID:24901509

  17. Major carcinogenic pathways identified by gene expression analysis of peritoneal mesotheliomas following chemical treatment in F344 rats

    EPA Science Inventory

    This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo a...

  18. Pathways Disrupted in Human ALS Motor Neurons Identified Through Genetic Correction of Mutant SOD1

    PubMed Central

    Kiskinis, Evangelos; Sandoe, Jackson; Williams, Luis A.; Boulting, Gabriella L.; Moccia, Rob; Wainger, Brian J.; Han, Steve; Peng, Theodore; Thams, Sebastian; Mikkilineni, Shravani; Mellin, Cassidy; Merkle, Florian T.; Davis-Dusenbery, Brandi N.; Ziller, Michael; Oakley, Derek; Ichida, Justin; Dicostanza, Stefania; Atwater, Nick; Maeder, Morgan L.; Goodwin, Mathew J.; Nemesh, James; Handsaker, Robert E.; Paull, Daniel; Noggle, Scott; McCarroll, Steven A.; Joung, J. Keith; Woolf, Clifford J.; Brown, Robert H; Eggan, Kevin

    2015-01-01

    Summary Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and whether they converge on common pathways to cause neuronal degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional and functional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered sub-cellular transport as well as activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that these pathways were perturbed in a manner dependent on the SOD1 mutation. Finally, interrogation of stem cell-derived motor neurons produced from ALS patients harboring a repeat expansion in C9orf72 indicates at least a subset of these changes are more broadly conserved in ALS. PMID:24704492

  19. Temporal retinal transcriptome and systems biology analysis identifies key pathways and hub genes in Staphylococcus aureus endophthalmitis

    PubMed Central

    Rajamani, Deepa; Singh, Pawan Kumar; Rottmann, Bruce G.; Singh, Natasha; Bhasin, Manoj K.; Kumar, Ashok

    2016-01-01

    Bacterial endophthalmitis remains a devastating inflammatory condition associated with permanent vision loss. Hence, assessing the host response in this disease may provide new targets for intervention. Using a mouse model of Staphylococcus aureus (SA) endophthalmitis and performing retinal transcriptome analysis, we discovered progressive changes in the expression of 1,234 genes. Gene ontology (GO) and pathway analyses revealed the major pathways impacted in endophthalmitis includes: metabolism, inflammatory/immune, antimicrobial, cell trafficking, and lipid biosynthesis. Among the immune/inflammation pathways, JAK/Stat and IL-17A signaling were the most significantly affected. Interactive network-based analyses identified 13 focus hub genes (IL-6, IL-1β, CXCL2, STAT3, NUPR1, Jun, CSF1, CYR61, CEBPB, IGF-1, EGFR1, SPP1, and TGM2) within these important pathways. The expression of hub genes confirmed by qRT-PCR, ELISA (IL-6, IL-1β, and CXCL2), and Western blot or immunostaining (CEBP, STAT3, NUPR1, and IGF1) showed strong correlation with transcriptome data. Since TLR2 plays an important role in SA endophthalmitis, counter regulation analysis of TLR2 ligand pretreated retina or the use of retinas from TLR2 knockout mice showed the down-regulation of inflammatory regulatory genes. Collectively, our study provides, for the first time, a comprehensive analysis of the transcriptomic response and identifies key pathways regulating retinal innate responses in staphylococcal endophthalmitis. PMID:26865111

  20. Temporal retinal transcriptome and systems biology analysis identifies key pathways and hub genes in Staphylococcus aureus endophthalmitis.

    PubMed

    Rajamani, Deepa; Singh, Pawan Kumar; Rottmann, Bruce G; Singh, Natasha; Bhasin, Manoj K; Kumar, Ashok

    2016-01-01

    Bacterial endophthalmitis remains a devastating inflammatory condition associated with permanent vision loss. Hence, assessing the host response in this disease may provide new targets for intervention. Using a mouse model of Staphylococcus aureus (SA) endophthalmitis and performing retinal transcriptome analysis, we discovered progressive changes in the expression of 1,234 genes. Gene ontology (GO) and pathway analyses revealed the major pathways impacted in endophthalmitis includes: metabolism, inflammatory/immune, antimicrobial, cell trafficking, and lipid biosynthesis. Among the immune/inflammation pathways, JAK/Stat and IL-17A signaling were the most significantly affected. Interactive network-based analyses identified 13 focus hub genes (IL-6, IL-1?, CXCL2, STAT3, NUPR1, Jun, CSF1, CYR61, CEBPB, IGF-1, EGFR1, SPP1, and TGM2) within these important pathways. The expression of hub genes confirmed by qRT-PCR, ELISA (IL-6, IL-1?, and CXCL2), and Western blot or immunostaining (CEBP, STAT3, NUPR1, and IGF1) showed strong correlation with transcriptome data. Since TLR2 plays an important role in SA endophthalmitis, counter regulation analysis of TLR2 ligand pretreated retina or the use of retinas from TLR2 knockout mice showed the down-regulation of inflammatory regulatory genes. Collectively, our study provides, for the first time, a comprehensive analysis of the transcriptomic response and identifies key pathways regulating retinal innate responses in staphylococcal endophthalmitis. PMID:26865111

  1. Changes in microRNAs associated with Twist-1 and Bcl-2 overexpression identify signaling pathways.

    PubMed

    Zhao, Nan; Sun, Bao-cun; Zhao, Xiu-lan; Wang, Yong; Sun, Hui-zhi; Dong, Xue-yi; Meng, Jie; Gu, Qiang

    2015-12-01

    Epithelial-mesenchymal transition (EMT) is regulated by multiple signal transduction pathways. Twist-1 is one of the most important transcription factors in these pathways. In a previous study, we found that Bcl-2 enhanced the role of Twist-1 in EMT. Coexpression of Twist-1 and Bcl-2 may play an import role in vasculogenic mimicry (VM) through regulation of EMT. Moreover, regulators of EMT and VM are known to be important targets for microRNAs (miRNAs). To better understand how these critical pathways are induced by coexpression of Twist-1 and Bcl-2, we performed a comprehensive comparative bioinformatics analysis using microarrays on HCCs that overexpressed Twist-1 and Bcl-2. Eleven miRNAs associated with coexpression of Twist-1 and Bcl-2 were selected from the comprehensive analysis of miRNA microarray and ChIP-seq analysis. Changes in miRNAs were associated with significant differences in the expression of genes involved in signal transduction pathways related to processes including tumor invasion, metastasis, angiogenesis, and tumor cell shape. We confirmed the role of Twist-1 and Bcl-2 coexpression in HCC cells using wound healing assays, invasion assays, and 3D Matrigel assays. Furthermore, the role of miR-27a as a crucial regulator of EMT and VM was confirmed in HCC cells by RT-PCR and western blot analysis. These findings provide evidence that Bcl-2 enhances the role of Twist-1 in VM and EMT through miRNAs. PMID:26341138

  2. Pubertal Effects on Adjustment in Girls: Moving from Demonstrating Effects to Identifying Pathways

    ERIC Educational Resources Information Center

    Graber, Julia A.; Brooks-Gunn, Jeanne; Warren, Michelle P.

    2006-01-01

    The present investigation examines mediated pathways from pubertal development to changes in depressive affect and aggression. Participants were 100 white girls who were between the ages of 10 and 14 (M=12.13, SD=0.80); girls were from well-educated, middle-to upper-middle class families, and attended private schools in a major northeastern urban

  3. Diverging Alternative Splicing Fingerprints in the Transforming Growth Factor-? Signaling Pathway Identified in Thoracic Aortic Aneurysms

    PubMed Central

    Kurtovic, Sanela; Paloschi, Valentina; Folkersen, Lasse; Gottfries, Johan; Franco-Cereceda, Anders; Eriksson, Per

    2011-01-01

    Impaired regulation of the transforming growth factor-? (TGF?) signaling pathway has been linked to thoracic aortic aneurysm (TAA). Previous work has indicated that differential splicing is a common phenomenon, potentially influencing the function of proteins. In the present study we investigated the occurrence of differential splicing in the TGF? pathway associated with TAA in patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV). Affymetrix human exon arrays were applied to 81 intima/media tissue samples from dilated (n = 51) and nondilated (n = 30) aortas of TAV and BAV patients. To analyze the occurrence of alternative splicing in the TGF? pathway, multivariate techniques, including principal component analysis and OPLS-DA (orthogonal partial least squares to latent structures discriminant analysis), were applied on all exons (n = 614) of the TGF? pathway. The scores plot, based on the splice index of individual exons, showed separate clusters of patients with both dilated and nondilated aorta, thereby illustrating the potential importance of alternative splicing in TAA. In total, differential splicing was detected in 187 exons. Furthermore, the pattern of alternative splicing is clearly differs between TAV and BAV patients. Differential splicing was specific for BAV and TAV patients in 40 and 86 exons, respectively, and splicings of 61 exons were shared between the two phenotypes. The occurrence of differential splicing was demonstrated in selected genes by reverse transcriptionpolymerase chain reaction. In summary, alternative splicing is a common feature of TAA formation. Our results suggest that dilatation in TAV and BAV patients has different alternative splicing fingerprints in the TGF? pathway. PMID:21448509

  4. A novel method to identify pathways associated with renal cell carcinoma based on a gene co-expression network.

    PubMed

    Ruan, Xiyun; Li, Hongyun; Liu, Bo; Chen, Jie; Zhang, Shibao; Sun, Zeqiang; Liu, Shuangqing; Sun, Fahai; Liu, Qingyong

    2015-08-01

    The aim of the present study was to develop a novel method for identifying pathways associated with renal cell carcinoma (RCC) based on a gene co-expression network. A framework was established where a co-expression network was derived from the database as well as various co-expression approaches. First, the backbone of the network based on differentially expressed (DE) genes between RCC patients and normal controls was constructed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The differentially co-expressed links were detected by Pearson's correlation, the empirical Bayesian (EB) approach and Weighted Gene Co-expression Network Analysis (WGCNA). The co-expressed gene pairs were merged by a rank-based algorithm. We obtained 842; 371; 2,883 and 1,595 co-expressed gene pairs from the co-expression networks of the STRING database, Pearson's correlation EB method and WGCNA, respectively. Two hundred and eighty-one differentially co-expressed (DC) gene pairs were obtained from the merged network using this novel method. Pathway enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the network enrichment analysis (NEA) method were performed to verify feasibility of the merged method. Results of the KEGG and NEA pathway analyses showed that the network was associated with RCC. The suggested method was computationally efficient to identify pathways associated with RCC and has been identified as a useful complement to traditional co-expression analysis. PMID:26058425

  5. International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways.

    PubMed

    Cordell, Heather J; Han, Younghun; Mells, George F; Li, Yafang; Hirschfield, Gideon M; Greene, Casey S; Xie, Gang; Juran, Brian D; Zhu, Dakai; Qian, David C; Floyd, James A B; Morley, Katherine I; Prati, Daniele; Lleo, Ana; Cusi, Daniele; Gershwin, M Eric; Anderson, Carl A; Lazaridis, Konstantinos N; Invernizzi, Pietro; Seldin, Michael F; Sandford, Richard N; Amos, Christopher I; Siminovitch, Katherine A

    2015-01-01

    Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist. PMID:26394269

  6. International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways

    PubMed Central

    Cordell, Heather J.; Han, Younghun; Mells, George F.; Li, Yafang; Hirschfield, Gideon M.; Greene, Casey S.; Xie, Gang; Juran, Brian D.; Zhu, Dakai; Qian, David C.; Floyd, James A. B.; Morley, Katherine I.; Prati, Daniele; Lleo, Ana; Cusi, Daniele; Schlicht, Erik M; Lammert, Craig; Atkinson, Elizabeth J; Chan, Landon L; de Andrade, Mariza; Balschun, Tobias; Mason, Andrew L; Myers, Robert P; Zhang, Jinyi; Milkiewicz, Piotr; Qu, Jia; Odin, Joseph A; Luketic, Velimir A; Bacon, Bruce R; Bodenheimer Jr, Henry C; Liakina, Valentina; Vincent, Catherine; Levy, Cynthia; Gregersen, Peter K; Almasio, Piero L; Alvaro, Domenico; Andreone, Pietro; Andriulli, Angelo; Barlassina, Cristina; Battezzati, Pier Maria; Benedetti, Antonio; Bernuzzi, Francesca; Bianchi, Ilaria; Bragazzi, Maria Consiglia; Brunetto, Maurizia; Bruno, Savino; Casella, Giovanni; Coco, Barbara; Colli, Agostino; Colombo, Massimo; Colombo, Silvia; Cursaro, Carmela; Crocè, Lory Saveria; Crosignani, Andrea; Donato, Maria Francesca; Elia, Gianfranco; Fabris, Luca; Ferrari, Carlo; Floreani, Annarosa; Foglieni, Barbara; Fontana, Rosanna; Galli, Andrea; Lazzari, Roberta; Macaluso, Fabio; Malinverno, Federica; Marra, Fabio; Marzioni, Marco; Mattalia, Alberto; Montanari, Renzo; Morini, Lorenzo; Morisco, Filomena; Hani S, Mousa; Muratori, Luigi; Muratori, Paolo; Niro, Grazia A; Palmieri, Vincenzo O; Picciotto, Antonio; Podda, Mauro; Portincasa, Piero; Ronca, Vincenzo; Rosina, Floriano; Rossi, Sonia; Sogno, Ilaria; Spinzi, Giancarlo; Spreafico, Marta; Strazzabosco, Mario; Tarallo, Sonia; Tarocchi, Mirko; Tiribelli, Claudio; Toniutto, Pierluigi; Vinci, Maria; Zuin, Massimo; Ch'ng, Chin Lye; Rahman, Mesbah; Yapp, Tom; Sturgess, Richard; Healey, Christopher; Czajkowski, Marek; Gunasekera, Anton; Gyawali, Pranab; Premchand, Purushothaman; Kapur, Kapil; Marley, Richard; Foster, Graham; Watson, Alan; Dias, Aruna; Subhani, Javaid; Harvey, Rory; McCorry, Roger; Ramanaden, David; Gasem, Jaber; Evans, Richard; Mathialahan, Thiriloganathan; Shorrock, Christopher; Lipscomb, George; Southern, Paul; Tibble, Jeremy; Gorard, David; Palegwala, Altaf; Jones, Susan; Carbone, Marco; Dawwas, Mohamed; Alexander, Graeme; Dolwani, Sunil; Prince, Martin; Foxton, Matthew; Elphick, David; Mitchison, Harriet; Gooding, Ian; Karmo, Mazn; Saksena, Sushma; Mendall, Mike; Patel, Minesh; Ede, Roland; Austin, Andrew; Sayer, Joanna; Hankey, Lorraine; Hovell, Christopher; Fisher, Neil; Carter, Martyn; Koss, Konrad; Piotrowicz, Andrzej; Grimley, Charles; Neal, David; Lim, Guan; Levi, Sass; Ala, Aftab; Broad, Andrea; Saeed, Athar; Wood, Gordon; Brown, Jonathan; Wilkinson, Mark; Gordon, Harriet; Ramage, John; Ridpath, Jo; Ngatchu, Theodore; Grover, Bob; Shaukat, Syed; Shidrawi, Ray; Abouda, George; Ali, Faiz; Rees, Ian; Salam, Imroz; Narain, Mark; Brown, Ashley; Taylor-Robinson, Simon; Williams, Simon; Grellier, Leonie; Banim, Paul; Das, Debashis; Chilton, Andrew; Heneghan, Michael; Curtis, Howard; Gess, Markus; Drake, Ian; Aldersley, Mark; Davies, Mervyn; Jones, Rebecca; McNair, Alastair; Srirajaskanthan, Raj; Pitcher, Maxton; Sen, Sambit; Bird, George; Barnardo, Adrian; Kitchen, Paul; Yoong, Kevin; Chirag, Oza; Sivaramakrishnan, Nurani; MacFaul, George; Jones, David; Shah, Amir; Evans, Chris; Saha, Subrata; Pollock, Katharine; Bramley, Peter; Mukhopadhya, Ashis; Fraser, Andrew; Mills, Peter; Shallcross, Christopher; Campbell, Stewart; Bathgate, Andrew; Shepherd, Alan; Dillon, John; Rushbrook, Simon; Przemioslo, Robert; Macdonald, Christopher; Metcalf, Jane; Shmueli, Udi; Davis, Andrew; Naqvi, Asifabbas; Lee, Tom; Ryder, Stephen D; Collier, Jane; Klass, Howard; Ninkovic, Mary; Cramp, Matthew; Sharer, Nicholas; Aspinall, Richard; Goggin, Patrick; Ghosh, Deb; Douds, Andrew; Hoeroldt, Barbara; Booth, Jonathan; Williams, Earl; Hussaini, Hyder; Stableforth, William; Ayres, Reuben; Thorburn, Douglas; Marshall, Eileen; Burroughs, Andrew; Mann, Steven; Lombard, Martin; Richardson, Paul; Patanwala, Imran; Maltby, Julia; Brookes, Matthew; Mathew, Ray; Vyas, Samir; Singhal, Saket; Gleeson, Dermot; Misra, Sharat; Butterworth, Jeff; George, Keith; Harding, Tim; Douglass, Andrew; Panter, Simon; Shearman, Jeremy; Bray, Gary; Butcher, Graham; Forton, Daniel; Mclindon, John; Cowan, Matthew; Whatley, Gregory; Mandal, Aditya; Gupta, Hemant; Sanghi, Pradeep; Jain, Sanjiv; Pereira, Steve; Prasad, Geeta; Watts, Gill; Wright, Mark; Neuberger, James; Gordon, Fiona; Unitt, Esther; Grant, Allister; Delahooke, Toby; Higham, Andrew; Brind, Alison; Cox, Mark; Ramakrishnan, Subramaniam; King, Alistair; Collins, Carole; Whalley, Simon; Li, Andy; Fraser, Jocelyn; Bell, Andrew; Wong, Voi Shim; Singhal, Amit; Gee, Ian; Ang, Yeng; Ransford, Rupert; Gotto, James; Millson, Charles; Bowles, Jane; Thomas, Caradog; Harrison, Melanie; Galaska, Roman; Kendall, Jennie; Whiteman, Jessica; Lawlor, Caroline; Gray, Catherine; Elliott, Keith; Mulvaney-Jones, Caroline; Hobson, Lucie; Van Duyvenvoorde, Greta; Loftus, Alison; Seward, Katie; Penn, Ruth; Maiden, Jane; Damant, Rose; Hails, Janeane; Cloudsdale, Rebecca; Silvestre, Valeria; Glenn, Sue; Dungca, Eleanor; Wheatley, Natalie; Doyle, Helen; Kent, Melanie; Hamilton, Caroline; Braim, Delyth; Wooldridge, Helen; Abrahams, Rachel; Paton, Alison; Lancaster, Nicola; Gibbins, Andrew; Hogben, Karen; Desousa, Phillipa; Muscariu, Florin; Musselwhite, Janine; McKay, Alexandra; Tan, LaiTing; Foale, Carole; Brighton, Jacqueline; Flahive, Kerry; Nambela, Estelle; Townshend, Paula; Ford, Chris; Holder, Sophie; Palmer, Caroline; Featherstone, James; Nasseri, Mariam; Sadeghian, Joy; Williams, Bronwen; Thomas, Carol; Rolls, Sally-Ann; Hynes, Abigail; Duggan, Claire; Jones, Sarah; Crossey, Mary; Stansfield, Glynis; MacNicol, Carolyn; Wilkins, Joy; Wilhelmsen, Elva; Raymode, Parizade; Lee, Hye-Jeong; Durant, Emma; Bishop, Rebecca; Ncube, Noma; Tripoli, Sherill; Casey, Rebecca; Cowley, Caroline; Miller, Richard; Houghton, Kathryn; Ducker, Samantha; Wright, Fiona; Bird, Bridget; Baxter, Gwen; Keggans, Janie; Hughes, Maggie; Grieve, Emma; Young, Karin; Williams, D; Ocker, Kate; Hines, Frances; Martin, Kirsty; Innes, Caron; Valliani, Talal; Fairlamb, Helen; Thornthwaite, Sarah; Eastick, Anne; Tanqueray, Elizabeth; Morrison, Jennifer; Holbrook, Becky; Browning, Julie; Walker, Kirsten; Congreave, Susan; Verheyden, Juliette; Slininger, Susan; Stafford, Lizzie; O'Donnell, Denise; Ainsworth, Mark; Lord, Susan; Kent, Linda; March, Linda; Dickson, Christine; Simpson, Diane; Longhurst, Beverley; Hayes, Maria; Shpuza, Ervin; White, Nikki; Besley, Sarah; Pearson, Sallyanne; Wright, Alice; Jones, Linda; Gunter, Emma; Dewhurst, Hannah; Fouracres, Anna; Farrington, Liz; Graves, Lyn; Marriott, Suzie; Leoni, Marina; Tyrer, David; Martin, Kate; Dali-kemmery, Lola; Lambourne, Victoria; Green, Marie; Sirdefield, Dawn; Amor, Kelly; Colley, Julie; Shinder, Bal; Jones, Jayne; Mills, Marisa; Carnahan, Mandy; Taylor, Natalie; Boulton, Kerenza; Tregonning, Julie; Brown, Carly; Clifford, Gayle; Archer, Emily; Hamilton, Maria; Curtis, Janette; Shewan, Tracey; Walsh, Sue; Warner, Karen; Netherton, Kimberley; Mupudzi, Mcdonald; Gunson, Bridget; Gitahi, Jane; Gocher, Denise; Batham, Sally; Pateman, Hilary; Desmennu, Senayon; Conder, Jill; Clement, Darren; Gallagher, Susan; Orpe, Jacky; Chan, PuiChing; Currie, Lynn; O'Donohoe, Lynn; Oblak, Metod; Morgan, Lisa; Quinn, Marie; Amey, Isobel; Baird, Yolanda; Cotterill, Donna; Cumlat, Lourdes; Winter, Louise; Greer, Sandra; Spurdle, Katie; Allison, Joanna; Dyer, Simon; Sweeting, Helen; Kordula, Jean; Gershwin, M. Eric; Anderson, Carl A.; Lazaridis, Konstantinos N.; Invernizzi, Pietro; Seldin, Michael F.; Sandford, Richard N.; Amos, Christopher I.; Siminovitch, Katherine A.

    2015-01-01

    Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10−8) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine–cytokine pathways, for which relevant therapies exist. PMID:26394269

  7. Rapamycin regulates biochemical metabolites

    PubMed Central

    Tucci, Paola; Porta, Giovanni; Agostini, Massimiliano; Antonov, Alexey; Garabadgiu, Alexander Vasilievich; Melino, Gerry; Willis, Anne E

    2013-01-01

    The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth, and deregulation of this pathway is associated with tumorigenesis. p53, and its less investigated family member p73, have been shown to interact closely with mTOR pathways through the transcriptional regulation of different target genes. To investigate the metabolic changes that occur upon inhibition of the mTOR pathway and the role of p73 in this response primary mouse embryonic fibroblast from control and TAp73?/? were treated with the macrocyclic lactone rapamycin. Extensive gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS/MS) analysis were used to obtain a rapamycin-dependent global metabolome profile from control or TAp73?/? cells. In total 289 metabolites involved in selective pathways were identified; 39 biochemical metabolites were found to be significantly altered, many of which are known to be associated with the cellular stress response. PMID:23839040

  8. Structural and Biochemical Studies of the C-Terminal Domain of Mouse Peptide-N-glycanase Identify it as a Mannose-Binding Module

    SciTech Connect

    Zhou,X.; Zhao, G.; Truglio, J.; Wang, L.; Li, G.; Lennarz, W.; Schindelin, H.

    2006-01-01

    The inability of certain N-linked glycoproteins to adopt their native conformation in the endoplasmic reticulum (ER) leads to their retrotranslocation into the cytosol and subsequent degradation by the proteasome. In this pathway the cytosolic peptide-N-glycanase (PNGase) cleaves the N-linked glycan chains off denatured glycoproteins. PNGase is highly conserved in eukaryotes and plays an important role in ER-associated protein degradation. In higher eukaryotes, PNGase has an N-terminal and a C-terminal extension in addition to its central catalytic domain, which is structurally and functionally related to transglutaminases. Although the N-terminal domain of PNGase is involved in protein-protein interactions, the function of the C-terminal domain has not previously been characterized. Here, we describe biophysical, biochemical, and crystallographic studies of the mouse PNGase C-terminal domain, including visualization of a complex between this domain and mannopentaose. These studies demonstrate that the C-terminal domain binds to the mannose moieties of N-linked oligosaccharide chains, and we further show that it enhances the activity of the mouse PNGase core domain, presumably by increasing the affinity of mouse PNGase for the glycan chains of misfolded glycoproteins.

  9. Structural and biochemical characterization of Chlamydia trachomatis hypothetical protein CT263 supports that menaquinone synthesis occurs through the futalosine pathway.

    PubMed

    Barta, Michael L; Thomas, Keisha; Yuan, Hongling; Lovell, Scott; Battaile, Kevin P; Schramm, Vern L; Hefty, P Scott

    2014-11-14

    The obligate intracellular human pathogen Chlamydia trachomatis is the etiological agent of blinding trachoma and sexually transmitted disease. Genomic sequencing of Chlamydia indicated this medically important bacterium was not exclusively dependent on the host cell for energy. In order for the electron transport chain to function, electron shuttling between membrane-embedded complexes requires lipid-soluble quinones (e.g. menaquionone or ubiquinone). The sources or biosynthetic pathways required to obtain these electron carriers within C. trachomatis are poorly understood. The 1.58 crystal structure of C. trachomatis hypothetical protein CT263 presented here supports a role in quinone biosynthesis. Although CT263 lacks sequence-based functional annotation, the crystal structure of CT263 displays striking structural similarity to 5'-methylthioadenosine nucleosidase (MTAN) enzymes. Although CT263 lacks the active site-associated dimer interface found in prototypical MTANs, co-crystal structures with product (adenine) or substrate (5'-methylthioadenosine) indicate that the canonical active site residues are conserved. Enzymatic characterization of CT263 indicates that the futalosine pathway intermediate 6-amino-6-deoxyfutalosine (kcat/Km = 1.8 10(3) M(-1) s(-1)), but not the prototypical MTAN substrates (e.g. S-adenosylhomocysteine and 5'-methylthioadenosine), is hydrolyzed. Bioinformatic analyses of the chlamydial proteome also support the futalosine pathway toward the synthesis of menaquinone in Chlamydiaceae. This report provides the first experimental support for quinone synthesis in Chlamydia. Menaquinone synthesis provides another target for agents to combat C. trachomatis infection. PMID:25253688

  10. Skin biopsy: Identifying and overcoming errors in the skin biopsy pathway.

    PubMed

    Stratman, Erik J; Elston, Dirk M; Miller, Stanley J

    2016-01-01

    The skin biopsy pathway involves numerous communication requirements, technical events, human handoffs, and cognitive decisions. Every step in the process has an error rate >0. To deliver the highest quality care, dermatologists obtaining skin biopsy specimens should implement systems in their office to minimize errors. This includes the prevention of wrong-site surgery, which in most instances involves accurate communication of the correct biopsy location to the performing surgeon. Part II of this continuing medical education article presents techniques for assessing and planning improvement to the skin biopsy pathway in your office, and provides a simple online quality improvement activity that allows Board-certified dermatologists the opportunity to potentially improve aspects of the skin biopsy process in their own practices, and in the process obtain Maintenance of Certification credit. PMID:26702795

  11. Global gene analysis identifying genes commonly regulated by the Ras/Raf/MEK and type I IFN pathways

    PubMed Central

    Komatsu, Y.; Hirasawa, K.; Christian, S.L.

    2015-01-01

    Oncolytic viruses exploit alterations in cancer cells to specifically infect cancer cells but not normal healthy cells. Previous work has shown that oncogenic Ras interferes with interferon (IFN) signaling to promote viral replication. Furthermore, inhibition of the Ras/Raf/MEK/ERK pathway at the level of Ras, MEK, or ERK was sufficient to restore IFN signaling. In order to identify genes that were commonly regulated by the inhibition of the Ras pathway and the IFN pathway, we treated NIH/3T3 cells that overexpress oncogenic Ras with the MEK inhibitor, U0126, or IFN-α for 6 h, and performed DNA microarray analysis (Gene Expression Omnibus accession number GSE49469). Here, we also provide additional information on the experimental and functional analysis of the genes responsive to U0126 and IFN. PMID:26484185

  12. Carrot-specific features of the phenylpropanoid pathway identified by feeding cultured cells with defined intermediates.

    PubMed

    Toffali, Ketti; Ceoldo, Stefania; Stocchero, Matteo; Levi, Marisa; Guzzo, Flavia

    2013-08-01

    Plants produce a vast array of secondary metabolites, many of which have important biological properties in animals when consumed as part of the diet. Interestingly, although the activities and benefits of plant secondary metabolites in animals are well established, comparatively little is known about the endogenous functions of these compounds in plants. One way to investigate the role of secondary products in plants is to modify the secondary metabolome and investigate the impact of such modifications on the phenotype. We have designed a novel feeding approach using different hydroxycinnamic acids (HCAs) and the cyanidin precursor dihydroquercetin (DHQ) to modify the metabolome of carrot R3M suspension cells. This strategy increased the accumulation of specific metabolites in a predictable way, and provided novel insights into the carrot phenylpropanoid pathway, suggesting that (a) cells use HCA hexose esters as substrates in the biosynthetic pathway leading to the accumulation of the various HCA derivatives and (b) p-coumaric acid derivative levels play a key roles in the regulation the flux of HCAs along the pathway. Moreover, this rapid strategy for metabolome modification does not depend on the availability of molecular tools or knowledge and can therefore be applied to any plant species. PMID:23759106

  13. Analysis of miRNA in Normal Appearing White Matter to Identify Altered CNS Pathways in Multiple Sclerosis

    PubMed Central

    Guerau-de-Arellano, Mireia; Liu, Yue; Meisen, Walter H; Pitt, David; Racke, Michael K; Lovett-Racke, Amy E

    2016-01-01

    Genetic studies suggest that the immune system is the greatest genetic contributor to multiple sclerosis (MS) susceptibility. Yet, these immune-related genes do not explain why inflammation is limited to the CNS in MS. We hypothesize that there is an underlying dysregulation in the CNS of MS patients that makes them more vulnerable to CNS inflammation. The sparsity of CNS-related genes associated with MS suggests that epigenetic changes in the CNS may play a role. Thus, a miRNA profiling study was performed in NAWM of MS patients and control subjects to determine if specific CNS pathways can be identified that may be altered due to miRNA-mediated post-transcriptional dysregulation. There were 15 differentially expressed miRNAs found in the MS patients’ NAWM. Pathway analysis indicated that the MAPK pathway and pathways associated with the blood-brain barrier were predicted to be significantly affected by these miRNAs. Using target predication and mRNA analysis, an inverse relationship was found between miR-191 and BDNF, SOX4, FZD5 and WSB1. The pathway and target analysis of the MS-associated miRNAs suggests that MS patients’ CNS is more prone to inflammation and less capable of repair, yet enriched in neuroprotective mechanisms. PMID:26894232

  14. Non-host disease resistance response in pea (Pisum sativum) pods: Biochemical function of DRR206 and phytoalexin pathway localization.

    PubMed

    Seneviratne, Herana Kamal; Dalisay, Doralyn S; Kim, Kye-Won; Moinuddin, Syed G A; Yang, Hong; Hartshorn, Christopher M; Davin, Laurence B; Lewis, Norman G

    2015-05-01

    Continually exposed to potential pathogens, vascular plants have evolved intricate defense mechanisms to recognize encroaching threats and defend themselves. They do so by inducing a set of defense responses that can help defeat and/or limit effects of invading pathogens, of which the non-host disease resistance response is the most common. In this regard, pea (Pisum sativum) pod tissue, when exposed to Fusarium solani f. sp. phaseoli spores, undergoes an inducible transcriptional activation of pathogenesis-related genes, and also produces (+)-pisatin, its major phytoalexin. One of the inducible pathogenesis-related genes is Disease Resistance Response-206 (DRR206), whose role in vivo was unknown. DRR206 is, however, related to the dirigent protein (DP) family. In this study, its biochemical function was investigated in planta, with the metabolite associated with its gene induction being pinoresinol monoglucoside. Interestingly, both pinoresinol monoglucoside and (+)-pisatin were co-localized in pea pod endocarp epidermal cells, as demonstrated using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. In addition, endocarp epidermal cells are also the site for both chalcone synthase and DRR206 gene expression. Taken together, these data indicate that both (+)-pisatin and pinoresinol monoglucoside function in the overall phytoalexin responses. PMID:25457488

  15. Solid-Phase Enrichment and Analysis of Azide-Labeled Natural Products: Fishing Downstream of Biochemical Pathways.

    PubMed

    Prez, Alexander J; Wesche, Frank; Adihou, Hlne; Bode, Helge B

    2016-01-11

    Many methods have been devised over the decades to trace precursors of specific molecules in cellular environments as, for example, in biosynthesis studies. The advent of click chemistry has facilitated the powerful combination of tracing and at the same time sieving the highly complex metabolome for compounds derived from simple or complex starting materials, especially when the click reaction takes place on a solid support. While the principle of solid-phase click reactions has already been successfully applied for selective protein and peptide enrichment, the successful enrichment of much smaller primary and secondary metabolites, showing great structural diversity and undergoing many different biosynthetic steps, has seen only little development. For bacterial secondary metabolism, a far broader tolerance for "clickable" precursors was observed than in ribosomal proteinogenesis, thus making this method a surprisingly valuable tool for the tracking and discovery of compounds within the cellular biochemical network. The implementation of this method has led to the identification of several new compounds from the bacterial genera Photorhabdus and Xenorhabdus, clearly proving its power. PMID:26626278

  16. Structural and Biochemical Characterization of Chlamydia trachomatis Hypothetical Protein CT263 Supports That Menaquinone Synthesis Occurs through the Futalosine Pathway*

    PubMed Central

    Barta, Michael L.; Thomas, Keisha; Yuan, Hongling; Lovell, Scott; Battaile, Kevin P.; Schramm, Vern L.; Hefty, P. Scott

    2014-01-01

    The obligate intracellular human pathogen Chlamydia trachomatis is the etiological agent of blinding trachoma and sexually transmitted disease. Genomic sequencing of Chlamydia indicated this medically important bacterium was not exclusively dependent on the host cell for energy. In order for the electron transport chain to function, electron shuttling between membrane-embedded complexes requires lipid-soluble quinones (e.g. menaquionone or ubiquinone). The sources or biosynthetic pathways required to obtain these electron carriers within C. trachomatis are poorly understood. The 1.58Å crystal structure of C. trachomatis hypothetical protein CT263 presented here supports a role in quinone biosynthesis. Although CT263 lacks sequence-based functional annotation, the crystal structure of CT263 displays striking structural similarity to 5′-methylthioadenosine nucleosidase (MTAN) enzymes. Although CT263 lacks the active site-associated dimer interface found in prototypical MTANs, co-crystal structures with product (adenine) or substrate (5′-methylthioadenosine) indicate that the canonical active site residues are conserved. Enzymatic characterization of CT263 indicates that the futalosine pathway intermediate 6-amino-6-deoxyfutalosine (kcat/Km = 1.8 × 103 m−1 s−1), but not the prototypical MTAN substrates (e.g. S-adenosylhomocysteine and 5′-methylthioadenosine), is hydrolyzed. Bioinformatic analyses of the chlamydial proteome also support the futalosine pathway toward the synthesis of menaquinone in Chlamydiaceae. This report provides the first experimental support for quinone synthesis in Chlamydia. Menaquinone synthesis provides another target for agents to combat C. trachomatis infection. PMID:25253688

  17. Computational Biophysical, Biochemical, and Evolutionary Signature of Human R-Spondin Family Proteins, the Member of Canonical Wnt/?-Catenin Signaling Pathway

    PubMed Central

    Sharma, Ashish Ranjan; Lee, Sang-Soo; Yoon, Jeong Kyo; George Priya Doss, C.; Song, Dong-Keun

    2014-01-01

    In human, Wnt/?-catenin signaling pathway plays a significant role in cell growth, cell development, and disease pathogenesis. Four human (Rspo)s are known to activate canonical Wnt/?-catenin signaling pathway. Presently, (Rspo)s serve as therapeutic target for several human diseases. Henceforth, basic understanding about the molecular properties of (Rspo)s is essential. We approached this issue by interpreting the biochemical and biophysical properties along with molecular evolution of (Rspo)s thorough computational algorithm methods. Our analysis shows that signal peptide length is roughly similar in (Rspo)s family along with similarity in aa distribution pattern. In Rspo3, four N-glycosylation sites were noted. All members are hydrophilic in nature and showed alike GRAVY values, approximately. Conversely, Rspo3 contains the maximum positively charged residues while Rspo4 includes the lowest. Four highly aligned blocks were recorded through Gblocks. Phylogenetic analysis shows Rspo4 is being rooted with Rspo2 and similarly Rspo3 and Rspo1 have the common point of origin. Through phylogenomics study, we developed a phylogenetic tree of sixty proteins (n = 60) with the orthologs and paralogs seed sequences. Protein-protein network was also illustrated. Results demonstrated in our study may help the future researchers to unfold significant physiological and therapeutic properties of (Rspo)s in various disease models. PMID:25276837

  18. Identification of Biochemical Pathways Associated with Lead Tolerance and Detoxification in Chrysopogon zizanioides L. Nash (Vetiver) by Metabolic Profiling.

    PubMed

    Pidatala, Venkataramana R; Li, Kefeng; Sarkar, Dibyendu; Ramakrishna, Wusirika; Datta, Rupali

    2016-03-01

    Lead (Pb) is a major urban pollutant, due to deteriorating lead-based paint in houses built before 1978. Phytoremediation is an inexpensive and effective technique for remediation of Pb-contaminated homes. Vetiver (Chrysopogon zizanioides), a noninvasive, fast-growing grass with high biomass, can tolerate and accumulate large quantities of Pb in its tissues. Lead is known to induce phytochelatins and antioxidative enzymes in vetiver; however, the overall impact of Pb stress on metabolic pathways of vetiver is unknown. In the current study, vetiver plants were treated with different concentrations of Pb in a hydroponic setup. Metabolites were extracted and analyzed using LC/MS/MS. Multivariate analysis of metabolites in both root and shoot tissue showed tremendous induction in key metabolic pathways including sugar metabolism, amino acid metabolism, and an increase in production of osmoprotectants, such as betaine and polyols, and metal-chelating organic acids. The data obtained provide a comprehensive insight into the overall stress response mechanisms in vetiver. PMID:26843403

  19. Oxygen and hydrogen peroxide in the early evolution of life on earth: in silico comparative analysis of biochemical pathways.

    PubMed

    Slesak, Ireneusz; Slesak, Halina; Kruk, Jerzy

    2012-08-01

    In the Universe, oxygen is the third most widespread element, while on Earth it is the most abundant one. Moreover, oxygen is a major constituent of all biopolymers fundamental to living organisms. Besides O(2), reactive oxygen species (ROS), among them hydrogen peroxide (H(2)O(2)), are also important reactants in the present aerobic metabolism. According to a widely accepted hypothesis, aerobic metabolism and many other reactions/pathways involving O(2) appeared after the evolution of oxygenic photosynthesis. In this study, the hypothesis was formulated that the Last Universal Common Ancestor (LUCA) was at least able to tolerate O(2) and detoxify ROS in a primordial environment. A comparative analysis was carried out of a number of the O(2)-and H(2)O(2)-involving metabolic reactions that occur in strict anaerobes, facultative anaerobes, and aerobes. The results indicate that the most likely LUCA possessed O(2)-and H(2)O(2)-involving pathways, mainly reactions to remove ROS, and had, at least in part, the components of aerobic respiration. Based on this, the presence of a low, but significant, quantity of H(2)O(2) and O(2) should be taken into account in theoretical models of the early Archean atmosphere and oceans and the evolution of life. It is suggested that the early metabolism involving O(2)/H(2)O(2) was a key adaptation of LUCA to already existing weakly oxic zones in Earth's primordial environment. PMID:22970865

  20. Predicting and analyzing DNA-binding domains using a systematic approach to identifying a set of informative physicochemical and biochemical properties

    PubMed Central

    2011-01-01

    Background Existing methods of predicting DNA-binding proteins used valuable features of physicochemical properties to design support vector machine (SVM) based classifiers. Generally, selection of physicochemical properties and determination of their corresponding feature vectors rely mainly on known properties of binding mechanism and experience of designers. However, there exists a troublesome problem for designers that some different physicochemical properties have similar vectors of representing 20 amino acids and some closely related physicochemical properties have dissimilar vectors. Results This study proposes a systematic approach (named Auto-IDPCPs) to automatically identify a set of physicochemical and biochemical properties in the AAindex database to design SVM-based classifiers for predicting and analyzing DNA-binding domains/proteins. Auto-IDPCPs consists of 1) clustering 531 amino acid indices in AAindex into 20 clusters using a fuzzy c-means algorithm, 2) utilizing an efficient genetic algorithm based optimization method IBCGA to select an informative feature set of size m to represent sequences, and 3) analyzing the selected features to identify related physicochemical properties which may affect the binding mechanism of DNA-binding domains/proteins. The proposed Auto-IDPCPs identified m=22 features of properties belonging to five clusters for predicting DNA-binding domains with a five-fold cross-validation accuracy of 87.12%, which is promising compared with the accuracy of 86.62% of the existing method PSSM-400. For predicting DNA-binding sequences, the accuracy of 75.50% was obtained using m=28 features, where PSSM-400 has an accuracy of 74.22%. Auto-IDPCPs and PSSM-400 have accuracies of 80.73% and 82.81%, respectively, applied to an independent test data set of DNA-binding domains. Some typical physicochemical properties discovered are hydrophobicity, secondary structure, charge, solvent accessibility, polarity, flexibility, normalized Van Der Waals volume, pK (pK-C, pK-N, pK-COOH and pK-a(RCOOH)), etc. Conclusions The proposed approach Auto-IDPCPs would help designers to investigate informative physicochemical and biochemical properties by considering both prediction accuracy and analysis of binding mechanism simultaneously. The approach Auto-IDPCPs can be also applicable to predict and analyze other protein functions from sequences. PMID:21342579

  1. Oxygen and Hydrogen Peroxide in the Early Evolution of Life on Earth: In silico Comparative Analysis of Biochemical Pathways

    PubMed Central

    ?lesak, Halina; Kruk, Jerzy

    2012-01-01

    Abstract In the Universe, oxygen is the third most widespread element, while on Earth it is the most abundant one. Moreover, oxygen is a major constituent of all biopolymers fundamental to living organisms. Besides O2, reactive oxygen species (ROS), among them hydrogen peroxide (H2O2), are also important reactants in the present aerobic metabolism. According to a widely accepted hypothesis, aerobic metabolism and many other reactions/pathways involving O2 appeared after the evolution of oxygenic photosynthesis. In this study, the hypothesis was formulated that the Last Universal Common Ancestor (LUCA) was at least able to tolerate O2 and detoxify ROS in a primordial environment. A comparative analysis was carried out of a number of the O2-and H2O2-involving metabolic reactions that occur in strict anaerobes, facultative anaerobes, and aerobes. The results indicate that the most likely LUCA possessed O2-and H2O2-involving pathways, mainly reactions to remove ROS, and had, at least in part, the components of aerobic respiration. Based on this, the presence of a low, but significant, quantity of H2O2 and O2 should be taken into account in theoretical models of the early Archean atmosphere and oceans and the evolution of life. It is suggested that the early metabolism involving O2/H2O2 was a key adaptation of LUCA to already existing weakly oxic zones in Earth's primordial environment. Key Words: Hydrogen peroxideOxygenOrigin of lifePhotosynthesisSuperoxide dismutaseSuperoxide reductase. Astrobiology 12, 775784. PMID:22970865

  2. Probing the coagulation pathway with aptamers identifies combinations that synergistically inhibit blood clot formation

    PubMed Central

    Bompiani, Kristin M; Lohrmann, Jens L; Pitoc, George A; Frederiksen, James W; Mackensen, George B; Sullenger, Bruce A

    2014-01-01

    SUMMARY Coordinated enzymatic reactions regulate blood clot generation. To explore the contributions of various coagulation enzymes in this process, we utilized a panel of aptamers against factors VIIa, IXa, Xa, and prothrombin. Each aptamer dose-dependently inhibited clot formation, yet none was able to completely impede this process in highly procoagulant settings. However several combinations of two aptamers synergistically impaired clot formation. One extremely potent aptamer combination was able to maintain human blood fluidity even during extracorporeal circulation, a highly procoagulant setting encountered during cardiopulmonary bypass surgery. Moreover, this aptamer cocktail could be rapidly reversed with antidotes to restore normal hemostasis, indicating that even highly potent aptamer combinations can be rapidly controlled. These studies highlight the potential utility of using sets of aptamers to probe the functions of proteins in molecular pathways for research and therapeutic ends. PMID:25065530

  3. High-Throughput Screen in Cryptococcus neoformans Identifies a Novel Molecular Scaffold That Inhibits Cell Wall Integrity Pathway Signaling

    PubMed Central

    2015-01-01

    Cryptococcus neoformans is one of the most important human fungal pathogens; however, no new therapies have been developed in over 50 years. Fungicidal activity is crucially important for an effective anticryptococal agent and, therefore, we screened 361,675 molecules against C. neoformans using an adenylate kinase release assay that specifically detects fungicidal activity. A set of secondary assays narrowed the set of hits to molecules that interfere with fungal cell wall integrity and identified three benzothioureas with low in vitro mammalian toxicity and good in vitro anticryptococcal (minimum inhibitory concentration = 4 μg/mL). This scaffold inhibits signaling through the cell wall integrity MAP kinase cascade. Structure–activity studies indicate that the thiocarbonyl moiety is crucial for activity. Genetic and biochemical data suggest that benzothioureas inhibit signaling upstream of the kinase cascade. Thus, the benzothioureas appear to be a promising new scaffold for further exploration in the search for new anticryptococcal agents. PMID:26807437

  4. A Genomewide Overexpression Screen Identifies Genes Involved in the Phosphatidylinositol 3-Kinase Pathway in the Human Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Koushik, Amrita B.; Welter, Brenda H.; Rock, Michelle L.

    2014-01-01

    Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen. PMID:24442890

  5. Whole-exome sequencing in obsessive-compulsive disorder identifies rare mutations in immunological and neurodevelopmental pathways.

    PubMed

    Cappi, C; Brentani, H; Lima, L; Sanders, S J; Zai, G; Diniz, B J; Reis, V N S; Hounie, A G; Conceição do Rosário, M; Mariani, D; Requena, G L; Puga, R; Souza-Duran, F L; Shavitt, R G; Pauls, D L; Miguel, E C; Fernandez, T V

    2016-01-01

    Studies of rare genetic variation have identified molecular pathways conferring risk for developmental neuropsychiatric disorders. To date, no published whole-exome sequencing studies have been reported in obsessive-compulsive disorder (OCD). We sequenced all the genome coding regions in 20 sporadic OCD cases and their unaffected parents to identify rare de novo (DN) single-nucleotide variants (SNVs). The primary aim of this pilot study was to determine whether DN variation contributes to OCD risk. To this aim, we evaluated whether there is an elevated rate of DN mutations in OCD, which would justify this approach toward gene discovery in larger studies of the disorder. Furthermore, to explore functional molecular correlations among genes with nonsynonymous DN SNVs in OCD probands, a protein-protein interaction (PPI) network was generated based on databases of direct molecular interactions. We applied Degree-Aware Disease Gene Prioritization (DADA) to rank the PPI network genes based on their relatedness to a set of OCD candidate genes from two OCD genome-wide association studies (Stewart et al., 2013; Mattheisen et al., 2014). In addition, we performed a pathway analysis with genes from the PPI network. The rate of DN SNVs in OCD was 2.51 × 10(-8) per base per generation, significantly higher than a previous estimated rate in unaffected subjects using the same sequencing platform and analytic pipeline. Several genes harboring DN SNVs in OCD were highly interconnected in the PPI network and ranked high in the DADA analysis. Nearly all the DN SNVs in this study are in genes expressed in the human brain, and a pathway analysis revealed enrichment in immunological and central nervous system functioning and development. The results of this pilot study indicate that further investigation of DN variation in larger OCD cohorts is warranted to identify specific risk genes and to confirm our preliminary finding with regard to PPI network enrichment for particular biological pathways and functions. PMID:27023170

  6. The Prognostic Value of the Apoptosis Pathway in Colorectal Cancer: A Review of the Literature on Biomarkers Identified by Immunohistochemistry

    PubMed Central

    Zeestraten, Eliane C.M.; Benard, Anne; Reimers, Marlies S.; Schouten, Philip C.; Liefers, Gerrit J.; van de Velde, Cornelis J.H.; Kuppen, Peter J.K.

    2013-01-01

    Research towards biomarkers that predict patient outcome in colorectal cancer (CRC) is rapidly expanding. However, none of these biomarkers have been recommended by the American Association of Clinical Oncology or the European Group on Tumor Markers. Current staging criteria result in substantial under-and over-treatment of CRC patients. Evasion of apoptosis, a characteristic feature of tumorigenesis, is known to correlate with patient outcome. We reviewed the literature on immunohistochemistry-based studies between 1998 and 2011 describing biomarkers in this pathway in CRC and identified 26 markers. Most frequently described were p53, Bcl-2, survivin, and the Fas and TRAILR1 receptors and their ligands. None of the studies reviewed provided sufficient support for implementing a single marker into current clinical practice. This is likely due to the complex biology of this pathway. We suggest focusing on the combination of key markers within the apoptosis pathway that together represent an ‘apoptotic tumor profile’, which better reflects the status of this pathway in a tumor. PMID:24179395

  7. Major carcinogenic pathways identified by gene expression analysis of peritoneal mesotheliomas following chemical treatment in F344 rats

    SciTech Connect

    Kim, Yongbaek; Thai-Vu Ton; De Angelo, Anthony B.; Morgan, Kevin; Devereux, Theodora R.; Anna, Colleen; Collins, Jennifer B.; Paules, Richard S.; Crosby, Lynn M.; Sills, Robert C. . E-mail: sills@niehs.nih.gov

    2006-07-15

    This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo arrays, with over 20,000 target genes, were used to evaluate o-NT- and BCA-induced RPMs, when compared to a non-transformed mesothelial cell line (Fred-PE). Analysis using Ingenuity Pathway Analysis software revealed 169 cancer-related genes that were categorized into binding activity, growth and proliferation, cell cycle progression, apoptosis, and invasion and metastasis. The microarray data were validated by positive correlation with quantitative real-time RT-PCR on 16 selected genes including igf1, tgfb3 and nov. Important carcinogenic pathways involved in RPM formation included insulin-like growth factor 1 (IGF-1), p38 MAPkinase, Wnt/{beta}-catenin and integrin signaling pathways. This study demonstrated that mesotheliomas in rats exposed to o-NT- and BCA were similar to mesotheliomas in humans, at least at the cellular and molecular level.

  8. Proteomic analysis identifies alterations in cellular morphology and cell death pathways in mouse brain after chronic corticosterone treatment.

    PubMed

    Skynner, Heather A; Amos, Doran P; Murray, Fraser; Salim, Kamran; Knowles, Michael R; Munoz-Sanjuan, Ignacio; Camargo, Luis M; Bonnert, Timothy P; Guest, Paul C

    2006-08-01

    Some patients with Major Depression and other neurological afflictions display hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. HPA hyperactivity may be due to impaired feedback inhibition and manifested as increased levels of circulating cortisol. Subcutaneous implants of corticosterone pellets were used to mimic this situation in mice to gain insight into any effects on brain function by comparative proteomic analysis using two-dimensional Differential In-Gel Electrophoresis. A total of 150 different protein spots were altered by corticosterone treatment in the hypothalamus, hippocampus and cerebral cortex. Of these, 117 spots were identified by matrix-assisted laser desorption/ionization-time of flight mass fingerprinting equating to 51 different proteins. Association of these corticosterone-modulated proteins with biological functions using the Ingenuity Pathways Analysis tool showed that cell morphology was significantly altered in the hippocampus and cerebral cortex, whereas the hypothalamus showed significant changes in cell death. Ingenuity Pathways Analysis of the canonical signaling pathways showed that glycolysis and gluconeogenesis were altered in the hypothalamus and the hippocampus and all three brain regions showed changes in phenylalanine, glutamate and nitrogen metabolism. Further elucidation of these pathways could lead to identification of biomarkers for the development of pharmacological therapies targeted at neuropsychiatric disorders. PMID:16797492

  9. Transport of dissolved organic carbon from soil to surface water: Identifying the transport pathways

    NASA Astrophysics Data System (ADS)

    Van Gaelen, Nele

    2013-04-01

    Over the last decades, increasing concentrations of dissolved organic carbon (DOC) have been found in surface waters. It has also become clear that land use is an important driver for DOC export. However, causal factors controlling this temporal and spatial variation are not clear. Efforts to model DOC export on a catchment scale are rare. In this research, we aim to determine the factors controlling variations in DOC concentration and quality in surface waters. Secondly, the importance of the different pathways (surface runoff, subsurface flow and groundwater flow) for the transport of dissolved organic matter from the soil to the surface water is investigated. Six headwater catchments (100 - 400 ha) were selected in Belgium, representing three different types of land use, namely forest, grassland and arable land. At the outlet of each catchment, a flow-proportional sampler has been collecting samples of base flow and peak discharge since January 2010. In addition, samples of groundwater, subsurface water and precipitation water were collected on a regular base in three of the catchments. Samples were analyzed for DOC, specific UV absorbance (SUVA) and dissolved silica (DSi). Elemental analysis was carried out using ICP-OES. Since 2012, precipitation water and a selection of river water samples was also analyzed for O and H isotopes. Overall, DOC concentrations were highest in forest catchments and lowest in grassland catchments. For all land use types, measured DOC concentrations were highest during peak discharge. The rise in DOC concentrations was associated with a change in DOC quality. During periods of greater discharge, higher SUVA values were measured, indicating DOC with higher aromaticity (humic and fulvic fractions) reaches the outlet. ICP and DSi results also showed a significant difference in geochemical composition of the river water if peak events are compared to base flow samples. During an event, Ca, Mg, Na, S and DSi concentrations were lowered, while K concentrations rose. Isotope analysis showed more heavy O an H isotopes during peak events than during baseflow. Results of the river water were combined with analysis of possible end-members in the catchments, using the groundwater, soil water and precipitation samples. An end-member-mixing-analysis (EMMA) gained more insight into the contributing pathways for the transport of organic matter from the soil to the surface water during base and peak flow. Furthermore, results from the different catchments were compared, and allowed to relate DOC transport to land use type. This is an important step towards a model describing DOC transport at the catchment scale.

  10. Adolescents Can Know Best: Using concept mapping to identify factors and pathways driving adolescent sexuality in Lima, Peru

    PubMed Central

    Bayer, Angela M.; Cabrera, Lilia Z.; Gilman, Robert H.; Hindin, Michelle J.; Tsui, Amy O.

    2011-01-01

    The primary objective of this study was to identify and describe individual- and environmental-level factors that Peruvian adolescents perceive to be related to adolescent sexuality. A series of concept mapping sessions were carried out from January-March 2006 with 63 15–17 year olds from a low-income community near Lima in order for adolescents to (1) brainstorm items that they thought were related to sexuality (2) sort, group and rate items to score their importance for sexuality-related outcomes, and (3) create pathways from the groups of items to engaging in sex. Brainstorming resulted in 61 items, which participants grouped into 11 clusters. The highest rated clusters were personal values, respect and confidence in relationships, future achievements and parent-child communication. The pathway of decision-making about having sex primarily contained items rated as only moderately important. This study identified important understudied factors, new perspectives on previously-recognized factors, and possible pathways to sexual behavior. These interesting, provocative findings underscore the importance of directly integrating adolescent voices into future sexual and reproductive health research, policies and programs that target this population. PMID:20382462

  11. Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways

    PubMed Central

    Rardin, Matthew J.; Newman, John C.; Held, Jason M.; Cusack, Michael P.; Sorensen, Dylan J.; Li, Biao; Schilling, Birgit; Mooney, Sean D.; Kahn, C. Ronald; Verdin, Eric; Gibson, Bradford W.

    2013-01-01

    Large-scale proteomic approaches have identified numerous mitochondrial acetylated proteins; however in most cases, their regulation by acetyltransferases and deacetylases remains unclear. Sirtuin 3 (SIRT3) is an NAD+-dependent mitochondrial protein deacetylase that has been shown to regulate a limited number of enzymes in key metabolic pathways. Here, we use a rigorous label-free quantitative MS approach (called MS1 Filtering) to analyze changes in lysine acetylation from mouse liver mitochondria in the absence of SIRT3. Among 483 proteins, a total of 2,187 unique sites of lysine acetylation were identified after affinity enrichment. MS1 Filtering revealed that lysine acetylation of 283 sites in 136 proteins was significantly increased in the absence of SIRT3 (at least twofold). A subset of these sites was independently validated using selected reaction monitoring MS. These data show that SIRT3 regulates acetylation on multiple proteins, often at multiple sites, across several metabolic pathways including fatty acid oxidation, ketogenesis, amino acid catabolism, and the urea and tricarboxylic acid cycles, as well as mitochondrial regulatory proteins. The widespread modification of key metabolic pathways greatly expands the number of known substrates and sites that are targeted by SIRT3 and establishes SIRT3 as a global regulator of mitochondrial protein acetylation with the capability of coordinating cellular responses to nutrient status and energy homeostasis. PMID:23576753

  12. Delays in the stroke thrombolysis pathway--identifying areas for improvement.

    PubMed

    Brewer, L; Arize, C; McCormack, J; Williams, D

    2014-05-01

    Despite international consensus on the benefits of thrombolysis for ischaemic stroke (IS), it remains underused. Guidelines now recommend a door-to-needle time of 60 minutes. We reviewed the rate and timeliness of thrombolysis for IS at our hospital. 323 stroke patients presented between January 2011 and April 2012.Thirty patients (10.6% of IS) were thrombolysed, mean age was 68.5 years (42 to 88) and 19 patients (63%) were male. Thirty-six patients (12.7% of IS) were not thrombolysed despite arriving within the time-window and symptom resolution was the commonest reason (15 patients; 42%). Despite most thrombolysed patients (42%) presenting to the Emergency Department during daytime working hours, there were delays at each step of the acute care pathway. The mean time for stroke team review was 23 minutes (5-50). The mean door-to-CT and the door-to-needle times were 60 minutes (25-95) and 92 minutes (46-130) respectively. In parallel with national stroke incentives, local audit can highlight barriers to uptake and efficiency within thrombolysis services. PMID:24908858

  13. Cohesion Group Approach for Evolutionary Analysis of Aspartokinase, an Enzyme That Feeds a Branched Network of Many Biochemical Pathways

    PubMed Central

    Lo, Chien-Chi; Bonner, Carol A.; Xie, Gary; D'Souza, Mark; Jensen, Roy A.

    2009-01-01

    Summary: Aspartokinase (Ask) exists within a variable network that supports the synthesis of 9 amino acids and a number of other important metabolites. Lysine, isoleucine, aromatic amino acids, and dipicolinate may arise from the ASK network or from alternative pathways. Ask proteins were subjected to cohesion group analysis, a methodology that sorts a given protein assemblage into groups in which evolutionary continuity is assured. Two subhomology divisions, ASKα and ASKβ, have been recognized. The ASKα subhomology division is the most ancient, being widely distributed throughout the Archaea and Eukarya and in some Bacteria. Within an indel region of about 75 amino acids near the N terminus, ASKβ sequences differ from ASKα sequences by the possession of a proposed ancient deletion. ASKβ sequences are present in most Bacteria and usually exhibit an in-frame internal translational start site that can generate a small Ask subunit that is identical to the C-terminal portion of the larger subunit of a heterodimeric unit. Particularly novel are ask genes embedded in gene contexts that imply specialization for ectoine (osmotic agent) or aromatic amino acids. The cohesion group approach is well suited for the easy recognition of relatively recent lateral gene transfer (LGT) events, and many examples of these are described. Given the current density of genome representation for Proteobacteria, it is possible to reconstruct more ancient landmark LGT events. Thus, a plausible scenario in which the three well-studied and iconic Ask homologs of Escherichia coli are not within the vertical genealogy of Gammaproteobacteria, but rather originated via LGT from a Bacteroidetes donor, is supported. PMID:19946135

  14. Identifying Vulnerable Pathways in Mycobacterium tuberculosis by Using a Knockdown Approach ?

    PubMed Central

    Carroll, Paul; Faray-Kele, Marie-Claire; Parish, Tanya

    2011-01-01

    We constructed recombinant strains of Mycobacterium tuberculosis in which expression of specific genes was downregulated to identify vulnerable drug targets. Growth phenotypes in macrophages and culture were used to rank targets: the dprE1, clpP1, and fadD32 operons were the best targets and glnA1, glnE, pknL, regX3, and senX3 were poor targets. PMID:21642404

  15. Identifying vulnerable pathways in Mycobacterium tuberculosis by using a knockdown approach.

    PubMed

    Carroll, Paul; Faray-Kele, Marie-Claire; Parish, Tanya

    2011-07-01

    We constructed recombinant strains of Mycobacterium tuberculosis in which expression of specific genes was downregulated to identify vulnerable drug targets. Growth phenotypes in macrophages and culture were used to rank targets: the dprE1, clpP1, and fadD32 operons were the best targets and glnA1, glnE, pknL, regX3, and senX3 were poor targets. PMID:21642404

  16. Cell-Based Assay To Identify Inhibitors of the Hfq-sRNA Regulatory Pathway

    PubMed Central

    El-Mowafi, Shaima A.; Alumasa, John N.; Ades, Sarah E.

    2014-01-01

    Noncoding small RNAs (sRNAs) act in conjunction with the RNA chaperone Hfq to regulate gene expression in bacteria. Because Hfq is required for virulence in several bacterial pathogens, the Hfq-sRNA system is an attractive target for antibiotic development. A reporter strain in which the expression of yellow fluorescent protein (YFP) is controlled by Hfq-sRNA was engineered to identify inhibitors of this system. A reporter that is targeted by Hfq in conjunction with the RybB sRNA was used in a genetic screen to identify inhibitors from a library of cyclic peptides produced in Escherichia coli using split-intein circular ligation of peptides and proteins (SICLOPPS), an intein-based technology. One cyclic peptide identified in this screen, RI20, inhibited Hfq-mediated repression of gene expression in conjunction with both RybB and an unrelated sRNA, MicF. Gel mobility shift assays showed that RI20 inhibited binding of Hfq to RybB and MicF with similar Ki values. These data suggest that RI20 inhibits Hfq activity by blocking interactions with sRNAs and provide a paradigm for inhibiting virulence genes in Gram-negative pathogens. PMID:25001303

  17. Integrated genome-wide association, coexpression network, and expression single nucleotide polymorphism analysis identifies novel pathway in allergic rhinitis

    PubMed Central

    2014-01-01

    Background Allergic rhinitis is a common disease whose genetic basis is incompletely explained. We report an integrated genomic analysis of allergic rhinitis. Methods We performed genome wide association studies (GWAS) of allergic rhinitis in 5633 ethnically diverse North American subjects. Next, we profiled gene expression in disease-relevant tissue (peripheral blood CD4+ lymphocytes) collected from subjects who had been genotyped. We then integrated the GWAS and gene expression data using expression single nucleotide (eSNP), coexpression network, and pathway approaches to identify the biologic relevance of our GWAS. Results GWAS revealed ethnicity-specific findings, with 4 genome-wide significant loci among Latinos and 1 genome-wide significant locus in the GWAS meta-analysis across ethnic groups. To identify biologic context for these results, we constructed a coexpression network to define modules of genes with similar patterns of CD4+ gene expression (coexpression modules) that could serve as constructs of broader gene expression. 6 of the 22 GWAS loci with P-value ≤ 1x10−6 tagged one particular coexpression module (4.0-fold enrichment, P-value 0.0029), and this module also had the greatest enrichment (3.4-fold enrichment, P-value 2.6 × 10−24) for allergic rhinitis-associated eSNPs (genetic variants associated with both gene expression and allergic rhinitis). The integrated GWAS, coexpression network, and eSNP results therefore supported this coexpression module as an allergic rhinitis module. Pathway analysis revealed that the module was enriched for mitochondrial pathways (8.6-fold enrichment, P-value 4.5 × 10−72). Conclusions Our results highlight mitochondrial pathways as a target for further investigation of allergic rhinitis mechanism and treatment. Our integrated approach can be applied to provide biologic context for GWAS of other diseases. PMID:25085501

  18. A metabolomics approach using juvenile cystic mice to identify urinary biomarkers and altered pathways in polycystic kidney disease

    PubMed Central

    Taylor, Sandra L.; Ganti, Sheila; Bukanov, Nikolay O.; Chapman, Arlene; Fiehn, Oliver; Osier, Michael; Kim, Kyoungmi

    2010-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and affects 1 in 1,000 individuals. Ultrasound is most often used to diagnose ADPKD; such a modality is only useful late in the disease after macroscopic cysts are present. There is accumulating evidence suggesting that there are common cellular and molecular mechanisms responsible for cystogenesis in human and murine PKD regardless of the genes mutated, and, in the case of complex metabolomic analysis, the use of a mouse model has distinct advantages for proof of principle over a human study. Therefore, in this study we utilized a urinary metabolomics-based investigation using gas chromatography-time of flight mass spectrometry to demonstrate that the cystic mouse can be discriminated from its wild-type counterpart by urine analysis alone. At day 26 of life, before there is serological evidence of kidney dysfunction, affected mice are distinguishable by urine metabolomic analysis; this finding persists through 45 days until 64 days, at which time body weight differences confound the results. Using functional score analysis and the KEGG pathway database, we identify several biologically relevant metabolic pathways which are altered very early in this disease, the most highly represented being the purine and galactose metabolism pathways. In addition, we identify several specific candidate biomarkers, including allantoic acid and adenosine, which are augmented in the urine of young cystic mice. These markers and pathway components, once extended to human disease, may prove useful as a noninvasive means of diagnosing cystic kidney diseases and to suggest novel therapeutic approaches. Thus, urine metabolomics has great diagnostic potential for cystic renal disorders and deserves further study. PMID:20130118

  19. How metal films de-wet substratesidentifying the kinetic pathways and energetic driving forces

    NASA Astrophysics Data System (ADS)

    McCarty, Kevin F.; Hamilton, John C.; Sato, Yu; Sa, Angela; Stumpf, Roland; de la Figuera, Juan; Thrmer, Konrad; Jones, Frank; Schmid, Andreas K.; Talin, A. Alec; Bartelt, Norman C.

    2009-04-01

    We study how single-crystal chromium films of uniform thickness on W(110) substrates are converted to arrays of three-dimensional (3D) Cr islands during annealing. We use low-energy electron microscopy (LEEM) to directly observe a kinetic pathway that produces trenches that expose the wetting layer. Adjacent film steps move simultaneously uphill and downhill relative to the staircase of atomic steps on the substrate. This step motion thickens the film regions where steps advance. Where film steps retract, the film thins, eventually exposing the stable wetting layer. Since our analysis shows that thick Cr films have a lattice constant close to bulk Cr, we propose that surface and interface stress provide a possible driving force for the observed morphological instability. Atomistic simulations and analytic elastic models show that surface and interface stress can cause a dependence of film energy on thickness that leads to an instability to simultaneous thinning and thickening. We observe that de-wetting is also initiated at bunches of substrate steps in two other systems, Ag/W(110) and Ag/Ru(0001). We additionally describe how Cr films are converted into patterns of unidirectional stripes as the trenches that expose the wetting layer lengthen along the W[001] direction. Finally, we observe how 3D Cr islands form directly during film growth at elevated temperature. The Cr mesas (wedges) form as Cr film steps advance down the staircase of substrate steps, another example of the critical role that substrate steps play in 3D island formation.

  20. Temporal Expression Profiling Identifies Pathways Mediating Effect of Causal Variant on Phenotype

    PubMed Central

    Gupta, Saumya; Radhakrishnan, Aparna; Raharja-Liu, Pandu; Lin, Gen; Steinmetz, Lars M.; Gagneur, Julien; Sinha, Himanshu

    2015-01-01

    Even with identification of multiple causal genetic variants for common human diseases, understanding the molecular processes mediating the causal variants effect on the disease remains a challenge. This understanding is crucial for the development of therapeutic strategies to prevent and treat disease. While static profiling of gene expression is primarily used to get insights into the biological bases of diseases, it makes differentiating the causative from the correlative effects difficult, as the dynamics of the underlying biological processes are not monitored. Using yeast as a model, we studied genome-wide gene expression dynamics in the presence of a causal variant as the sole genetic determinant, and performed allele-specific functional validation to delineate the causal effects of the genetic variant on the phenotype. Here, we characterized the precise genetic effects of a functional MKT1 allelic variant in sporulation efficiency variation. A mathematical model describing meiotic landmark events and conditional activation of MKT1 expression during sporulation specified an early meiotic role of this variant. By analyzing the early meiotic genome-wide transcriptional response, we demonstrate an MKT1-dependent role of novel modulators, namely, RTG1/3, regulators of mitochondrial retrograde signaling, and DAL82, regulator of nitrogen starvation, in additively effecting sporulation efficiency. In the presence of functional MKT1 allele, better respiration during early sporulation was observed, which was dependent on the mitochondrial retrograde regulator, RTG3. Furthermore, our approach showed that MKT1 contributes to sporulation independent of Puf3, an RNA-binding protein that steady-state transcription profiling studies have suggested to mediate MKT1-pleiotropic effects during mitotic growth. These results uncover interesting regulatory links between meiosis and mitochondrial retrograde signaling. In this study, we highlight the advantage of analyzing allele-specific transcriptional dynamics of mediating genes. Applications in higher eukaryotes can be valuable for inferring causal molecular pathways underlying complex dynamic processes, such as development, physiology and disease progression. PMID:26039065

  1. Temporal expression profiling identifies pathways mediating effect of causal variant on phenotype.

    PubMed

    Gupta, Saumya; Radhakrishnan, Aparna; Raharja-Liu, Pandu; Lin, Gen; Steinmetz, Lars M; Gagneur, Julien; Sinha, Himanshu

    2015-06-01

    Even with identification of multiple causal genetic variants for common human diseases, understanding the molecular processes mediating the causal variants' effect on the disease remains a challenge. This understanding is crucial for the development of therapeutic strategies to prevent and treat disease. While static profiling of gene expression is primarily used to get insights into the biological bases of diseases, it makes differentiating the causative from the correlative effects difficult, as the dynamics of the underlying biological processes are not monitored. Using yeast as a model, we studied genome-wide gene expression dynamics in the presence of a causal variant as the sole genetic determinant, and performed allele-specific functional validation to delineate the causal effects of the genetic variant on the phenotype. Here, we characterized the precise genetic effects of a functional MKT1 allelic variant in sporulation efficiency variation. A mathematical model describing meiotic landmark events and conditional activation of MKT1 expression during sporulation specified an early meiotic role of this variant. By analyzing the early meiotic genome-wide transcriptional response, we demonstrate an MKT1-dependent role of novel modulators, namely, RTG1/3, regulators of mitochondrial retrograde signaling, and DAL82, regulator of nitrogen starvation, in additively effecting sporulation efficiency. In the presence of functional MKT1 allele, better respiration during early sporulation was observed, which was dependent on the mitochondrial retrograde regulator, RTG3. Furthermore, our approach showed that MKT1 contributes to sporulation independent of Puf3, an RNA-binding protein that steady-state transcription profiling studies have suggested to mediate MKT1-pleiotropic effects during mitotic growth. These results uncover interesting regulatory links between meiosis and mitochondrial retrograde signaling. In this study, we highlight the advantage of analyzing allele-specific transcriptional dynamics of mediating genes. Applications in higher eukaryotes can be valuable for inferring causal molecular pathways underlying complex dynamic processes, such as development, physiology and disease progression. PMID:26039065

  2. Meta-analyses identify 13 novel loci associated with age at menopause and highlights DNA repair and immune pathways

    PubMed Central

    Stolk, Lisette; Perry, John RB; Chasman, Daniel I; He, Chunyan; Mangino, Massimo; Sulem, Patrick; Barbalic, Maja; Broer, Linda; Byrne, Enda M; Ernst, Florian; Esko, Tnu; Franceschini, Nora; Gudbjartsson, Daniel F; Hottenga, Jouke-Jan; Kraft, Peter; McArdle, Patick F; Porcu, Eleonora; Shin, So-Youn; Smith, Albert V; van Wingerden, Sophie; Zhai, Guangju; Zhuang, Wei V; Albrecht, Eva; Alizadeh, Behrooz Z; Aspelund, Thor; Bandinelli, Stefania; Lauc, Lovorka Barac; Beckmann, Jacques S; Boban, Mladen; Boerwinkle, Eric; Broekmans, Frank J; Burri, Andrea; Campbell, Harry; Chanock, Stephen J; Chen, Constance; Cornelis, Marilyn C; Corre, Tanguy; Coviello, Andrea D; dAdamo, Pio; Davies, Gail; de Faire, Ulf; de Geus, Eco JC; Deary, Ian J; Dedoussis, George VZ; Deloukas, Panagiotis; Ebrahim, Shah; Eiriksdottir, Gudny; Emilsson, Valur; Eriksson, Johan G; Fauser, Bart CJM; Ferreli, Liana; Ferrucci, Luigi; Fischer, Krista; Folsom, Aaron R; Garcia, Melissa E; Gasparini, Paolo; Gieger, Christian; Glazer, Nicole; Grobbee, Diederick E; Hall, Per; Haller, Toomas; Hankinson, Susan E; Hass, Merli; Hayward, Caroline; Heath, Andrew C; Hofman, Albert; Ingelsson, Erik; Janssens, A Cecile JW; Johnson, Andrew D; Karasik, David; Kardia, Sharon LR; Keyzer, Jules; Kiel, Douglas P; Kolcic, Ivana; Kutalik, Zoltn; Lahti, Jari; Lai, Sandra; Laisk, Triin; Laven, Joop SE; Lawlor, Debbie A; Liu, Jianjun; Lopez, Lorna M; Louwers, Yvonne V; Magnusson, Patrik KE; Marongiu, Mara; Martin, Nicholas G; Klaric, Irena Martinovic; Masciullo, Corrado; McKnight, Barbara; Medland, Sarah E; Melzer, David; Mooser, Vincent; Navarro, Pau; Newman, Anne B; Nyholt, Dale R; Onland-Moret, N. Charlotte; Palotie, Aarno; Par, Guillaume; Parker, Alex N; Pedersen, Nancy L; Peeters, Petra HM; Pistis, Giorgio; Plump, Andrew S; Polasek, Ozren; Pop, Victor JM; Psaty, Bruce M; Rikknen, Katri; Rehnberg, Emil; Rotter, Jerome I; Rudan, Igor; Sala, Cinzia; Salumets, Andres; Scuteri, Angelo; Singleton, Andrew; Smith, Jennifer A; Snieder, Harold; Soranzo, Nicole; Stacey, Simon N; Starr, John M; Stathopoulou, Maria G; Stirrups, Kathleen; Stolk, Ronald P; Styrkarsdottir, Unnur; Sun, Yan V; Tenesa, Albert; Thorand, Barbara; Toniolo, Daniela; Tryggvadottir, Laufey; Tsui, Kim; Ulivi, Sheila; van Dam, Rob M; van der Schouw, Yvonne T; van Gils, Carla H; van Nierop, Peter; Vink, Jacqueline M; Visscher, Peter M; Voorhuis, Marlies; Waeber, Grard; Wallaschofski, Henri; Wichmann, H Erich; Widen, Elisabeth; Gent, Colette JM Wijnands-van; Willemsen, Gonneke; Wilson, James F; Wolffenbuttel, Bruce HR; Wright, Alan F; Yerges-Armstrong, Laura M; Zemunik, Tatijana; Zgaga, Lina; Zillikens, M. Carola; Zygmunt, Marek; Arnold, Alice M; Boomsma, Dorret I; Buring, Julie E.; Crisponi, Laura; Demerath, Ellen W; Gudnason, Vilmundur; Harris, Tamara B; Hu, Frank B; Hunter, David J; Launer, Lenore J; Metspalu, Andres; Montgomery, Grant W; Oostra, Ben A; Ridker, Paul M; Sanna, Serena; Schlessinger, David; Spector, Tim D; Stefansson, Kari; Streeten, Elizabeth A; Thorsteinsdottir, Unnur; Uda, Manuela; Uitterlinden, Andr G; van Duijn, Cornelia M; Vlzke, Henry; Murray, Anna; Murabito, Joanne M; Visser, Jenny A; Lunetta, Kathryn L

    2011-01-01

    To identify novel loci for age at natural menopause, we performed a meta-analysis of 22 genome-wide association studies in 38,968 women of European descent, with replication in up to 14,435 women. In addition to four known loci, we identified 13 new age at natural menopause loci (P < 5 10?8). The new loci included genes implicated in DNA repair (EXO1, HELQ, UIMC1, FAM175A, FANCI, TLK1, POLG, PRIM1) and immune function (IL11, NLRP11, BAT2). Gene-set enrichment pathway analyses using the full GWAS dataset identified exodeoxyribonuclease, NF?B signalling and mitochondrial dysfunction as biological processes related to timing of menopause. PMID:22267201

  3. Meta-analyses identify 13 loci associated with age at menopause and highlight DNA repair and immune pathways.

    PubMed

    Stolk, Lisette; Perry, John R B; Chasman, Daniel I; He, Chunyan; Mangino, Massimo; Sulem, Patrick; Barbalic, Maja; Broer, Linda; Byrne, Enda M; Ernst, Florian; Esko, Tnu; Franceschini, Nora; Gudbjartsson, Daniel F; Hottenga, Jouke-Jan; Kraft, Peter; McArdle, Patrick F; Porcu, Eleonora; Shin, So-Youn; Smith, Albert V; van Wingerden, Sophie; Zhai, Guangju; Zhuang, Wei V; Albrecht, Eva; Alizadeh, Behrooz Z; Aspelund, Thor; Bandinelli, Stefania; Lauc, Lovorka Barac; Beckmann, Jacques S; Boban, Mladen; Boerwinkle, Eric; Broekmans, Frank J; Burri, Andrea; Campbell, Harry; Chanock, Stephen J; Chen, Constance; Cornelis, Marilyn C; Corre, Tanguy; Coviello, Andrea D; d'Adamo, Pio; Davies, Gail; de Faire, Ulf; de Geus, Eco J C; Deary, Ian J; Dedoussis, George V Z; Deloukas, Panagiotis; Ebrahim, Shah; Eiriksdottir, Gudny; Emilsson, Valur; Eriksson, Johan G; Fauser, Bart C J M; Ferreli, Liana; Ferrucci, Luigi; Fischer, Krista; Folsom, Aaron R; Garcia, Melissa E; Gasparini, Paolo; Gieger, Christian; Glazer, Nicole; Grobbee, Diederick E; Hall, Per; Haller, Toomas; Hankinson, Susan E; Hass, Merli; Hayward, Caroline; Heath, Andrew C; Hofman, Albert; Ingelsson, Erik; Janssens, A Cecile J W; Johnson, Andrew D; Karasik, David; Kardia, Sharon L R; Keyzer, Jules; Kiel, Douglas P; Kolcic, Ivana; Kutalik, Zoltn; Lahti, Jari; Lai, Sandra; Laisk, Triin; Laven, Joop S E; Lawlor, Debbie A; Liu, Jianjun; Lopez, Lorna M; Louwers, Yvonne V; Magnusson, Patrik K E; Marongiu, Mara; Martin, Nicholas G; Klaric, Irena Martinovic; Masciullo, Corrado; McKnight, Barbara; Medland, Sarah E; Melzer, David; Mooser, Vincent; Navarro, Pau; Newman, Anne B; Nyholt, Dale R; Onland-Moret, N Charlotte; Palotie, Aarno; Par, Guillaume; Parker, Alex N; Pedersen, Nancy L; Peeters, Petra H M; Pistis, Giorgio; Plump, Andrew S; Polasek, Ozren; Pop, Victor J M; Psaty, Bruce M; Rikknen, Katri; Rehnberg, Emil; Rotter, Jerome I; Rudan, Igor; Sala, Cinzia; Salumets, Andres; Scuteri, Angelo; Singleton, Andrew; Smith, Jennifer A; Snieder, Harold; Soranzo, Nicole; Stacey, Simon N; Starr, John M; Stathopoulou, Maria G; Stirrups, Kathleen; Stolk, Ronald P; Styrkarsdottir, Unnur; Sun, Yan V; Tenesa, Albert; Thorand, Barbara; Toniolo, Daniela; Tryggvadottir, Laufey; Tsui, Kim; Ulivi, Sheila; van Dam, Rob M; van der Schouw, Yvonne T; van Gils, Carla H; van Nierop, Peter; Vink, Jacqueline M; Visscher, Peter M; Voorhuis, Marlies; Waeber, Grard; Wallaschofski, Henri; Wichmann, H Erich; Widen, Elisabeth; Wijnands-van Gent, Colette J M; Willemsen, Gonneke; Wilson, James F; Wolffenbuttel, Bruce H R; Wright, Alan F; Yerges-Armstrong, Laura M; Zemunik, Tatijana; Zgaga, Lina; Zillikens, M Carola; Zygmunt, Marek; Arnold, Alice M; Boomsma, Dorret I; Buring, Julie E; Crisponi, Laura; Demerath, Ellen W; Gudnason, Vilmundur; Harris, Tamara B; Hu, Frank B; Hunter, David J; Launer, Lenore J; Metspalu, Andres; Montgomery, Grant W; Oostra, Ben A; Ridker, Paul M; Sanna, Serena; Schlessinger, David; Spector, Tim D; Stefansson, Kari; Streeten, Elizabeth A; Thorsteinsdottir, Unnur; Uda, Manuela; Uitterlinden, Andr G; van Duijn, Cornelia M; Vlzke, Henry; Murray, Anna; Murabito, Joanne M; Visser, Jenny A; Lunetta, Kathryn L

    2012-03-01

    To newly identify loci for age at natural menopause, we carried out a meta-analysis of 22 genome-wide association studies (GWAS) in 38,968 women of European descent, with replication in up to 14,435 women. In addition to four known loci, we identified 13 loci newly associated with age at natural menopause (at P < 5 10(-8)). Candidate genes located at these newly associated loci include genes implicated in DNA repair (EXO1, HELQ, UIMC1, FAM175A, FANCI, TLK1, POLG and PRIM1) and immune function (IL11, NLRP11 and PRRC2A (also known as BAT2)). Gene-set enrichment pathway analyses using the full GWAS data set identified exoDNase, NF-?B signaling and mitochondrial dysfunction as biological processes related to timing of menopause. PMID:22267201

  4. Analysis of genomic aberrations and gene expression profiling identifies novel lesions and pathways in myeloproliferative neoplasms.

    PubMed

    Rice, K L; Lin, X; Wolniak, K; Ebert, B L; Berkofsky-Fessler, W; Buzzai, M; Sun, Y; Xi, C; Elkin, P; Levine, R; Golub, T; Gilliland, D G; Crispino, J D; Licht, J D; Zhang, W

    2011-11-01

    Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis, are myeloproliferative neoplasms (MPNs) with distinct clinical features and are associated with the JAK2V617F mutation. To identify genomic anomalies involved in the pathogenesis of these disorders, we profiled 87 MPN patients using Affymetrix 250K single-nucleotide polymorphism (SNP) arrays. Aberrations affecting chr9 were the most frequently observed and included 9pLOH (n=16), trisomy 9 (n=6) and amplifications of 9p13.3-23.3 (n=1), 9q33.1-34.13 (n=1) and 9q34.13 (n=6). Patients with trisomy 9 were associated with elevated JAK2V617F mutant allele burden, suggesting that gain of chr9 represents an alternative mechanism for increasing JAK2V617F dosage. Gene expression profiling of patients with and without chr9 abnormalities (+9, 9pLOH), identified genes potentially involved in disease pathogenesis including JAK2, STAT5B and MAPK14. We also observed recurrent gains of 1p36.31-36.33 (n=6), 17q21.2-q21.31 (n=5) and 17q25.1-25.3 (n=5) and deletions affecting 18p11.31-11.32 (n=8). Combined SNP and gene expression analysis identified aberrations affecting components of a non-canonical PRC2 complex (EZH1, SUZ12 and JARID2) and genes comprising a 'HSC signature' (MLLT3, SMARCA2 and PBX1). We show that NFIB, which is amplified in 7/87 MPN patients and upregulated in PV CD34+ cells, protects cells from apoptosis induced by cytokine withdrawal. PMID:22829077

  5. Analysis of genomic aberrations and gene expression profiling identifies novel lesions and pathways in myeloproliferative neoplasms

    PubMed Central

    Rice, K L; Lin, X; Wolniak, K; Ebert, B L; Berkofsky-Fessler, W; Buzzai, M; Sun, Y; Xi, C; Elkin, P; Levine, R; Golub, T; Gilliland, D G; Crispino, J D; Licht, J D; Zhang, W

    2011-01-01

    Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis, are myeloproliferative neoplasms (MPNs) with distinct clinical features and are associated with the JAK2V617F mutation. To identify genomic anomalies involved in the pathogenesis of these disorders, we profiled 87 MPN patients using Affymetrix 250K single-nucleotide polymorphism (SNP) arrays. Aberrations affecting chr9 were the most frequently observed and included 9pLOH (n=16), trisomy 9 (n=6) and amplifications of 9p13.323.3 (n=1), 9q33.134.13 (n=1) and 9q34.13 (n=6). Patients with trisomy 9 were associated with elevated JAK2V617F mutant allele burden, suggesting that gain of chr9 represents an alternative mechanism for increasing JAK2V617F dosage. Gene expression profiling of patients with and without chr9 abnormalities (+9, 9pLOH), identified genes potentially involved in disease pathogenesis including JAK2, STAT5B and MAPK14. We also observed recurrent gains of 1p36.3136.33 (n=6), 17q21.2q21.31 (n=5) and 17q25.125.3 (n=5) and deletions affecting 18p11.3111.32 (n=8). Combined SNP and gene expression analysis identified aberrations affecting components of a non-canonical PRC2 complex (EZH1, SUZ12 and JARID2) and genes comprising a HSC signature' (MLLT3, SMARCA2 and PBX1). We show that NFIB, which is amplified in 7/87 MPN patients and upregulated in PV CD34+ cells, protects cells from apoptosis induced by cytokine withdrawal. PMID:22829077

  6. Integrated Genomics Identifies Five Medulloblastoma Subtypes with Distinct Genetic Profiles, Pathway Signatures and Clinicopathological Features

    PubMed Central

    Kool, Marcel; Koster, Jan; Bunt, Jens; Hasselt, Nancy E.; Lakeman, Arjan; van Sluis, Peter; Troost, Dirk; Meeteren, Netteke Schouten-van; Caron, Huib N.; Cloos, Jacqueline; Mri?, Alan; Ylstra, Bauke; Grajkowska, Wieslawa; Hartmann, Wolfgang; Pietsch, Torsten; Ellison, David; Clifford, Steven C.; Versteeg, Rogier

    2008-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Despite recent improvements in cure rates, prediction of disease outcome remains a major challenge and survivors suffer from serious therapy-related side-effects. Recent data showed that patients with WNT-activated tumors have a favorable prognosis, suggesting that these patients could be treated less intensively, thereby reducing the side-effects. This illustrates the potential benefits of a robust classification of medulloblastoma patients and a detailed knowledge of associated biological mechanisms. Methods and Findings To get a better insight into the molecular biology of medulloblastoma we established mRNA expression profiles of 62 medulloblastomas and analyzed 52 of them also by comparative genomic hybridization (CGH) arrays. Five molecular subtypes were identified, characterized by WNT signaling (A; 9 cases), SHH signaling (B; 15 cases), expression of neuronal differentiation genes (C and D; 16 and 11 cases, respectively) or photoreceptor genes (D and E; both 11 cases). Mutations in ?-catenin were identified in all 9 type A tumors, but not in any other tumor. PTCH1 mutations were exclusively identified in type B tumors. CGH analysis identified several fully or partly subtype-specific chromosomal aberrations. Monosomy of chromosome 6 occurred only in type A tumors, loss of 9q mostly occurred in type B tumors, whereas chromosome 17 aberrations, most common in medulloblastoma, were strongly associated with type C or D tumors. Loss of the inactivated X-chromosome was highly specific for female cases of type C, D and E tumors. Gene expression levels faithfully reflected the chromosomal copy number changes. Clinicopathological features significantly different between the 5 subtypes included metastatic disease and age at diagnosis and histology. Metastatic disease at diagnosis was significantly associated with subtypes C and D and most strongly with subtype E. Patients below 3 yrs of age had type B, D, or E tumors. Type B included most desmoplastic cases. We validated and confirmed the molecular subtypes and their associated clinicopathological features with expression data from a second independent series of 46 medulloblastomas. Conclusions The new medulloblastoma classification presented in this study will greatly enhance the understanding of this heterogeneous disease. It will enable a better selection and evaluation of patients in clinical trials, and it will support the development of new molecular targeted therapies. Ultimately, our results may lead to more individualized therapies with improved cure rates and a better quality of life. PMID:18769486

  7. Enhanced Sequential Search Methodology for Identifying Cost-Optimal Building Pathways

    SciTech Connect

    Horowitz, S.; Christensen, C.; Brandemuehl, M.; Krarti, M.

    2008-06-01

    The BEopt software is a building energy optimization tool that generates a cost-optimal path of building designs from a reference building up to zero-net energy. It employs a sequential search methodology to account for complex energy interactions between building efficiency measures. Enhancement strategies to this search methodology are developed to increase accuracy (ability to identify the true cost-optimal curve) and speed (number of required energy simulations). A test suite of optimizations is used to gauge the effectiveness of each strategy. Combinations of strategies are assembled into packages, ranging from conservative to aggressive, with so up to 71% fewer required simulations are required.

  8. Neuro magnetic resonance spectroscopy using wavelet decomposition and statistical testing identifies biochemical changes in people with spinal cord injury and pain.

    PubMed

    Stanwell, Peter; Siddall, Philip; Keshava, Nirmal; Cocuzzo, Daniel; Ramadan, Saadallah; Lin, Alexander; Herbert, David; Craig, Ashley; Tran, Yvonne; Middleton, James; Gautam, Shiva; Cousins, Michael; Mountford, Carolyn

    2010-11-01

    Spinal cord injury (SCI) can be accompanied by chronic pain, the mechanisms for which are poorly understood. Here we report that magnetic resonance spectroscopy measurements from the brain, collected at 3T, and processed using wavelet-based feature extraction and classification algorithms, can identify biochemical changes that distinguish control subjects from subjects with SCI as well as subdividing the SCI group into those with and without chronic pain. The results from control subjects (n=10) were compared to those with SCI (n=10). The SCI cohort was made up of subjects with chronic neuropathic pain (n=5) and those without chronic pain (n=5). The wavelet-based decomposition of frequency domain MRS signals employs statistical significance testing to identify features best suited to discriminate different classes. Moreover, the features benefit from careful attention to the post-processing of the spectroscopy data prior to the comparison of the three cohorts. The spectroscopy data, from the thalamus, best distinguished control subjects without SCI from those with SCI with a sensitivity and specificity of 0.9 (Percentage of Correct Classification). The spectroscopy data obtained from the prefrontal cortex and anterior cingulate cortex both distinguished between SCI subjects with chronic neuropathic pain and those without pain with a sensitivity and specificity of 1.0. In this study, where two underlying mechanisms co-exist (i.e. SCI and pain), the thalamic changes appear to be linked more strongly to SCI, while the anterior cingulate cortex and prefrontal cortex changes appear to be specifically linked to the presence of pain. PMID:20600973

  9. Heterologous Production of the Marine Myxobacterial Antibiotic Haliangicin and Its Unnatural Analogues Generated by Engineering of the Biochemical Pathway

    PubMed Central

    Sun, Yuwei; Feng, Zhiyang; Tomura, Tomohiko; Suzuki, Akira; Miyano, Seishi; Tsuge, Takashi; Mori, Hitoshi; Suh, Joo-Won; Iizuka, Takashi; Fudou, Ryosuke; Ojika, Makoto

    2016-01-01

    Despite their fastidious nature, marine myxobacteria have considerable genetic potential to produce novel secondary metabolites. The marine myxobacterium Haliangium ochraceum SMP-2 produces the antifungal polyketide haliangicin (1), but its productivity is unsatisfactory. The biosynthetic gene cluster hli (47.8 kbp) associated with 1 was identified and heterologously expressed in Myxococcus xanthus to permit the production of 1 with high efficiency (tenfold greater amount and threefold faster in growth speed compared with the original producer), as well as the generation of bioactive unnatural analogues of 1 through gene manipulation. A unique acyl-CoA dehydrogenase was found to catalyse an unusual γ,δ-dehydrogenation of the diketide starter unit, leading to the formation of the terminal alkene moiety of 1. Biological evaluation of the analogues obtained through this study revealed that their bioactivities (anti-oomycete and cytotoxic activities) can be modified by manipulating the vinyl epoxide at the terminus opposite the β-methoxyacrylate pharmacophore. PMID:26915413

  10. Heterologous Production of the Marine Myxobacterial Antibiotic Haliangicin and Its Unnatural Analogues Generated by Engineering of the Biochemical Pathway.

    PubMed

    Sun, Yuwei; Feng, Zhiyang; Tomura, Tomohiko; Suzuki, Akira; Miyano, Seishi; Tsuge, Takashi; Mori, Hitoshi; Suh, Joo-Won; Iizuka, Takashi; Fudou, Ryosuke; Ojika, Makoto

    2016-01-01

    Despite their fastidious nature, marine myxobacteria have considerable genetic potential to produce novel secondary metabolites. The marine myxobacterium Haliangium ochraceum SMP-2 produces the antifungal polyketide haliangicin (1), but its productivity is unsatisfactory. The biosynthetic gene cluster hli (47.8?kbp) associated with 1 was identified and heterologously expressed in Myxococcus xanthus to permit the production of 1 with high efficiency (tenfold greater amount and threefold faster in growth speed compared with the original producer), as well as the generation of bioactive unnatural analogues of 1 through gene manipulation. A unique acyl-CoA dehydrogenase was found to catalyse an unusual ?,?-dehydrogenation of the diketide starter unit, leading to the formation of the terminal alkene moiety of 1. Biological evaluation of the analogues obtained through this study revealed that their bioactivities (anti-oomycete and cytotoxic activities) can be modified by manipulating the vinyl epoxide at the terminus opposite the ?-methoxyacrylate pharmacophore. PMID:26915413

  11. Structural and Biochemical Characterization of the Salicylyl-acyltranferase SsfX3 from a Tetracycline Biosynthetic Pathway

    SciTech Connect

    Pickens, Lauren B.; Sawaya, Michael R.; Rasool, Huma; Pashkov, Inna; Yeates, Todd O.; Tang, Yi

    2012-03-14

    SsfX3 is a GDSL family acyltransferase that transfers salicylate to the C-4 hydroxyl of a tetracycline intermediate in the penultimate step during biosynthesis of the anticancer natural product SF2575. The C-4 salicylate takes the place of the more common C-4 dimethylamine functionality, making SsfX3 the first acyltransferase identified to act on a tetracycline substrate. The crystal structure of SsfX3 was determined at 2.5 {angstrom}, revealing two distinct domains as follows: an N-terminal {beta}-sandwich domain that resembles a carbohydrate-binding module, and a C-terminal catalytic domain that contains the atypical {alpha}/{beta}-hydrolase fold found in the GDSL hydrolase family of enzymes. The active site lies at one end of a large open binding pocket, which is spatially defined by structural elements from both the N- and C-terminal domains. Mutational analysis in the putative substrate binding pocket identified residues from both domains that are important for binding the acyl donor and acceptor. Furthermore, removal of the N-terminal carbohydrate-binding module-like domain rendered the stand-alone {alpha}/{beta}-hydrolase domain inactive. The additional noncatalytic module is therefore proposed to be required to define the binding pocket and provide sufficient interactions with the spatially extended tetracyclic substrate. SsfX3 was also demonstrated to accept a variety of non-native acyl groups. This relaxed substrate specificity toward the acyl donor allowed the chemoenzymatic biosynthesis of C-4-modified analogs of the immediate precursor to the bioactive SF2575; these were used to assay the structure activity relationships at the C-4 position.

  12. Gain-of-function assays in the axolotl (Ambystoma mexicanum) to identify signaling pathways that induce and regulate limb regeneration.

    PubMed

    Lee, Jangwoo; Aguilar, Cristian; Gardiner, David

    2013-01-01

    The adult salamander has been studied as a model for regeneration of complex tissues for many decades. Only recently with the development of gain-of-function assays for regeneration, has it been possible to screen for and assay the function of the multitude of signaling factors that have been identified in studies of embryonic development and tumorigenesis. Given the conservation of function of these regulatory pathways controlling growth and pattern formation, it is now possible to use the functional assays in the salamander to test the ability of endogenous as well as small-molecule signaling factors to induce a regenerative response. PMID:24029949

  13. Evolution of a Pathogen: A Comparative Genomics Analysis Identifies a Genetic Pathway to Pathogenesis in Acinetobacter

    PubMed Central

    Sahl, Jason W.; Gillece, John D.; Schupp, James M.; Waddell, Victor G.; Driebe, Elizabeth M.; Engelthaler, David M.; Keim, Paul

    2013-01-01

    Acinetobacter baumannii is an emergent and global nosocomial pathogen. In addition to A. baumannii, other Acinetobacter species, especially those in the Acinetobacter calcoaceticus-baumannii (Acb) complex, have also been associated with serious human infection. Although mechanisms of attachment, persistence on abiotic surfaces, and pathogenesis in A. baumannii have been identified, the genetic mechanisms that explain the emergence of A. baumannii as the most widespread and virulent Acinetobacter species are not fully understood. Recent whole genome sequencing has provided insight into the phylogenetic structure of the genus Acinetobacter. However, a global comparison of genomic features between Acinetobacter spp. has not been described in the literature. In this study, 136 Acinetobacter genomes, including 67 sequenced in this study, were compared to identify the acquisition and loss of genes in the expansion of the Acinetobacter genus. A whole genome phylogeny confirmed that A. baumannii is a monophyletic clade and that the larger Acb complex is also a well-supported monophyletic group. The whole genome phylogeny provided the framework for a global genomic comparison based on a blast score ratio (BSR) analysis. The BSR analysis demonstrated that specific genes have been both lost and acquired in the evolution of A. baumannii. In addition, several genes associated with A. baumannii pathogenesis were found to be more conserved in the Acb complex, and especially in A. baumannii, than in other Acinetobacter genomes; until recently, a global analysis of the distribution and conservation of virulence factors across the genus was not possible. The results demonstrate that the acquisition of specific virulence factors has likely contributed to the widespread persistence and virulence of A. baumannii. The identification of novel features associated with transcriptional regulation and acquired by clades in the Acb complex presents targets for better understanding the evolution of pathogenesis and virulence in the expansion of the genus. PMID:23365658

  14. Novel glycosylation pathways of retroviral envelope proteins identified with avian reticuloendotheliosis virus.

    PubMed Central

    Tsai, W P; Oroszlan, S

    1988-01-01

    Previously, we identified two mature glycoproteins, gp90, the surface glycoprotein, and gp20, the transmembrane protein, from avian reticuloendotheliosis virus and an avian reticuloendotheliosis virus env gene-encoded intracellular polyprotein gPr77env, but the precise relationship of gPr77env to the mature envelope proteins was not determined (W.-P. Tsai, T.D. Copeland, and S. Oroszlan, Virology 155:567-583, 1986). In the present study, using metabolic labeling of viral proteins with [35S]cysteine, radioimmunoprecipitation, and carbohydrate structure analysis, we have identified a higher-molecular-weight endo-H-resistant env gene-encoded polyprotein designated gPr115env in addition to the endo-H-sensitive gPr77env. It appears that gPr77env is the primary polyprotein precursor, modified with mannosyloligosaccharides that are processed into sialic-acid-rich extraordinarily large complex-type carbohydrates (up to 17 kilodaltons for each N-linked site) on the gp90 domain but not on the gPr22 domain. In this process, gPr77env is converted into the apparently endo-H-resistant secondary polyprotein, gPr115env, which is rapidly processed into gp90 and gPr22. The proteolytic processing which occurs only after the appearance of an endo-H resistant precursor is now clearly demonstrated for a retrovirus. Some important aspects of carbohydrate structure, including the site-specific glycosylation, as well as the intracellular location and nature of the potential enzyme involved in the proteolytic cleavage of gPr115env are discussed. Images PMID:2841469

  15. Exploring critical pathways for urban water management to identify robust strategies under deep uncertainties.

    PubMed

    Urich, Christian; Rauch, Wolfgang

    2014-12-01

    Long-term projections for key drivers needed in urban water infrastructure planning such as climate change, population growth, and socio-economic changes are deeply uncertain. Traditional planning approaches heavily rely on these projections, which, if a projection stays unfulfilled, can lead to problematic infrastructure decisions causing high operational costs and/or lock-in effects. New approaches based on exploratory modelling take a fundamentally different view. Aim of these is, to identify an adaptation strategy that performs well under many future scenarios, instead of optimising a strategy for a handful. However, a modelling tool to support strategic planning to test the implication of adaptation strategies under deeply uncertain conditions for urban water management does not exist yet. This paper presents a first step towards a new generation of such strategic planning tools, by combing innovative modelling tools, which coevolve the urban environment and urban water infrastructure under many different future scenarios, with robust decision making. The developed approach is applied to the city of Innsbruck, Austria, which is spatially explicitly evolved 20 years into the future under 1000 scenarios to test the robustness of different adaptation strategies. Key findings of this paper show that: (1) Such an approach can be used to successfully identify parameter ranges of key drivers in which a desired performance criterion is not fulfilled, which is an important indicator for the robustness of an adaptation strategy; and (2) Analysis of the rich dataset gives new insights into the adaptive responses of agents to key drivers in the urban system by modifying a strategy. PMID:25240118

  16. A systems biology approach to identify intelligence quotient score-related genomic regions, and pathways relevant to potential therapeutic treatments.

    PubMed

    Zhao, Min; Kong, Lei; Qu, Hong

    2014-01-01

    Although the intelligence quotient (IQ) is the most popular intelligence test in the world, little is known about the underlying biological mechanisms that lead to the differences in human. To improve our understanding of cognitive processes and identify potential biomarkers, we conducted a comprehensive investigation of 158 IQ-related genes selected from the literature. A genomic distribution analysis demonstrated that IQ-related genes were enriched in seven regions of chromosome 7 and the X chromosome. In addition, these genes were enriched in target lists of seven transcription factors and sixteen microRNAs. Using a network-based approach, we further reconstructed an IQ-related pathway from known human pathway interaction data. Based on this reconstructed pathway, we incorporated enriched drugs and described the importance of dopamine and norepinephrine systems in IQ-related biological process. These findings not only reveal several testable genes and processes related to IQ scores, but also have potential therapeutic implications for IQ-related mental disorders. PMID:24566931

  17. A systems biology approach to identify intelligence quotient score-related genomic regions, and pathways relevant to potential therapeutic treatments

    PubMed Central

    Zhao, Min; Kong, Lei; Qu, Hong

    2014-01-01

    Although the intelligence quotient (IQ) is the most popular intelligence test in the world, little is known about the underlying biological mechanisms that lead to the differences in human. To improve our understanding of cognitive processes and identify potential biomarkers, we conducted a comprehensive investigation of 158 IQ-related genes selected from the literature. A genomic distribution analysis demonstrated that IQ-related genes were enriched in seven regions of chromosome 7 and the X chromosome. In addition, these genes were enriched in target lists of seven transcription factors and sixteen microRNAs. Using a network-based approach, we further reconstructed an IQ-related pathway from known human pathway interaction data. Based on this reconstructed pathway, we incorporated enriched drugs and described the importance of dopamine and norepinephrine systems in IQ-related biological process. These findings not only reveal several testable genes and processes related to IQ scores, but also have potential therapeutic implications for IQ-related mental disorders. PMID:24566931

  18. RNAi screen identifies a role for adaptor protein AP-3 in sorting to the regulated secretory pathway

    PubMed Central

    Asensio, Cdric S.; Sirkis, Daniel W.

    2010-01-01

    The regulated release of proteins depends on their inclusion within large dense-core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network (TGN), but the mechanism for protein sorting to this regulated secretory pathway (RSP) and the cytosolic machinery involved in this process have remained poorly understood. Using an RNA interference screen in Drosophila melanogaster S2 cells, we now identify a small number of genes, including several subunits of the heterotetrameric adaptor protein AP-3, which are required for sorting to the RSP. In mammalian neuroendocrine cells, loss of AP-3 dysregulates exocytosis due to a primary defect in LDCV formation. Previous work implicated AP-3 in the endocytic pathway, but we find that AP-3 promotes sorting to the RSP within the biosynthetic pathway at the level of the TGN. Although vesicles with a dense core still form in the absence of AP-3, they contain substantially less synaptotagmin 1, indicating that AP-3 concentrates the proteins required for regulated exocytosis. PMID:21149569

  19. Using End-Member Mixing Analysis to Identify Transport Pathways in Agricultural Watersheds

    NASA Astrophysics Data System (ADS)

    Essaid, H.

    2013-12-01

    Concentrations of naturally occurring solutes were monitored in six agricultural watersheds as part of the Agricultural Chemical Sources, Transport and Fate study of the U.S. Geological Survey National Water Quality Assessment Program. The watersheds, located in Indiana (IN), Iowa (IA), Maryland (MD), Nebraska (NE), Mississippi (MS) and Washington (WA), ranged in size from 5 to 1300 square kilometers, and agricultural land use accounted for 60 - 98% of the watershed area. Concentrations of chloride, calcium, magnesium, sodium, potassium, fluoride, sulfate and silica in stream water were analyzed using principal components analysis (PCA) to identify the dominant processes controlling stream water chemistry. Concentrations measured in wells, streambed piezometers, unsaturated zone lysimeters, overland flow, and tile drains were then used to project the chemistry of these flow compartments/components onto the stream chemistry PCA transformation. This facilitated the identification of end members explaining observed stream chemistry, and the source of the water for these end members. The majority of the variance in stream chemistry could be explained by the mixing of three end-members: 94% for Leary Weber Ditch, IN; 84% for South Fork Iowa River, IA; 91% for Morgan Creek, MD; 81% for Maple Creek, NE; 92% for Tommie Bayou, MS; and, 97% for DR2, WA. The chemistry of overland flow and tile drainage was also contained within the end member concentrations. The end members were generally represented by two groundwater components and a very low solute concentration component (precipitation or irrigation water) that diluted the groundwater signal. Generally, one groundwater component represented recently recharged water that resembled the concentration of water in the deepest unsaturated zone lysimeters, and the other groundwater component was representative of regional groundwater conditions. Compared to regional groundwater, the recent groundwater component had lower concentrations of solutes derived from rock weathering (such as silica) and higher concentrations of solutes derived from the land surface (such as sulfate, nitrate and phosphorous).The two groundwater components represented shallow versus deep groundwater (as in IN, MD and WA), oxic versus anoxic GW (as in IA and MS), and groundwater from two different rock types (as in NE where groundwater from loess had higher solute concentrations than water from unconsolidated deposits). Examination of stream water chemistry for nested drainages within a single watershed indicated that the contribution from the deep (often anoxic) GW component increased as watershed size increased. The conclusions from this principal components and end-member-mixing analysis are being used to help identify and quantify the sources of nitrogen transport to the streams.

  20. Alternate dissociation pathways identified in charge-reduced protein complex ions.

    PubMed

    Pagel, Kevin; Hyung, Suk-Joon; Ruotolo, Brandon T; Robinson, Carol V

    2010-06-15

    Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes. PMID:20481443

  1. Soluble Signals from Cells Identified at the Cell Wall Establish a Developmental Pathway in Carrot.

    PubMed Central

    McCabe, P. F.; Valentine, T. A.; Forsberg, L. S.; Pennell, R. I.

    1997-01-01

    Cells in a plant differentiate according to their positions and use cell-cell communication to assess these positions. Similarly, single cells in suspension cultures can develop into somatic embryos, and cell-cell communication is thought to control this process. The monoclonal antibody JIM8 labels an epitope on cells in specific positions in plants. JIM8 also labels certain cells in carrot embryogenic suspension cultures. We have used JIM8 and secondary antibodies coupled to paramagnetic beads to label and immunomagnetically sort single cells in a carrot embryogenic suspension culture into pure populations. Cells in the JIM8(+) population develop into somatic embryos, whereas cells in the JIM8(-) population do not form somatic embryos. However, certain cells in JIM8(+) cultures (state B cells) undergo asymmetric divisions, resulting in daughter cells (state C cells) that do not label with JIM8 and that sort to JIM8(-) cultures. State C cells are competent to form somatic embryos, and we show here that a conditioned growth medium from a culture of JIM8(+) cells allows state C cells in a JIM8(-) culture to go on and develop into somatic embryos. JIM8 labels cells in suspension cultures at the cell wall. Therefore, a cell with a role in cell-cell communication and early cell fate selection can be identified by an epitope in its cell wall. PMID:12237357

  2. Identifying Cellular Pathways Modulated by Drosophila Palmitoyl-protein Thioesterase 1 Function

    PubMed Central

    Saja, Stephanie; Buff, Haley; Smith, Alexis C.; Williams, Tiffany S.; Korey, Christopher A.

    2010-01-01

    Infantile-onset Neuronal Ceroid Lipofuscinosis (INCL) is a severe pediatric neurodegenerative disorder produced by mutations in the gene encoding palmitoyl-protein thioesterase 1 (Ppt1). This enzyme is responsible for the removal of a palmitate post-translational modification from an unknown set of substrate proteins. To better understand the function of Ppt1 in neurons, we performed an unbiased dominant loss-of-function genetic modifier screen in Drosophila using a previously characterized Ppt1 gain-of-function system. The enhancers and suppressors identified in our screen make novel connections between Ppt1 and genes involved in cellular trafficking and the modulation of synaptic growth. We further support the relevance of our screen by demonstrating that Garland cells from Ppt1 loss-of-function mutants have defects in endocytic trafficking. Endocytic tracer uptake and ultrastructural analysis of these non-neuronal cells points to Ppt1 playing a role in modulating the early stages of vesicle formation. This work lays the groundwork for further experimental exploration of these processes to better understand their contributions to the INCL disease process. PMID:20206262

  3. Ectopic pregnancy as a model to identify endometrial genes and signaling pathways important in decidualization and regulated by local trophoblast.

    PubMed

    Duncan, W Colin; Shaw, Julie L V; Burgess, Stewart; McDonald, Sarah E; Critchley, Hilary O D; Horne, Andrew W

    2011-01-01

    The endometrium in early pregnancy undergoes decidualization and functional changes induced by local trophoblast, which are not fully understood. We hypothesized that endometrium from tubal ectopic pregnancy (EP) could be interrogated to identify novel genes and pathways involved in these processes. Gestation-matched endometrium was collected from women with EP (n = 11) and intrauterine pregnancies (IUP) (n = 13). RNA was extracted from the tissue. In addition, tissues were prepared for histological analysis for degree of decidualization. We compared a) the samples from EP that were decidualized (n = 6) with non-decidualized samples (n = 5), and b) the decidualized EP (n = 6) with decidualization-matched IUP (n = 6) samples using an Affymetrix gene array platform, with Ingenuity Pathway Analysis, combined with quantitative RT-PCR. Expression of PRL and IGFBP1 was used to confirm the degree of decidualization in each group. There were no differences in PRL or IGFBP1 expression in the decidualization-matched samples but a marked reduction (P<0.001) in the non-decidualized samples. Decidualization was associated with increased expression of 428 genes including SCARA5 (181-fold), DKK1 (71-fold) and PROK1 (32-fold), and decreased expression of 230 genes including MMP-7 (35-fold) and SFRP4 (21-fold). The top canonical pathways associated with these differentially expressed genes were Natural Killer Cell and Wnt/b-Catenin signaling. Local trophoblast was associated with much less alteration of endometrial gene expression with an increase in 56 genes, including CSH1 (8-fold), and a reduction in 29 genes including CRISP3 (8-fold). The top associated canonical pathway was Antigen Presentation. The study of endometrium from tubal EP may promote novel insights into genes involved in decidualization and those influenced by factors from neighboring trophoblast. This has afforded unique information not highlighted by previous studies and adds to our understanding of the endometrium in early pregnancy. PMID:21858178

  4. Ectopic Pregnancy as a Model to Identify Endometrial Genes and Signaling Pathways Important in Decidualization and Regulated by Local Trophoblast

    PubMed Central

    Burgess, Stewart; McDonald, Sarah E.; Critchley, Hilary O. D.; Horne, Andrew W.

    2011-01-01

    The endometrium in early pregnancy undergoes decidualization and functional changes induced by local trophoblast, which are not fully understood. We hypothesized that endometrium from tubal ectopic pregnancy (EP) could be interrogated to identify novel genes and pathways involved in these processes. Gestation-matched endometrium was collected from women with EP (n = 11) and intrauterine pregnancies (IUP) (n = 13). RNA was extracted from the tissue. In addition, tissues were prepared for histological analysis for degree of decidualization. We compared a) the samples from EP that were decidualized (n = 6) with non-decidualized samples (n = 5), and b) the decidualized EP (n = 6) with decidualization-matched IUP (n = 6) samples using an Affymetrix gene array platform, with Ingenuity Pathway Analysis, combined with quantitative RT-PCR. Expression of PRL and IGFBP1 was used to confirm the degree of decidualization in each group. There were no differences in PRL or IGFBP1 expression in the decidualization-matched samples but a marked reduction (P<0.001) in the non-decidualized samples. Decidualization was associated with increased expression of 428 genes including SCARA5 (181-fold), DKK1 (71-fold) and PROK1 (32-fold), and decreased expression of 230 genes including MMP-7 (35-fold) and SFRP4 (21-fold). The top canonical pathways associated with these differentially expressed genes were Natural Killer Cell and Wnt/b-Catenin signaling. Local trophoblast was associated with much less alteration of endometrial gene expression with an increase in 56 genes, including CSH1 (8-fold), and a reduction in 29 genes including CRISP3 (8-fold). The top associated canonical pathway was Antigen Presentation. The study of endometrium from tubal EP may promote novel insights into genes involved in decidualization and those influenced by factors from neighboring trophoblast. This has afforded unique information not highlighted by previous studies and adds to our understanding of the endometrium in early pregnancy. PMID:21858178

  5. Filaggrin-stratified transcriptomic analysis of pediatric skin identifies mechanistic pathways in patients with atopic dermatitis

    PubMed Central

    Cole, Christian; Kroboth, Karin; Schurch, Nicholas J.; Sandilands, Aileen; Sherstnev, Alexander; O'Regan, Grainne M.; Watson, Rosemarie M.; Irwin McLean, W.H.; Barton, Geoffrey J.; Irvine, Alan D.; Brown, Sara J.

    2014-01-01

    Background Atopic dermatitis (AD; eczema) is characterized by a widespread abnormality in cutaneous barrier function and propensity to inflammation. Filaggrin is a multifunctional protein and plays a key role in skin barrier formation. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a highly significant risk factor for atopic disease, but the molecular mechanisms leading to dermatitis remain unclear. Objective We sought to interrogate tissue-specific variations in the expressed genome in the skin of children with AD and to investigate underlying pathomechanisms in atopic skin. Methods We applied single-molecule direct RNA sequencing to analyze the whole transcriptome using minimal tissue samples. Uninvolved skin biopsy specimens from 26 pediatric patients with AD were compared with site-matched samples from 10 nonatopic teenage control subjects. Cases and control subjects were screened for FLG genotype to stratify the data set. Results Two thousand four hundred thirty differentially expressed genes (false discovery rate, P< .05) were identified, ofwhich 211 were significantly upregulated and 490 downregulated by greater than 2-fold. Gene ontology terms for extracellular space and defense response were enriched, whereas lipid metabolic processes were downregulated. The subset of FLG wild-type cases showed dysregulation of genes involved with lipid metabolism, whereas filaggrin haploinsufficiency affected global gene expression and was characterized by a type 1 interferonmediated stress response. Conclusion These analyses demonstrate the importance of extracellular space and lipid metabolism in atopic skin pathology independent of FLG genotype, whereas an aberrant defense response is seen in subjects with FLG mutations. Genotype stratification of the large data set has facilitated functional interpretation and might guide future therapy development. PMID:24880632

  6. Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators.

    PubMed

    Hieronymus, Haley; Lamb, Justin; Ross, Kenneth N; Peng, Xiao P; Clement, Cristina; Rodina, Anna; Nieto, Maria; Du, Jinyan; Stegmaier, Kimberly; Raj, Srilakshmi M; Maloney, Katherine N; Clardy, Jon; Hahn, William C; Chiosis, Gabriela; Golub, Todd R

    2006-10-01

    Although androgen receptor (AR)-mediated signaling is central to prostate cancer, the ability to modulate AR signaling states is limited. Here we establish a chemical genomic approach for discovery and target prediction of modulators of cancer phenotypes, as exemplified by AR signaling. We first identify AR activation inhibitors, including a group of structurally related compounds comprising celastrol, gedunin, and derivatives. To develop an in silico approach for target pathway identification, we apply a gene expression-based analysis that classifies HSP90 inhibitors as having similar activity to celastrol and gedunin. Validating this prediction, we demonstrate that celastrol and gedunin inhibit HSP90 activity and HSP90 clients, including AR. Broadly, this work identifies new modes of HSP90 modulation through a gene expression-based strategy. PMID:17010675

  7. Using Ambystoma mexicanum (Mexican axolotl) embryos, chemical genetics, and microarray analysis to identify signaling pathways associated with tissue regeneration.

    PubMed

    Ponomareva, Larissa V; Athippozhy, Antony; Thorson, Jon S; Voss, S Randal

    2015-12-01

    Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies, and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here, we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration. PMID:26092703

  8. Label-free Quantitative Urinary Proteomics Identifies the Arginase Pathway as a New Player in Congenital Obstructive Nephropathy*

    PubMed Central

    Lacroix, Chrystelle; Caubet, Cécile; Gonzalez-de-Peredo, Anne; Breuil, Benjamin; Bouyssié, David; Stella, Alexandre; Garrigues, Luc; Le Gall, Caroline; Raevel, Anthony; Massoubre, Angelique; Klein, Julie; Decramer, Stéphane; Sabourdy, Frédérique; Bandin, Flavio; Burlet-Schiltz, Odile; Monsarrat, Bernard; Schanstra, Joost-Peter; Bascands, Jean-Loup

    2014-01-01

    Obstructive nephropathy is a frequently encountered situation in newborns. In previous studies, the urinary peptidome has been analyzed for the identification of clinically useful biomarkers of obstructive nephropathy. However, the urinary proteome has not been explored yet and should allow additional insight into the pathophysiology of the disease. We have analyzed the urinary proteome of newborns (n = 5/group) with obstructive nephropathy using label free quantitative nanoLC-MS/MS allowing the identification and quantification of 970 urinary proteins. We next focused on proteins exclusively regulated in severe obstructive nephropathy and identified Arginase 1 as a potential candidate molecule involved in the development of obstructive nephropathy, located at the crossroad of pro- and antifibrotic pathways. The reduced urinary abundance of Arginase 1 in obstructive nephropathy was verified in independent clinical samples using both Western blot and MRM analysis. These data were confirmed in situ in kidneys obtained from a mouse obstructive nephropathy model. In addition, we also observed increased expression of Arginase 2 and increased total arginase activity in obstructed mouse kidneys. mRNA expression analysis of the related arginase pathways indicated that the pro-fibrotic arginase-related pathway is activated during obstructive nephropathy. Taken together we have identified a new actor in the development of obstructive nephropathy in newborns using quantitative urinary proteomics and shown its involvement in an in vivo model of disease. The present study demonstrates the relevance of such a quantitative urinary proteomics approach with clinical samples for a better understanding of the pathophysiology and for the discovery of potential therapeutic targets. PMID:25205225

  9. Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing

    PubMed Central

    Sadsad, Rosemarie; Martinez, Elena; Jelfs, Peter; Hill-Cawthorne, Grant A.; Gilbert, Gwendolyn L.; Marais, Ben J.; Sintchenko, Vitali

    2016-01-01

    Background Improved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways. Methods We sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants. Results Isolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade. Conclusion Whole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster. PMID:26938641

  10. Comparative transcriptome profiling of the injured zebrafish and mouse hearts identifies miRNA-dependent repair pathways

    PubMed Central

    Crippa, Stefania; Nemir, Mohamed; Ounzain, Samir; Ibberson, Mark; Berthonneche, Corinne; Sarre, Alexandre; Boisset, Gaëlle; Maison, Damien; Harshman, Keith; Xenarios, Ioannis; Diviani, Dario; Schorderet, Daniel; Pedrazzini, Thierry

    2016-01-01

    Aims The adult mammalian heart has poor regenerative capacity. In contrast, the zebrafish heart retains a robust capacity for regeneration into adulthood. These distinct responses are consequences of a differential utilization of evolutionary-conserved gene regulatory networks in the damaged heart. To systematically identify miRNA-dependent networks controlling cardiac repair following injury, we performed comparative gene and miRNA profiling of the cardiac transcriptome in adult mice and zebrafish. Methods and results Using an integrated approach, we show that 45 miRNA-dependent networks, involved in critical biological pathways, are differentially modulated in the injured zebrafish vs. mouse hearts. We study, more particularly, the miR-26a-dependent response. Therefore, miR-26a is down-regulated in the fish heart after injury, whereas its expression remains constant in the mouse heart. Targets of miR-26a involve activators of the cell cycle and Ezh2, a component of the polycomb repressive complex 2 (PRC2). Importantly, PRC2 exerts repressive functions on negative regulators of the cell cycle. In cultured neonatal cardiomyocytes, inhibition of miR-26a stimulates, therefore, cardiomyocyte proliferation. Accordingly, miR-26a knockdown prolongs the proliferative window of cardiomyocytes in the post-natal mouse heart. Conclusions This novel strategy identifies a series of miRNAs and associated pathways, in particular miR-26a, which represent attractive therapeutic targets for inducing repair in the injured heart. PMID:26857418

  11. Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use.

    PubMed

    Sun, Jing; Li, Ning; Oh, Kyu-Seon; Dutta, Bhaskar; Vayttaden, Sharat J; Lin, Bin; Ebert, Thomas S; De Nardo, Dominic; Davis, Joie; Bagirzadeh, Rustam; Lounsbury, Nicolas W; Pasare, Chandrashekhar; Latz, Eicke; Hornung, Veit; Fraser, Iain D C

    2016-01-01

    Toll-like receptors (TLRs) are a major class of pattern recognition receptors, which mediate the responses of innate immune cells to microbial stimuli. To systematically determine the roles of proteins in canonical TLR signaling pathways, we conducted an RNA interference (RNAi)-based screen in human and mouse macrophages. We observed a pattern of conserved signaling module dependencies across species, but found notable species-specific requirements at the level of individual proteins. Among these, we identified unexpected differences in the involvement of members of the interleukin-1 receptor-associated kinase (IRAK) family between the human and mouse TLR pathways. Whereas TLR signaling in mouse macrophages depended primarily on IRAK4 and IRAK2, with little or no role for IRAK1, TLR signaling and proinflammatory cytokine production in human macrophages depended on IRAK1, with knockdown of IRAK4 or IRAK2 having less of an effect. Consistent with species-specific roles for these kinases, IRAK4 orthologs failed to rescue signaling in IRAK4-deficient macrophages from the other species, and only mouse macrophages required the kinase activity of IRAK4 to mediate TLR responses. The identification of a critical role for IRAK1 in TLR signaling in humans could potentially explain the association of IRAK1 with several autoimmune diseases. Furthermore, this study demonstrated how systematic screening can be used to identify important characteristics of innate immune responses across species, which could optimize therapeutic targeting to manipulate human TLR-dependent outputs. PMID:26732763

  12. Adaptative biochemical pathways and regulatory networks in Klebsiella oxytoca BAS-10 producing a biotechnologically relevant exopolysaccharide during Fe(III)-citrate fermentation

    PubMed Central

    2012-01-01

    Background A bacterial strain previously isolated from pyrite mine drainage and named BAS-10 was tentatively identified as Klebsiella oxytoca. Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) and showing unusual physiological characteristics. In fact, under this growth condition, BAS-10 produces an exopolysaccharide (EPS) having a high rhamnose content and metal-binding properties, whose biotechnological applications were proven as very relevant. Results Further phylogenetic analysis, based on 16S rDNA sequence, definitively confirmed that BAS-10 belongs to K. oxytoca species. In order to rationalize the biochemical peculiarities of this unusual enterobacteriun, combined 2D-Differential Gel Electrophoresis (2D-DIGE) analysis and mass spectrometry procedures were used to investigate its proteomic changes: i) under aerobic or anaerobic cultivation with Fe(III)-citrate as sole carbon source; ii) under anaerobic cultivations using Na(I)-citrate or Fe(III)-citrate as sole carbon source. Combining data from these differential studies peculiar levels of outer membrane proteins, key regulatory factors of carbon and nitrogen metabolism and enzymes involved in TCA cycle and sugar biosynthesis or required for citrate fermentation and stress response during anaerobic growth on Fe(III)-citrate were revealed. The protein differential regulation seems to ensure efficient cell growth coupled with EPS production by adapting metabolic and biochemical processes in order to face iron toxicity and to optimize energy production. Conclusion Differential proteomics provided insights on the molecular mechanisms necessary for anaeorobic utilization of Fe(III)-citrate in a biotechnologically promising enterobacteriun, also revealing genes that can be targeted for the rational design of high-yielding EPS producer strains. PMID:23176641

  13. A Pathway Closely Related to the d-Tagatose Pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis

    PubMed Central

    Van der Heiden, Edwige; Lebrun, Sarah; Freichels, Rgine; Brans, Alain; Vastenavond, Christian M.; Galleni, Moreno; Joris, Bernard

    2013-01-01

    We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. PMID:23524682

  14. A pathway-based approach for identifying biomarkers of tumor progression to trastuzumab-resistant breast cancer.

    PubMed

    Nam, Seungyoon; Chang, Hae Ryung; Jung, Hae Rim; Gim, Youme; Kim, Nam Youl; Grailhe, Regis; Seo, Haeng Ran; Park, Hee Seo; Balch, Curt; Lee, Jinhyuk; Park, Inhae; Jung, So Youn; Jeong, Kyung-Chae; Powis, Garth; Liang, Han; Lee, Eun Sook; Ro, Jungsil; Kim, Yon Hui

    2015-01-28

    Although trastuzumab is a successful targeted therapy for breast cancer patients with tumors expressing HER2 (ERBB2), many patients eventually progress to drug resistance. Here, we identified subpathways differentially expressed between trastuzumab-resistant vs. -sensitive breast cancer cells, in conjunction with additional transcriptomic preclinical and clinical gene datasets, to rigorously identify overexpressed, resistance-associated genes. From this approach, we identified 32 genes reproducibly upregulated in trastuzumab resistance. 25 genes were upregulated in drug-resistant JIMT-1 cells, which also downregulated HER2 protein by >80% in the presence of trastuzumab. 24 genes were downregulated in trastuzumab-sensitive SKBR3 cells. Trastuzumab sensitivity was restored by siRNA knockdown of these genes in the resistant cells, and overexpression of 5 of the 25 genes was found in at least one of five refractory HER2?+?breast cancer. In summary, our rigorous computational approach, followed by experimental validation, significantly implicate ATF4, CHEK2, ENAH, ICOSLG, and RAD51 as potential biomarkers of trastuzumab resistance. These results provide further proof-of-concept of our methodology for successfully identifying potential biomarkers and druggable signal pathways involved in tumor progression to drug resistance. PMID:25449779

  15. Transcriptomic Analysis in a Drosophila Model Identifies Previously Implicated and Novel Pathways in the Therapeutic Mechanism in Neuropsychiatric Disorders

    PubMed Central

    Singh, Priyanka; Mohammad, Farhan; Sharma, Abhay

    2011-01-01

    We have taken advantage of a newly described Drosophila model to gain insights into the potential mechanism of antiepileptic drugs (AEDs), a group of drugs that are widely used in the treatment of several neurological and psychiatric conditions besides epilepsy. In the recently described Drosophila model that is inspired by pentylenetetrazole (PTZ) induced kindling epileptogenesis in rodents, chronic PTZ treatment for 7?days causes a decreased climbing speed and an altered CNS transcriptome, with the latter mimicking gene expression alterations reported in epileptogenesis. In the model, an increased climbing speed is further observed 7?days after withdrawal from chronic PTZ. We used this post-PTZ withdrawal regime to identify potential AED mechanism. In this regime, treatment with each of the five AEDs tested, namely, ethosuximide, gabapentin, vigabatrin, sodium valproate, and levetiracetam, resulted in rescuing of the altered climbing behavior. The AEDs also normalized PTZ withdrawal induced transcriptomic perturbation in fly heads; whereas AED untreated flies showed a large number of up- and down-regulated genes which were enriched in several processes including gene expression and cell communication, the AED treated flies showed differential expression of only a small number of genes that did not enrich gene expression and cell communication processes. Gene expression and cell communication related upregulated genes in AED untreated flies overrepresented several pathways spliceosome, RNA degradation, and ribosome in the former category, and inositol phosphate metabolism, phosphatidylinositol signaling, endocytosis, and hedgehog signaling in the latter. Transcriptome remodeling effect of AEDs was overall confirmed by microarray clustering that clearly separated the profiles of AED treated and untreated flies. Besides being consistent with previously implicated pathways, our results provide evidence for a role of other pathways in psychiatric drug mechanism. Overall, we provide an amenable model to understand neuropsychiatric mechanism in cellular and molecular terms. PMID:21503142

  16. Biochemical characterization and selective inhibition of ?-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway.

    PubMed

    Harrison, Peter J; Newgas, Sophie A; Descombes, Flora; Shepherd, Sarah A; Thompson, Andrew J; Bugg, Timothy D H

    2015-10-01

    The first three enzymatic steps of the strigolactone biosynthetic pathway catalysed by ?-carotene cis-trans isomerase Dwarf27 (D27) from Oryza sativa and carotenoid cleavage dioxygenases CCD7 and CCD8 from Arabidopsis thaliana have been reconstituted in vitro, and kinetic assays have been developed for each enzyme, in order to develop selective enzyme inhibitors. Recombinant OsD27 shows a UV-visible ?max at 422 nm and is inactivated by silver(I) acetate, consistent with the presence of an iron-sulfur cluster that is used in catalysis. OsD27 and AtCCD7 are not inhibited by hydroxamic acids that cause shoot branching in planta, but OsD27 is partially inhibited by terpene-like hydroxamic acids. The reaction catalysed by AtCCD8 is shown to be a two-step kinetic mechanism using pre-steady-state kinetic analysis. Kinetic evidence is presented for acid-base catalysis in the CCD8 catalytic cycle and the existence of an essential cysteine residue in the CCD8 active site. AtCCD8 is inhibited in a time-dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 ?m) that cause a shoot branching phenotype in A. thaliana, and selective inhibition of CCD8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 ?m). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8. PMID:26257333

  17. Genetic and Biochemical Characterization of a 2-Pyrone-4,6-Dicarboxylic Acid Hydrolase Involved in the Protocatechuate 4,5-Cleavage Pathway of Sphingomonas paucimobilis SYK-6

    PubMed Central

    Masai, Eiji; Shinohara, Shouji; Hara, Hirofumi; Nishikawa, Seiji; Katayama, Yoshihiro; Fukuda, Masao

    1999-01-01

    Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds with guaiacyl moieties, which are converted into protocatechuate by the actions of lignin degradation enzymes in this strain. Protocatechuate is a key metabolite in the SYK-6 degradation of lignin compounds with guaiacyl moieties, and it is thought that it degrades to pyruvate and oxaloacetate via the protocatechuate 4,5-cleavage pathway. In a 10.5-kb EcoRI fragment carrying the protocatechuate 4,5-dioxygenase gene (ligAB) (Y. Noda, S. Nishikawa, K. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. J. Bacteriol. 172:2704–2709, 1990), we found the ligI gene encoding 2-pyrone-4,6-dicarboxylic acid (PDC) hydrolase. PDC hydrolase is a member of this pathway and catalyzes the interconversion between PDC and 4-carboxy-2-hydroxymuconic acid (CHM). The ligI gene is thought to be transcribed divergently from ligAB and consists of an 879-bp open reading frame encoding a polypeptide with a molecular mass of 32,737 Da. The ligI gene product (LigI), expressed in Escherichia coli, was purified to near-homogeneity and was estimated to be a monomer (31.6 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9. The optimum pH for hydrolysis of PDC is 8.5, the optimum pH for synthesis of PDC is 6.0 to 7.5, and the Km values for PDC and CHM are 74 and 49 μM, respectively. LigI activity was inhibited by the addition of thiol reagents, suggesting that the cysteine residue is a catalytic site. LigI is more resistant to metal ion inhibition than the PDC hydrolases of Pseudomonas ochraceae (K. Maruyama, J. Biochem. 93:557–565, 1983) and Comamonas testosteroni (P. J. Kersten, S. Dagley, J. W. Whittaker, D. M. Arciero, and J. D. Lipscomb, J. Bacteriol. 152:1154–1162, 1982). The insertional inactivation of the ligI gene in S. paucimobilis SYK-6 led to the complete loss of PDC hydrolase activity and to a growth defect on vanillic acid; it did not affect growth on syringic acid. These results indicate that the ligI gene is essential for the growth of SYK-6 on vanillic acid but is not responsible for the growth of SYK-6 on syringic acid. PMID:9864312

  18. Long-term consequences of pubertal timing for youth depression: Identifying personal and contextual pathways of risk

    PubMed Central

    RUDOLPH, KAREN D.; TROOP-GORDON, WENDY; LAMBERT, SHARON F.; NATSUAKI, MISAKI N.

    2015-01-01

    This research explored sex differences in the pathways linking pubertal timing to depression across 4 years. A sample of 167 youth (M age = 12.41 years, SD = 1.19) and their caregivers completed measures of puberty and semistructured interviews of interpersonal stress and youth depression. Youth reported on psychological (negative self-focus, anxious arousal) and social–behavioral (coping) characteristics; parents reported on youths’ social–behavioral characteristics (withdrawal/social problems) and deviant peer affiliations. Early maturation predicted stable high trajectories of depression in girls; although early maturing boys showed low initial levels of depression, they did not differ from girls by the final wave of the study. Latent growth curve analyses identified several psychological, social–behavioral, and interpersonal pathways accounting for the contribution of pubertal timing to initial and enduring risk for depression in girls as well as emerging risk for depression in boys. These findings provide novel insight into multilevel processes accounting for sex differences in depression across the adolescent transition. PMID:25422971

  19. Identifying Yersinia YopH-targeted signal transduction pathways that impair neutrophil responses during in vivo murine infection

    PubMed Central

    Rolán, Hortensia G.; Durand, Enrique A.; Mecsas, Joan

    2013-01-01

    Summary Identifying molecular targets of Yersinia virulence effectors, or Yops, during animal infection is challenging because few cells are targeted by Yops in an infected organ and isolating these sparse effector-containing cells is difficult. YopH, a tyrosine phosphatase, is essential for full virulence of Yersinia. Investigating the YopH-targeted signal-transduction pathway(s) in neutrophils during infection of a murine host, we find that several host proteins, including the essential signaling adapter SLP-76, are dephosphorylated in the presence of YopH in neutrophils isolated from infected tissues. YopH inactivated PRAM-1/SKAP-HOM and the SLP-76/Vav/PLCγ2 signal-transduction axes, leading to an inhibition of calcium response in isolated neutrophils. Consistent with a failure to mount a calcium response, IL-10 production was reduced in neutrophils containing YopH from infected tissues. Finally, a yopH mutant survived better in the absence of neutrophils, indicating that neutrophil inactivation by YopH by targeting PRAM-1/SKAP-HOM and SLP-76/Vav/PLCγ2 signaling hubs may be critical for Yersinia survival. PMID:24034616

  20. Neuronal Expression of the Human Neuropeptide S Receptor NPSR1 Identifies NPS-Induced Calcium Signaling Pathways

    PubMed Central

    Erdmann, Frank; Kgler, Sebastian; Blaesse, Peter; Lange, Maren D.; Skryabin, Boris V.; Pape, Hans-Christian; Jngling, Kay

    2015-01-01

    The neuropeptide S (NPS) system was discovered as a novel neurotransmitter system a decade ago and has since been identified as a key player in the modulation of fear and anxiety. Genetic variations of the human NPS receptor (NPSR1) have been associated with pathologies like panic disorders. However, details on the molecular fundamentals of NPSR1 activity in neurons remained elusive. We expressed NPSR1 in primary hippocampal cultures. Using single-cell calcium imaging we found that NPSR1 stimulation induced calcium mobilization from the endoplasmic reticulum via activation of IP3 and ryanodine receptors. Store-operated calcium channels were activated in a downstream process mediating entry of extracellular calcium. We provide the first detailed analysis of NPSR1 activity and the underlying intracellular pathways with respect to calcium mobilization in neurons. PMID:25714705

  1. Pathway-Centric Integrative Analysis Identifies RRM2 as a Prognostic Marker in Breast Cancer Associated with Poor Survival and Tamoxifen Resistance123

    PubMed Central

    Putluri, Nagireddy; Maity, Suman; Kommangani, Ramakrishna; Creighton, Chad J.; Putluri, Vasanta; Chen, Fengju; Nanda, Sarmishta; Bhowmik, Salil Kumar; Terunuma, Atsushi; Dorsey, Tiffany; Nardone, Agostina; Fu, Xiaoyong; Shaw, Chad; Sarkar, Tapasree Roy; Schiff, Rachel; Lydon, John P.; OMalley, Bert W.; Ambs, Stefan; Das, Gokul M.; Michailidis, George; Sreekumar, Arun

    2014-01-01

    Breast cancer (BCa) molecular subtypes include luminal A, luminal B, normal-like, HER-2enriched, and basal-like tumors, among which luminal B and basal-like cancers are highly aggressive. Biochemical pathways associated with patient survival or treatment response in these more aggressive subtypes are not well understood. With the limited availability of pathologically verified clinical specimens, cell line models are routinely used for pathway-centric studies. We measured the metabolome of luminal and basal-like BCa cell lines using mass spectrometry, linked metabolites to biochemical pathways using Gene Set Analysis, and developed a novel rank-based method to select pathways on the basis of their enrichment in patient-derived omics data sets and prognostic relevance. Key mediators of the pathway were then characterized for their role in disease progression. Pyrimidine metabolism was altered in luminal versus basal BCa, whereas the combined expression of its associated genes or expression of one key gene, ribonucleotide reductase subunit M2 (RRM2) alone, associated significantly with decreased survival across all BCa subtypes, as well as in luminal patients resistant to tamoxifen. Increased RRM2 expression in tamoxifen-resistant patients was verified using tissue microarrays, whereas the metabolic products of RRM2 were higher in tamoxifen-resistant cells and in xenograft tumors. Both genetic and pharmacological inhibition of this key enzyme in tamoxifen-resistant cells significantly decreased proliferation, reduced expression of cell cycle genes, and sensitized the cells to tamoxifen treatment. Our study suggests for evaluating RRM2-associated metabolites as noninvasive markers for tamoxifen resistance and its pharmacological inhibition as a novel approach to overcome tamoxifen resistance in BCa. PMID:25016594

  2. DrGaP: A Powerful Tool for Identifying Driver Genes and Pathways in Cancer Sequencing Studies

    PubMed Central

    Hua, Xing; Xu, Haiming; Yang, Yaning; Zhu, Jun; Liu, Pengyuan; Lu, Yan

    2013-01-01

    Cancers are caused by the accumulation of genomic alterations. Driver mutations are required for the cancer phenotype, whereas passenger mutations are irrelevant to tumor development and accumulate through DNA replication. A major challenge facing the field of cancer genome sequencing is to identify cancer-associated genes with mutations that drive the cancer phenotype. Here, we describe a powerful and flexible statistical framework for identifying driver genes and driver signaling pathways in cancer genome-sequencing studies. Biological knowledge of the mutational process in tumors is fully integrated into our statistical models and includes such variables as the length of protein-coding regions, transcript isoforms, variation in mutation types, differences in background mutation rates, the redundancy of genetic code, and multiple mutations in one gene. This framework provides several significant features that are not addressed or naively obtained by previous methods. In particular, on the observation of low prevalence of somatic mutations in individual tumors, we propose a heuristic strategy to estimate the mixture proportion of chi-square distribution of likelihood ratio test (LRT) statistics. This provides significantly increased statistical power compared to regular LRT. Through a combination of simulation and analysis of TCGA cancer sequencing study data, we demonstrate high accuracy and sensitivity in our methods. Our statistical methods and several auxiliary bioinformatics tools have been incorporated into a computational tool, DrGaP. The newly developed tool is immediately applicable to cancer genome-sequencing studies and will lead to a more complete identification of altered driver genes and driver signaling pathways in cancer. PMID:23954162

  3. Pathway-based analysis of GWAs data identifies association of sex determination genes with susceptibility to testicular germ cell tumors.

    PubMed

    Koster, Roelof; Mitra, Nandita; D'Andrea, Kurt; Vardhanabhuti, Saran; Chung, Charles C; Wang, Zhaoming; Loren Erickson, R; Vaughn, David J; Litchfield, Kevin; Rahman, Nazneen; Greene, Mark H; McGlynn, Katherine A; Turnbull, Clare; Chanock, Stephen J; Nathanson, Katherine L; Kanetsky, Peter A

    2014-11-15

    Genome-wide association (GWA) studies of testicular germ cell tumor (TGCT) have identified 18 susceptibility loci, some containing genes encoding proteins important in male germ cell development. Deletions of one of these genes, DMRT1, lead to male-to-female sex reversal and are associated with development of gonadoblastoma. To further explore genetic association with TGCT, we undertook a pathway-based analysis of SNP marker associations in the Penn GWAs (349 TGCT cases and 919 controls). We analyzed a custom-built sex determination gene set consisting of 32 genes using three different methods of pathway-based analysis. The sex determination gene set ranked highly compared with canonical gene sets, and it was associated with TGCT (FDRG = 2.28 10(-5), FDRM = 0.014 and FDRI = 0.008 for Gene Set Analysis-SNP (GSA-SNP), Meta-Analysis Gene Set Enrichment of Variant Associations (MAGENTA) and Improved Gene Set Enrichment Analysis for Genome-wide Association Study (i-GSEA4GWAS) analysis, respectively). The association remained after removal of DMRT1 from the gene set (FDRG = 0.0002, FDRM = 0.055 and FDRI = 0.009). Using data from the NCI GWA scan (582 TGCT cases and 1056 controls) and UK scan (986 TGCT cases and 4946 controls), we replicated these findings (NCI: FDRG = 0.006, FDRM = 0.014, FDRI = 0.033, and UK: FDRG = 1.04 10(-6), FDRM = 0.016, FDRI = 0.025). After removal of DMRT1 from the gene set, the sex determination gene set remains associated with TGCT in the NCI (FDRG = 0.039, FDRM = 0.050 and FDRI = 0.055) and UK scans (FDRG = 3.00 10(-5), FDRM = 0.056 and FDRI = 0.044). With the exception of DMRT1, genes in the sex determination gene set have not previously been identified as TGCT susceptibility loci in these GWA scans, demonstrating the complementary nature of a pathway-based approach for genome-wide analysis of TGCT. PMID:24943593

  4. Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: effects of CREB pathway inhibition.

    PubMed

    Pistollato, Francesca; Louisse, Jochem; Scelfo, Bibiana; Mennecozzi, Milena; Accordi, Benedetta; Basso, Giuseppe; Gaspar, John Antonydas; Zagoura, Dimitra; Barilari, Manuela; Palosaari, Taina; Sachinidis, Agapios; Bremer-Hoffmann, Susanne

    2014-10-15

    According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro. Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2(+) neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations. PMID:25150140

  5. Mutations in PRDM5 in Brittle Cornea Syndrome Identify a Pathway Regulating Extracellular Matrix Development and Maintenance

    PubMed Central

    Burkitt Wright, EmmaM.M.; Spencer, HelenL.; Daly, SarahB.; Manson, ForbesD.C.; Zeef, LeoA.H.; Urquhart, Jill; Zoppi, Nicoletta; Bonshek, Richard; Tosounidis, Ioannis; Mohan, Meyyammai; Madden, Colm; Dodds, Annabel; Chandler, KateE.; Banka, Siddharth; Au, Leon; Clayton-Smith, Jill; Khan, Naz; Biesecker, LeslieG.; Wilson, Meredith; Rohrbach, Marianne; Colombi, Marina; Giunta, Cecilia; Black, GraemeC.M.

    2011-01-01

    Extreme corneal fragility and thinning, which have a high risk of catastrophic spontaneous rupture, are the cardinal features of brittle cornea syndrome (BCS), an autosomal-recessive generalized connective tissue disorder. Enucleation is frequently the only management option for this condition, resulting in blindness and psychosocial distress. Even when the cornea remains grossly intact, visual function could also be impaired by a high degree of myopia and keratoconus. Deafness is another common feature and results in combined sensory deprivation. Using autozygosity mapping, we identified mutations in PRDM5 in families with BCS. We demonstrate that regulation of expression of extracellular matrix components, particularly fibrillar collagens, by PRDM5 is a key molecular mechanism that underlies corneal fragility in BCS and controls normal corneal development and maintenance. ZNF469, encoding a zinc finger protein of hitherto undefined function, has been identified as a quantitative trait locus for central corneal thickness, and mutations in this gene have been demonstrated in Tunisian Jewish and Palestinian kindreds with BCS. We show that ZNF469 and PRDM5, two genes that when mutated cause BCS, participate in the same regulatory pathway. PMID:21664999

  6. Mutations in PRDM5 in brittle cornea syndrome identify a pathway regulating extracellular matrix development and maintenance.

    PubMed

    Burkitt Wright, Emma M M; Spencer, Helen L; Daly, Sarah B; Manson, Forbes D C; Zeef, Leo A H; Urquhart, Jill; Zoppi, Nicoletta; Bonshek, Richard; Tosounidis, Ioannis; Mohan, Meyyammai; Madden, Colm; Dodds, Annabel; Chandler, Kate E; Banka, Siddharth; Au, Leon; Clayton-Smith, Jill; Khan, Naz; Biesecker, Leslie G; Wilson, Meredith; Rohrbach, Marianne; Colombi, Marina; Giunta, Cecilia; Black, Graeme C M

    2011-06-10

    Extreme corneal fragility and thinning, which have a high risk of catastrophic spontaneous rupture, are the cardinal features of brittle cornea syndrome (BCS), an autosomal-recessive generalized connective tissue disorder. Enucleation is frequently the only management option for this condition, resulting in blindness and psychosocial distress. Even when the cornea remains grossly intact, visual function could also be impaired by a high degree of myopia and keratoconus. Deafness is another common feature and results in combined sensory deprivation. Using autozygosity mapping, we identified mutations in PRDM5 in families with BCS. We demonstrate that regulation of expression of extracellular matrix components, particularly fibrillar collagens, by PRDM5 is a key molecular mechanism that underlies corneal fragility in BCS and controls normal corneal development and maintenance. ZNF469, encoding a zinc finger protein of hitherto undefined function, has been identified as a quantitative trait locus for central corneal thickness, and mutations in this gene have been demonstrated in Tunisian Jewish and Palestinian kindreds with BCS. We show that ZNF469 and PRDM5, two genes that when mutated cause BCS, participate in the same regulatory pathway. PMID:21664999

  7. Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway.

    PubMed

    Xing, Mengtan; Yang, Mingrui; Huo, Wei; Feng, Feng; Wei, Leizhen; Jiang, Wenxia; Ning, Shaokai; Yan, Zhenxin; Li, Wen; Wang, Qingsong; Hou, Mei; Dong, Chunxia; Guo, Rong; Gao, Ge; Ji, Jianguo; Zha, Shan; Lan, Li; Liang, Huanhuan; Xu, Dongyi

    2015-01-01

    Non-homologous end joining (NHEJ) is a major pathway to repair DNA double-strand breaks (DSBs), which can display different types of broken ends. However, it is unclear how NHEJ factors organize to repair diverse types of DNA breaks. Here, through systematic analysis of the human NHEJ factor interactome, we identify PAXX as a direct interactor of Ku. The crystal structure of PAXX is similar to those of XRCC4 and XLF. Importantly, PAXX-deficient cells are sensitive to DSB-causing agents. Moreover, epistasis analysis demonstrates that PAXX functions together with XLF in response to ionizing radiation-induced complex DSBs, whereas they function redundantly in response to Topo2 inhibitor-induced simple DSBs. Consistently, PAXX and XLF coordinately promote the ligation of complex but not simple DNA ends in vitro. Altogether, our data identify PAXX as a new NHEJ factor and provide insight regarding the organization of NHEJ factors responding to diverse types of DSB ends. PMID:25670504

  8. Systems-Based Analysis of the Sarcocystis neurona Genome Identifies Pathways That Contribute to a Heteroxenous Life Cycle

    PubMed Central

    Blazejewski, Tomasz; Nursimulu, Nirvana; Pszenny, Viviana; Dangoudoubiyam, Sriveny; Namasivayam, Sivaranjani; Chiasson, Melissa A.; Chessman, Kyle; Tonkin, Michelle; Swapna, Lakshmipuram S.; Hung, Stacy S.; Bridgers, Joshua; Ricklefs, Stacy M.; Boulanger, Martin J.; Dubey, Jitender P.; Porcella, Stephen F.; Kissinger, Jessica C.; Howe, Daniel K.

    2015-01-01

    ABSTRACT Sarcocystis neurona is a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystis is one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystis species. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many dense-granule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts. PMID:25670772

  9. Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway

    PubMed Central

    Xing, Mengtan; Yang, Mingrui; Huo, Wei; Feng, Feng; Wei, Leizhen; Jiang, Wenxia; Ning, Shaokai; Yan, Zhenxin; Li, Wen; Wang, Qingsong; Hou, Mei; Dong, Chunxia; Guo, Rong; Gao, Ge; Ji, Jianguo; Zha, Shan; Lan, Li; Liang, Huanhuan; Xu, Dongyi

    2015-01-01

    Non-homologous end joining (NHEJ) is a major pathway to repair DNA double-strand breaks (DSBs), which can display different types of broken ends. However, it is unclear how NHEJ factors organize to repair diverse types of DNA breaks. Here, through systematic analysis of the human NHEJ factor interactome, we identify PAXX as a direct interactor of Ku. The crystal structure of PAXX is similar to those of XRCC4 and XLF. Importantly, PAXX-deficient cells are sensitive to DSB-causing agents. Moreover, epistasis analysis demonstrates that PAXX functions together with XLF in response to ionizing radiation-induced complex DSBs, whereas they function redundantly in response to Topo2 inhibitor-induced simple DSBs. Consistently, PAXX and XLF coordinately promote the ligation of complex but not simple DNA ends in vitro. Altogether, our data identify PAXX as a new NHEJ factor and provide insight regarding the organization of NHEJ factors responding to diverse types of DSB ends. PMID:25670504

  10. Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma

    PubMed Central

    Guichard, Ccile; Amaddeo, Giuliana; Imbeaud, Sandrine; Ladeiro, Yannick; Pelletier, Laura; Maad, Ichrafe Ben; Calderaro, Julien; Bioulac-Sage, Paulette; Letexier, Mlanie; Degos, Franoise; Clment, Bruno; Balabaud, Charles; Chevet, Eric; Laurent, Alexis; Couchy, Gabrielle; Letouz, Eric; Calvo, Fabien; Zucman-Rossi, Jessica

    2012-01-01

    Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. High-resolution copy number analysis of 125 tumors of which 24 were subjected to whole-exome sequencing identified 135 homozygous deletions and 994 somatic gene mutations with predicted functional consequences. We identified new recurrent alterations in 6 genes (ARID1A, RPS6KA3, NFE2L2, IRF2, CDH8 and PROKR2) not previously described in HCC. Functional analyses demonstrated tumor suppressor properties for IRF2 whose inactivation, exclusively found in hepatitis B virus related tumors, leads to impaired TP53 function. Alternatively, inactivation of proteins involved in chromatin remodeling was frequent and predominant in alcohol related tumors. Moreover, activation of the oxidative stress metabolism and inactivation of RPS6KA3 were new pathways associated with WNT/?-catenin activation, thereby suggesting a cooperative effect in tumorigenesis. This study shows the dramatic somatic genetic diversity in HCC, it reveals interactions between oncogene and tumor suppressor gene mutations markedly related to specific risk factors. PMID:22561517

  11. A mouse mammary gland involution mRNA signature identifies biological pathways potentially associated with breast cancer metastasis.

    PubMed

    Stein, Torsten; Salomonis, Nathan; Nuyten, Dimitry S A; van de Vijver, Marc J; Gusterson, Barry A

    2009-06-01

    Mouse mammary gland involution resembles a wound healing response with suppressed inflammation. Wound healing and inflammation are also associated with tumour development, and a 'wound-healing' gene expression signature can predict metastasis formation and survival. Recent studies have shown that an involuting mammary gland stroma can promote metastasis. It could therefore be hypothesised that gene expression signatures from an involuting mouse mammary gland may provide new insights into the physiological pathways that promote breast cancer progression. Indeed, using the HOPACH clustering method, the human orthologues of genes that were differentially regulated at day 3 of mammary gland involution and showed prolonged expression throughout the first 4 days of involution distinguished breast cancers in the NKI 295 breast cancer dataset with low and high metastatic activity. Most strikingly, genes associated with copper ion homeostasis and with HIF-1 promoter binding sites were the most over-represented, linking this signature to hypoxia. Further, six out of the ten mRNAs with strongest up-regulation in cancers with poor survival code for secreted factors, identifying potential candidates that may be involved in stromal/matrix-enhanced metastasis formation/breast cancer development. This method therefore identified biological processes that occur during mammary gland involution, which may be critical in promoting breast cancer metastasis that could form a basis for future investigation, and supports a role for copper in breast cancer development. PMID:19408105

  12. Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

    SciTech Connect

    Pistollato, Francesca; Louisse, Jochem; Scelfo, Bibiana; Mennecozzi, Milena; Accordi, Benedetta; Basso, Giuseppe; Gaspar, John Antonydas; Zagoura, Dimitra; Barilari, Manuela; Palosaari, Taina; Sachinidis, Agapios; Bremer-Hoffmann, Susanne

    2014-10-15

    According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro. Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2{sup +} neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations. - Highlights: • HESCs derived neuronal cells serve as benchmark for iPSC based neuronal toxicity test development. • Comparisons between hESCs and hiPSCs demonstrated variability of the epigenetic state • CREB pathway modulation have been explored in relation to the neurotoxicant exposure KG-501 • hiPSC might be promising tools to translate theoretical AoPs into toxicological in vitro tests.

  13. An automated RNA-Seq analysis pipeline to identify and visualize differentially expressed genes and pathways in CHO cells.

    PubMed

    Chen, Chun; Le, Huong; Goudar, Chetan T

    2015-01-01

    Recent advances in RNA-Seq based comparative transcriptomics have opened up a unique opportunity to understand the mechanisms of different phenotypes in bioprocessing-related cell lines including Chinese hamster ovary (CHO) cells. However, simple and powerful tools are needed to translate large data sets into biologically relevant information that can be leveraged for genetic engineering and cell culture medium and process development. While tools exist to perform specific tasks associated with transcriptomics analysis, integrated end to end solutions that span the entire spectrum of raw data processing to visualization of gene expression changes on canonical pathways are rare. Additionally, these are not automated and require substantial user intervention. To address this gap, we have developed an automated RNA-Seq analysis pipeline in R which leverages the latest public domain statistical advances in transcriptomics data analysis. This pipeline reads RNA-Seq gene count data, identifies differentially expressed genes and differentially expressed pathways, and provides multiple intuitive visualizations as outputs. By using two publicly available CHO RNA-Seq datasets, we have demonstrated the utility of this pipeline. Subsequently, this pipeline was used to demonstrate transcriptomic similarity between laboratory- and pilot-scale bioreactors, helping make a case for the suitability of the lab-scale bioreactor as a scaled-down model. Automated end to end RNA-Seq data analysis approaches such as the one presented in this study will shorten the time required from acquiring sequencing data to biological interpretation of the results and can help accelerate the adoption of RNA-Seq analysis and thus mechanism-driven approaches for cell line and bioprocess optimization. PMID:26150012

  14. MelanomaDB: A Web Tool for Integrative Analysis of Melanoma Genomic Information to Identify Disease-Associated Molecular Pathways

    PubMed Central

    Trevarton, Alexander J.; Mann, Michael B.; Knapp, Christoph; Araki, Hiromitsu; Wren, Jonathan D.; Stones-Havas, Steven; Black, Michael A.; Print, Cristin G.

    2013-01-01

    Despite on-going research, metastatic melanoma survival rates remain low and treatment options are limited. Researchers can now access a rapidly growing amount of molecular and clinical information about melanoma. This information is becoming difficult to assemble and interpret due to its dispersed nature, yet as it grows it becomes increasingly valuable for understanding melanoma. Integration of this information into a comprehensive resource to aid rational experimental design and patient stratification is needed. As an initial step in this direction, we have assembled a web-accessible melanoma database, MelanomaDB, which incorporates clinical and molecular data from publically available sources, which will be regularly updated as new information becomes available. This database allows complex links to be drawn between many different aspects of melanoma biology: genetic changes (e.g., mutations) in individual melanomas revealed by DNA sequencing, associations between gene expression and patient survival, data concerning drug targets, biomarkers, druggability, and clinical trials, as well as our own statistical analysis of relationships between molecular pathways and clinical parameters that have been produced using these data sets. The database is freely available at http://genesetdb.auckland.ac.nz/melanomadb/about.html. A subset of the information in the database can also be accessed through a freely available web application in the Illumina genomic cloud computing platform BaseSpace at http://www.biomatters.com/apps/melanoma-profiler-for-research. The MelanomaDB database illustrates dysregulation of specific signaling pathways across 310 exome-sequenced melanomas and in individual tumors and identifies the distribution of somatic variants in melanoma. We suggest that MelanomaDB can provide a context in which to interpret the tumor molecular profiles of individual melanoma patients relative to biological information and available drug therapies. PMID:23875173

  15. Whole Blood Transcriptomics and Urinary Metabolomics to Define Adaptive Biochemical Pathways of High-Intensity Exercise in 50-60 Year Old Masters Athletes

    PubMed Central

    Mukherjee, Kamalika; Edgett, Brittany A.; Burrows, Harrison W.; Castro, Cecilia; Griffin, Julian L.; Schwertani, Adel Giaid; Gurd, Brendon J.; Funk, Colin D.

    2014-01-01

    Exercise is beneficial for a variety of age-related disorders. However, the molecular mechanisms mediating the beneficial adaptations to exercise in older adults are not well understood. The aim of the current study was to utilize a dual approach to characterize the genetic and metabolic adaptive pathways altered by exercise in veteran athletes and age-matched untrained individuals. Two groups of 5060 year old males: competitive cyclists (athletes, n?=?9; VO2peak 59.15.2 mlkg?1min?1; peak aerobic power 38339 W) and untrained, minimally active individuals (controls, n?=?8; VO2peak 35.99.7 mlkg?1min?1; peak aerobic power 23057 W) were examined. All participants completed an acute bout of submaximal endurance exercise, and blood and urine samples pre- and post-exercise were analyzed for gene expression and metabolic changes utilizing genome-wide DNA microarray analysis and NMR spectroscopy-based metabolomics, respectively. Our results indicate distinct differences in gene and metabolite expression involving energy metabolism, lipids, insulin signaling and cardiovascular function between the two groups. These findings may lead to new insights into beneficial signaling pathways of healthy aging and help identify surrogate markers for monitoring exercise and training load. PMID:24643011

  16. Genome-wide coexpression of steroid receptors in the mouse brain: Identifying signaling pathways and functionally coordinated regions

    PubMed Central

    Lelieveldt, Boudewijn P. F.; Grefhorst, Aldo; van Weert, Lisa T. C. M.; Mol, Isabel M.; Sips, Hetty C. M.; van den Heuvel, José K.; Datson, Nicole A.; Visser, Jenny A.; Meijer, Onno C.

    2016-01-01

    Steroid receptors are pleiotropic transcription factors that coordinate adaptation to different physiological states. An important target organ is the brain, but even though their effects are well studied in specific regions, brain-wide steroid receptor targets and mediators remain largely unknown due to the complexity of the brain. Here, we tested the idea that novel aspects of steroid action can be identified through spatial correlation of steroid receptors with genome-wide mRNA expression across different regions in the mouse brain. First, we observed significant coexpression of six nuclear receptors (NRs) [androgen receptor (Ar), estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2), glucocorticoid receptor (Gr), mineralocorticoid receptor (Mr), and progesterone receptor (Pgr)] with sets of steroid target genes that were identified in single brain regions. These coexpression relationships were also present in distinct other brain regions, suggestive of as yet unidentified coordinate regulation of brain regions by, for example, glucocorticoids and estrogens. Second, coexpression of a set of 62 known NR coregulators and the six steroid receptors in 12 nonoverlapping mouse brain regions revealed selective downstream pathways, such as Pak6 as a mediator for the effects of Ar and Gr on dopaminergic transmission. Third, Magel2 and Irs4 were identified and validated as strongly responsive targets to the estrogen diethylstilbestrol in the mouse hypothalamus. The brain- and genome-wide correlations of mRNA expression levels of six steroid receptors that we provide constitute a rich resource for further predictions and understanding of brain modulation by steroid hormones. PMID:26811448

  17. Genome-wide coexpression of steroid receptors in the mouse brain: Identifying signaling pathways and functionally coordinated regions.

    PubMed

    Mahfouz, Ahmed; Lelieveldt, Boudewijn P F; Grefhorst, Aldo; van Weert, Lisa T C M; Mol, Isabel M; Sips, Hetty C M; van den Heuvel, José K; Datson, Nicole A; Visser, Jenny A; Reinders, Marcel J T; Meijer, Onno C

    2016-03-01

    Steroid receptors are pleiotropic transcription factors that coordinate adaptation to different physiological states. An important target organ is the brain, but even though their effects are well studied in specific regions, brain-wide steroid receptor targets and mediators remain largely unknown due to the complexity of the brain. Here, we tested the idea that novel aspects of steroid action can be identified through spatial correlation of steroid receptors with genome-wide mRNA expression across different regions in the mouse brain. First, we observed significant coexpression of six nuclear receptors (NRs) [androgen receptor (Ar), estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2), glucocorticoid receptor (Gr), mineralocorticoid receptor (Mr), and progesterone receptor (Pgr)] with sets of steroid target genes that were identified in single brain regions. These coexpression relationships were also present in distinct other brain regions, suggestive of as yet unidentified coordinate regulation of brain regions by, for example, glucocorticoids and estrogens. Second, coexpression of a set of 62 known NR coregulators and the six steroid receptors in 12 nonoverlapping mouse brain regions revealed selective downstream pathways, such as Pak6 as a mediator for the effects of Ar and Gr on dopaminergic transmission. Third, Magel2 and Irs4 were identified and validated as strongly responsive targets to the estrogen diethylstilbestrol in the mouse hypothalamus. The brain- and genome-wide correlations of mRNA expression levels of six steroid receptors that we provide constitute a rich resource for further predictions and understanding of brain modulation by steroid hormones. PMID:26811448

  18. Pathway-based Genome-Wide Association Analysis Identified the Importance of EphrinA-EphR pathway for Femoral Neck Bone Geometry

    PubMed Central

    Chen, Yuan; Xiong, Dong-Hai; Guo, Yan-Fang; Pan, Feng; Zhou, Qi; Zhang, Feng; Deng, Hong-Wen

    2009-01-01

    Femoral neck (FN) bone geometry is an important predictor of bone strength with high heritability. Previous studies have revealed certain candidate genes for FN bone geometry. However, the majority of the underlying genetic factors remain to be discovered. In this study, pathway-based genome-wide association analysis was performed to explore the joint effects of genes within biological pathways on FN bone geometry variations in a cohort of 1,000 unrelated U.S. whites. Nominal significant associations (nominal p value < 0.05) were observed between 76 pathways and a key FN bone geometry variable - section modulus (Z), biomechanically indicative of bone strength subject to bending. Among them, EphrinA-EphR pathway was most significantly associated with FN Z even after multiple testing adjustments (pFWER value = 0.035). The association of EphrinA-EphR pathway with FN Z was also observed in an independent sample from Framingham Osteoporosis Study. Overall, these results suggest the significant genetic contribution of EphrinA-EphR pathway to femoral neck bone geometry. PMID:19786129

  19. Refining prognosis and identifying targetable pathways for high-risk endometrial cancer; a TransPORTEC initiative.

    PubMed

    Stelloo, Ellen; Bosse, Tjalling; Nout, Remi A; MacKay, Helen J; Church, David N; Nijman, Hans W; Leary, Alexandra; Edmondson, Richard J; Powell, Melanie E; Crosbie, Emma J; Kitchener, Henry C; Mileshkin, Linda; Pollock, Pamela M; Smit, Vincent T; Creutzberg, Carien L

    2015-06-01

    This study aimed to investigate whether molecular analysis can be used to refine risk assessment, direct adjuvant therapy, and identify actionable alterations in high-risk endometrial cancer. TransPORTEC, an international consortium related to the PORTEC3 trial, was established for translational research in high-risk endometrial cancer. In this explorative study, routine molecular analyses were used to detect prognostic subgroups: p53 immunohistochemistry, microsatellite instability and POLE proofreading mutation. Furthermore, DNA was analyzed for hotspot mutations in 13 additional genes (BRAF, CDKNA2, CTNNB1, FBXW7, FGFR2, FGFR3, FOXL2, HRAS, KRAS, NRAS, PIK3CA, PPP2R1A, and PTEN) and protein expression of ER, PR, PTEN, and ARID1a was analyzed. Rates of distant metastasis, recurrence-free, and overall survival were calculated using the Kaplan-Meier method and log-rank test. In total, samples of 116 high-risk endometrial cancer patients were included: 86 endometrioid; 12 serous; and 18 clear cell. For endometrioid, serous, and clear cell cancers, 5-year recurrence-free survival rates were 68%, 27%, and 50% (P=0.014) and distant metastasis rates 23%, 64%, and 50% (P=0.001), respectively. Four prognostic subgroups were identified: (1) a group of p53-mutant tumors; (2) microsatellite instable tumors; (3) POLE proofreading-mutant tumors; and (4) a group with no specific molecular profile (NSMP). In group 3 (POLE-mutant; n=14) and group 2 (microsatellite instable; n=19) patients, no distant metastasis occurred, compared with 50% distant metastasis rate in group 1 (p53-mutant; n=36) and 39% in group 4 (NSMP; P<0.001). Five-year recurrence-free survival was 93% and 95% for group 3 (POLE-mutant) and group 2 (microsatellite instable) vs 42% (group 1, p53-mutant) and 52% (group 4, NSMP; P<0.001). Targetable FBXW7 and FGFR2 mutations (6%), alterations in the PI3K-AKT pathway (60%) and hormone receptor positivity (45%) were frequently found. In conclusion, molecular analysis of high-risk endometrial cancer identifies four distinct prognostic subgroups, with potential therapeutic implications. High frequencies of targetable alterations were identified and may serve as targets for individualized treatment. PMID:25720322

  20. The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance.

    PubMed

    Conte, Ivan; Merella, Stefania; Garcia-Manteiga, Jose Manuel; Migliore, Chiara; Lazarevic, Dejan; Carrella, Sabrina; Marco-Ferreres, Raquel; Avellino, Raffaella; Davidson, Nathan Paul; Emmett, Warren; Sanges, Remo; Bockett, Nicholas; Van Heel, David; Meroni, Germana; Bovolenta, Paola; Stupka, Elia; Banfi, Sandro

    2014-07-01

    Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development. PMID:24895435

  1. Gene expression profiling and candidate gene resequencing identifies pathways and mutations important for malignant transformation caused by leukemogenic fusion genes.

    PubMed

    Novak, Rachel L; Harper, David P; Caudell, David; Slape, Christopher; Beachy, Sarah H; Aplan, Peter D

    2012-12-01

    NUP98-HOXD13 (NHD13) and CALM-AF10 (CA10) are oncogenic fusion proteins produced by recurrent chromosomal translocations in patients with acute myeloid leukemia (AML). Transgenic mice that express these fusions develop AML with a long latency and incomplete penetrance, suggesting that collaborating genetic events are required for leukemic transformation. We employed genetic techniques to identify both preleukemic abnormalities in healthy transgenic mice as well as collaborating events leading to leukemic transformation. Candidate gene resequencing revealed that 6 of 27 (22%) CA10 AMLs spontaneously acquired a Ras pathway mutation and 8 of 27 (30%) acquired an Flt3 mutation. Two CA10 AMLs acquired an Flt3 internal-tandem duplication, demonstrating that these mutations can be acquired in murine as well as human AML. Gene expression profiles revealed a marked upregulation of Hox genes, particularly Hoxa5, Hoxa9, and Hoxa10 in both NHD13 and CA10 mice. Furthermore, mir196b, which is embedded within the Hoxa locus, was overexpressed in both CA10 and NHD13 samples. In contrast, the Hox cofactors Meis1 and Pbx3 were differentially expressed; Meis1 was increased in CA10 AMLs but not NHD13 AMLs, whereas Pbx3 was consistently increased in NHD13 but not CA10 AMLs. Silencing of Pbx3 in NHD13 cells led to decreased proliferation, increased apoptosis, and decreased colony formation invitro, suggesting a previously unexpected role for Pbx3 in leukemic transformation. PMID:22885519

  2. A Kinetic-Model-Based Approach to Identify Malfunctioning Components in Signal Transduction Pathways from Artificial Clinical Data

    PubMed Central

    Li, Xianhua; Ribaudo, Nicholas; Huang, Zuyi (Jacky)

    2015-01-01

    Detection of malfunctioning reactions or molecules from clinical data is essential for disease treatments. In order to find an alternative to the existing oversimplistic mathematical models, a kinetic model is developed in this work to infer the malfunctioning reactions/molecules by quantifying the similarity between the clinical profile and the output profiles predicted from the model in which certain reactions/molecules malfunction. The new approach was tested in IL-6 and TNF-?/NF-?B signaling pathway, for four abnormal conditions including up/downregulation of single reaction rate constants and up/downregulation of single molecules. Since limited quantitative clinical data were available, the IL-6 ODE model was used to generate artificial clinical data for the abnormal steady-state value shown in two key molecules: nuclear STAT3 and SOCS3. Similarly, the TNF-?/NF-?B model was used to obtain the data in which abnormal oscillation dynamic was shown in the profile of NF-?B. The results show that the approach developed in this study was able to successfully identify the malfunctioning reactions and molecules from the clinical data. It was also found that this new approach was noise-robust and that it managed to reveal unique solution for the faulty components in a network. PMID:26697484

  3. Mutations in TLR/MYD88 pathway identify a subset of young chronic lymphocytic leukemia patients with favorable outcome.

    PubMed

    Martnez-Trillos, Alejandra; Pinyol, Magda; Navarro, Alba; Aymerich, Marta; Jares, Pedro; Juan, Manel; Rozman, Mara; Colomer, Dolors; Delgado, Julio; Gin, Eva; Gonzlez-Daz, Marcos; Hernndez-Rivas, Jess M; Colado, Enrique; Rayn, Consolacin; Payer, Angel R; Terol, Maria Jos; Navarro, Blanca; Quesada, Victor; Puente, Xos S; Rozman, Ciril; Lpez-Otn, Carlos; Campo, Elas; Lpez-Guillermo, Armando; Villamor, Neus

    2014-06-12

    Mutations in Toll-like receptor (TLR) and myeloid differentiation primary response 88 (MYD88) genes have been found in chronic lymphocytic leukemia (CLL) at low frequency. We analyzed the incidence, clinicobiological characteristics, and outcome of patients with TLR/MYD88 mutations in 587 CLL patients. Twenty-three patients (3.9%) had mutations, 19 in MYD88 (one with concurrent IRAK1 mutation), 2 TLR2 (one with concomitant TLR6 mutation), 1 IRAK1, and 1 TLR5. No mutations were found in IRAK2 and IRAK4. TLR/MYD88-mutated CLL overexpressed genes of the nuclear factor ?B pathway. Patients with TLR/MYD88 mutations were significantly younger (83% age ?50 years) than those with no mutations. TLR/MYD88 mutations were the most frequent in young patients. Patients with mutated TLR/MYD88 CLL had a higher frequency of mutated IGHV and low expression of CD38 and ZAP-70. Overall survival (OS) was better in TLR/MYD88-mutated than unmutated patients in the whole series (10-year OS, 100% vs 62%; P = .002), and in the subset of patients age ?50 years (100% vs 70%; P = .02). In addition, relative OS of TLR/MYD88-mutated patients was similar to that in the age- and gender-matched population. In summary, TLR/MYD88 mutations identify a population of young CLL patients with favorable outcome. PMID:24782504

  4. Suppressors of cdc25p overexpression identify two pathways that influence the G2/M checkpoint in fission yeast.

    PubMed Central

    Forbes, K C; Humphrey, T; Enoch, T

    1998-01-01

    Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25(+)), which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3(+)). This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. We show that rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees and that cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA. PMID:9832516

  5. The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance

    PubMed Central

    Conte, Ivan; Merella, Stefania; Garcia-Manteiga, Jose Manuel; Migliore, Chiara; Lazarevic, Dejan; Carrella, Sabrina; Marco-Ferreres, Raquel; Avellino, Raffaella; Davidson, Nathan Paul; Emmett, Warren; Sanges, Remo; Bockett, Nicholas; Van Heel, David; Meroni, Germana; Bovolenta, Paola; Stupka, Elia; Banfi, Sandro

    2014-01-01

    Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development. PMID:24895435

  6. Extragenic Suppressors of the Arabidopsis Det1 Mutant Identify Elements of Flowering-Time and Light-Response Regulatory Pathways

    PubMed Central

    Pepper, A. E.; Chory, J.

    1997-01-01

    Light regulation of seedling morphogenesis is mediated by photoreceptors that perceive red, far-red, blue and UV light. Photomorphogenetic mutants of Arabidopsis have identified several of the primary photoreceptors, as well as a set of negative regulators of seedling photomorphogenesis, including DET1, that appear to act downstream of the photoreceptors. To study the regulatory context in which DET1 acts to repress photomorphogenesis, we used a simple morphological screen to isolate extragenic mutations in six loci, designated ted (for reversal of the det phenotype), that partially or fully suppress the seedling morphological phenotype of det1-1. Genetic analyses indicate that mutations in the ted4 and ted5 loci identify new alleles of the previously described photomorphogenetic loci hy1 and hy5, respectively. Molecular analyses indicate that the ted mutations partially suppress the dark-grown gene expression phenotype of det1-1, and that the mechanism of suppression does not involve direct remediation of the splicing defect caused by the det1-1 mutation. The ted mutations also partially suppress the light-grown morphological phenotype of mature det1-1 plants, and ted1 and ted2 suppress a daylength insensitivity phenotype of det1. TED1, TED2 and TED3 are newly described genes, whose function appears closely associated with that of DET1. In addition, alleles of ted1 are associated with a moderate late-flowering phenotype, suggesting that TED1 plays a role in the pathways that regulate both seedling morphogenesis and the initiation of flowering. PMID:9093863

  7. Construction and analysis of biochemical networks

    NASA Astrophysics Data System (ADS)

    Binns, Michael; Theodoropoulos, Constantinos

    2012-09-01

    Bioprocesses are being implemented for a range of different applications including the production of fuels, chemicals and drugs. Hence, it is becoming increasingly important to understand and model how they function and how they can be modified or designed to give the optimal performance. Here we discuss the construction and analysis of biochemical networks which are the first logical steps towards this goal. The construction of a reaction network is possible through reconstruction: extracting information from literature and from databases. This can be supplemented by reaction prediction methods which can identify steps which are missing from the current knowledge base. Analysis of biochemical systems generally requires some experimental input but can be used to identify important reactions and targets for enhancing the performance of the organism involved. Metabolic flux, pathway and metabolic control analysis can be used to determine the limits, capabilities and potential targets for enhancement respectively.

  8. HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444 issue 3)

    SciTech Connect

    Wang, Xiaozhen; Lu, Guang; Li, Li; Yi, Juan; Yan, Kaowen; Wang, Yaqing; Zhu, Baili; Kuang, Jingyu; Lin, Ming; Zhang, Sha; Shao, Genze

    2014-02-14

    Highlights: • The 2000–2634 aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

  9. HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444, isse 4)

    SciTech Connect

    Wang, Xiaozhen; Lu, Guang; Li, Li; Yi, Juan; Yan, Kaowen; Wang, Yaqing; Zhu, Baili; Kuang, Jingyu; Lin, Ming; Zhang, Sha; Shao, Genze

    2014-02-21

    Highlights: • The 2000–2634aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1 binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

  10. Identifying putative candidate genes and pathways involved in immune responses to porcine reproductive and respiratory syndrome virus (PRRSV) infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Differences in gene expression were compared between RNAs from lungs of high (HR) and low (LR) PRRSV burden pigs using the swine protein-annotated long oligonucleotide microarray, the Pigoligoarray. Pathway analyses were carried out to determine biological processes, pathways and networks that diffe...

  11. Hydrograph Separations can Identify Contaminant-Specific Pathways for Conservation Targeting in a Tile-Drained Watershed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Water quality issues continue to vex agriculture, partly due to perceived tradeoffs among different contaminants. Yet, agricultural water quality is influenced by contaminant-transport pathways unique to each watershed that are not well defined. More accurate information on contaminant pathways coul...

  12. Subpathway-GMir: identifying miRNA-mediated metabolic subpathways by integrating condition-specific genes, microRNAs, and pathway topologies

    PubMed Central

    Sun, Zeguo; Han, Junwei; Zhang, Chunlong; Yang, Haixiu; Shang, Desi; Su, Fei; Shi, Xinrui; Li, Shang; Li, Chunquan; Li, Xia

    2015-01-01

    MicroRNAs (miRNAs) regulate disease-relevant metabolic pathways. However, most current pathway identification methods fail to consider miRNAs in addition to genes when analyzing pathways. We developed a powerful method called Subpathway-GMir to construct miRNA-regulated metabolic pathways and to identify miRNA-mediated subpathways by considering condition-specific genes, miRNAs, and pathway topologies. We used Subpathway-GMir to analyze two liver hepatocellular carcinomas (LIHC), one stomach adenocarcinoma (STAD), and one type 2 diabetes (T2D) data sets. Results indicate that Subpathway-GMir is more effective in identifying phenotype-associated metabolic pathways than other methods and our results are reproducible and robust. Subpathway-GMir provides a flexible platform for identifying abnormal metabolic subpathways mediated by miRNAs, and may help to clarify the roles that miRNAs play in a variety of diseases. The Subpathway-GMir method has been implemented as a freely available R package. PMID:26472186

  13. Subpathway-GMir: identifying miRNA-mediated metabolic subpathways by integrating condition-specific genes, microRNAs, and pathway topologies.

    PubMed

    Feng, Li; Xu, Yanjun; Zhang, Yunpeng; Sun, Zeguo; Han, Junwei; Zhang, Chunlong; Yang, Haixiu; Shang, Desi; Su, Fei; Shi, Xinrui; Li, Shang; Li, Chunquan; Li, Xia

    2015-11-17

    MicroRNAs (miRNAs) regulate disease-relevant metabolic pathways. However, most current pathway identification methods fail to consider miRNAs in addition to genes when analyzing pathways. We developed a powerful method called Subpathway-GMir to construct miRNA-regulated metabolic pathways and to identify miRNA-mediated subpathways by considering condition-specific genes, miRNAs, and pathway topologies. We used Subpathway-GMir to analyze two liver hepatocellular carcinomas (LIHC), one stomach adenocarcinoma (STAD), and one type 2 diabetes (T2D) data sets. Results indicate that Subpathway-GMir is more effective in identifying phenotype-associated metabolic pathways than other methods and our results are reproducible and robust. Subpathway-GMir provides a flexible platform for identifying abnormal metabolic subpathways mediated by miRNAs, and may help to clarify the roles that miRNAs play in a variety of diseases. The Subpathway-GMir method has been implemented as a freely available R package. PMID:26472186

  14. Morphological covariance in anatomical MRI scans can identify discrete neural pathways in the brain and their disturbances in persons with neuropsychiatric disorders.

    PubMed

    Bansal, Ravi; Hao, Xuejun; Peterson, Bradley S

    2015-05-01

    We hypothesize that coordinated functional activity within discrete neural circuits induces morphological organization and plasticity within those circuits. Identifying regions of morphological covariation that are independent of morphological covariation in other regions therefore may therefore allow us to identify discrete neural systems within the brain. Comparing the magnitude of these variations in individuals who have psychiatric disorders with the magnitude of variations in healthy controls may allow us to identify aberrant neural pathways in psychiatric illnesses. We measured surface morphological features by applying nonlinear, high-dimensional warping algorithms to manually defined brain regions. We transferred those measures onto the surface of a unit sphere via conformal mapping and then used spherical wavelets and their scaling coefficients to simplify the data structure representing these surface morphological features of each brain region. We used principal component analysis (PCA) to calculate covariation in these morphological measures, as represented by their scaling coefficients, across several brain regions. We then assessed whether brain subregions that covaried in morphology, as identified by large eigenvalues in the PCA, identified specific neural pathways of the brain. To do so, we spatially registered the subnuclei for each eigenvector into the coordinate space of a Diffusion Tensor Imaging dataset; we used these subnuclei as seed regions to track and compare fiber pathways with known fiber pathways identified in neuroanatomical atlases. We applied these procedures to anatomical MRI data in a cohort of 82 healthy participants (42 children, 18 males, age 10.5 2.43 years; 40 adults, 22 males, age 32.42 10.7 years) and 107 participants with Tourette's Syndrome (TS) (71 children, 59 males, age 11.19 2.2 years; 36 adults, 21 males, age 37.34 10.9 years). We evaluated the construct validity of the identified covariation in morphology using DTI data from a different set of 20 healthy adults (10 males, mean age 29.7 7.7 years). The PCA identified portions of structures that covaried across the brain, the eigenvalues measuring the magnitude of the covariation in morphology along the respective eigenvectors. Our results showed that the eigenvectors, and the DTI fibers tracked from their associated brain regions, corresponded with known neural pathways in the brain. In addition, the eigenvectors that captured morphological covariation across regions, and the principal components along those eigenvectors, identified neural pathways with aberrant morphological features associated with TS. These findings suggest that covariations in brain morphology can identify aberrant neural pathways in specific neuropsychiatric disorders. PMID:25700952

  15. EDdb: a web resource for eating disorder and its application to identify an extended adipocytokine signaling pathway related to eating disorder.

    PubMed

    Zhao, Min; Li, XiaoMo; Qu, Hong

    2013-12-01

    Eating disorder is a group of physiological and psychological disorders affecting approximately 1% of the female population worldwide. Although the genetic epidemiology of eating disorder is becoming increasingly clear with accumulated studies, the underlying molecular mechanisms are still unclear. Recently, integration of various high-throughput data expanded the range of candidate genes and started to generate hypotheses for understanding potential pathogenesis in complex diseases. This article presents EDdb (Eating Disorder database), the first evidence-based gene resource for eating disorder. Fifty-nine experimentally validated genes from the literature in relation to eating disorder were collected as the core dataset. Another four datasets with 2824 candidate genes across 601 genome regions were expanded based on the core dataset using different criteria (e.g., protein-protein interactions, shared cytobands, and related complex diseases). Based on human protein-protein interaction data, we reconstructed a potential molecular sub-network related to eating disorder. Furthermore, with an integrative pathway enrichment analysis of genes in EDdb, we identified an extended adipocytokine signaling pathway in eating disorder. Three genes in EDdb (ADIPO (adiponectin), TNF (tumor necrosis factor) and NR3C1 (nuclear receptor subfamily 3, group C, member 1)) link the KEGG (Kyoto Encyclopedia of Genes and Genomes) "adipocytokine signaling pathway" with the BioCarta "visceral fat deposits and the metabolic syndrome" pathway to form a joint pathway. In total, the joint pathway contains 43 genes, among which 39 genes are related to eating disorder. As the first comprehensive gene resource for eating disorder, EDdb ( http://eddb.cbi.pku.edu.cn ) enables the exploration of gene-disease relationships and cross-talk mechanisms between related disorders. Through pathway statistical studies, we revealed that abnormal body weight caused by eating disorder and obesity may both be related to dysregulation of the novel joint pathway of adipocytokine signaling. In addition, this joint pathway may be the common pathway for body weight regulation in complex human diseases related to unhealthy lifestyle. PMID:24302289

  16. Analysis of TETRAKETIDE α-PYRONE REDUCTASE function in Arabidopsis thaliana reveals a previously unknown, but conserved, biochemical pathway in sporopollenin monomer biosynthesis.

    PubMed

    Grienenberger, Etienne; Kim, Sung Soo; Lallemand, Benjamin; Geoffroy, Pierrette; Heintz, Dimitri; Souza, Clarice de Azevedo; Heitz, Thierry; Douglas, Carl J; Legrand, Michel

    2010-12-01

    The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield α-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide α-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated α-pyrone compounds that were previously unknown to be sporopollenin precursors. PMID:21193572

  17. Analysis of TETRAKETIDE α-PYRONE REDUCTASE Function in Arabidopsis thaliana Reveals a Previously Unknown, but Conserved, Biochemical Pathway in Sporopollenin Monomer Biosynthesis[C][W

    PubMed Central

    Grienenberger, Etienne; Kim, Sung Soo; Lallemand, Benjamin; Geoffroy, Pierrette; Heintz, Dimitri; Souza, Clarice de Azevedo; Heitz, Thierry; Douglas, Carl J.; Legrand, Michel

    2010-01-01

    The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield α-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide α-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated α-pyrone compounds that were previously unknown to be sporopollenin precursors. PMID:21193572

  18. Biochemical insights from population studies with genetics and metabolomics.

    PubMed

    Suhre, Karsten; Raffler, Johannes; Kastenmller, Gabi

    2016-01-01

    Genome-wide association studies with concentrations of hundreds of small molecules in samples collected from thousands of individuals (mGWAS) access otherwise inaccessible natural genetic experiments and their influence on the metabolic capacities of the human body. By sampling the natural metabolic and genetic variability that is present in the general population, mGWAS identified over 150 associations between genetic variants and variation in the metabolic composition of human body fluids. Many of these genetic variants were found to be located in enzyme or transporter coding genes, whose functions match the biochemical nature of the associated metabolites. Associations identified by mGWAS can reveal novel biochemical knowledge, such as the function of uncharacterized genes, the biochemical identity of small molecules, and the structure of entire biochemical pathways. Here we review findings of recent mGWAS and discuss concrete examples of how their results can be interpreted in a biochemical context. We describe online resources that are available for mining mGWAS results. In this context, we present two concepts that also find more general applications in the field of metabolomics: strengthening of associations by looking at ratios between metabolite pairs and reconstruction of metabolic pathways by Gaussian graphical modeling. PMID:26432701

  19. Phosphoproteomic Analysis of Leukemia Cells under Basal and Drug-treated Conditions Identifies Markers of Kinase Pathway Activation and Mechanisms of Resistance*

    PubMed Central

    Alcolea, Maria P.; Casado, Pedro; Rodrguez-Prados, Juan-Carlos; Vanhaesebroeck, Bart; Cutillas, Pedro R.

    2012-01-01

    Protein kinase signaling is fundamental to cell homeostasis and is deregulated in all cancers but varies between patients. Understanding the mechanisms underlying this heterogeneity is critical for personalized targeted therapies. Here, we used a recently established LC-MS/MS platform to profile protein phosphorylation in acute myeloid leukemia cell lines with different sensitivities to kinase inhibitors. The compounds used in this study were originally developed to target Janus kinase, phosphatidylinositol 3-kinase, and MEK. After further validation of the technique, we identified several phosphorylation sites that were inhibited by these compounds but whose intensities did not always correlate with growth inhibition sensitivity. In contrast, several hundred phosphorylation sites that correlated with sensitivity/resistance were not in general inhibited by the compounds. These results indicate that markers of pathway activity may not always be reliable indicators of sensitivity of cancer cells to inhibitors that target such pathways, because the activity of parallel kinases can contribute to resistance. By mining our data we identified protein kinase C isoforms as one of such parallel pathways being more active in resistant cells. Consistent with the view that several parallel kinase pathways were contributing to resistance, inhibitors that target protein kinase C, MEK, and Janus kinase potentiated each other in arresting the proliferation of multidrug-resistant cells. Untargeted/unbiased approaches, such as the one described here, to quantify the activity of the intended target kinase pathway in concert with the activities of parallel kinase pathways will be invaluable to personalize therapies based on kinase inhibitors. PMID:22547687

  20. NAViGaTing the Micronome Using Multiple MicroRNA Prediction Databases to Identify Signalling Pathway-Associated MicroRNAs

    PubMed Central

    Shirdel, Elize A.; Xie, Wing; Mak, Tak W.; Jurisica, Igor

    2011-01-01

    Background MicroRNAs are a class of small RNAs known to regulate gene expression at the transcript level, the protein level, or both. Since microRNA binding is sequence-based but possibly structure-specific, work in this area has resulted in multiple databases storing predicted microRNA:target relationships computed using diverse algorithms. We integrate prediction databases, compare predictions to in vitro data, and use cross-database predictions to model the microRNA:transcript interactome referred to as the micronome to study microRNA involvement in well-known signalling pathways as well as associations with disease. We make this data freely available with a flexible user interface as our microRNA Data Integration Portal mirDIP (http://ophid.utoronto.ca/mirDIP). Results mirDIP integrates prediction databases to elucidate accurate microRNA:target relationships. Using NAViGaTOR to produce interaction networks implicating microRNAs in literature-based, KEGG-based and Reactome-based pathways, we find these signalling pathway networks have significantly more microRNA involvement compared to chance (p<0.05), suggesting microRNAs co-target many genes in a given pathway. Further examination of the micronome shows two distinct classes of microRNAs; universe microRNAs, which are involved in many signalling pathways; and intra-pathway microRNAs, which target multiple genes within one signalling pathway. We find universe microRNAs to have more targets (p<0.0001), to be more studied (p<0.0002), and to have higher degree in the KEGG cancer pathway (p<0.0001), compared to intra-pathway microRNAs. Conclusions Our pathway-based analysis of mirDIP data suggests microRNAs are involved in intra-pathway signalling. We identify two distinct classes of microRNAs, suggesting a hierarchical organization of microRNAs co-targeting genes both within and between pathways, and implying differential involvement of universe and intra-pathway microRNAs at the disease level. PMID:21364759

  1. Transcriptome Analysis in Prenatal IGF1-Deficient Mice Identifies Molecular Pathways and Target Genes Involved in Distal Lung Differentiation

    PubMed Central

    Hernndez-Porras, Isabel; Lpez, Icar Paula; De Las Rivas, Javier; Pichel, Jos Garca

    2013-01-01

    Background Insulin-like Growth Factor 1 (IGF1) is a multifunctional regulator of somatic growth and development throughout evolution. IGF1 signaling through IGF type 1 receptor (IGF1R) controls cell proliferation, survival and differentiation in multiple cell types. IGF1 deficiency in mice disrupts lung morphogenesis, causing altered prenatal pulmonary alveologenesis. Nevertheless, little is known about the cellular and molecular basis of IGF1 activity during lung development. Methods/Principal Findings Prenatal Igf1?/? mutant mice with a C57Bl/6J genetic background displayed severe disproportional lung hypoplasia, leading to lethal neonatal respiratory distress. Immuno-histological analysis of their lungs showed a thickened mesenchyme, alterations in extracellular matrix deposition, thinner smooth muscles and dilated blood vessels, which indicated immature and delayed distal pulmonary organogenesis. Transcriptomic analysis of Igf1?/? E18.5 lungs using RNA microarrays identified deregulated genes related to vascularization, morphogenesis and cellular growth, and to MAP-kinase, Wnt and cell-adhesion pathways. Up-regulation of immunity-related genes was verified by an increase in inflammatory markers. Increased expression of Nfib and reduced expression of Klf2, Egr1 and Ctgf regulatory proteins as well as activation of ERK2 MAP-kinase were corroborated by Western blot. Among IGF-system genes only IGFBP2 revealed a reduction in mRNA expression in mutant lungs. Immuno-staining patterns for IGF1R and IGF2, similar in both genotypes, correlated to alterations found in specific cell compartments of Igf1?/? lungs. IGF1 addition to Igf1?/? embryonic lungs cultured ex vivo increased airway septa remodeling and distal epithelium maturation, processes accompanied by up-regulation of Nfib and Klf2 transcription factors and Cyr61 matricellular protein. Conclusions/Significance We demonstrated the functional tissue specific implication of IGF1 on fetal lung development in mice. Results revealed novel target genes and gene networks mediators of IGF1 action on pulmonary cellular proliferation, differentiation, adhesion and immunity, and on vascular and distal epithelium maturation during prenatal lung development. PMID:24391734

  2. A comparative analysis of transcriptomic, biochemical, and physiological responses to elevated ozone identifies species-specific mechanisms of resilience in legume crops

    PubMed Central

    Yendrek, Craig R.; Koester, Robert P.

    2015-01-01

    Current concentrations of tropospheric ozone ([O3]) pollution negatively impact plant metabolism, which can result in decreased crop yields. Interspecific variation in the physiological response of plants to elevated [O3] exists; however, the underlying cellular responses explaining species-specific differences are largely unknown. Here, a physiological screen has been performed on multiple varieties of legume species. Three varieties of garden pea (Pisum sativum L.) were resilient to elevated [O3]. Garden pea showed no change in photosynthetic capacity or leaf longevity when exposed to elevated [O3], in contrast to varieties of soybean (Glycine max (L.) Merr.) and common bean (Phaseolus vulgaris L.). Global transcriptomic and targeted biochemical analyses were then done to examine the mechanistic differences in legume responses to elevated [O3]. In all three species, there was an O3-mediated reduction in specific leaf weight and total non-structural carbohydrate content, as well as increased abundance of respiration-related transcripts. Differences specific to garden pea included a pronounced increase in the abundance of GLUTATHIONE REDUCTASE transcript, as well as greater contents of foliar glutathione, apoplastic ascorbate, and sucrose in elevated [O3]. These results suggest that garden pea may have had greater capacity for detoxification, which prevented net losses in CO2 fixation in an elevated [O3] environment. PMID:26324463

  3. A comparative analysis of transcriptomic, biochemical, and physiological responses to elevated ozone identifies species-specific mechanisms of resilience in legume crops.

    PubMed

    Yendrek, Craig R; Koester, Robert P; Ainsworth, Elizabeth A

    2015-12-01

    Current concentrations of tropospheric ozone ([O3]) pollution negatively impact plant metabolism, which can result in decreased crop yields. Interspecific variation in the physiological response of plants to elevated [O3] exists; however, the underlying cellular responses explaining species-specific differences are largely unknown. Here, a physiological screen has been performed on multiple varieties of legume species. Three varieties of garden pea (Pisum sativum L.) were resilient to elevated [O3]. Garden pea showed no change in photosynthetic capacity or leaf longevity when exposed to elevated [O3], in contrast to varieties of soybean (Glycine max (L.) Merr.) and common bean (Phaseolus vulgaris L.). Global transcriptomic and targeted biochemical analyses were then done to examine the mechanistic differences in legume responses to elevated [O3]. In all three species, there was an O3-mediated reduction in specific leaf weight and total non-structural carbohydrate content, as well as increased abundance of respiration-related transcripts. Differences specific to garden pea included a pronounced increase in the abundance of GLUTATHIONE REDUCTASE transcript, as well as greater contents of foliar glutathione, apoplastic ascorbate, and sucrose in elevated [O3]. These results suggest that garden pea may have had greater capacity for detoxification, which prevented net losses in CO2 fixation in an elevated [O3] environment. PMID:26324463

  4. Use of an Activated Beta-Catenin to Identify Wnt Pathway Target Genes in Caenorhabditis elegans, Including a Subset of Collagen Genes Expressed in Late Larval Development

    PubMed Central

    Jackson, Belinda M.; Abete-Luzi, Patricia; Krause, Michael W.; Eisenmann, David M.

    2014-01-01

    The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin?dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle. PMID:24569038

  5. PROFILING GENE EXPRESSION IN HUMAN H295R ADRENOCORTICAL CARCINOMA CELLS AND RAT TESTES TO IDENTIFY PATHWAYS OF TOXICITY FOR CONAZOLE FUNGICIDES

    EPA Science Inventory

    Profiling Gene Expression in Human H295R Adrenocortical Carcinoma Cells and Rat Testes to Identify Pathways of Toxicity for Conazole Fungicides
    Ren1, H., Schmid1, J., Retief2, J., Turpaz2, Y.,Zhang3, X.,Jones3, P., Newsted3, J.,Giesy3, J., Wolf1, D.,Wood1, C., Bao1, W., Dix1, ...

  6. Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics

    SciTech Connect

    Kleinstreuer, N.C.; Smith, A.M.; West, P.R.; Conard, K.R.; Fontaine, B.R.; Weir-Hauptman, A.M.; Palmer, J.A.; Knudsen, T.B.; Dix, D.J.; Donley, E.L.R.; Cezar, G.G.; University of Wisconsin-Madison, Madison, WI 53706

    2011-11-15

    Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast Trade-Mark-Sign chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism, pantothenate and CoA biosynthesis, glutathione metabolism, and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information, including Stemina Biomarker Discovery's predictive DevTox Registered-Sign model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity. -- Highlights: Black-Right-Pointing-Pointer We tested 11 environmental compounds in a hESC metabolomics platform. Black-Right-Pointing-Pointer Significant changes in secreted small molecule metabolites were observed. Black-Right-Pointing-Pointer Perturbed mass features map to pathways critical for normal development and pregnancy. Black-Right-Pointing-Pointer Arginine, proline, nicotinate, nicotinamide and glutathione pathways were affected.

  7. A Novel Proteomics-Based Clinical Diagnostics Technology Identifies Heterogeneity in Activated Signaling Pathways in Gastric Cancers

    PubMed Central

    Kim, Sung; Liu, Xinjun; Lee, Tani; Kim, Kyoung-Mee; Do, In-Gu; Park, Joon Oh; Park, Se Hoon; Jang, Jiryeon; Hoe, Nicholas; Harvie, Gulia; Kuller, Anne; Jain, Anjali; Meyer, Gary; Leesman, Glen; Park, Young Suk; Choi, Min Gew; Sohn, Tae Sung; Bae, Jae Moon; Lim, Ho Yeong; Singh, Sharat; Kang, Won Ki

    2013-01-01

    Purpose The aim of this study was to utilize the proteomics-based Collaborative Enzyme Enhanced Reactive (CEER) immunoassay to investigate protein tyrosine phosphorylations as diagnostic markers in gastric cancers (GCs). Experimental Design Protein lysates from fresh-frozen 434 advanced stage GCs were analyzed for phosphorylation of HER1, HER2, p95HER2, HER3, cMET, IGF1R and PI3K. The pathway activation patterns were segregated based on the tumor HER2 status. Hierarchical clustering was utilized to determine pathway coactivations in GCs. Prognostic value of pathway activation patterns was determined by correlating disease-free survival times of the various GC subgroups using Kaplan-Meier survival analysis. CEER was also used to determine the presence of tyrosine phosphorylated signaling cascades in circulating tumor cells (CTCs) and ascites tumor cells (ATCs). Results Utilizing a novel diagnostics immunoassay, CEER, we demonstrate the presence of p95HER2 and concomitantly activated signaling pathways in GC tumor tissues, CTCs and ATCs isolated from GC patients for the first time. p95HER2 is expressed in ?77% of HER2(+) GCs. Approximately 54% of GCs have an activated HER1, HER2, HER3, cMET or IGF1R and demonstrate a poorer prognosis than those where these receptor tyrosine kinases (RTKs) are not activated. Hierarchical clustering of RTKs reveals co-clustering of phosphorylated HER1:cMET, HER2:HER3 and IGF1R-PI3K. Coactivation of HER1 with cMET renders GCs with a shorter disease-free survival as compared to only cMET activated GCs. Conclusions Our study highlights the utility of a novel companion diagnostics technology, CEER that has strong implications for drug development and therapeutic monitoring. CEER is used to provide an increased understanding of activated signaling pathways in advanced GCs that can significantly improve their clinical management through accurate patient selection for targeted therapeutics. PMID:23372746

  8. Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways

    NASA Astrophysics Data System (ADS)

    Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

    2015-05-01

    We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

  9. Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways.

    PubMed

    Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

    2015-05-01

    We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation. PMID:25692658

  10. A Synthetic Lethal Screen Identifies DNA Repair Pathways that Sensitize Cancer Cells to Combined ATR Inhibition and Cisplatin Treatments

    PubMed Central

    Mohni, Kareem N.; Thompson, Petria S.; Luzwick, Jessica W.; Glick, Gloria G.; Pendleton, Christopher S.; Lehmann, Brian D.; Pietenpol, Jennifer A.; Cortez, David

    2015-01-01

    The DNA damage response kinase ATR may be a useful cancer therapeutic target. ATR inhibition synergizes with loss of ERCC1, ATM, XRCC1 and DNA damaging chemotherapy agents. Clinical trials have begun using ATR inhibitors in combination with cisplatin. Here we report the first synthetic lethality screen with a combination treatment of an ATR inhibitor (ATRi) and cisplatin. Combination treatment with ATRi/cisplatin is synthetically lethal with loss of the TLS polymerase ? and 53BP1. Other DNA repair pathways including homologous recombination and mismatch repair do not exhibit synthetic lethal interactions with ATRi/cisplatin, even though loss of some of these repair pathways sensitizes cells to cisplatin as a single-agent. We also report that ATRi strongly synergizes with PARP inhibition, even in homologous recombination-proficient backgrounds. Lastly, ATR inhibitors were able to resensitize cisplatin-resistant cell lines to cisplatin. These data provide a comprehensive analysis of DNA repair pathways that exhibit synthetic lethality with ATR inhibitors when combined with cisplatin chemotherapy, and will help guide patient selection strategies as ATR inhibitors progress into the cancer clinic. PMID:25965342

  11. Perturbation of cellular proteostasis networks identifies pathways that modulate precursor and intermediate but not mature levels of frataxin

    PubMed Central

    Nabhan, Joseph F.; Gooch, Renea L.; Piatnitski Chekler, Eugene L.; Pierce, Betsy; Bulawa, Christine E.

    2015-01-01

    Friedreichs Ataxia is a genetic disease caused by expansion of an intronic trinucleotide repeat in the frataxin (FXN) gene yielding diminished FXN expression and consequently disease. Since increasing FXN protein levels is desirable to ameliorate pathology, we explored the role of major cellular proteostasis pathways and mitochondrial proteases in FXN processing and turnover. We targeted p97/VCP, the ubiquitin proteasome pathway (UPP), and autophagy with chemical inhibitors in cell lines and patient-derived cells. p97 inhibition by DBeQ increased precursor FXN levels, while UPP and autophagic flux modulators had variable effects predominantly on intermediate FXN. Our data suggest that these pathways cannot be modulated to influence mature functional FXN levels. We also targeted known mitochondrial proteases by RNA interference and discovered a novel protease PITRM1 that regulates intermediate FXN levels. Treatment with the aforementioned chemical and genetic modulators did not have a differential effect in patient cells containing lower amounts of FXN. Interestingly, a number of treatments caused a change in total amount of FXN protein, without an effect on mature FXN. Our results imply that regulation of FXN protein levels is complex and that total amounts can be modulated chemically and genetically without altering the absolute amount of mature FXN protein. PMID:26671574

  12. A Synthetic Lethal Screen Identifies DNA Repair Pathways that Sensitize Cancer Cells to Combined ATR Inhibition and Cisplatin Treatments.

    PubMed

    Mohni, Kareem N; Thompson, Petria S; Luzwick, Jessica W; Glick, Gloria G; Pendleton, Christopher S; Lehmann, Brian D; Pietenpol, Jennifer A; Cortez, David

    2015-01-01

    The DNA damage response kinase ATR may be a useful cancer therapeutic target. ATR inhibition synergizes with loss of ERCC1, ATM, XRCC1 and DNA damaging chemotherapy agents. Clinical trials have begun using ATR inhibitors in combination with cisplatin. Here we report the first synthetic lethality screen with a combination treatment of an ATR inhibitor (ATRi) and cisplatin. Combination treatment with ATRi/cisplatin is synthetically lethal with loss of the TLS polymerase ζ and 53BP1. Other DNA repair pathways including homologous recombination and mismatch repair do not exhibit synthetic lethal interactions with ATRi/cisplatin, even though loss of some of these repair pathways sensitizes cells to cisplatin as a single-agent. We also report that ATRi strongly synergizes with PARP inhibition, even in homologous recombination-proficient backgrounds. Lastly, ATR inhibitors were able to resensitize cisplatin-resistant cell lines to cisplatin. These data provide a comprehensive analysis of DNA repair pathways that exhibit synthetic lethality with ATR inhibitors when combined with cisplatin chemotherapy, and will help guide patient selection strategies as ATR inhibitors progress into the cancer clinic. PMID:25965342

  13. Perturbation of cellular proteostasis networks identifies pathways that modulate precursor and intermediate but not mature levels of frataxin.

    PubMed

    Nabhan, Joseph F; Gooch, Renea L; Piatnitski Chekler, Eugene L; Pierce, Betsy; Bulawa, Christine E

    2015-01-01

    Friedreich's Ataxia is a genetic disease caused by expansion of an intronic trinucleotide repeat in the frataxin (FXN) gene yielding diminished FXN expression and consequently disease. Since increasing FXN protein levels is desirable to ameliorate pathology, we explored the role of major cellular proteostasis pathways and mitochondrial proteases in FXN processing and turnover. We targeted p97/VCP, the ubiquitin proteasome pathway (UPP), and autophagy with chemical inhibitors in cell lines and patient-derived cells. p97 inhibition by DBeQ increased precursor FXN levels, while UPP and autophagic flux modulators had variable effects predominantly on intermediate FXN. Our data suggest that these pathways cannot be modulated to influence mature functional FXN levels. We also targeted known mitochondrial proteases by RNA interference and discovered a novel protease PITRM1 that regulates intermediate FXN levels. Treatment with the aforementioned chemical and genetic modulators did not have a differential effect in patient cells containing lower amounts of FXN. Interestingly, a number of treatments caused a change in total amount of FXN protein, without an effect on mature FXN. Our results imply that regulation of FXN protein levels is complex and that total amounts can be modulated chemically and genetically without altering the absolute amount of mature FXN protein. PMID:26671574

  14. Expression-based network biology identifies alteration in key regulatory pathways of type 2 diabetes and associated risk/complications.

    PubMed

    Sengupta, Urmi; Ukil, Sanchaita; Dimitrova, Nevenka; Agrawal, Shipra

    2009-01-01

    Type 2 diabetes mellitus (T2D) is a multifactorial and genetically heterogeneous disease which leads to impaired glucose homeostasis and insulin resistance. The advanced form of disease causes acute cardiovascular, renal, neurological and microvascular complications. Thus there is a constant need to discover new and efficient treatment against the disease by seeking to uncover various novel alternate signalling mechanisms that can lead to diabetes and its associated complications. The present study allows detection of molecular targets by unravelling their role in altered biological pathways during diabetes and its associated risk factors and complications. We have used an integrated functional networks concept by merging co-expression network and interaction network to detect the transcriptionally altered pathways and regulations involved in the disease. Our analysis reports four novel significant networks which could lead to the development of diabetes and other associated dysfunctions. (a) The first network illustrates the up regulation of TGFBRII facilitating oxidative stress and causing the expression of early transcription genes via MAPK pathway leading to cardiovascular and kidney related complications. (b) The second network demonstrates novel interactions between GAPDH and inflammatory and proliferation candidate genes i.e., SUMO4 and EGFR indicating a new link between obesity and diabetes. (c) The third network portrays unique interactions PTPN1 with EGFR and CAV1 which could lead to an impaired vascular function in diabetic nephropathy condition. (d) Lastly, from our fourth network we have inferred that the interaction of beta-catenin with CDH5 and TGFBR1 through Smad molecules could contribute to endothelial dysfunction. A probability of emergence of kidney complication might be suggested in T2D condition. An experimental investigation on this aspect may further provide more decisive observation in drug target identification and better understanding of the pathophysiology of T2D and its complications. PMID:19997558

  15. Transcript Profiling of Paoenia ostii during Artificial Chilling Induced Dormancy Release Identifies Activation of GA Pathway and Carbohydrate Metabolism

    PubMed Central

    Liu, Chunying; Zhang, Yang; Zheng, Guosheng

    2013-01-01

    Endo-dormant flower buds must pass through a period of chilling to reinitiate growth and subsequent flowering, which is a major obstacle to the forcing culture of tree peony in winter. Customized cDNA microarray (8×15 K element) was used to investigate gene expression profiling in tree peony ‘Feng Dan Bai’ buds during 24 d chilling treatment at 0–4°C. According to the morphological changes after the whole plants were transferred to green house, endo-dormancy was released after 18 d chilling treatment, and prolonged chilling treatment increased bud break rate. Pearson correlation hierarchical clustering of sample groups was highly consistent with the dormancy transitions revealed by morphological changes. Totally 3,174 significantly differentially-expressed genes (P<0.05) were observed through endo-dormancy release process, of which the number of up-regulated (1,611) and that of down-regulated (1,563) was almost the same. Functional annotation of differentially-expressed genes revealed that cellular process, metabolic process, response to stimulus, regulation of biological process and development process were well-represented. Hierarchical clustering indicated that activation of genes involved in carbohydrate metabolism (Glycolysis, Citrate cycle and Pentose phosphate pathway), energy metabolism and cell growth. Based on the results of GO analysis, totally 51 probes presented in the microarray were associated with GA response and GA signaling pathway, and 22 of them were differently expressed. The expression profiles also revealed that the genes of GA biosynthesis, signaling and response involved in endo-dormancy release. We hypothesized that activation of GA pathway played a central role in the regulation of dormancy release in tree peony. PMID:23405132

  16. Utilising conservative tracers and spatial surveys to identify controls on pathways and DOC exports in an Arctic catchment.

    NASA Astrophysics Data System (ADS)

    Lessels, J. S.; Tetzlaff, D.; Dinsmore, K. J.; Street, L. E.; Dean, J.; Washbourne, I. J.; Billett, M. F.; Baxter, R.; Subke, J. A.; Wookey, P. A.

    2014-12-01

    Dissolved organic carbon (DOC) is typically the predominant form of carbon exported from headwater streams, it therefore represents a major carbon export from Arctic catchments. The projected deepening of thaw depth in permafrost regions, due to an increase in air temperature, may have a significant effect on the amount of DOC exported from these systems. However, quantification of the impacts of climate driven changes on DOC export are still highly uncertain. Understanding the processes controlling DOC export is therefore crucial in predicting the potential impact of projected environmental changes. The controls of DOC production and transport are heavily influenced by soil and vegetation, which are highly variable across the landscape. To completely understand these systems information regarding spatial variability of plants, soils and thaw depths must be taken into account. In this study sub-weekly sampling of DOC was undertaken throughout 2014 in a headwater (<1 km2) catchment in the Northwest Territories, Canada. Spatial surveys of soil properties, active thaw depth and normalised difference vegetation index (NDVI) were collected and used in conjunction with conservative stable water isotopes tracers and major ions to understand sources, flow pathways and timing of DOC exports from the catchment. Stable isotope tracers act as fingerprints of water allowing sources and pathways to be assessed. Observations reveal changing DOC concentrations throughout the season as the active layer deepens and the connectivity of the soils to the stream network throughout the catchment increases. Linking the DOC data with the conservative tracer response improves the identification of carbon pathways and fluxes from the soils; preliminary analysis indicates DOC is being delivered via deeper more mineral soils later in the season. The results indicate that the active layer depth has a strong influence on the amount of DOC exported from the system, independent of the amount of carbon stored in these deeper soils.

  17. Functional and biochemical consequences of abrogating the activation of multiple diverse early signaling pathways in Kit. Role for Src kinase pathway in Kit-induced cooperation with erythropoietin receptor.

    PubMed

    Tan, Bai Lin; Hong, Li; Munugalavadla, Veerendra; Kapur, Reuben

    2003-03-28

    Kit receptor tyrosine kinase and erythropoietin receptor (Epo-R) cooperate in regulating blood cell development. Mice that lack the expression of Kit or Epo-R die in utero of severe anemia. Stimulation of Kit by its ligand, stem cell factor activates several distinct early signaling pathways, including phospholipase C gamma, phosphatidylinositol 3-kinase, Src kinase, Grb2, and Grb7. The role of these pathways in Kit-induced growth, proliferation, or cooperation with Epo-R is not known. We demonstrate that inactivation of any one of these early signaling pathways in Kit significantly impairs growth and proliferation. However, inactivation of the Src pathway demonstrated the most profound defect. Combined stimulation with Epo also resulted in impaired cooperation between Src-defective Kit mutant and Epo-R and, to a lesser extent, with Kit mutants defective in the activation of phosphatidylinositol 3-kinase or Grb2. The impaired cooperation between the Src-defective Kit mutant and Epo-R was associated with reduced transphosphorylation of Epo-R and expression of c-Myc. Remarkably, restoration of only the Src pathway in a Kit receptor defective in the activation of all early signaling pathways demonstrated a 50% correction in proliferation in response to Kit stimulation and completely restored the cooperation with Epo-R. These data demonstrate an essential role for Src pathway in regulating growth, proliferation, and cooperation with Epo-R downstream from Kit. PMID:12486028

  18. Three-Dimensional Pharmacophore Design and Biochemical Screening Identifies Substituted 1,2,4-Triazoles as Inhibitors of the Annexin A2–S100A10 Protein Interaction

    PubMed Central

    Reddy, Tummala R K; Li, Chan; Fischer, Peter M; Dekker, Lodewijk V

    2012-01-01

    Abstract Protein interactions are increasingly appreciated as targets in small-molecule drug discovery. The interaction between the adapter protein S100A10 and its binding partner annexin A2 is a potentially important drug target. To obtain small-molecule starting points for inhibitors of this interaction, a three-dimensional pharmacophore model was constructed from the X-ray crystal structure of the complex between S100A10 and annexin A2. The pharmacophore model represents the favourable hydrophobic and hydrogen bond interactions between the two partners, as well as spatial and receptor site constraints (excluded volume spheres). Using this pharmacophore model, UNITY flex searches were carried out on a 3D library of 0.7 million commercially available compounds. This resulted in 568 hit compounds. Subsequently, GOLD docking studies were performed on these hits, and a set of 190 compounds were purchased and tested biochemically for inhibition of the protein interaction. Three compounds of similar chemical structure were identified as genuine inhibitors of the binding of annexin A2 to S100A10. The binding modes predicted by GOLD were in good agreement with their UNITY-generated conformations. We synthesised a series of analogues revealing areas critical for binding. Thus computational predictions and biochemical screening can be used successfully to derive novel chemical classes of protein–protein interaction blockers. PMID:22644793

  19. Identifying viable regulatory and innovation pathways for regenerative medicine: a case study of cultured red blood cells.

    PubMed

    Mittra, J; Tait, J; Mastroeni, M; Turner, M L; Mountford, J C; Bruce, K

    2015-01-25

    The creation of red blood cells for the blood transfusion markets represents a highly innovative application of regenerative medicine with a medium term (5-10 year) prospect for first clinical studies. This article describes a case study analysis of a project to derive red blood cells from human embryonic stem cells, including the systemic challenges arising from (i) the selection of appropriate and viable regulatory protocols and (ii) technological constraints related to stem cell manufacture and scale up to clinical Good Manufacturing Practice (GMP) standard. The method used for case study analysis (Analysis of Life Science Innovation Systems (ALSIS)) is also innovative, demonstrating a new approach to social and natural science collaboration to foresight product development pathways. Issues arising along the development pathway include cell manufacture and scale-up challenges, affected by regulatory demands emerging from the innovation ecosystem (preclinical testing and clinical trials). Our discussion reflects on the efforts being made by regulators to adapt the current pharmaceuticals-based regulatory model to an allogeneic regenerative medicine product and the broader lessons from this case study for successful innovation and translation of regenerative medicine therapies, including the role of methodological and regulatory innovation in future development in the field. PMID:25094050

  20. Patient-Based Transcriptome-Wide Analysis Identify Interferon and Ubiquination Pathways as Potential Predictors of Influenza A Disease Severity

    PubMed Central

    Hoang, Long Truong; Tolfvenstam, Thomas; Ooi, Eng Eong; Khor, Chiea Chuen; Naim, Ahmand Nazri Mohamed; Ho, Eliza Xin Pei; Ong, Swee Hoe; Wertheim, Heiman F.; Fox, Annette; Van Vinh Nguyen, Chau; Nghiem, Ngoc My; Ha, Tuan Manh; Thi Ngoc Tran, Anh; Tambayah, Paul; Lin, Raymond; Sangsajja, Chariya; Manosuthi, Weerawat; Chuchottaworn, Chareon; Sansayunh, Piamlarp; Chotpitayasunondh, Tawee; Suntarattiwong, Piyarat; Chokephaibulkit, Kulkanya; Puthavathana, Pilaipan; de Jong, Menno D.; Farrar, Jeremy; van Doorn, H. Rogier; Hibberd, Martin Lloyd

    2014-01-01

    Background The influenza A virus is an RNA virus that is responsible for seasonal epidemics worldwide with up to five million cases of severe illness and 500,000 deaths annually according to the World Health Organization estimates. The factors associated with severe diseases are not well defined, but more severe disease is more often seen among persons aged >65 years, infants, pregnant women, and individuals of any age with underlying health conditions. Methodology/Principal Findings Using gene expression microarrays, the transcriptomic profiles of influenza-infected patients with severe (N?=?11), moderate (N?=?40) and mild (N?=?83) symptoms were compared with the febrile patients of unknown etiology (N?=?73). We found that influenza-infected patients, regardless of their clinical outcomes, had a stronger induction of antiviral and cytokine responses and a stronger attenuation of NK and T cell responses in comparison with those with unknown etiology. More importantly, we found that both interferon and ubiquitination signaling were strongly attenuated in patients with the most severe outcomes in comparison with those with moderate and mild outcomes, suggesting the protective roles of these pathways in disease pathogenesis. Conclusion/Significances The attenuation of interferon and ubiquitination pathways may associate with the clinical outcomes of influenza patients. PMID:25365328

  1. Evaluation of microbial triglyceride oil purification requirements for the CelTherm process: an efficient biochemical pathway to renewable fuels and chemicals.

    PubMed

    Linnen, Michael; Seames, Wayne; Kubatova, Alena; Menon, Suresh; Alisala, Kashinatham; Hash, Sara

    2014-10-01

    CelTherm is a biochemical process to produce renewable fuels and chemicals from lignocellulosic biomass. The present study's objective was to determine the level of treatment/purity of the microbial triacylglyceride oil (TAG) necessary to facilitate fuel production. After a unique microbe aerobically synthesizes TAG from biomass-derived sugars, the microbes were harvested and dried then crude TAG was chemically extracted from the residual biomass. Some TAGs were further purified to hydrotreating process requirements. Both grades were then noncatalytically cracked into a petroleum-like intermediate characterized by gas chromatography. Experiments were repeated using refined soybean oil for comparison to previous studies. The products from crude microbial TAG cracking were then further refined into a jet fuel product. Fuel tests indicate that this jet fuel corresponds to specifications for JP-8 military turbine fuel. It was thus concluded that the crude microbial TAG is a suitable feedstock with no further purification required, demonstrating CelTherm's commercial potential. PMID:24781206

  2. Experimentally-Derived Fibroblast Gene Signatures Identify Molecular Pathways Associated with Distinct Subsets of Systemic Sclerosis Patients in Three Independent Cohorts

    PubMed Central

    Johnson, Michael E.; Mahoney, J. Matthew; Taroni, Jaclyn; Sargent, Jennifer L.; Marmarelis, Eleni; Wu, Ming-Ru; Varga, John; Hinchcliff, Monique E.; Whitfield, Michael L.

    2015-01-01

    Genome-wide expression profiling in systemic sclerosis (SSc) has identified four intrinsic subsets of disease (fibroproliferative, inflammatory, limited, and normal-like), each of which shows deregulation of distinct signaling pathways; however, the full set of pathways contributing to this differential gene expression has not been fully elucidated. Here we examine experimentally derived gene expression signatures in dermal fibroblasts for thirteen different signaling pathways implicated in SSc pathogenesis. These data show distinct and overlapping sets of genes induced by each pathway, allowing for a better understanding of the molecular relationship between profibrotic and immune signaling networks. Pathway-specific gene signatures were analyzed across a compendium of microarray datasets consisting of skin biopsies from three independent cohorts representing 80 SSc patients, 4 morphea, and 26 controls. IFN? signaling showed a strong association with early disease, while TGF? signaling spanned the fibroproliferative and inflammatory subsets, was associated with worse MRSS, and was higher in lesional than non-lesional skin. The fibroproliferative subset was most strongly associated with PDGF signaling, while the inflammatory subset demonstrated strong activation of innate immune pathways including TLR signaling upstream of NF-?B. The limited and normal-like subsets did not show associations with fibrotic and inflammatory mediators such as TGF? and TNF?. The normal-like subset showed high expression of genes associated with lipid signaling, which was absent in the inflammatory and limited subsets. Together, these data suggest a model by which IFN? is involved in early disease pathology, and disease severity is associated with active TGF? signaling. PMID:25607805

  3. Method for identifying subsurface fluid migration and drainage pathways in and among oil and gas reservoirs using 3-D and 4-D seismic imaging

    DOEpatents

    Anderson, Roger N.; Boulanger, Albert; Bagdonas, Edward P.; Xu, Liqing; He, Wei

    1996-01-01

    The invention utilizes 3-D and 4-D seismic surveys as a means of deriving information useful in petroleum exploration and reservoir management. The methods use both single seismic surveys (3-D) and multiple seismic surveys separated in time (4-D) of a region of interest to determine large scale migration pathways within sedimentary basins, and fine scale drainage structure and oil-water-gas regions within individual petroleum producing reservoirs. Such structure is identified using pattern recognition tools which define the regions of interest. The 4-D seismic data sets may be used for data completion for large scale structure where time intervals between surveys do not allow for dynamic evolution. The 4-D seismic data sets also may be used to find variations over time of small scale structure within individual reservoirs which may be used to identify petroleum drainage pathways, oil-water-gas regions and, hence, attractive drilling targets. After spatial orientation, and amplitude and frequency matching of the multiple seismic data sets, High Amplitude Event (HAE) regions consistent with the presence of petroleum are identified using seismic attribute analysis. High Amplitude Regions are grown and interconnected to establish plumbing networks on the large scale and reservoir structure on the small scale. Small scale variations over time between seismic surveys within individual reservoirs are identified and used to identify drainage patterns and bypassed petroleum to be recovered. The location of such drainage patterns and bypassed petroleum may be used to site wells.

  4. Method for identifying subsurface fluid migration and drainage pathways in and among oil and gas reservoirs using 3-D and 4-D seismic imaging

    DOEpatents

    Anderson, R.N.; Boulanger, A.; Bagdonas, E.P.; Xu, L.; He, W.

    1996-12-17

    The invention utilizes 3-D and 4-D seismic surveys as a means of deriving information useful in petroleum exploration and reservoir management. The methods use both single seismic surveys (3-D) and multiple seismic surveys separated in time (4-D) of a region of interest to determine large scale migration pathways within sedimentary basins, and fine scale drainage structure and oil-water-gas regions within individual petroleum producing reservoirs. Such structure is identified using pattern recognition tools which define the regions of interest. The 4-D seismic data sets may be used for data completion for large scale structure where time intervals between surveys do not allow for dynamic evolution. The 4-D seismic data sets also may be used to find variations over time of small scale structure within individual reservoirs which may be used to identify petroleum drainage pathways, oil-water-gas regions and, hence, attractive drilling targets. After spatial orientation, and amplitude and frequency matching of the multiple seismic data sets, High Amplitude Event (HAE) regions consistent with the presence of petroleum are identified using seismic attribute analysis. High Amplitude Regions are grown and interconnected to establish plumbing networks on the large scale and reservoir structure on the small scale. Small scale variations over time between seismic surveys within individual reservoirs are identified and used to identify drainage patterns and bypassed petroleum to be recovered. The location of such drainage patterns and bypassed petroleum may be used to site wells. 22 figs.

  5. A lin-45 raf enhancer screen identifies eor-1, eor-2 and unusual alleles of Ras pathway genes in Caenorhabditis elegans.

    PubMed Central

    Rocheleau, Christian E; Howard, Robyn M; Goldman, Alissa P; Volk, Mandy L; Girard, Laura J; Sundaram, Meera V

    2002-01-01

    In Caenorhabditis elegans, the Ras/Raf/MEK/ERK signal transduction pathway controls multiple processes including excretory system development, P12 fate specification, and vulval cell fate specification. To identify positive regulators of Ras signaling, we conducted a genetic screen for mutations that enhance the excretory system and egg-laying defects of hypomorphic lin-45 raf mutants. This screen identified unusual alleles of several known Ras pathway genes, including a mutation removing the second SH3 domain of the sem-5/Grb2 adaptor, a temperature-sensitive mutation in the helical hairpin of let-341/Sos, a gain-of-function mutation affecting a potential phosphorylation site of the lin-1 Ets domain transcription factor, a dominant-negative allele of ksr-1, and hypomorphic alleles of sur-6/PP2A-B, sur-2/Mediator, and lin-25. In addition, this screen identified multiple alleles of two newly identified genes, eor-1 and eor-2, that play a relatively weak role in vulval fate specification but positively regulate Ras signaling during excretory system development and P12 fate specification. The spectrum of identified mutations argues strongly for the specificity of the enhancer screen and for a close involvement of eor-1 and eor-2 in Ras signaling. PMID:12019228

  6. The need for complex 3D culture models to unravel novel pathways and identify accurate biomarkers in breast cancer.

    PubMed

    Weigelt, Britta; Ghajar, Cyrus M; Bissell, Mina J

    2014-04-01

    The recent cataloging of the genomic aberrations in breast cancer has revealed the diversity and complexity of the disease at the genetic level. To unravel the functional consequences of specific repertoires of mutations and copy number changes on signaling pathways in breast cancer, it is crucial to develop model systems that truly recapitulate the disease. Here we discuss the three-dimensional culture models currently being used or recently developed for the study of normal mammary epithelial cells and breast cancer, including primary tumors and dormancy. We discuss the insights gained from these models in regards to cell signaling and potential therapeutic strategies, and the challenges that need to be met for the generation of heterotypic breast cancer model systems that are amenable for high-throughput approaches. PMID:24412474

  7. An Arrayed RNA Interference Genome-Wide Screen Identifies Candidate Genes Involved in the MicroRNA 21 Biogenesis Pathway

    PubMed Central

    Shum, David; Bhinder, Bhavneet; Ramirez, Christina N.; Radu, Constantin; Calder, Paul A.; Beauchamp, Lesslie; Farazi, T.; Landthaler, M.; Tuschi, T.; Magdaleno, Susan

    2013-01-01

    Abstract MicroRNAs (miRNAs) are evolutionary conserved noncoding molecules that regulate gene expression. They influence a number of diverse biological functions, such as development and differentiation. However, their dysregulation has been shown to be associated with disease states, such as cancer. Genes and pathways regulating their biogenesis remain unknown and are highly sought after. For this purpose, we have validated a multiplexed high-content assay strategy to screen for such modulators. Here, we describe its implementation that makes use of a cell-based gain-of-function reporter assay monitoring enhanced green fluorescent protein expression under the control of miRNA 21 (miR-21); combined with measures of both cell metabolic activities through the use of Alamar Blue and cell death through imaged Hoechst-stained nuclei. The strategy was validated using a panel of known genes and enabled us to successfully progress to and complete an arrayed genome-wide short interfering RNA (siRNA) screen against the Ambion Silencer Select v4.0 library containing 64,755 siRNA duplexes covering 21,565 genes. We applied a high-stringency hit analysis method, referred to as the BhinderDjaballah analysis method, leading to the nomination of 1,273 genes as candidate inhibitors of the miR-21 biogenesis pathway; after several iterations eliminating those genes with only one active duplex and those enriched in seed sequence mediated off-target effects. Biological classifications revealed four major control junctions among them vesicular transport via clathrin-mediated endocytosis. Altogether, our screen has uncovered a number of novel candidate regulators that are potentially good druggable targets allowing for the discovery and development of small molecules for regulating miRNA function. PMID:23153064

  8. Seasonal induction of alternative principal pathway for rose flower scent.

    PubMed

    Hirata, Hiroshi; Ohnishi, Toshiyuki; Tomida, Kensuke; Ishida, Haruka; Kanda, Momoyo; Sakai, Miwa; Yoshimura, Jin; Suzuki, Hideyuki; Ishikawa, Takamasa; Dohra, Hideo; Watanabe, Naoharu

    2016-01-01

    Ecological adaptations to seasonal changes are often observed in the phenotypic traits of plants and animals, and these adaptations are usually expressed through the production of different biochemical end products. In this study, ecological adaptations are observed in a biochemical pathway without alteration of the end products. We present an alternative principal pathway to the characteristic floral scent compound 2-phenylethanol (2PE) in roses. The new pathway is seasonally induced in summer as a heat adaptation that uses rose phenylpyruvate decarboxylase (RyPPDC) as a novel enzyme. RyPPDC transcript levels and the resulting production of 2PE are increased time-dependently under high temperatures. The novel summer pathway produces levels of 2PE that are several orders of magnitude higher than those produced by the previously known pathway. Our results indicate that the alternative principal pathway identified here is a seasonal adaptation for managing the weakened volatility of summer roses. PMID:26831950

  9. Seasonal induction of alternative principal pathway for rose flower scent

    PubMed Central

    Hirata, Hiroshi; Ohnishi, Toshiyuki; Tomida, Kensuke; Ishida, Haruka; Kanda, Momoyo; Sakai, Miwa; Yoshimura, Jin; Suzuki, Hideyuki; Ishikawa, Takamasa; Dohra, Hideo; Watanabe, Naoharu

    2016-01-01

    Ecological adaptations to seasonal changes are often observed in the phenotypic traits of plants and animals, and these adaptations are usually expressed through the production of different biochemical end products. In this study, ecological adaptations are observed in a biochemical pathway without alteration of the end products. We present an alternative principal pathway to the characteristic floral scent compound 2-phenylethanol (2PE) in roses. The new pathway is seasonally induced in summer as a heat adaptation that uses rose phenylpyruvate decarboxylase (RyPPDC) as a novel enzyme. RyPPDC transcript levels and the resulting production of 2PE are increased time-dependently under high temperatures. The novel summer pathway produces levels of 2PE that are several orders of magnitude higher than those produced by the previously known pathway. Our results indicate that the alternative principal pathway identified here is a seasonal adaptation for managing the weakened volatility of summer roses. PMID:26831950

  10. Biochemical characterization of recombinant β-carbonic anhydrase (PgiCAb) identified in the genome of the oral pathogenic bacterium Porphyromonas gingivalis.

    PubMed

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; AlOthman, Zeid; Osman, Sameh M; Supuran, Claudiu T; Capasso, Clemente

    2015-06-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β-, γ-, δ- and ζ-CAs are ubiquitous metalloenzymes present in prokaryotes and eukaryotes. CAs started to be investigated in detail only recently in pathogenic bacteria, in the search for antibiotics with a novel mechanism of action, since it has been demonstrated that in many such organisms they are essential for the life cycle of the organism. CA inhibition leads to growth impairment or growth defects in several pathogenic bacteria. The microbiota of the human oral mucosa consists of a myriad of bacterial species, Porphyromonas gingivalis being one of them and the major pathogen responsible for the development of chronic periodontitis. The genome of P. gingivalis encodes for a β- and a γ-CAs. Recently, our group purified the recombinant γ-CA (named PgiCA) which was shown to possess a significant catalytic activity for the reaction that converts CO2 to bicarbonate and protons, with a kcat of 4.1 × 10(5 )s(-1) and a kcat/Km of 5.4 × 10(7 )M(-1 )× s(-1). We have also investigated its inhibition profile with a range of inorganic anions such as thiocyanate, cyanide, azide, hydrogen sulfide, sulfamate and trithiocarbonate. Here, we describe the cloning, purification and kinetic parameters of the other class of CA identified in the genome of P. gingivalis, the β-CA, named PgiCAb. This enzyme has a good catalytic activity, with a kcat of 2.8 × 10(5 )s(-1) and a kcat/Km of 1.5 × 10(7 )M(-1 )× s(-1). PgiCAb was also inhibited by the clinically used sulfonamide acetazolamide, with an inhibition constant of 214 nM. The role of CAs as possible virulence factors of P. gingivalis is poorly understood at the moment but their good catalytic activity and the fact that they might be inhibited by a large number of compounds, which may pave the way for finding inhibitors with antibacterial activity that may elucidate these phenomena and lead to novel antibiotics. PMID:25032746

  11. The awesome power of yeast biochemical genomics.

    PubMed

    Carlson, M

    2000-02-01

    A new genomic strategy for identifying the gene encoding any biochemical activity has recently been developed, in which an array of individual yeast strains expressing a genomic set of open reading frames fused to glutathione S-transferase can be assayed for a biochemical activity of interest. Designated 'biochemical genomics', this approach represents an innovative application of genomic information. PMID:10652525

  12. Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

    PubMed

    Trefely, Sophie; Khoo, Poh-Sim; Krycer, James R; Chaudhuri, Rima; Fazakerley, Daniel J; Parker, Benjamin L; Sultani, Ghazal; Lee, James; Stephan, Jean-Philippe; Torres, Eric; Jung, Kenneth; Kuijl, Coenraad; James, David E; Junutula, Jagath R; Stöckli, Jacqueline

    2015-10-23

    The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling. PMID:26342081

  13. Proteomic Profiling Identifies Dysregulated Pathways in Small Cell Lung Cancer and Novel Therapeutic Targets Including PARP1

    PubMed Central

    Byers, Lauren Averett; Wang, Jing; Nilsson, Monique B.; Fujimoto, Junya; Saintigny, Pierre; Yordy, John; Giri, Uma; Peyton, Michael; Fan, You Hong; Diao, Lixia; Masrorpour, Fatemeh; Shen, Li; Liu, Wenbin; Duchemann, Boris; Tumula, Praveen; Bhardwaj, Vikas; Welsh, James; Weber, Stephanie; Glisson, Bonnie S.; Kalhor, Neda; Wistuba, Ignacio I.; Girard, Luc; Lippman, Scott M.; Mills, Gordon B.; Coombes, Kevin R.; Weinstein, John N.; Minna, John D.; Heymach, John V.

    2013-01-01

    Small cell lung cancer (SCLC) is an aggressive malignancy distinct from non-small cell lung cancer (NSCLC) in its metastatic potential and treatment response. Using an integrative proteomic and transcriptomic analysis, we investigated molecular differences contributing to the distinct clinical behavior of SCLC and NSCLC. SCLC demonstrated lower levels of several receptor tyrosine kinases and decreased activation of PI3K and Ras/MEK pathways, but significantly increased levels of E2F1-regulated factors including EZH2, thymidylate synthase, apoptosis mediators, and DNA repair proteins. Additionally, poly (ADP-ribose) polymerase 1 (PARP1), a DNA repair protein and E2F1 co-activator, was highly expressed at the mRNA and protein levels in SCLC. SCLC growth was inhibited by PARP1 and EZH2 knockdown. Furthermore, SCLC was significantly more sensitive to PARP inhibitors than NSCLC, and PARP inhibition downregulated key components of the DNA repair machinery and enhanced the efficacy of chemotherapy. PMID:22961666

  14. Stromal Transcriptional Profiles Reveal Hierarchies of Anatomical Site, Serum Response and Disease and Identify Disease Specific Pathways

    PubMed Central

    Parsonage, Greg N.; Legault, Holly M.; OToole, Margot; Pearson, Mark J.; Thomas, Andrew M.; Scheel-Toellner, Dagmar; Raza, Karim; Buckley, Christopher D.; Falciani, Francesco

    2015-01-01

    Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through _3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts. PMID:25807374

  15. Specific inhibitors for identifying pathways for methane production from carbon monoxide by a nonadapted anaerobic mixed culture.

    PubMed

    Navarro, Silvia Sancho; Cimpoia, Ruxandra; Bruant, Guillaume; Guiot, Serge R

    2014-06-01

    Specific inhibitors such as 2-bromoethanesulfonate (BES) and vancomycin were employed in activity batch tests to decipher metabolic pathways that are preferentially used by a mixed anaerobic consortium (sludge from an anaerobic digester) to transform carbon monoxide (CO) into methane (CH4). We first evaluated the inhibitory effect of both BES and vancomycin on the microbial community, as well as the efficiency and stability of vancomycin at 35 C, over time. The activity tests with CO2-H2, CO, glucose, acetate, formate, propionate, butyrate, methanol, and ethanol showed that vancomycin does not inhibit some Gram-negative bacteria, and 50 mmol/L BES effectively blocks CH4 production in the sludge. However, when sludge was incubated with propionate, butyrate, methanol, or ethanol as the sole energy and carbon source, methanogenesis was only partially inhibited by BES. Separate tests showed that 0.07 mmol/L vancomycin is enough to maintain its inhibitory efficiency and stability in the population for at least 32 days at 35 C. Using the inhibitors above, it was demonstrated that CO conversion to CH4 is an indirect, 2-step process, in which the CO is converted first to acetate and subsequently to CH4. PMID:24896194

  16. Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors

    PubMed Central

    Schneider, Anna; Corona, Angela; Spöring, Imke; Jordan, Mareike; Buchholz, Bernd; Maccioni, Elias; Di Santo, Roberto; Bodem, Jochen; Tramontano, Enzo; Wöhrl, Birgitta M.

    2016-01-01

    We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs. PMID:26850643

  17. Identifying Resistance Mechanisms against Five Tyrosine Kinase Inhibitors Targeting the ERBB/RAS Pathway in 45 Cancer Cell Lines

    PubMed Central

    Szsz, A. Marcell; Sztupinszki, Zsfia; Lik, Istvn; Szendr?i, Attila; Schfer, Reinhold; Gy?rffy, Balzs

    2013-01-01

    Because of the low overall response rates of 1047% to targeted cancer therapeutics, there is an increasing need for predictive biomarkers. We aimed to identify genes predicting response to five already approved tyrosine kinase inhibitors. We tested 45 cancer cell lines for sensitivity to sunitinib, erlotinib, lapatinib, sorafenib and gefitinib at the clinically administered doses. A resistance matrix was determined, and gene expression profiles of the subsets of resistant vs. sensitive cell lines were compared. Triplicate gene expression signatures were obtained from the caArray project. Significance analysis of microarrays and rank products were applied for feature selection. Ninety-five genes were also measured by RT-PCR. In case of four sunitinib resistance associated genes, the results were validated in clinical samples by immunohistochemistry. A list of 63 top genes associated with resistance against the five tyrosine kinase inhibitors was identified. Quantitative RT-PCR analysis confirmed 45 of 63 genes identified by microarray analysis. Only two genes (ANXA3 and RAB25) were related to sensitivity against more than three inhibitors. The immunohistochemical analysis of sunitinib-treated metastatic renal cell carcinomas confirmed the correlation between RAB17, LGALS8, and EPCAM and overall survival. In summary, we determined predictive biomarkers for five tyrosine kinase inhibitors, and validated sunitinib resistance biomarkers by immunohistochemistry in an independent patient cohort. PMID:23555683

  18. The roles of IP3 receptor in energy metabolic pathways and reactive oxygen species homeostasis revealed by metabolomic and biochemical studies.

    PubMed

    Wen, He; Xu, Wen Jun; Jin, Xing; Oh, Sehyun; Phan, Chau Hong Duc; Song, Jayoung; Lee, Sang Kook; Park, Sunghyouk

    2015-11-01

    Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are calcium channels modulating important calcium-mediated processes. Recent studies implicate IP(3)R in cell metabolism, but specific evidence is missing regarding IP(3)R's effects on actual metabolic pathways and key energy metabolites. Here, we applied metabolomics and molecular biology to compare DT40 cell lines devoid of IP(3)R (KO) and its wild-type (WT) counterpart. NMR and LC-MS metabolomic data showed that the KO cell line has a very different basic energy metabolism from the WT cell line, showing enhanced Warburg effect. In particular, the KO cells exhibited significant perturbation in energy charge, reduced glutathione and NADPH ratios with slower cellular growth rate. Subsequent flow cytometry results showed that the KO cell line has a higher level of general reactive oxygen species (ROS) but has a lower level of peroxynitrites. This ROS disturbance could be explained by observing lower expression of superoxide dismutase 2 (SOD2) and unchanged expression of catalase. The higher ROS seems to be involved in the slower growth rate of the KO cells, with an ROS scavenger increasing their growth rate. However, the KO and WT cell lines did not show any noticeable differences in AMPK and phosphorylated AMPK levels, suggesting possible saturation of AMPK-mediated metabolic regulatory circuit in both cells. Overall, our study reveals IP3R's roles in ROS homeostasis and metabolic pathways as well as the effects of its KO on cellular phenotypes. PMID:26235438

  19. Structure/Function Analysis of a Type III Polyketide Synthase in the Brown Alga Ectocarpus siliculosus Reveals a Biochemical Pathway in Phlorotannin Monomer Biosynthesis[W

    PubMed Central

    Meslet-Cladire, Laurence; Delage, Ludovic; Leroux, Cdric J.-J.; Goulitquer, Sophie; Leblanc, Catherine; Creis, Emeline; Gall, Erwan Ar; Stiger-Pouvreau, Valrie; Czjzek, Mirjam; Potin, Philippe

    2013-01-01

    Brown algal phlorotannins are structural analogs of condensed tannins in terrestrial plants and, like plant phenols, they have numerous biological functions. Despite their importance in brown algae, phlorotannin biosynthetic pathways have been poorly characterized at the molecular level. We found that a predicted type III polyketide synthase in the genome of the brown alga Ectocarpus siliculosus, PKS1, catalyzes a major step in the biosynthetic pathway of phlorotannins (i.e., the synthesis of phloroglucinol monomers from malonyl-CoA). The crystal structure of PKS1 at 2.85- resolution provided a good quality electron density map showing a modified Cys residue, likely connected to a long chain acyl group. An additional pocket not found in other known type III PKSs contains a reaction product that might correspond to a phloroglucinol precursor. In vivo, we also found a positive correlation between the phloroglucinol content and the PKS III gene expression level in cells of a strain of Ectocarpus adapted to freshwater during its reacclimation to seawater. The evolution of the type III PKS gene family in Stramenopiles suggests a lateral gene transfer event from an actinobacterium. PMID:23983220

  20. Structure/function analysis of a type iii polyketide synthase in the brown alga Ectocarpus siliculosus reveals a biochemical pathway in phlorotannin monomer biosynthesis.

    PubMed

    Meslet-Cladire, Laurence; Delage, Ludovic; Leroux, Cdric J-J; Goulitquer, Sophie; Leblanc, Catherine; Creis, Emeline; Gall, Erwan Ar; Stiger-Pouvreau, Valrie; Czjzek, Mirjam; Potin, Philippe

    2013-08-01

    Brown algal phlorotannins are structural analogs of condensed tannins in terrestrial plants and, like plant phenols, they have numerous biological functions. Despite their importance in brown algae, phlorotannin biosynthetic pathways have been poorly characterized at the molecular level. We found that a predicted type III polyketide synthase in the genome of the brown alga Ectocarpus siliculosus, PKS1, catalyzes a major step in the biosynthetic pathway of phlorotannins (i.e., the synthesis of phloroglucinol monomers from malonyl-CoA). The crystal structure of PKS1 at 2.85- resolution provided a good quality electron density map showing a modified Cys residue, likely connected to a long chain acyl group. An additional pocket not found in other known type III PKSs contains a reaction product that might correspond to a phloroglucinol precursor. In vivo, we also found a positive correlation between the phloroglucinol content and the PKS III gene expression level in cells of a strain of Ectocarpus adapted to freshwater during its reacclimation to seawater. The evolution of the type III PKS gene family in Stramenopiles suggests a lateral gene transfer event from an actinobacterium. PMID:23983220

  1. Noncanonical PAR3 activation by factor Xa identifies a novel pathway for Tie2 activation and stabilization of vascular integrity

    PubMed Central

    Stavenuiter, Fabian

    2014-01-01

    Endothelial barrier protective effects of activated protein C (APC) require the endothelial protein C receptor (EPCR), protease-activated receptor (PAR) 1, and PAR3. In contrast, PAR1 and PAR3 activation by thrombin results in barrier disruption. Noncanonical PAR1 and PAR3 activation by APC vs canonical activation by thrombin provides an explanation for the functional selectivity of these proteases. Here we found that factor Xa (FXa) activated PAR1 at canonical Arg41 similar to thrombin but cleaved PAR3 at noncanonical Arg41 similar to APC. This unique PAR1-PAR3 activation profile permitted the identification of noncanonical PAR3 activation as a novel activation pathway for barrier protective tunica intima endothelial receptor tyrosine kinase 2 (Tie2). APC, FXa, and the noncanonical PAR3 tethered-ligand peptide induced prolonged activation of Tie2, whereas thrombin and the canonical PAR3 tethered-ligand peptide did not. Tie2 activation by FXa required PAR3 and EPCR. FXa and the noncanonical PAR3 tethered-ligand peptide induced Tie2- and PAR3-dependent upregulation of tight-junction-associated protein zona occludens 1 (ZO-1), translocation of ZO-1 to cell-cell borders, and the formation of typical ZO-1 honeycomb patterns that are indicative of tight-junction stabilization. These data provide intriguing novel insights into the diversification of functional selectivity of protease signaling achievable by canonical and noncanonical PAR activation, such as the activation of vascular-protective Tie2 by noncanonical PAR3 activation. PMID:25320242

  2. A Genome-Wide Association Study of the Maize Hypersensitive Defense Response Identifies Genes That Cluster in Related Pathways

    PubMed Central

    Venkata, Bala P.; Marla, Sandeep; Ji, Jiabing; Gachomo, Emma; Chu, Kevin; Negeri, Adisu; Benson, Jacqueline; Nelson, Rebecca; Bradbury, Peter; Nielsen, Dahlia; Holland, James B.; Balint-Kurti, Peter J.; Johal, Gurmukh

    2014-01-01

    Much remains unknown of molecular events controlling the plant hypersensitive defense response (HR), a rapid localized cell death that limits pathogen spread and is mediated by resistance (R-) genes. Genetic control of the HR is hard to quantify due to its microscopic and rapid nature. Natural modifiers of the ectopic HR phenotype induced by an aberrant auto-active R-gene (Rp1-D21), were mapped in a population of 3,381 recombinant inbred lines from the maize nested association mapping population. Joint linkage analysis was conducted to identify 32 additive but no epistatic quantitative trait loci (QTL) using a linkage map based on more than 7000 single nucleotide polymorphisms (SNPs). Genome-wide association (GWA) analysis of 26.5 million SNPs was conducted after adjusting for background QTL. GWA identified associated SNPs that colocalized with 44 candidate genes. Thirty-six of these genes colocalized within 23 of the 32 QTL identified by joint linkage analysis. The candidate genes included genes predicted to be in involved programmed cell death, defense response, ubiquitination, redox homeostasis, autophagy, calcium signalling, lignin biosynthesis and cell wall modification. Twelve of the candidate genes showed significant differential expression between isogenic lines differing for the presence of Rp1-D21. Low but significant correlations between HR-related traits and several previously-measured disease resistance traits suggested that the genetic control of these traits was substantially, though not entirely, independent. This study provides the first system-wide analysis of natural variation that modulates the HR response in plants. PMID:25166276

  3. Metabolic Engineering of Light and Dark Biochemical Pathways in Wild-Type and Mutant Strains of Synechocystis PCC 6803 for Maximal, 24-Hour Production of Hydrogen Gas

    SciTech Connect

    Ely, Roger L.; Chaplen, Frank W.R.

    2014-03-11

    This project used the cyanobacterial species Synechocystis PCC 6803 to pursue two lines of inquiry, with each line addressing one of the two main factors affecting hydrogen (H2) production in Synechocystis PCC 6803: NADPH availability and O2 sensitivity. H2 production in Synechocystis PCC 6803 requires a very high NADPH:NADP+ ratio, that is, the NADP pool must be highly reduced, which can be problematic because several metabolic pathways potentially can act to raise or lower NADPH levels. Also, though the [NiFe]-hydrogenase in PCC 6803 is constitutively expressed, it is reversibly inactivated at very low O2 concentrations. Largely because of this O2 sensitivity and the requirement for high NADPH levels, a major portion of overall H2 production occurs under anoxic conditions in the dark, supported by breakdown of glycogen or other organic substrates accumulated during photosynthesis. Also, other factors, such as N or S limitation, pH changes, presence of other substances, or deletion of particular respiratory components, can affect light or dark H2 production. Therefore, in the first line of inquiry, under a number of culture conditions with wild type (WT) Synechocystis PCC 6803 cells and a mutant with impaired type I NADPH-dehydrogenase (NDH-1) function, we used H2 production profiling and metabolic flux analysis, with and without specific inhibitors, to examine systematically the pathways involved in light and dark H2 production. Results from this work provided rational bases for metabolic engineering to maximize photobiological H2 production on a 24-hour basis. In the second line of inquiry, we used site-directed mutagenesis to create mutants with hydrogenase enzymes exhibiting greater O2 tolerance. The research addressed the following four tasks: 1. Evaluate the effects of various culture conditions (N, S, or P limitation; light/dark; pH; exogenous organic carbon) on H2 production profiles of WT cells and an NDH-1 mutant; 2. Conduct metabolic flux analyses for enhanced H2 production profiles using selected culture conditions and inhibitors of specific pathways in WT cells and an NDH-1 mutant; 3. Create Synechocystis PCC 6803 mutant strains with modified hydrogenases exhibiting increased O2 tolerance and greater H2 production; and 4. Integrate enhanced hydrogenase mutants and culture and metabolic factor studies to maximize 24-hour H2 production.

  4. Global transcriptome analysis identifies regulated transcripts and pathways activated during oogenesis and early embryogenesis in Atlantic cod.

    PubMed

    Kleppe, Lene; Edvardsen, Rolf Brudvik; Furmanek, Tomasz; Taranger, Geir Lasse; Wargelius, Anna

    2014-07-01

    The molecular mechanisms underlying oogenesis and maternally controlled embryogenesis in fish are not fully understood, especially in marine species. Our aim was to study the egg and embryo transcriptome during oogenesis and early embryogenesis in Atlantic cod. Follicles from oogenesis stages (pre-, early-, and late-vitellogenic), ovulated eggs, and two embryonic stages (blastula, gastrula) were collected from broodstock fish and fertilized eggs. Gene expression profiles were measured in a 44?K oligo microarray consisting of 23,000 cod genes. Hundreds of differentially expressed genes (DEGs) were identified in the follicle stages investigated, implicating a continuous accumulation and degradation of polyadenylated transcripts throughout oogenesis. Very few DEGs were identified from ovulated egg to blastula, showing a more stable maternal RNA pool in early embryonic stages. The highest induction of expression was observed between blastula and gastrula, signifying the onset of zygotic transcription. During early vitellogenesis, several of the most upregulated genes are linked to nervous system signaling, suggesting increasing requirements for ovarian synaptic signaling to stimulate the rapid growth of oocytes. Highly upregulated genes during late vitellogenesis are linked to protein processing, fat metabolism, osmoregulation, and arrested meiosis. One of the genes with the highest upregulation in the ovulated egg is involved in oxidative phosphorylation, reflecting increased energy requirements during fertilization and the first rapid cell divisions of early embryogenesis. In conclusion, this study provides a large-scale presentation of the Atlantic cod's maternally controlled transcriptome in ovarian follicles through oogenesis, ovulated eggs, and early embryos. PMID:24687555

  5. Exome sequencing of desmoplastic melanoma identifies recurrent NFKBIE promoter mutations and diverse activating mutations in the MAPK pathway.

    PubMed

    Shain, A Hunter; Garrido, Maria; Botton, Thomas; Talevich, Eric; Yeh, Iwei; Sanborn, J Zachary; Chung, Jongsuk; Wang, Nicholas J; Kakavand, Hojabr; Mann, Graham J; Thompson, John F; Wiesner, Thomas; Roy, Ritu; Olshen, Adam B; Gagnon, Alexander; Gray, Joe W; Huh, Nam; Hur, Joe S; Busam, Klaus J; Scolyer, Richard A; Cho, Raymond J; Murali, Rajmohan; Bastian, Boris C

    2015-10-01

    Desmoplastic melanoma is an uncommon variant of melanoma with sarcomatous histology, distinct clinical behavior and unknown pathogenesis. We performed low-coverage genome and high-coverage exome sequencing of 20 desmoplastic melanomas, followed by targeted sequencing of 293 genes in a validation cohort of 42 cases. A high mutation burden (median of 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers. Mutation patterns strongly implicate ultraviolet radiation as the dominant mutagen, indicating a superficially located cell of origin. Newly identified alterations included recurrent promoter mutations of NFKBIE, encoding NF-?B inhibitor ? (I?B?), in 14.5% of samples. Common oncogenic mutations in melanomas, in particular in BRAF (encoding p.Val600Glu) and NRAS (encoding p.Gln61Lys or p.Gln61Arg), were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS and PIK3CA, some of which are candidates for targeted therapies. PMID:26343386

  6. Large-scale association analyses identify new loci influencing glycemic traits and provide insight into the underlying biological pathways

    PubMed Central

    Scott, Robert A; Lagou, Vasiliki; Welch, Ryan P; Wheeler, Eleanor; Montasser, May E; Luan, Jianan; Mgi, Reedik; Strawbridge, Rona J; Rehnberg, Emil; Gustafsson, Stefan; Kanoni, Stavroula; Rasmussen-Torvik, Laura J; Yengo, Loc; Lecoeur, Cecile; Shungin, Dmitry; Sanna, Serena; Sidore, Carlo; Johnson, Paul C D; Jukema, J Wouter; Johnson, Toby; Mahajan, Anubha; Verweij, Niek; Thorleifsson, Gudmar; Hottenga, Jouke-Jan; Shah, Sonia; Smith, Albert V; Sennblad, Bengt; Gieger, Christian; Salo, Perttu; Perola, Markus; Timpson, Nicholas J; Evans, David M; Pourcain, Beate St; Wu, Ying; Andrews, Jeanette S; Hui, Jennie; Bielak, Lawrence F; Zhao, Wei; Horikoshi, Momoko; Navarro, Pau; Isaacs, Aaron; OConnell, Jeffrey R; Stirrups, Kathleen; Vitart, Veronique; Hayward, Caroline; Esko, Tnu; Mihailov, Evelin; Fraser, Ross M; Fall, Tove; Voight, Benjamin F; Raychaudhuri, Soumya; Chen, Han; Lindgren, Cecilia M; Morris, Andrew P; Rayner, Nigel W; Robertson, Neil; Rybin, Denis; Liu, Ching-Ti; Beckmann, Jacques S; Willems, Sara M; Chines, Peter S; Jackson, Anne U; Kang, Hyun Min; Stringham, Heather M; Song, Kijoung; Tanaka, Toshiko; Peden, John F; Goel, Anuj; Hicks, Andrew A; An, Ping; Mller-Nurasyid, Martina; Franco-Cereceda, Anders; Folkersen, Lasse; Marullo, Letizia; Jansen, Hanneke; Oldehinkel, Albertine J; Bruinenberg, Marcel; Pankow, James S; North, Kari E; Forouhi, Nita G; Loos, Ruth J F; Edkins, Sarah; Varga, Tibor V; Hallmans, Gran; Oksa, Heikki; Antonella, Mulas; Nagaraja, Ramaiah; Trompet, Stella; Ford, Ian; Bakker, Stephan J L; Kong, Augustine; Kumari, Meena; Gigante, Bruna; Herder, Christian; Munroe, Patricia B; Caulfield, Mark; Antti, Jula; Mangino, Massimo; Small, Kerrin; Miljkovic, Iva; Liu, Yongmei; Atalay, Mustafa; Kiess, Wieland; James, Alan L; Rivadeneira, Fernando; Uitterlinden, Andre G; Palmer, Colin N A; Doney, Alex S F; Willemsen, Gonneke; Smit, Johannes H; Campbell, Susan; Polasek, Ozren; Bonnycastle, Lori L; Hercberg, Serge; Dimitriou, Maria; Bolton, Jennifer L; Fowkes, Gerard R; Kovacs, Peter; Lindstrm, Jaana; Zemunik, Tatijana; Bandinelli, Stefania; Wild, Sarah H; Basart, Hanneke V; Rathmann, Wolfgang; Grallert, Harald; Maerz, Winfried; Kleber, Marcus E; Boehm, Bernhard O; Peters, Annette; Pramstaller, Peter P; Province, Michael A; Borecki, Ingrid B; Hastie, Nicholas D; Rudan, Igor; Campbell, Harry; Watkins, Hugh; Farrall, Martin; Stumvoll, Michael; Ferrucci, Luigi; Waterworth, Dawn M; Bergman, Richard N; Collins, Francis S; Tuomilehto, Jaakko; Watanabe, Richard M; de Geus, Eco J C; Penninx, Brenda W; Hofman, Albert; Oostra, Ben A; Psaty, Bruce M; Vollenweider, Peter; Wilson, James F; Wright, Alan F; Hovingh, G Kees; Metspalu, Andres; Uusitupa, Matti; Magnusson, Patrik K E; Kyvik, Kirsten O; Kaprio, Jaakko; Price, Jackie F; Dedoussis, George V; Deloukas, Panos; Meneton, Pierre; Lind, Lars; Boehnke, Michael; Shuldiner, Alan R; van Duijn, Cornelia M; Morris, Andrew D; Toenjes, Anke; Peyser, Patricia A; Beilby, John P; Krner, Antje; Kuusisto, Johanna; Laakso, Markku; Bornstein, Stefan R; Schwarz, Peter E H; Lakka, Timo A; Rauramaa, Rainer; Adair, Linda S; Smith, George Davey; Spector, Tim D; Illig, Thomas; de Faire, Ulf; Hamsten, Anders; Gudnason, Vilmundur; Kivimaki, Mika; Hingorani, Aroon; Keinanen-Kiukaanniemi, Sirkka M; Saaristo, Timo E; Boomsma, Dorret I; Stefansson, Kari; van der Harst, Pim; Dupuis, Jose; Pedersen, Nancy L; Sattar, Naveed; Harris, Tamara B; Cucca, Francesco; Ripatti, Samuli; Salomaa, Veikko; Mohlke, Karen L; Balkau, Beverley; Froguel, Philippe; Pouta, Anneli; Jarvelin, Marjo-Riitta; Wareham, Nicholas J; Bouatia-Naji, Nabila; McCarthy, Mark I; Franks, Paul W; Meigs, James B; Teslovich, Tanya M; Florez, Jose C; Langenberg, Claudia; Ingelsson, Erik; Prokopenko, Inga; Barroso, Ins

    2012-01-01

    Through genome-wide association meta-analyses of up to 133,010 individuals of European ancestry without diabetes, including individuals newly genotyped using the Metabochip, we have raised the number of confirmed loci influencing glycemic traits to 53, of which 33 also increase type 2 diabetes risk (q < 0.05). Loci influencing fasting insulin showed association with lipid levels and fat distribution, suggesting impact on insulin resistance. Gene-based analyses identified further biologically plausible loci, suggesting that additional loci beyond those reaching genome-wide significance are likely to represent real associations. This conclusion is supported by an excess of directionally consistent and nominally significant signals between discovery and follow-up studies. Functional follow-up of these newly discovered loci will further improve our understanding of glycemic control. PMID:22885924

  7. Global transcriptome analysis identifies regulated transcripts and pathways activated during oogenesis and early embryogenesis in atlantic cod

    PubMed Central

    Kleppe, Lene; Edvardsen, Rolf Brudvik; Furmanek, Tomasz; Taranger, Geir Lasse; Wargelius, Anna

    2014-01-01

    The molecular mechanisms underlying oogenesis and maternally controlled embryogenesis in fish are not fully understood, especially in marine species. Our aim was to study the egg and embryo transcriptome during oogenesis and early embryogenesis in Atlantic cod. Follicles from oogenesis stages (pre-, early-, and late-vitellogenic), ovulated eggs, and two embryonic stages (blastula, gastrula) were collected from broodstock fish and fertilized eggs. Gene expression profiles were measured in a 44 K oligo microarray consisting of 23,000 cod genes. Hundreds of differentially expressed genes (DEGs) were identified in the follicle stages investigated, implicating a continuous accumulation and degradation of polyadenylated transcripts throughout oogenesis. Very few DEGs were identified from ovulated egg to blastula, showing a more stable maternal RNA pool in early embryonic stages. The highest induction of expression was observed between blastula and gastrula, signifying the onset of zygotic transcription. During early vitellogenesis, several of the most upregulated genes are linked to nervous system signaling, suggesting increasing requirements for ovarian synaptic signaling to stimulate the rapid growth of oocytes. Highly upregulated genes during late vitellogenesis are linked to protein processing, fat metabolism, osmoregulation, and arrested meiosis. One of the genes with the highest upregulation in the ovulated egg is involved in oxidative phosphorylation, reflecting increased energy requirements during fertilization and the first rapid cell divisions of early embryogenesis. In conclusion, this study provides a large-scale presentation of the Atlantic cod's maternally controlled transcriptome in ovarian follicles through oogenesis, ovulated eggs, and early embryos. Mol. Reprod. Dev. 81: 619–635, 2014. © 2014 Wiley Periodicals, Inc. PMID:24687555

  8. Combined transcriptome and proteome analysis identifies pathways and markers associated with the establishment of rapeseed microspore-derived embryo development.

    PubMed

    Joosen, Ronny; Cordewener, Jan; Supena, Ence Darmo Jaya; Vorst, Oscar; Lammers, Michiel; Maliepaard, Chris; Zeilmaker, Tieme; Miki, Brian; America, Twan; Custers, Jan; Boutilier, Kim

    2007-05-01

    Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants. PMID:17384159

  9. RNA Sequencing Identifies Upregulated Kyphoscoliosis Peptidase and Phosphatidic Acid Signaling Pathways in Muscle Hypertrophy Generated by Transgenic Expression of Myostatin Propeptide

    PubMed Central

    Miao, Yuanxin; Yang, Jinzeng; Xu, Zhong; Jing, Lu; Zhao, Shuhong; Li, Xinyun

    2015-01-01

    Myostatin (MSTN), a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type littermate controls with a high-throughput RNA sequencing method. The results show that 132 genes were significantly differentially expressed between transgenic mice and wild-type control mice; 97 of these genes were up-regulated, and 35 genes were down-regulated in the skeletal muscle. Several genes that had not been reported to be involved in muscle hypertrophy were identified, including up-regulated myosin binding protein H (mybph), and zinc metallopeptidase STE24 (Zmpste24). In addition, kyphoscoliosis peptidase (Ky), which plays a vital role in muscle growth, was also up-regulated in the transgenic mice. Interestingly, a pathway analysis based on grouping the differentially expressed genes uncovered that cardiomyopathy-related pathways and phosphatidic acid (PA) pathways (Dgki, Dgkz, Plcd4) were up-regulated. Increased PA signaling may increase mTOR signaling, resulting in skeletal muscle growth. The findings of the RNA sequencing analysis help to understand the molecular mechanisms of muscle hypertrophy caused by MSTN inhibition. PMID:25860951

  10. Ion permeation in the NanC porin from Escherichia coli: free energy calculations along pathways identified by coarse-grain simulations.

    PubMed

    Dreyer, Jens; Strodel, Paul; Ippoliti, Emiliano; Finnerty, Justin; Eisenberg, Bob; Carloni, Paolo

    2013-10-31

    Using the X-ray structure of a recently discovered bacterial protein, the N-acetylneuraminic acid-inducible channel (NanC), we investigate computationally K(+) and Cl(-) ions' permeation. We identify ion permeation pathways that are likely to be populated using coarse-grain Monte Carlo simulations. Next, we use these pathways as reaction coordinates for umbrella sampling-based free energy simulations. We find distinct tubelike pathways connecting specific binding sites for K(+) and, more pronounced, for Cl(-) ions. Both ions permeate the porin preserving almost all of their first hydration shell. The calculated free energy barriers are G(#) ? 4 kJ/mol and G(#) ? 8 kJ/mol for Cl(-) and K(+), respectively. Within the approximations associated with these values, discussed in detail in this work, we suggest that the porin is slightly selective for Cl(-) versus K(+). Our suggestion is consistent with the experimentally observed weak Cl(-) over K(+) selectivity. A rationale for the latter is suggested by a comparison with previous calculations on strongly anion selective porins. PMID:24147565

  11. RNA sequencing identifies upregulated kyphoscoliosis peptidase and phosphatidic acid signaling pathways in muscle hypertrophy generated by transgenic expression of myostatin propeptide.

    PubMed

    Miao, Yuanxin; Yang, Jinzeng; Xu, Zhong; Jing, Lu; Zhao, Shuhong; Li, Xinyun

    2015-01-01

    Myostatin (MSTN), a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type littermate controls with a high-throughput RNA sequencing method. The results show that 132 genes were significantly differentially expressed between transgenic mice and wild-type control mice; 97 of these genes were up-regulated, and 35 genes were down-regulated in the skeletal muscle. Several genes that had not been reported to be involved in muscle hypertrophy were identified, including up-regulated myosin binding protein H (mybph), and zinc metallopeptidase STE24 (Zmpste24). In addition, kyphoscoliosis peptidase (Ky), which plays a vital role in muscle growth, was also up-regulated in the transgenic mice. Interestingly, a pathway analysis based on grouping the differentially expressed genes uncovered that cardiomyopathy-related pathways and phosphatidic acid (PA) pathways (Dgki, Dgkz, Plcd4) were up-regulated. Increased PA signaling may increase mTOR signaling, resulting in skeletal muscle growth. The findings of the RNA sequencing analysis help to understand the molecular mechanisms of muscle hypertrophy caused by MSTN inhibition. PMID:25860951

  12. Analysis of the Protein Kinase A-Regulated Proteome of Cryptococcus neoformans Identifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation

    PubMed Central

    Geddes, J. M. H.; Caza, M.; Croll, D.; Stoynov, N.; Foster, L. J.

    2016-01-01

    ABSTRACT The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningitis in immunocompromised individuals. The expression of virulence factors, including capsule and melanin, is in part regulated by the cyclic-AMP/protein kinase A (cAMP/PKA) signal transduction pathway. In this study, we investigated the influence of PKA on the composition of the intracellular proteome to obtain a comprehensive understanding of the regulation that underpins virulence. Through quantitative proteomics, enrichment and bioinformatic analyses, and an interactome study, we uncovered a pattern of PKA regulation for proteins associated with translation, the proteasome, metabolism, amino acid biosynthesis, and virulence-related functions. PKA regulation of the ubiquitin-proteasome pathway in C. neoformans showed a striking parallel with connections between PKA and protein degradation in chronic neurodegenerative disorders and other human diseases. Further investigation of proteasome function with the inhibitor bortezomib revealed an impact on capsule production as well as hypersusceptibility for strains with altered expression or activity of PKA. Parallel studies with tunicamycin also linked endoplasmic reticulum stress with capsule production and PKA. Taken together, the data suggest a model whereby expression of PKA regulatory and catalytic subunits and the activation of PKA influence proteostasis and the function of the endoplasmic reticulum to control the elaboration of the polysaccharide capsule. Overall, this study revealed both broad and conserved influences of the cAMP/PKA pathway on the proteome and identified proteostasis as a potential therapeutic target for the treatment of cryptococcosis. PMID:26758180

  13. Ordering events of biochemical evolution.

    PubMed

    Cunchillos, C; Lecointre, G

    2007-05-01

    Metabolic pathways exhibit structures resulting from an evolutionary process. Pathways have been inherited through time with modification, from the earliest periods of life. It is possible to compare the structure of pathways as done in comparative anatomy, i.e. for inferring ancestral pathways or parts of it (ancestral enzymatic functions), using standard phylogenetic reconstruction. Thus a phylogenetic tree of pathways provides a relative ordering of the rise of enzymatic functions. It even becomes possible to order the birth of each complete pathway in time. This particular "DNA-free" conceptual approach to evolutionary biochemistry is reviewed, gathering all the justifications given for it. Then, the method of assigning a given pathway to a time span of biochemical development is revisited. The previous method used an implicit "clock" of metabolic development that is difficult to justify. We develop a new clock-free approach, using functional biochemical arguments. Results of the two methods are not significantly different; our method is just more precise. This suggests that the clock assumed in the first method does not provoke any important artefact in describing the development of biochemical evolution. It is just unnecessary to postulate it. As a result, most of the amino acid metabolic pathways develop forwards, confirming former models of amino acid catabolism evolution, but not those for amino acid anabolism. The order of appearance of sectors of universal cellular metabolism is: (1) amino acid catabolism, (2) amino acid anabolism and closure of the urea cycle, (3) glycolysis and glycogenesis, (4) closure of the pentose-phosphate cycle, (5) closure of the Krebs cycle and fatty acids metabolism, (6) closure of the Calvin cycle. PMID:17408843

  14. Heterogeneous activation of the TGF? pathway in glioblastomas identified by gene expression-based classification using TGF?-responsive genes

    PubMed Central

    Xu, Xie L; Kapoun, Ann M

    2009-01-01

    Background TGF? has emerged as an attractive target for the therapeutic intervention of glioblastomas. Aberrant TGF? overproduction in glioblastoma and other high-grade gliomas has been reported, however, to date, none of these reports has systematically examined the components of TGF? signaling to gain a comprehensive view of TGF? activation in large cohorts of human glioma patients. Methods TGF? activation in mammalian cells leads to a transcriptional program that typically affects 510% of the genes in the genome. To systematically examine the status of TGF? activation in high-grade glial tumors, we compiled a gene set of transcriptional response to TGF? stimulation from tissue culture and in vivo animal studies. These genes were used to examine the status of TGF? activation in high-grade gliomas including a large cohort of glioblastomas. Unsupervised and supervised classification analysis was performed in two independent, publicly available glioma microarray datasets. Results Unsupervised and supervised classification using the TGF?-responsive gene list in two independent glial tumor gene expression data sets revealed various levels of TGF? activation in these tumors. Among glioblastomas, one of the most devastating human cancers, two subgroups were identified that showed distinct TGF? activation patterns as measured from transcriptional responses. Approximately 62% of glioblastoma samples analyzed showed strong TGF? activation, while the rest showed a weak TGF? transcriptional response. Conclusion Our findings suggest heterogeneous TGF? activation in glioblastomas, which may cause potential differences in responses to anti-TGF? therapies in these two distinct subgroups of glioblastomas patients. PMID:19192267

  15. Translational research in brain metastasis is identifying molecular pathways that may lead to the development of new therapeutic strategies

    PubMed Central

    Gril, Brunilde; Evans, Lynda; Palmieri, Diane; Steeg, Patricia S.

    2010-01-01

    Central nervous system (CNS) or brain metastasis is an emerging area of interest in organ-specific metastasis research. Lung and breast cancers are the most common types of primary tumors to develop brain metastases. This disease complication contributes significantly to the morbidity and mortality of both of these common cancers; as such, brain metastasis is designated an unmet medical need by the US Food and Drug Administration. Recently, an increase in incidence of CNS disease has been noted in the literature for breast cancer, while it has been an ongoing major complication from lung cancer. Progress in treating brain metastases has been hampered by a lack of model systems, a lack of human tissue samples, and the exclusion of brain metastatic patients from many clinical trials. While each of those is significant, the major impediment to effectively treating brain metastatic disease is the bloodbrain barrier (BBB). This barrier excludes most chemotherapeutics from the brain and creates a sanctuary site for metastatic tumors. Recent findings on the biology of this disease and translational leads identified by molecular studies are discussed in this article. PMID:20303257

  16. Time-resolved Studies of IsdG Protein Identify Molecular Signposts along the Non-canonical Heme Oxygenase Pathway.

    PubMed

    Streit, Bennett R; Kant, Ravi; Tokmina-Lukaszewska, Monika; Celis, Arianna I; Machovina, Melodie M; Skaar, Eric P; Bothner, Brian; DuBois, Jennifer L

    2016-01-01

    IsdGs are heme monooxygenases that break open the tetrapyrrole, releasing the iron, and thereby allowing bacteria expressing this protein to use heme as a nutritional iron source. Little is currently known about the mechanism by which IsdGs degrade heme, although the products differ from those generated by canonical heme oxygenases. A synthesis of time-resolved techniques, including in proteo mass spectrometry and conventional and stopped-flow UV/visible spectroscopy, was used in conjunction with analytical methods to define the reaction steps mediated by IsdG from Staphylococcus aureus and their time scales. An apparent meso-hydroxyheme (forming with k = 0.6 min(-1), pH 7.4, 10 mm ascorbate, 10 μm IsdG-heme, 22 °C) was identified as a likely common intermediate with the canonical heme oxygenases. Unlike heme oxygenases, this intermediate does not form with added H2O2 nor does it convert to verdoheme and CO. Rather, the next observable intermediates (k = 0.16 min(-1)) were a set of formyloxobilin isomers, similar to the mycobilin products of the IsdG homolog from Mycobacterium tuberculosis (MhuD). These converted in separate fast and slow phases to β-/δ-staphylobilin isomers and formaldehyde (CH2O). Controlled release of this unusual C1 product may support IsdG's dual role as both an oxygenase and a sensor of heme availability in S. aureus. PMID:26534961

  17. YlxM Is a Newly Identified Accessory Protein That Influences the Function of Signal Recognition Particle Pathway Components in Streptococcus mutans

    PubMed Central

    Williams, Matthew L.; Crowley, Paula J.; Hasona, Adnan

    2014-01-01

    Streptococcus mutans is a cariogenic oral pathogen whose virulence is determined largely by its membrane composition. The signal recognition particle (SRP) protein-targeting pathway plays a pivotal role in membrane biogenesis. S. mutans SRP pathway mutants demonstrate growth defects, cannot contend with environmental stress, and exhibit multiple changes in membrane composition. This study sought to define a role for ylxM, which in S. mutans and numerous other bacteria resides directly upstream of the ffh gene, encoding a major functional element of the bacterial SRP. YlxM was observed as a produced protein in S. mutans. Its predicted helix-turn-helix motif suggested that it has a role as a transcriptional regulator of components within the SRP pathway; however, no evidence of transcriptional regulation was found. Instead, capture enzyme-linked immunosorbent assay (ELISA), affinity chromatography, and bio-layer interferometry (BLI) demonstrated that S. mutans YlxM interacts with the SRP components Ffh and small cytoplasmic RNA (scRNA) but not with the SRP receptor FtsY. In the absence of FtsY, YlxM increased the GTP hydrolysis activity of Ffh alone and in complex with scRNA. However, in the presence of FtsY, YlxM caused an overall diminution of net GTPase activity. Thus, YlxM appears to modulate GTP hydrolysis, a process necessary for proper recycling of SRP pathway components. The presence of YlxM conferred a significant competitive growth advantage under nonstress and acid stress conditions when wild-type and ylxM mutant strains were cultured together. Our results identify YlxM as a component of the S. mutans SRP and suggest a regulatory function affecting GTPase activity. PMID:24659773

  18. SFGD: a comprehensive platform for mining functional information from soybean transcriptome data and its use in identifying acyl-lipid metabolism pathways

    PubMed Central

    2014-01-01

    Background Soybean (Glycine max L.) is one of the world’s most important leguminous crops producing high-quality protein and oil. Increasing the relative oil concentration in soybean seeds is many researchers’ goal, but a complete analysis platform of functional annotation for the genes involved in the soybean acyl-lipid pathway is still lacking. Following the success of soybean whole-genome sequencing, functional annotation has become a major challenge for the scientific community. Whole-genome transcriptome analysis is a powerful way to predict genes with biological functions. It is essential to build a comprehensive analysis platform for integrating soybean whole-genome sequencing data, the available transcriptome data and protein information. This platform could also be used to identify acyl-lipid metabolism pathways. Description In this study, we describe our construction of the Soybean Functional Genomics Database (SFGD) using Generic Genome Browser (Gbrowse) as the core platform. We integrated microarray expression profiling with 255 samples from 14 groups’ experiments and mRNA-seq data with 30 samples from four groups’ experiments, including spatial and temporal transcriptome data for different soybean development stages and environmental stresses. The SFGD includes a gene co-expression regulatory network containing 23,267 genes and 1873 miRNA-target pairs, and a group of acyl-lipid pathways containing 221 enzymes and more than 1550 genes. The SFGD also provides some key analysis tools, i.e. BLAST search, expression pattern search and cis-element significance analysis, as well as gene ontology information search and single nucleotide polymorphism display. Conclusion The SFGD is a comprehensive database integrating genome and transcriptome data, and also for soybean acyl-lipid metabolism pathways. It provides useful toolboxes for biologists to improve the accuracy and robustness of soybean functional genomics analysis, further improving understanding of gene regulatory networks for effective crop improvement. The SFGD is publically accessible at http://bioinformatics.cau.edu.cn/SFGD/, with all data available for downloading. PMID:24712981

  19. Gene-centric analysis identifies variants associated with interleukin-6 levels and shared pathways with other inflammation markers.

    PubMed

    Shah, Tina; Zabaneh, Delilah; Gaunt, Tom; Swerdlow, Daniel I; Shah, Sonia; Talmud, Philippa J; Day, Ian N; Whittaker, John; Holmes, Michael V; Sofat, Reecha; Humphries, Steve E; Kivimaki, Mika; Kumari, Meena; Hingorani, Aroon D; Casas, Juan P

    2013-04-01

    BACKGROUND- Inflammatory cytokine interleukin-6 (IL-6), a possible risk factor for coronary heart disease, has an estimated heritability of >60%, but to date few genetic variants influencing IL-6 levels are known. METHODS AND RESULTS- We used the ITMAT-Broad-Care (IBC) HumanCVD disease BeadChip in the Whitehall II study (N=4911) and British Women's Heart and Health Study (N=3445) to identify single-nucleotide polymorphisms associated with circulating IL-6 levels. Twenty-two single-nucleotide polymorphisms from 7 loci (IL6R/TDRD10, HLA-DRB1, BUD13, SEZ6L, IL1RN, TRIB3, and ABO) were associated with IL-6 (P<10(-5)), although none were associated with the IL6 gene itself. With the exception of TRIB3, all loci have been previously reported in genome-wide association studies for autoimmune and cardiovascular diseases. Fourteen single-nucleotide polymorphisms in the IL6R region in high-linkage disequilibrium (r(2)>0.9) with a nonsynonymous variant, rs2228145, were also associated with IL-6 and C-reactive protein concentration (P<10(-5)). An IL-6-specific weighted allele score explained 2% of the variance of log IL-6 levels (P=2.4410(-22)) in Whitehall II and 1% (P=1.910(-8)) in British Women's Heart and Health Studies. CONCLUSIONS- Multiple common genetic variants of modest effect influence IL-6 concentration. Several loci contain single-nucleotide polymorphisms, exhibiting overlapping associations with autoimmune and cardiovascular disorders and other circulating biomarkers. Genetic variants associated with IL-6 provide important tools for probing the causal relevance of IL-6 signaling in a range of cardiometabolic diseases. PMID:23505291

  20. A System Biology Approach to Identify Regulatory Pathways Underlying the Neuroendocrine Control of Female Puberty in Rats and Nonhuman Primates

    PubMed Central

    Lomniczi, Alejandro; Wright, Hollis; Castellano, Juan Manuel; Sonmez, Kemal; Ojeda, Sergio R.

    2014-01-01

    Puberty is a major developmental milestone controlled by the interaction of genetic factors and environmental cues of mostly metabolic and circadian nature. An increased pulsatile release of the decapeptide gonadotropin releasing hormone (GnRH) from hypothalamic neurosecretory neurons is required for both the initiation and progression of the pubertal process. This increase is brought about by coordinated changes that occur in neuronal and glial networks associated with GnRH neurons. These changes ultimately result in increased neuronal and glial stimulatory inputs to the GnRH neuronal network and a reduction of transsynaptic inhibitory influences. While some of the major players controlling pubertal GnRH secretion have been identified using gene-centric approaches, much less is known about the system-wide control of the overall process. Because the pubertal activation of GnRH release involves a diversity of cellular phenotypes, and a myriad of intracellular and cell-to-cell signaling molecules, it appears that the overall process is controlled by a highly coordinated and interactive regulatory system involving hundreds, if not thousands, of gene products. In this article we will discuss emerging evidence suggesting that these genes are arranged as functionally connected networks organized, both internally and across sub-networks, in a hierarchical fashion. According to this concept, the core of these networks is composed of transcriptional regulators that, by directing expression of downstream subordinate genes, provide both stability and coordination to the cellular networks involved in initiating the pubertal process. The integrative response of these gene networks to external inputs is postulated to be coordinated by epigenetic mechanisms. PMID:23998662

  1. Small-molecule vasopressin-2 receptor antagonist identified by a g-protein coupled receptor "pathway" screen.

    PubMed

    Yangthara, Buranee; Mills, Aaron; Chatsudthipong, Varanuj; Tradtrantip, Lukmanee; Verkman, A S

    2007-07-01

    G-protein-coupled receptors (GPCRs) such as the vasopressin-2 receptor (V(2)R) are an important class of drug targets. We developed an efficient screen for GPCR-induced cAMP elevation using as read-out cAMP activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. Fischer rat thyroid cells expressing CFTR and a halide-sensing yellow fluorescent protein (H148Q/I152L) were transfected with V(2)R. Increased cell Cl(-) conductance after agonist-induced cAMP elevation was assayed using a plate reader from cell fluorescence after solution I(-) addition. The Z' factor for the assay was approximately 0.7 with the V(2)R agonist [deamino-Cys1, Val4, d-Arg8]-vasopressin (1 nM) as positive control. Primary screening of 50,000 small molecules yielded a novel, 5-aryl-4-benzoyl-3-hydroxy-1-(2-arylethyl)-2H-pyrrol-2-one class of V(2)R antagonists that are unrelated structurally to known V(2)R antagonists. The most potent compound, V(2)R(inh)-02, which was identified by screening 35 structural analogs, competitively inhibited V(2)R-induced cAMP elevation with K(i) value of approximately 70 nM and fully displaced radiolabeled vasopressin in binding experiments. V(2)R(inh)-02 did not inhibit forskolin or beta(2)-adrenergic receptor-induced cAMP production and was more than 50 times more potent for V(2)R than for V(1a)R. The favorable in vitro properties of the pyrrol-2-one antagonists suggests their potential usefulness in aquaretic applications. The CFTR-linked cAMP assay developed here is applicable for efficient, high-throughput identification of modulators of cAMP-coupled GPCRs. PMID:17435162

  2. The use of conditional probability functions and potential source contribution functions to identify source regions and advection pathways of hydrocarbon emissions in Houston, Texas

    NASA Astrophysics Data System (ADS)

    Xie, Yulong; Berkowitz, Carl M.

    In this study, we demonstrate the utility of conditional probability functions (CPFs), potential source contribution functions (PSCFs), and hierarchical clustering analysis (HAC) to identify the source region and transport pathways of hydrocarbons measured at five photochemical assessment monitoring stations (PAMS) near the Houston Ship Channel from June to October 2003. In contrast to scatter plots, which only show the pair-wise correlation of species, commonality in CPF figures shows both correlation and information on the source region of the species in question. In this study, we use over 50 hourly volatile organic compound (VOC) concentrations and surface wind observations to show that VOCs with similar CPF patterns likely have common transport pathways. This was established with the multivariate technique, which uses the hierarchical clustering analysis to define clusters of VOCs having similar CPF patterns. This method revealed that alkenes, and in particular those with geometric isomers such as cis-/ trans-2-butene and cis-/ trans-2-pentene, have similar CPF patterns and hence, a common area of origin. The alkane isomers often show CPF patterns among themselves, and similarly, aromatic compounds often show similar patterns. We also show how calculated trajectory information can be used in the PSCF analysis to produce a graphic picture that identifies specific geographic areas associated with a given VOC (or other pollutant). The use of these techniques in the chemically and meteorologically complex environment of Houston, Texas, suggests its further utility in other areas with relatively simpler conditions.

  3. CSGene: a literature-based database for cell senescence genes and its application to identify critical cell aging pathways and associated diseases.

    PubMed

    Zhao, M; Chen, L; Qu, H

    2016-01-01

    Cell senescence is a cellular process in which normal diploid cells cease to replicate and is a major driving force for human cancers and aging-associated diseases. Recent studies on cell senescence have identified many new genetic components and pathways that control cell aging. However, there is no comprehensive resource for cell senescence that integrates various genetic studies and relationships with cell senescence, and the risk associated with complex diseases such as cancer is still unexplored. We have developed the first literature-based gene resource for exploring cell senescence genes, CSGene. We complied 504 experimentally verified genes from public data resources and published literature. Pathway analyses highlighted the prominent roles of cell senescence genes in the control of rRNA gene transcription and unusual rDNA repeat that constitute a center for the stability of the whole genome. We also found a strong association of cell senescence with HIV-1 infection and viral carcinogenesis that are mainly related to promoter/enhancer binding and chromatin modification processes. Moreover, pan-cancer mutation and network analysis also identified common cell aging mechanisms in cancers and uncovered a highly modular network structure. These results highlight the utility of CSGene for elucidating the complex cellular events of cell senescence. PMID:26775705

  4. Robust Selection Algorithm (RSA) for Multi-Omic Biomarker Discovery; Integration with Functional Network Analysis to Identify miRNA Regulated Pathways in Multiple Cancers

    PubMed Central

    Sehgal, Vasudha; Seviour, Elena G.; Moss, Tyler J.; Mills, Gordon B.; Azencott, Robert; Ram, Prahlad T.

    2015-01-01

    MicroRNAs (miRNAs) play a crucial role in the maintenance of cellular homeostasis by regulating the expression of their target genes. As such, the dysregulation of miRNA expression has been frequently linked to cancer. With rapidly accumulating molecular data linked to patient outcome, the need for identification of robust multi-omic molecular markers is critical in order to provide clinical impact. While previous bioinformatic tools have been developed to identify potential biomarkers in cancer, these methods do not allow for rapid classification of oncogenes versus tumor suppressors taking into account robust differential expression, cutoffs, p-values and non-normality of the data. Here, we propose a methodology, Robust Selection Algorithm (RSA) that addresses these important problems in big data omics analysis. The robustness of the survival analysis is ensured by identification of optimal cutoff values of omics expression, strengthened by p-value computed through intensive random resampling taking into account any non-normality in the data and integration into multi-omic functional networks. Here we have analyzed pan-cancer miRNA patient data to identify functional pathways involved in cancer progression that are associated with selected miRNA identified by RSA. Our approach demonstrates the way in which existing survival analysis techniques can be integrated with a functional network analysis framework to efficiently identify promising biomarkers and novel therapeutic candidates across diseases. PMID:26505200

  5. Microarray analysis in a cell death resistant glioma cell line to identify signaling pathways and novel genes controlling resistance and malignancy.

    PubMed

    Seznec, Janina; Naumann, Ulrike

    2011-01-01

    Glioblastoma multiforme (GBM) is a lethal type of cancer mainly resistant to radio- and chemotherapy. Since the tumor suppressor p53 functions as a transcription factor regulating the expression of genes involved in growth inhibition, DNA repair and apoptosis, we previously assessed whether specific differences in the modulation of gene expression are responsible for the anti-tumor properties of a dominant positive p53, chimeric tumor suppressor (CTS)-1. CTS-1 is based on the sequence of p53 and designed to resist various mechanisms of inactivation which limit the activity of p53. To identify CTS-1-regulated cell death-inducing genes, we generated a CTS-1-resistant glioma cell line (229R). We used Affymetrix whole-genome microarray expression analysis to analyze alterations in gene expression and identified a variety of CTS-1 regulated genes involved in cancer-linked processes. 313 genes were differentially expressed in Adeno-CTS-1 (Ad-CTS-1)-infected and 700 genes in uninfected 229R cells compared to matching parental cells. Ingenuity Pathway Analysis (IPA) determined a variety of differentially expressed genes in Ad-CTS-1-infected cells that were members of the intracellular networks with central tumor-involved players such as nuclear factor kappa B (NF-?B), protein kinase B (PKB/AKT) or transforming growth factor beta (TGF-?). Differentially regulated genes include secreted factors as well as intracellular proteins and transcription factors regulating not only cell death, but also processes such as tumor cell motility and immunity. This work gives an overview of the pathways differentially regulated in the resistant versus parental glioma cells and might be helpful to identify candidate genes which could serve as targets to develop novel glioma specific therapy strategies. PMID:24212935

  6. 40 CFR 158.2010 - Biochemical pesticides data requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Biochemical pesticides data...) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Biochemical Pesticides § 158.2010 Biochemical pesticides... required to support registration of biochemical pesticides. Sections 158.2080 through 158.2084 identify...

  7. 40 CFR 158.2010 - Biochemical pesticides data requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Biochemical pesticides data...) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Biochemical Pesticides § 158.2010 Biochemical pesticides... required to support registration of biochemical pesticides. Sections 158.2080 through 158.2084 identify...

  8. 40 CFR 158.2010 - Biochemical pesticides data requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Biochemical pesticides data...) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Biochemical Pesticides § 158.2010 Biochemical pesticides... required to support registration of biochemical pesticides. Sections 158.2080 through 158.2084 identify...

  9. 40 CFR 158.2010 - Biochemical pesticides data requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Biochemical pesticides data...) PESTICIDE PROGRAMS DATA REQUIREMENTS FOR PESTICIDES Biochemical Pesticides § 158.2010 Biochemical pesticides... required to support registration of biochemical pesticides. Sections 158.2080 through 158.2084 identify...

  10. siRNA screening identifies differences in the Fanconi anemia pathway in BALB/c-Trp53+/- with susceptibility versus C57BL/6-Trp53+/- mice with resistance to mammary tumors.

    PubMed

    Bhringer, M; Obermeier, K; Griner, N; Waldraff, D; Dickinson, E; Eirich, K; Schindler, D; Hagen, M; Jerry, D J; Wiesmller, L

    2013-11-28

    BALB/c mice heterozygous for Trp53 develop a high proportion of spontaneous mammary tumors, a phenotype distinct from other mouse strains. BALB/c-Trp53+/- female mice, thus, resemble the hereditary Li-Fraumeni syndrome (LFS) characterized by early-onset of breast cancer, even though LFS involves TP53 mutations, which may involve not only loss- but also gain-of-function. Previous analysis of tumors in BALB/c-Trp53+/- females showed frequent loss of heterozygosity involving the wild-type allele of Trp53 and displayed characteristics indicative of mitotic recombination. Critical involvement of DNA double-strand break (DSB) repair dysfunction, particularly of homologous recombination (HR), was also noticed in the etiology of human breast cancer. To better define functional alterations in BALB/c-Trp53+/- mice, we applied a fluorescence-based DSB repair assay on mouse embryonic fibroblasts (MEFs) from BALB/c-Trp53+/- versus C57BL/6J-Trp53+/- mice. This approach revealed deregulation of HR but not non-homologous end-joining (NHEJ) in BALB/c-Trp53+/-, which was further confirmed for mammary epithelial cells. Screening of a small interfering RNA-library targeting DSB repair, recombination, replication and signaling genes, identified 25 genes causing differences between homologous DSB repair in the two strains upon silencing. Interactome analysis of the hits revealed clustering of replication-related and fanconi anemia (FA)/breast cancer susceptibility (BRCA) genes. Further dissection of the functional change in BALB/c-Trp53+/- by immunofluorescence microscopy of nuclear 53BP1, Replication protein A (RPA) and Rad51 foci uncovered differences in crosslink and replication-associated repair. Chromosome breakage, G2 arrest and biochemical analyses indicated a FA pathway defect downstream of FancD2 associated with reduced levels of BRCA2. Consistent with polygenic models for BRCA, mammary carcinogenesis in BALB/c-Trp53+/- mice may, therefore, be promoted by a BRCA modifier allele in the FA pathway in the context of partial p53 loss-of-function. PMID:23435420

  11. The Use of Conditional Probability Functions and Potential Source Contribution Functions to Identify Source Regions and Advection Pathways of Hydrocarbon Emissions in Houston, Texas

    SciTech Connect

    Xie, YuLong; Berkowitz, Carl M.

    2007-09-01

    In this study, we demonstrate the utility of conditional probability functions (CPFs), potential source contribution functions (PSCFs), and hierarchical clustering analysis to identify the source region and transport pathways of hydrocarbons measured at five photochemical assessment monitoring stations (PAMS) near the Houston ship channel from June to October 2003. Over 50 volatile organic compound (VOC) concentrations were measured on the hourly collected samples. Routine surface observations of wind directions measured at each of the receptor sites were used extensively. We show that VOCs with similar CPF patterns likely have common transport pathways. This was established with the multivariate technique, which uses the hierarchical clustering analysis to allow clusters of groups of VOCs to form with similar CPF patterns. This method revealed that alkenes, and in particular those with geometric isomers such as cis-/trans-2-butene and cis-/trans-2-pentene, have similar CPF patterns. The alkane isomers often show CPF patterns among themselves, and similarly, aromatic compounds often show similar patterns among themselves too. We also show how trajectory information can be used in conjunction with the PSCF analysis to produce a graphic analysis suggesting specific source areas for a given VOC. The use of these techniques in the chemically and meteorologically complex environment of Houston, Texas, suggests its further utility in other areas with relatively simpler conditions.

  12. A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway

    PubMed Central

    Anania, Veronica G.; Bustos, Daisy J.; Lill, Jennie R.; Kirkpatrick, Donald S.; Coscoy, Laurent

    2013-01-01

    The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor ? (TCR?) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCR? occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCR? in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCR? is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCR? peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCR? remains elusive. These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms. PMID:23431445

  13. High Throughput Sequencing Identifies MicroRNAs Mediating α-Synuclein Toxicity by Targeting Neuroactive-Ligand Receptor Interaction Pathway in Early Stage of Drosophila Parkinson's Disease Model

    PubMed Central

    Kong, Yan; Liang, Xijun; Liu, Lin; Zhang, Dongdong; Wan, Chao; Gan, Zhenji; Yuan, Liudi

    2015-01-01

    Parkinson’s disease (PD) is a prevalent neurodegenerative disorder with pathological features including death of dopaminergic neurons in the substantia nigra and intraneuronal accumulations of Lewy bodies. As the main component of Lewy bodies, α-synuclein is implicated in PD pathogenesis by aggregation into insoluble filaments. However, the detailed mechanisms underlying α-synuclein induced neurotoxicity in PD are still elusive. MicroRNAs are ~20nt small RNA molecules that fine-tune gene expression at posttranscriptional level. A plethora of miRNAs have been found to be dysregulated in the brain and blood cells of PD patients. Nevertheless, the detailed mechanisms and their in vivo functions in PD still need further investigation. By using Drosophila PD model expressing α-synuclein A30P, we examined brain miRNA expression with high-throughput small RNA sequencing technology. We found that five miRNAs (dme-miR-133-3p, dme-miR-137-3p, dme-miR-13b-3p, dme-miR-932-5p, dme-miR-1008-5p) were upregulated in PD flies. Among them, miR-13b, miR-133, miR-137 are brain enriched and highly conserved from Drosophila to humans. KEGG pathway analysis using DIANA miR-Path demonstrated that neuroactive-ligand receptor interaction pathway was most likely affected by these miRNAs. Interestingly, miR-137 was predicted to regulate most of the identified targets in this pathway, including dopamine receptor (DopR, D2R), γ-aminobutyric acid (GABA) receptor (GABA-B-R1, GABA-B-R3) and N-methyl-D-aspartate (NMDA) receptor (Nmdar2). The validation experiments showed that the expression of miR-137 and its targets was negatively correlated in PD flies. Further experiments using luciferase reporter assay confirmed that miR-137 could act on specific sites in 3’ UTR region of D2R, Nmdar2 and GABA-B-R3, which downregulated significantly in PD flies. Collectively, our findings indicate that α-synuclein could induce the dysregulation of miRNAs, which target neuroactive ligand-receptor interaction pathway in vivo. We believe it will help us further understand the contribution of miRNAs to α-synuclein neurotoxicity and provide new insights into the pathogenesis driving PD. PMID:26361355

  14. Hyperoxia-Induced Protein Alterations in Renal Rat Tissue: A Quantitative Proteomic Approach to Identify Hyperoxia-Induced Effects in Cellular Signaling Pathways

    PubMed Central

    Hinkelbein, Jochen; Böhm, Lennert; Spelten, Oliver; Sander, David; Soltész, Stefan; Braunecker, Stefan

    2015-01-01

    Introduction. In renal tissue as well as in other organs, supranormal oxygen pressure may lead to deleterious consequences on a cellular level. Additionally, hyperoxia-induced effect in cells and related free radicals may potentially contribute to renal failure. The aim of this study was to analyze time-dependent alterations of rat kidney protein expression after short-term normobaric hyperoxia using proteomics and bioinformatic approaches. Material and Methods. N = 36 Wistar rats were randomized into six different groups: three groups with normobaric hyperoxia (exposure to 100% oxygen for 3 h) and three groups with normobaric normoxia (NN; room air). After hyperoxia exposure, kidneys were removed immediately, after 3 days and after 7 days. Kidney lysates were analyzed by two-dimensional gel electrophoresis followed by peptide mass fingerprinting using tandem mass spectrometry. Statistical analysis was performed with DeCyder 2D software (p < 0.01). Biological functions of differential regulated proteins were studied using functional network analysis (Ingenuity Pathways Analysis and PathwayStudio). Results. Expression of 14 proteins was significantly altered (p < 0.01): eight proteins (MEP1A_RAT, RSSA_RAT, F16P1_RAT, STML2_RAT, BPNT1_RAT, LGMN_RAT, ATPA_RAT, and VDAC1_RAT) were downregulated and six proteins (MTUS1_RAT, F16P1_RAT, ACTG_RAT, ACTB_RAT, 2ABA_RAT, and RAB1A_RAT) were upregulated. Bioinformatic analyses revealed an association of regulated proteins with inflammation. Conclusions. Significant alterations in renal protein expression could be demonstrated for up to 7 days even after short-term hyperoxia. The identified proteins indicate an association with inflammation signaling cascades. MEP1A and VDAC1 could be promising candidates to identify hyperoxic injury in kidney cells. PMID:26106253

  15. ReactionFlow: an interactive visualization tool for causality analysis in biological pathways

    PubMed Central

    2015-01-01

    Background Molecular and systems biologists are tasked with the comprehension and analysis of incredibly complex networks of biochemical interactions, called pathways, that occur within a cell. Through interviews with domain experts, we identified four common tasks that require an understanding of the causality within pathways, that is, the downstream and upstream relationships between proteins and biochemical reactions, including: visualizing downstream consequences of perturbing a protein; finding the shortest path between two proteins; detecting feedback loops within the pathway; and identifying common downstream elements from two or more proteins. Results We introduce ReactionFlow, a visual analytics application for pathway analysis that emphasizes the structural and causal relationships amongst proteins, complexes, and biochemical reactions within a given pathway. To support the identified causality analysis tasks, user interactions allow an analyst to filter, cluster, and select pathway components across linked views. Animation is used to highlight the flow of activity through a pathway. Conclusions We evaluated ReactionFlow by providing our application to two domain experts who have significant experience with biomolecular pathways, after which we conducted a series of in-depth interviews focused on each of the four causality analysis tasks. Their feedback leads us to believe that our techniques could be useful to researchers who must be able to understand and analyze the complex nature of biological pathways. ReactionFlow is available at https://github.com/CreativeCodingLab/ReactionFlow. PMID:26361502

  16. Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1S6K pathway signaling

    PubMed Central

    Lindquist, Robert A.; Ottina, Kathleen A.; Wheeler, Douglas B.; Hsu, Peggy P.; Thoreen, Carson C.; Guertin, David A.; Ali, Siraj M.; Sengupta, Shomit; Shaul, Yoav D.; Lamprecht, Michael R.; Madden, Katherine L.; Papallo, Adam R.; Jones, Thouis R.; Sabatini, David M.; Carpenter, Anne E.

    2011-01-01

    The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls cell growth in response to nutrient availability and growth factors. TORC1 signaling is hyperactive in cancer, and regulators of TORC1 signaling may represent therapeutic targets for human diseases. To identify novel regulators of TORC1 signaling, we performed a genome-scale RNA interference screen on microarrays of Drosophila melanogaster cells expressing human RPS6, a TORC1 effector whose phosphorylated form we detected by immunofluorescence. Our screen revealed that the TORC1S6KRPS6 signaling axis is regulated by many subcellular components, including the Class I vesicle coat (COPI), the spliceosome, the proteasome, the nuclear pore, and the translation initiation machinery. Using additional RNAi reagents, we confirmed 70 novel genes as significant on-target regulators of RPS6 phosphorylation, and we characterized them with extensive secondary assays probing various arms of the TORC1 pathways, identifying functional relationships among those genes. We conclude that cell-based microarrays are a useful platform for genome-scale and secondary screening in Drosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling. PMID:21239477

  17. Integrating Bayesian variable selection with Modular Response Analysis to infer biochemical network topology

    PubMed Central

    2013-01-01

    Background Recent advancements in genetics and proteomics have led to the acquisition of large quantitative data sets. However, the use of these data to reverse engineer biochemical networks has remained a challenging problem. Many methods have been proposed to infer biochemical network topologies from different types of biological data. Here, we focus on unraveling network topologies from steady state responses of biochemical networks to successive experimental perturbations. Results We propose a computational algorithm which combines a deterministic network inference method termed Modular Response Analysis (MRA) and a statistical model selection algorithm called Bayesian Variable Selection, to infer functional interactions in cellular signaling pathways and gene regulatory networks. It can be used to identify interactions among individual molecules involved in a biochemical pathway or reveal how different functional modules of a biological network interact with each other to exchange information. In cases where not all network components are known, our method reveals functional interactions which are not direct but correspond to the interaction routes through unknown elements. Using computer simulated perturbation responses of signaling pathways and gene regulatory networks from the DREAM challenge, we demonstrate that the proposed method is robust against noise and scalable to large networks. We also show that our method can infer network topologies using incomplete perturbation datasets. Consequently, we have used this algorithm to explore the ERBB regulated G1/S transition pathway in certain breast cancer cells to understand the molecular mechanisms which cause these cells to become drug resistant. The algorithm successfully inferred many well characterized interactions of this pathway by analyzing experimentally obtained perturbation data. Additionally, it identified some molecular interactions which promote drug resistance in breast cancer cells. Conclusions The proposed algorithm provides a robust, scalable and cost effective solution for inferring network topologies from biological data. It can potentially be applied to explore novel pathways which play important roles in life threatening disease like cancer. PMID:23829771

  18. A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.

    PubMed

    Lecat, Sandra; Matthes, Hans W D; Pepperkok, Rainer; Simpson, Jeremy C; Galzi, Jean-Luc

    2015-05-01

    Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. PMID:25759509

  19. Genomic and Biochemical Analysis of the Diaminopimelate and Lysine Biosynthesis Pathway in Verrucomicrobium spinosum: Identification and Partial Characterization of L,L-Diaminopimelate Aminotransferase and UDP-N-Acetylmuramoylalanyl-D-glutamyl-2,6-meso-Diaminopimelate Ligase

    PubMed Central

    Nachar, Victoria R.; Savka, Francisco C.; McGroty, Sean E.; Donovan, Katherine A.; North, Rachel A.; Dobson, Renwick C. J.; Buckley, Larry J.; Hudson, Andr O.

    2012-01-01

    The Gram-negative bacterium Verrucomicrobium spinosum has attracted interest in recent years following the sequencing and annotation of its genome. Comparative genomic analysis of V. spinosum using diaminopimelate/lysine metabolic genes from Chlamydia trachomatis suggests that V. spinosum employs the L,L-diaminopimelate aminotransferase (DapL) pathway for diaminopimelate/lysine biosynthesis. The open reading frame corresponding to the putative dapL ortholog was cloned and the recombinant enzyme was shown to possess L,L-diaminopimelate aminotransferase activity in vitro. In vivo analysis using functional complementation confirmed that the dapL ortholog was able to functionally complement an E. coli mutant that confers auxotrophy for diaminopimelate and lysine. In addition to its role in lysine biosynthesis, the intermediate diaminopimelate has an integral role in peptidoglycan biosynthesis. To this end, the UDP-N-acetylmuramoylalanyl-d-glutamyl-2,6-meso-diaminopimelate ligase ortholog was also identified, cloned, and was shown to possess meso-diaminopimelate ligase activity in vivo. The L,L-diaminopimelate aminotransferase pathway has been experimentally confirmed in several bacteria, some of which are deemed pathogenic to animals. Since animals, and particularly humans, lack the genetic machinery for the synthesis of diaminopimelate/lysine de novo, the enzymes involved in this pathway are attractive targets for development of antibiotics. Whether dapL is an essential gene in any bacteria is currently not known. V. spinosum is an excellent candidate to investigate the essentiality of dapL, since the bacterium employs the DapL pathway for lysine and cell wall biosynthesis, is non-pathogenic to humans, facile to grow, and can be genetically manipulated. PMID:22783236

  20. Use of peanut lectin and rat mammary stem cell lines to identify a cellular differentiation pathway for the alveolar cell in the rat mammary gland.

    PubMed

    Rudland, P S

    1992-10-01

    The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar-like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently-conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein-secreting vacuolated cells. PNL-staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125I]-labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structures of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase-conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end-buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat beta-casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end-buds to bind PNL without the necessity for their desialylation similar to that seen in casein secretory alveoli of lactating rats. In conclusion the different interconverting cell types of Rama 25 which form a pathway to casein-secretory cells in vitro are thus equated with recognisable epithelial cell types in vivo. These results suggest that casein-secretory cells in vivo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli. PMID:1522129

  1. ChIP-Seq and RNA-Seq Analyses Identify Components of the Wnt and Fgf Signaling Pathways as Prep1 Target Genes in Mouse Embryonic Stem Cells

    PubMed Central

    Laurent, Audrey; Calabrese, Manuela; Warnatz, Hans-Jrg; Yaspo, Marie-Laure; Tkachuk, Vsevolod; Torres, Miguel; Blasi, Francesco; Penkov, Dmitry

    2015-01-01

    The Prep1 (Pknox1) homeodomain transcription factor is essential at multiple stages of embryo development. In the E11.5 embryo trunk, we previously estimated that Prep1 binds about 3,300 genomic sites at a highly specific decameric consensus sequence, mainly governing basal cellular functions. We now show that in embryonic stem (ES) cells Prep1 binding pattern only partly overlaps that of the embryo trunk, with about 2,000 novel sites. Moreover, in ES cells Prep1 still binds mostly to promoters, as in total embryo trunk but, among the peaks bound exclusively in ES cells, the percentage of enhancers was three-fold higher. RNA-seq identifies about 1800 genes down-regulated in Prep1-/- ES cells which belong to gene ontology categories not enriched in the E11.5 Prep1i/i differentiated embryo, including in particular essential components of the Wnt and Fgf pathways. These data agree with aberrant Wnt and Fgf expression levels in the Prep1-/- ES cells with a deficient embryoid bodies (EBs) formation and differentiation. Re-establishment of the Prep1 level rescues the phenotype. PMID:25875616

  2. Network analysis of gene expression in peripheral blood identifies mTOR and NF-κB pathways involved in antipsychotic-induced extrapyramidal symptoms.

    PubMed

    Mas, S; Gassó, P; Parellada, E; Bernardo, M; Lafuente, A

    2015-10-01

    To identify the candidate genes for pharmacogenetic studies of antipsychotic (AP)-induced extrapyramidal symptoms (EPS), we propose a systems biology analytical approach, based on protein-protein interaction network construction and functional annotation analysis, of changes in gene expression (Human Genome U219 Array Plate) induced by treatment with risperidone or paliperidone in peripheral blood. 12 AP-naïve patients with first-episode psychosis participated in the present study. Our analysis revealed that, in response to AP treatment, constructed networks were enriched for different biological processes in patients without EPS (ubiquitination, protein folding and adenosine triphosphate (ATP) metabolism) compared with those presenting EPS (insulin receptor signaling, lipid modification, regulation of autophagy and immune response). Moreover, the observed differences also involved specific pathways, such as anaphase promoting complex /cdc20, prefoldin/CCT/triC and ATP synthesis in no-EPS patients, and mammalian target of rapamycin and NF-κB kinases in patients with EPS. Our results showing different patterns of gene expression in EPS patients, offer new and valuable markers for pharmacogenetic studies. PMID:25623440

  3. eIF5A1/RhoGDIα pathway: a novel therapeutic target for treatment of spinal cord injury identified by a proteomics approach

    PubMed Central

    Liu, Wei; Shang, Fei-Fei; Xu, Yang; Belegu, Visar; Xia, Lei; Zhao, Wei; Liu, Ran; Wang, Wei; Liu, Jin; Li, Chen-Yun; Wang, Ting-Hua

    2015-01-01

    Spinal cord injury (SCI) is frequently accompanied by a degree of spontaneous functional recovery. The underlying mechanisms through which such recovery is generated remain elusive. In this study, we observed a significant spontaneous motor function recovery 14 to 28 days after spinal cord transection (SCT) in rats. Using a comparative proteomics approach, caudal to the injury, we detected difference in 20 proteins. Two of these proteins, are eukaryotic translation initiation factor 5A1 (eIF5A1) that is involved in cell survival and proliferation, and Rho GDP dissociation inhibitor alpha (RhoGDIα), a member of Rho GDI family that is involved in cytoskeletal reorganization. After confirming the changes in expression levels of these two proteins following SCT, we showed that in vivo eIF5A1 up-regulation and down-regulation significantly increased and decreased, respectively, motor function recovery. In vitro, eIF5A1 overexpression in primary neurons increased cell survival and elongated neurite length while eIF5A1 knockdown reversed these results. We found that RhoGDIα up-regulation and down-regulation rescues the effect of eIF5A1 down-regulation and up-regulation both in vivo and in vitro. Therefore, we have identified eIF5A1/RhoGDIα pathway as a new therapeutic target for treatment of spinal cord injured patients. PMID:26593060

  4. The G alpha subunit G alpha 4 couples to pterin receptors and identifies a signaling pathway that is essential for multicellular development in Dictyostelium.

    PubMed Central

    Hadwiger, J A; Lee, S; Firtel, R A

    1994-01-01

    In this paper, we show that the G alpha subunit G alpha 4 couples to pterin receptors and identifies a signalling pathway that is essential for multicellular development in Dictyostelium. G alpha 4 is developmentally regulated, is essential for proper morphogenesis and spore production, and functions cell nonautonomously. We show that G alpha 4 is coupled to receptors (alpha FAR) that activate chemotaxis and adenylyl and guanylyl cyclases in response to folate during the early stages of development and to a late class of folate receptors (beta FAR) that have different specificities for pterins. G alpha 4 is preferentially expressed in cells randomly distributed within the aggregate that are a component of the anterior-like cell population, and it is not detectably expressed in prespore cells. Our results suggest that an endogenous factor, possibly a pterin, produced during multicellular development is a requisite signal for multicellular development, acting through G alpha 4. We propose that the G alpha 4-expressing cells function as a regulatory cell type controlling prespore cell fate, possibly in response to an endogenous pterin. Our results indicate that G alpha 4 and G alpha 2 have parallel functions in mediating cellular responses to folate (pterins) and cAMP, respectively. Images PMID:7937994

  5. Inference of Longevity-Related Genes from a Robust Coexpression Network of Seed Maturation Identifies Regulators Linking Seed Storability to Biotic Defense-Related Pathways.

    PubMed

    Righetti, Karima; Vu, Joseph Ly; Pelletier, Sandra; Vu, Benoit Ly; Glaab, Enrico; Lalanne, David; Pasha, Asher; Patel, Rohan V; Provart, Nicholas J; Verdier, Jerome; Leprince, Olivier; Buitink, Julia

    2015-10-01

    Seed longevity, the maintenance of viability during storage, is a crucial factor for preservation of genetic resources and ensuring proper seedling establishment and high crop yield. We used a systems biology approach to identify key genes regulating the acquisition of longevity during seed maturation of Medicago truncatula. Using 104 transcriptomes from seed developmental time courses obtained in five growth environments, we generated a robust, stable coexpression network (MatNet), thereby capturing the conserved backbone of maturation. Using a trait-based gene significance measure, a coexpression module related to the acquisition of longevity was inferred from MatNet. Comparative analysis of the maturation processes in M. truncatula and Arabidopsis thaliana seeds and mining Arabidopsis interaction databases revealed conserved connectivity for 87% of longevity module nodes between both species. Arabidopsis mutant screening for longevity and maturation phenotypes demonstrated high predictive power of the longevity cross-species network. Overrepresentation analysis of the network nodes indicated biological functions related to defense, light, and auxin. Characterization of defense-related wrky3 and nf-x1-like1 (nfxl1) transcription factor mutants demonstrated that these genes regulate some of the network nodes and exhibit impaired acquisition of longevity during maturation. These data suggest that seed longevity evolved by co-opting existing genetic pathways regulating the activation of defense against pathogens. PMID:26410298

  6. Integrated analysis of oral tongue squamous cell carcinoma identifies key variants and pathways linked to risk habits, HPV, clinical parameters and tumor recurrence

    PubMed Central

    Krishnan, Neeraja; Gupta, Saurabh; Palve, Vinayak; Varghese, Linu; Pattnaik, Swetansu; Jain, Prach; Khyriem, Costerwell; Hariharan, Arun; Dhas, Kunal; Nair, Jayalakshmi; Pareek, Manisha; Prasad, Venkatesh; Siddappa, Gangotri; Suresh, Amritha; Kekatpure, Vikram; Kuriakose, Moni; Panda, Binay

    2015-01-01

    Oral tongue squamous cell carcinomas (OTSCC) are a homogeneous group of tumors characterized by aggressive behavior, early spread to lymph nodes and a higher rate of regional failure. Additionally, the incidence of OTSCC among younger population (<50yrs) is on the rise; many of whom lack the typical associated risk factors of alcohol and/or tobacco exposure. We present data on single nucleotide variations (SNVs), indels, regions with loss of heterozygosity (LOH), and copy number variations (CNVs) from fifty-paired oral tongue primary tumors and link the significant somatic variants with clinical parameters, epidemiological factors including human papilloma virus (HPV) infection and tumor recurrence. Apart from the frequent somatic variants harbored in TP53, CASP8, RASA1, NOTCH and CDKN2A genes, significant amplifications and/or deletions were detected in chromosomes 6-9, and 11 in the tumors. Variants in CASP8 and CDKN2A were mutually exclusive. CDKN2A, PIK3CA, RASA1 and DMD variants were exclusively linked to smoking, chewing, HPV infection and tumor stage. We also performed a whole-genome gene expression study that identified matrix metalloproteases to be highly expressed in tumors and linked pathways involving arachidonic acid and NF-k-B to habits and distant metastasis, respectively. Functional knockdown studies in cell lines demonstrated the role of CASP8 in a HPV-negative OTSCC cell line. Finally, we identified a 38-gene minimal signature that predicts tumor recurrence using an ensemble machine-learning method. Taken together, this study links molecular signatures to various clinical and epidemiological factors in a homogeneous tumor population with a relatively high HPV prevalence. PMID:26834999

  7. Stability of open pathways

    PubMed Central

    Flach, Edward H.; Schnell, Santiago

    2010-01-01

    We consider the steady state of an open biochemical pathway, with controlled flow. Previously we have shown that the steady state of open enzyme catalysed reactions may be unstable, which discourages the application of the quasi-steady-state approximation (QSSA) (IEE Proc. Syst. Biol. 153 (2006) 187). Here we examine basic open biochemical pathway structures, to see the stability of their steady states. Following De Leenheer et al. (J. Math. Chem. 41 (2007) 295), we employ the Gershgorin circle theorem, which elegantly assesses stability. This is the key tool for our analysis. Once we have the linear stability matrix laid out in a suitable form, the application of the method is straightforward. We find that in open biochemical pathways, simple chains, branches and loops always have stable steady states. We conclude that simple open pathways are stable. PMID:20875827

  8. Integrated RNA-Seq and sRNA-Seq Analysis Identifies Chilling and Freezing Responsive Key Molecular Players and Pathways in Tea Plant (Camellia sinensis)

    PubMed Central

    Zheng, Chao; Zhao, Lei; Wang, Yu; Shen, Jiazhi; Zhang, Yinfei; Jia, Sisi; Li, Yusheng; Ding, Zhaotang

    2015-01-01

    Tea [Camellia sinensis (L) O. Kuntze, Theaceae] is one of the most popular non-alcoholic beverages worldwide. Cold stress is one of the most severe abiotic stresses that limit tea plants’ growth, survival and geographical distribution. However, the genetic regulatory network and signaling pathways involved in cold stress responses in tea plants remain unearthed. Using RNA-Seq, DGE and sRNA-Seq technologies, we performed an integrative analysis of miRNA and mRNA expression profiling and their regulatory network of tea plants under chilling (4℃) and freezing (-5℃) stress. Differentially expressed (DE) miRNA and mRNA profiles were obtained based on fold change analysis, miRNAs and target mRNAs were found to show both coherent and incoherent relationships in the regulatory network. Furthermore, we compared several key pathways (e.g., ‘Photosynthesis’), GO terms (e.g., ‘response to karrikin’) and transcriptional factors (TFs, e.g., DREB1b/CBF1) which were identified as involved in the early chilling and/or freezing response of tea plants. Intriguingly, we found that karrikins, a new group of plant growth regulators, and β-primeverosidase (BPR), a key enzyme functionally relevant with the formation of tea aroma might play an important role in both early chilling and freezing response of tea plants. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-Seq and sRNA-Seq analysis. This is the first study to simultaneously profile the expression patterns of both miRNAs and mRNAs on a genome-wide scale to elucidate the molecular mechanisms of early responses of tea plants to cold stress. In addition to gaining a deeper insight into the cold resistant characteristics of tea plants, we provide a good case study to analyse mRNA/miRNA expression and profiling of non-model plant species using next-generation sequencing technology. PMID:25901577

  9. Simulation studies in biochemical signaling and enzyme reactions

    NASA Astrophysics Data System (ADS)

    Nelatury, Sudarshan R.; Vagula, Mary C.

    2014-06-01

    Biochemical pathways characterize various biochemical reaction schemes that involve a set of species and the manner in which they are connected. Determination of schematics that represent these pathways is an important task in understanding metabolism and signal transduction. Examples of these Pathways are: DNA and protein synthesis, and production of several macro-molecules essential for cell survival. A sustained feedback mechanism arises in gene expression and production of mRNA that lead to protein synthesis if the protein so synthesized serves as a transcription factor and becomes a repressor of the gene expression. The cellular regulations are carried out through biochemical networks consisting of reactions and regulatory proteins. Systems biology is a relatively new area that attempts to describe the biochemical pathways analytically and develop reliable mathematical models for the pathways. A complete understanding of chemical reaction kinetics is prohibitively hard thanks to the nonlinear and highly complex mechanisms that regulate protein formation, but attempting to numerically solve some of the governing differential equations seems to offer significant insight about their biochemical picture. To validate these models, one can perform simple experiments in the lab. This paper introduces fundamental ideas in biochemical signaling and attempts to take first steps into the understanding of biochemical oscillations. Initially, the two-pool model of calcium is used to describe the dynamics behind the oscillations. Later we present some elementary results showing biochemical oscillations arising from solving differential equations of Elowitz and Leibler using MATLAB software.

  10. Serum Biochemical Phenotypes in the Domestic Dog.

    PubMed

    Chang, Yu-Mei; Hadox, Erin; Szladovits, Balazs; Garden, Oliver A

    2016-01-01

    The serum or plasma biochemical profile is essential in the diagnosis and monitoring of systemic disease in veterinary medicine, but current reference intervals typically take no account of breed-specific differences. Breed-specific hematological phenotypes have been documented in the domestic dog, but little has been published on serum biochemical phenotypes in this species. Serum biochemical profiles of dogs in which all measurements fell within the existing reference intervals were retrieved from a large veterinary database. Serum biochemical profiles from 3045 dogs were retrieved, of which 1495 had an accompanying normal glucose concentration. Sixty pure breeds plus a mixed breed control group were represented by at least 10 individuals. All analytes, except for sodium, chloride and glucose, showed variation with age. Total protein, globulin, potassium, chloride, creatinine, cholesterol, total bilirubin, ALT, CK, amylase, and lipase varied between sexes. Neutering status significantly impacted all analytes except albumin, sodium, calcium, urea, and glucose. Principal component analysis of serum biochemical data revealed 36 pure breeds with distinctive phenotypes. Furthermore, comparative analysis identified 23 breeds with significant differences from the mixed breed group in all biochemical analytes except urea and glucose. Eighteen breeds were identified by both principal component and comparative analysis. Tentative reference intervals were generated for breeds with a distinctive phenotype identified by comparative analysis and represented by at least 120 individuals. This is the first large-scale analysis of breed-specific serum biochemical phenotypes in the domestic dog and highlights potential genetic components of biochemical traits in this species. PMID:26919479

  11. Serum Biochemical Phenotypes in the Domestic Dog

    PubMed Central

    Chang, Yu-Mei; Hadox, Erin; Szladovits, Balazs; Garden, Oliver A.

    2016-01-01

    The serum or plasma biochemical profile is essential in the diagnosis and monitoring of systemic disease in veterinary medicine, but current reference intervals typically take no account of breed-specific differences. Breed-specific hematological phenotypes have been documented in the domestic dog, but little has been published on serum biochemical phenotypes in this species. Serum biochemical profiles of dogs in which all measurements fell within the existing reference intervals were retrieved from a large veterinary database. Serum biochemical profiles from 3045 dogs were retrieved, of which 1495 had an accompanying normal glucose concentration. Sixty pure breeds plus a mixed breed control group were represented by at least 10 individuals. All analytes, except for sodium, chloride and glucose, showed variation with age. Total protein, globulin, potassium, chloride, creatinine, cholesterol, total bilirubin, ALT, CK, amylase, and lipase varied between sexes. Neutering status significantly impacted all analytes except albumin, sodium, calcium, urea, and glucose. Principal component analysis of serum biochemical data revealed 36 pure breeds with distinctive phenotypes. Furthermore, comparative analysis identified 23 breeds with significant differences from the mixed breed group in all biochemical analytes except urea and glucose. Eighteen breeds were identified by both principal component and comparative analysis. Tentative reference intervals were generated for breeds with a distinctive phenotype identified by comparative analysis and represented by at least 120 individuals. This is the first large-scale analysis of breed-specific serum biochemical phenotypes in the domestic dog and highlights potential genetic components of biochemical traits in this species. PMID:26919479

  12. A combined remote sensing and modeling based approach to identify sustainable pathways for urban and peri-urban agriculture in China

    NASA Astrophysics Data System (ADS)

    Wattenbach, M.; Delgado, J. M.; Roessner, S.; Bochow, M.; Güntner, A.; Kropp, J.; Cantu Ros, A. G.; Hattermann, F.; Kolbe, T.; Sodoudi, S.; Cubasch, U. Ulrich; Zeitz, J.; Ross, L.; Böckel, K.; Fang, C.; Bo, L.; Pan, G.

    2012-04-01

    As the world's biggest economy, China is becoming the biggest consumer of resources globally. Given this trend, the over-proportional fast increase in urbanization presents China with fundamental problems. Among the most urgent ones is the increasing loss of agricultural land as urbanization takes place in the most productive regions along the coast. The latter is being responsible for a shift in agriculture production towards climatically less favorable areas. At the same time, the loss of green areas in and around growing cities is increasing the effect of the urban heat island. The perception of the potential risks related to this phenomenon, in the context of climate change, has led the Shanghai city administration to increase its urban-greening efforts, expanding the per capita area of green from 1m2 in 1990 to 12.5m2 in 2008. In this context, this paper aims at identifying the influence of urban and peri-urban agriculture (UPA) on the sustainability of the urban regions of Shanghai and Nanjing. In particular, it focuses on the effects of UPA on the greenhouse gas (GHG) emissions, soil nutrients and water balances, local climate and the structure and functions of the urbanized areas. We propose an interdisciplinary framework combining remote sensing, model simulations and GHG field observations and targeted at identifying "win-win" strategies for sustainable planning pathways showing high potentials for UPA. The framework is based on spatial scenario modeling, automatic classification of urban structure types and on a prototype of a high-quality spatial database consisting of a 3D city model. Dynamic boundary conditions for climate and urban development are provided by state of the art models. These approaches meet the needs of stakeholders and planners in China. A special emphasis is put on interdependencies between small holder farming in the urban and peri-urban zone and climate change adaptation and mitigation strategies focusing on improved management of local water and nutrient cycles. The whole database generated will be structured and made accessible for planners and stakeholders in the form of a 3D city visualization model.

  13. Logic models of pathway biology.

    PubMed

    Watterson, Steven; Marshall, Stephen; Ghazal, Peter

    2008-05-01

    Living systems seamlessly perform complex information processing and control tasks using combinatorially complex sets of biochemical reactions. Drugs that therapeutically modulate the biological processes of disease are developed using single protein target strategies, often with limited knowledge of the complex underlying role of the targets. Approaches that attempt to consider the combinatorial complexity from the outset might help identify any causal relationships that could lead to undesirable or adverse side effects earlier in the development pipeline. Such approaches, in particular logic methodologies, might also aid pathway selection and multiple target strategies during the drug discovery phase. Here, we describe the use of logic as a tractable and informative approach to modelling biological pathways that can allow us to improve our understanding of the dependencies in complex biological processes. PMID:18468563

  14. A Global Genomic and Genetic Strategy to Identify, Validate and Use Gene Signatures of Xenobiotic-Responsive Transcription Factors in Prediction of Pathway Activation in the Mouse Liver

    EPA Science Inventory

    Many drugs and environmentally-relevant chemicals activate xenobiotic-responsive transcription factors. Identification of target genes of these factors would be useful in predicting pathway activation in in vitro chemical screening as well as their involvement in disease states. ...

  15. Proteomics and pathway analysis identifies JNK signaling as critical for high linear energy transfer radiation-induced apoptosis in non-small lung cancer cells.

    PubMed

    Sthl, Sara; Fung, Eva; Adams, Christopher; Lengqvist, Johan; Mrk, Birgitta; Stenerlw, Bo; Lewensohn, Rolf; Lehti, Janne; Zubarev, Roman; Viktorsson, Kristina

    2009-05-01

    During the past decade, we have witnessed an explosive increase in generation of large proteomics data sets, not least in cancer research. There is a growing need to extract and correctly interpret information from such data sets to generate biologically relevant hypotheses. A pathway search engine (PSE) has recently been developed as a novel tool intended to meet these requirements. Ionizing radiation (IR) is an anticancer treatment modality that triggers multiple signal transduction networks. In this work, we show that high linear energy transfer (LET) IR induces apoptosis in a non-small cell lung cancer cell line, U-1810, whereas low LET IR does not. PSE was applied to study changes in pathway status between high and low LET IR to find pathway candidates of importance for high LET-induced apoptosis. Such pathways are potential clinical targets, and they were further validated in vitro. We used an unsupervised shotgun proteomics approach where high resolution mass spectrometry coupled to nanoflow liquid chromatography determined the identity and relative abundance of expressed proteins. Based on the proteomics data, PSE suggested the JNK pathway (p = 6.10(-6)) as a key event in response to high LET IR. In addition, the Fas pathway was found to be activated (p = 3.10(-5)) and the p38 pathway was found to be deactivated (p = 0.001) compared with untreated cells. Antibody-based analyses confirmed that high LET IR caused an increase in phosphorylation of JNK. Moreover pharmacological inhibition of JNK blocked high LET-induced apoptotic signaling. In contrast, neither an activation of p38 nor a role for p38 in high LET IR-induced apoptotic signaling was found. We conclude that, in contrast to conventional low LET IR, high LET IR can trigger activation of the JNK pathway, which in turn is critical for induction of apoptosis in these cells. Thus PSE predictions were largely confirmed, and PSE was proven to be a useful hypothesis-generating tool. PMID:19168796

  16. Applying meta-pathway analyses through metagenomics to identify the functional properties of the major bacterial communities of a single spontaneous cocoa bean fermentation process sample.

    PubMed

    Illeghems, Koen; Weckx, Stefan; De Vuyst, Luc

    2015-09-01

    A high-resolution functional metagenomic analysis of a representative single sample of a Brazilian spontaneous cocoa bean fermentation process was carried out to gain insight into its bacterial community functioning. By reconstruction of microbial meta-pathways based on metagenomic data, the current knowledge about the metabolic capabilities of bacterial members involved in the cocoa bean fermentation ecosystem was extended. Functional meta-pathway analysis revealed the distribution of the metabolic pathways between the bacterial members involved. The metabolic capabilities of the lactic acid bacteria present were most associated with the heterolactic fermentation and citrate assimilation pathways. The role of Enterobacteriaceae in the conversion of substrates was shown through the use of the mixed-acid fermentation and methylglyoxal detoxification pathways. Furthermore, several other potential functional roles for Enterobacteriaceae were indicated, such as pectinolysis and citrate assimilation. Concerning acetic acid bacteria, metabolic pathways were partially reconstructed, in particular those related to responses toward stress, explaining their metabolic activities during cocoa bean fermentation processes. Further, the in-depth metagenomic analysis unveiled functionalities involved in bacterial competitiveness, such as the occurrence of CRISPRs and potential bacteriocin production. Finally, comparative analysis of the metagenomic data with bacterial genomes of cocoa bean fermentation isolates revealed the applicability of the selected strains as functional starter cultures. PMID:25998815

  17. The Use of Item Analysis for Improvement of Biochemical Teaching

    ERIC Educational Resources Information Center

    Nagata, Ryoichi

    2004-01-01

    Item analysis was used to find out which biochemical explanations need to be improved in biochemical teaching, not which items are to be discarded, improved, or reusable in biochemical examinations. The analysis revealed the basic facts of which less able students had more misunderstanding than able students. Identifying these basic facts helps

  18. THE PHYTOCHROMES: A BIOCHEMICAL MECHANISM OF SIGNALING IN SIGHT?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biochemical mechanism by which the phytochrome family of plant sensory photoreceptors transmit perceived informational light signals downstream to transduction pathway components is undetermined. However, the recent sequencing of the entire genome of the cyanobacterium Synechocystis has reveale...

  19. Exchange reactions catalyzed by group-transferring enzymes oppose the quantitation and the unravelling of the identify of the pentose pathway.

    PubMed

    Flanigan, I; Collins, J G; Arora, K K; MacLeod, J K; Williams, J F

    1993-04-01

    1. The distributions and rates of transfer of carbon isotopes from a selection of specifically labelled ketosugar-phosphate substrates by exchange reactions catalyzed by the pentose and photosynthetic carbon-reduction-pathway group-transferring enzymes transketolase, transaldolase and aldolase have been measured using 13C-NMR spectroscopy. 2. The rates of these exchange reactions were 5, 4 and 1.5 mumol min-1 mg-1 for transketolase exchange, transaldolase exchange and aldolase exchange, respectively. 3. A comparison of the exchange capacities contributed by the activities of these enzymes in three in vitro liver preparations with the maximum non-oxidative pentose pathway flux rates of the preparations shows that transketolase and aldolase exchanges exceeded flux by 9-19 times in liver cytosol and acetone powder enzyme preparations and by 5 times in hepatocytes. Transaldolase was less effective in the comparison of exchange versus flux rates: transaldolase exchange exceeded flux by 1.6 and 5 in catalysis by liver cytosol and acetone powder preparations, respectively, but was only 0.6 times the flux in hepatocytes. 4. Values of group enzyme exchange and pathway flux rates in the above three preparations are important because of the feature role of liver and of these particular preparations in the establishment, elucidation and measurement of a proposed reaction scheme for the fat-cell-type pentose pathway in biochemistry. 5. It is the claim of this paper that the excess of exchange rate activity (particularly transketolase exchange) over pathway flux will overturn attempts to unravel, using isotopically labelled sugar substrates, the identity, reaction sequence and quantitative contribution of the pentose pathway to glucose metabolism. 6. The transketolase exchange reactions relative to the pentose pathway flux rates in normal, regenerating and foetal liver, Morris hepatomas, mammary carcinoma, melanoma, colonic epithelium, spinach chloroplasts and epididymal fat tissue show that transketolase exchange may exceed flux in these tissues by factors ranging over 5-600 times. 7. The confusion of pentose pathway theory by the effects of transketolase exchange action is illustrated by the 13C-NMR spectrum of the hexose 6-phosphate products of ribose 5-phosphate dissimilation, formed after 30 min of liver enzyme action, and shows 13C-labelling in carbons 1 and 3 of glucose 6-phosphate with ratios which range over 2.1-6.4 rather than the mandatory value of 2 which is imposed by the theoretical mechanism of the pathway. PMID:8477719

  20. Metabolomic profile of glycolysis and the pentose phosphate pathway identifies the central role of glucose-6-phosphate dehydrogenase in clear cell-renal cell carcinoma

    PubMed Central

    Sanguedolce, Francesca; Cagiano, Simona; Bufo, Pantaleo; Lastilla, Gaetano; Maiorano, Eugenio; Ribatti, Domenico; Giglio, Andrea; Serino, Grazia; Vavallo, Antonio; Bettocchi, Carlo; Selvaggi, Francesco Paolo; Battaglia, Michele; Ditonno, Pasquale

    2015-01-01

    The analysis of cancer metabolome has shown that proliferating tumor cells require a large quantities of different nutrients in order to support their high rate of proliferation. In this study we analyzed the metabolic profile of glycolysis and the pentose phosphate pathway (PPP) in human clear cell-renal cell carcinoma (ccRCC) and evaluate the role of these pathways in sustaining cell proliferation, maintenance of NADPH levels, and production of reactive oxygen species (ROS). Metabolomic analysis showed a clear signature of increased glucose uptake and utilization in ccRCC tumor samples. Elevated levels of glucose-6-phosphate dehydrogenase (G6PDH) in association with higher levels of PPP-derived metabolites, suggested a prominent role of this pathway in RCC-associated metabolic alterations. G6PDH inhibition, caused a significant decrease in cancer cell survival, a decrease in NADPH levels, and an increased production of ROS, suggesting that the PPP plays an important role in the regulation of ccRCC redox homeostasis. Patients with high levels of glycolytic enzymes had reduced progression-free and cancer-specific survivals as compared to subjects with low levels. Our data suggest that oncogenic signaling pathways may promote ccRCC through rerouting the sugar metabolism. Blocking the flux through this pathway may serve as a novel therapeutic target. PMID:25945836

  1. Endothelial cells and cathepsins: Biochemical and biomechanical regulation.

    PubMed

    Platt, Manu O; Shockey, W Andrew

    2016-03-01

    Cathepsins are mechanosensitive proteases that are regulated not only by biochemical factors, but are also responsive to biomechanical forces in the cardiovascular system that regulate their expression and activity to participate in cardiovascular tissue remodeling. Their elastinolytic and collagenolytic activity have been implicated in atherosclerosis, abdominal aortic aneurysms, and in heart valve disease, all of which are lined by endothelial cells that are the mechanosensitive monolayer of cells that sense and respond to fluid shear stress as the blood flows across the surfaces of the arteries and valve leaflets. Inflammatory cytokine signaling is integrated with biomechanical signaling pathways by the endothelial cells to transcribe, translate, and activate either the cysteine cathepsins to remodel the tissue or to express their inhibitors to maintain healthy cardiovascular tissue structure. Other cardiovascular diseases should now be included in the study of the cysteine cathepsin activation because of the additional biochemical cues they provide that merges with the already existing hemodynamics driving cardiovascular disease. Sickle cell disease causes a chronic inflammation including elevated TNFα and increased numbers of circulating monocytes that alter the biochemical stimulation while the more viscous red blood cells due to the sickling of hemoglobin alters the hemodynamics and is associated with accelerated elastin remodeling causing pediatric strokes. HIV-mediated cardiovascular disease also occurs earlier in than the broader population and the influence of HIV-proteins and antiretrovirals on endothelial cells must be considered to understand these accelerated mechanisms in order to identify new therapeutic targets for prevention. PMID:26458976

  2. CSNK1A1 and Gli2 as Novel Targets Identified Through an Integrative Analysis of Gene Expression Data, Protein-Protein Interaction and Pathways Networks in Glioblastoma Tumors: Can These Two Be Antagonistic Proteins?

    PubMed Central

    Mishra, Seema

    2014-01-01

    Glioblastoma (GBM) is the malignant form of glioma, and the interplay of different pathways working in concert in GBM development and progression needs to be fully understood. Wnt signaling and sonic hedgehog (SHH) signaling pathways, having basic similarities, are among the major pathways aberrantly activated in GBM, and hence, need to be targeted. It becomes imperative, therefore, to explore the functioning of these pathways in context of each other in GBM. An integrative approach may help provide new biological insights, as well as solve the problem of identifying common drug targets for simultaneous targeting of these pathways. The beauty of this approach is that it can recapitulate several known facts, as well as decipher new emerging patterns, identifying those targets that could be missed when relying on one type of data at a time. This approach can be easily extended to other systems to discover key patterns in the functioning of signaling molecules. Studies were designed to assess the relationship between significant differential expression of genes of the Wnt (Wnt/?-catenin canonical and Wnt non-canonical) and SHH signaling pathways and their connectivity patterns in interaction and signaling networks. Further, the aim was to decipher underlying mechanistic patterns that may be involved in a more specific way and to generate a ranked list of genes that can be used as markers or drug targets. These studies predict that Wnt pathway plays a relatively more pro-active role than the SHH pathway in GBM. Further, CTNNB1, CSNK1A1, and Gli2 proteins may act as key drug targets common to these pathways. While CTNNB1 is a widely studied molecule in the context of GBM, the likely roles of CSNK1A1 and Gli2 are found to be relatively novel. It is surmised that Gli2 may be antagonistic to CSNK1A1, preventing the phosphorylation of CTNNB1 and SMO proteins in Wnt and SHH signaling pathway, respectively, by CSNK1A1, and thereby, aberrant activation. New insights into the possible behavior of these pathway molecules relative to each other in GBM reveal some key interesting patterns. PMID:25374452

  3. A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein.

    PubMed Central

    Rebay, I; Chen, F; Hsiao, F; Kolodziej, P A; Kuang, B H; Laverty, T; Suh, C; Voas, M; Williams, A; Rubin, G M

    2000-01-01

    The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Previously, we have shown that ectopic expression of a constitutively active protein (yan(ACT)) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, we have performed a genetic screen to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan(ACT). Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes we recovered are four RTK pathway components, rolled (MAPK), son-of-sevenless, Star, and pointed, and two genes, eyes absent and string, that have not been implicated previously in RTK signaling events. We also isolated mutations in five previously uncharacterized genes, one of which, split ends, we have characterized molecularly and have shown to encode a member of the RRM family of RNA-binding proteins. PMID:10655223

  4. Symptoms of Major Depressive Disorder Subsequent to Child Maltreatment: Examining Change across Multiple Levels of Analysis to Identify Transdiagnostic Risk Pathways

    PubMed Central

    Shenk, Chad E.; Griffin, Amanda M.; O’Donnell, Kieran J.

    2016-01-01

    Major depressive disorder (MDD) is a prevalent psychiatric condition in the child maltreatment population. However, not all children who have been maltreated will develop MDD or MDD symptoms, suggesting the presence of unique risk pathways that explain how certain children develop MDD symptoms when others do not. The current study tested several candidate risk pathways to MDD symptoms following child maltreatment: 1) neuroendocrine, 2) autonomic, 3) affective, and 4) emotion regulation. Female adolescents (N=110; Age range: 14–19) were recruited into a substantiated child maltreatment or comparison condition and completed a laboratory stressor, saliva samples, and measures of emotion regulation, negative affect, and MDD symptoms. MDD symptoms were reassessed eighteen months later. Mediational modeling revealed that emotion regulation was the only significant indirect effect of the relationship between child maltreatment and subsequent MDD symptoms, demonstrating that children exposed to maltreatment had greater difficulties managing affective states that in turn led to more severe MDD symptoms. These results highlight the importance of emotion dysregulation as a central risk pathway to MDD following child maltreatment. Areas of future research and implications for optimizing prevention and clinical intervention through the direct targeting of transdiagnostic risk pathways are discussed. PMID:26535940

  5. Robust simplifications of multiscale biochemical networks

    PubMed Central

    Radulescu, Ovidiu; Gorban, Alexander N; Zinovyev, Andrei; Lilienbaum, Alain

    2008-01-01

    Background Cellular processes such as metabolism, decision making in development and differentiation, signalling, etc., can be modeled as large networks of biochemical reactions. In order to understand the functioning of these systems, there is a strong need for general model reduction techniques allowing to simplify models without loosing their main properties. In systems biology we also need to compare models or to couple them as parts of larger models. In these situations reduction to a common level of complexity is needed. Results We propose a systematic treatment of model reduction of multiscale biochemical networks. First, we consider linear kinetic models, which appear as "pseudo-monomolecular" subsystems of multiscale nonlinear reaction networks. For such linear models, we propose a reduction algorithm which is based on a generalized theory of the limiting step that we have developed in [1]. Second, for non-linear systems we develop an algorithm based on dominant solutions of quasi-stationarity equations. For oscillating systems, quasi-stationarity and averaging are combined to eliminate time scales much faster and much slower than the period of the oscillations. In all cases, we obtain robust simplifications and also identify the critical parameters of the model. The methods are demonstrated for simple